Middleton's Allergy, Principles and Practice [Mosby, 6ed.] 0721682332

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MIDDLETON'S ALLERGY Principles & Practice Sixth edition With more than 683 illustrations and 35 color plates Edited by

N. Franklin Adkinson Jr. MD Professor of Medicine and Training Program Director, Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, The Hopkins Bayview Medical Campus, Baltimore, Maryland

John W. Yunginger MD Emeritus Consultant in Pediatrics and Internal Medicine (Allergy), Mayo Clinic and Foundation, Emeritus Professor of Pediatrics, Mayo Medical School, Rochester, Minnesota William W. Busse MD Charles E. Reed Professor of Medicine, University of Wisconsin, Hospital and Clinics, Head, Allergy and Clinical Immunology, Department of Medicine, University of Wisconsin School of Medicine, Madison, Wisconsin Bruce S. Bochner MD Professor of Medicine and Director, Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, The Hopkins Bayview Medical Campus, Baltimore, Maryland Stephen T. Holgate MD, DSc, FRCP, FMed Sci MRC Clinical Professor of Immunopharmacology, Southampton General Hospital School of Medicine, University of Southampton, Southampton, United Kingdom F. Estelle R. Simons MD, FRCPC Professor and Head, Section of Allergy and Clinical Immunology, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, Manitoba, Canada

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The Curtis Center Independence Square West Philadelphia, Pennsylvania 19106 MIDDLETON'S ALLERGY PRINCIPLES & PRACTICE, 6th EDITION 0-323-01425-9 Copyright © 2003, Mosby, Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Health Sciences Rights Department in Philadelphia, PA, USA: phone: (+1) 215 238 7869, fax: (+1) 215 238 2239, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://www.elsevier.com), by selecting ‘Customer Support’ and then ‘Obtaining Permissions’.

NOTICE Medicine is an ever-changing field. Standard safety precautions must be followed, but as new research and clinical experience broaden our knowledge, changes in treatment and drug therapy may become necessary or appropriate. Readers are advised to check the most current product information provided by the manufacturer of each drug to be administered to verify the recommended dose, the method and duration of administration, and contraindications. It is the responsibility of the treating physician, relying on experience and knowledge of the patient, to determine dosages and the best treatment for each individual patient. Neither the publisher nor the editors assume any liability for any injury and/or damage to persons or property arising from this publication.

Previous editions copyrighted 1978, 1983, 1988, 1993, 1998 International Standard Book Number 0-323-01425-9 Acquisitions Editor: Catherine Carroll Developmental Editor: Jennifer Shreiner Publishing Services Manager: Patricia Tannian Senior Project Manager: Anne Altepeter Book Design Manager: Gail Morey Hudson Cover Design: Jen Brockett Printed in the United States of America Last digit is the print number: 9 8 7 6 5 4 3 2 1

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Contributors

N. Franklin Adkinson Jr. MD

Professor of Medicine and Training Program Director, Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, The Hopkins Bayview Medical Campus, Baltimore, Maryland, USA Cheryl Adolphson MS Senior Science Writer, Department of Immunology, Mayo Clinic and Foundation, Rochester, Minnesota, USA Rafeul Alam MD, PhD Veda and Chauncey Ritter Chair in Immunology, Professor and Director, Division of Allergy and Immunology, National Jewish Medical and Research Center, Denver, Colorado, USA Philip W. Askenase MD Chief, Section of Allergy and Clinical Immunology, Internal Medicine, Yale University School of Medicine, Attending Physician, Yale–New Haven Medical Center, New Haven, Connecticut, USA Bharat Awsare MD Fellow, Division of Critical Care, Pulmonary, Allergic, and Immunologic Diseases, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA

Claus Bachert MD, PhD Professor of Surgery, Ear, Nose, and Throat Department, University Hospital Ghent, Ghent, Belgium Mark Ballow MD Professor of Pediatrics, State University of New York, Buffalo School of Medicine and Biomedical Sciences, Director, Allergy/Clinical Immunology Fellowship Training Program Chief, Allergy, Clinical Immunology, and Pediatric Rheumatology Division, Children's Hospital of Buffalo, Buffalo, New York, USA Peter J. Barnes DM, DSc, FRCP Professor of Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom Neal P. Barney MD Associate Professor of Ophthalmology, Ophthalmology Department, University of Wisconsin, Associate Professor of Medicine and Visual Sciences, Ophthalmology and Visual Sciences Department, University of Wisconsin Hospital and Clinics, Madison, Wisconsin, USA

Vikas Batra MD Fellow, Division of Critical Care, Pulmonary, Allergic, and Immunologic Diseases, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Bruce S. Bochner MD Professor of Medicine and Director, Division of Allergy and Clinical Immunology, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Mark Boguniewicz MD Senior Faculty Member, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, USA Larry Borish MD Associate Professor of Medicine, Asthma and Allergic Disease Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA Louis-Philipe Boulet MD, FRCPC, FCCP Professor of Medicine, Université Laval, Respirologist, Centre de Pneumologie,

Institut de Cardiologie et de Pneumologie de L'Université Laval, Sainte-Foy, Quebec, Canada Jean Bousquet MD, PhD Professor of Respiratory Medicine, Université Montpellier, Professor, CHU Montpellier, Montpellier, France William M.L. Bowerfind MD Research Fellow, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

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David H. Broide MD, ChB Professor of Medicine, Department of Medicine, University of California—San Diego, La Jolla, California, USA Rebecca Hatcher Buckley MD J. Buren Sidbury Professor of Pediatrics, Department of Pediatrics and Immunology, Duke University Medical Center, Chief, Division of Allergy and Immunology, Department of Pediatrics,

Duke University Medical Center, Durham, North Carolina, USA Robert K. Bush MD Professor of Medicine, University of Wisconsin—Madison, Chief of Allergy, William S. Middleton VA Hospital, Madison, Wisconsin, USA William W. Busse MD Professor of Medicine, University of Wisconsin Hospital and Clinics, Head, Allergy and Clinical Immunology, Department of Medicine, University of Wisconsin School of Medicine, Madison, Wisconsin William J. Calhoun MD Associate Professor of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA Lisa Cameron PhD Postdoctoral Fellow, Arizona Respiratory Center, Functional Genomic Lab, University of Arizona, Tucson, Arizona, USA Brendan J. Canning PhD

Associate Professor of Medicine, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Margaretha L. Casselbrant MD, PhD Professor of Otolaryngology, University of Pittsburgh School of Medicine, Director of Clinical Research and Education, Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania, USA Pota Christodoulopoulos MSc Postdoctoral Fellow, Meakins Christie Laboratories, McGill University, Montreal, Quebec, Canada Martin K. Church M PHARM, PhD, DSc Professor of Experimental Immunopharmacology, Dermatopharmacology Unit, University of Southampton, Southampton, Hampshire, United Kingdom Lauren Cohn MD Assistant Professor, Department of Internal Medicine, Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, Attending Physician,

Yale–New Haven Medical Center, New Haven, Connecticut, USA Ronina Covar MD Staff Physician, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, USA Pascal Demoly MD Associate Professor, Allergology and Respiratory Medicine, Université Montpellier, Associate Professor, CHU Montpellier, Montpellier, France William K. Dolen MD Professor of Pediatrics and Medicine, Allergy-Immunology Section, Medical College of Georgia, Augusta, Georgia, USA Ian Dransfield MD Reader, Centre for Inflammation Research, Edinburgh University Medical School, Edinburgh, United Kingdom Ronald Eccles DSc Professor, Common Cold Centre,

Cardiff University, Cardiff, United Kingdom Alan M. Edwards MB, BChur Clinical Assistant, The David Hide Asthma and Allergy Research Centre, St. Mary's Hospital, Newport, Isle of Wight, United Kingdom Robert E. Esch PhD Vice President, Research and Development, Greer Laboratories, Inc., Lenoir, North Carolina, USA John V. Fahy MD Associate Professor of Medicine, Department of Medicine and the Cardiovascular Research Institute, University of California—San Francisco, Moffitt Long Hospital, Division of Pulmonary and Critical Care Medicine, University of California—San Francisco, San Francisco, California, USA Reuben Falkoff MD, PhD Staff Allergist, Kaiser Permanente Medical Center, San Diego, California, USA Jordan N. Fink MD Professor of Pediatrics and Medicine (Allergy/Immunology), Departments of Pediatrics and Medicine,

Medical College of Wisconsin, Milwaukee, Wisconsin, USA

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James E. Fish MD Adjunct Professor, Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania, USA, Senior Distinguished Scientist, Aventis, Inc., Bridgewater, New Jersey, USA Thomas A. Fleisher MD Clinical Professor of Pediatrics, Department of Pediatrics, Uniformed Services University of the Health Sciences, Chief, Department of Laboratory Medicine, Warren G. Magnusen Clinical Center, National Institutes of Health, Bethesda, Maryland, USA Michael M. Frank MD Samuel L. Katz Professor and Chairman of Pediatrics and Professor of Medicine and Immunology, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA

Allison D. Fryer PhD Associate Professor of Physiology, Environmental Health Sciences, The Johns Hopkins University School of Public Health, Baltimore, Maryland, USA Deborah Gentile MD Division of Allergy, Asthma, and Immunology, Department of Pediatrics, Allegheny General Hospital, Pittsburgh, Pennsylvania, USA James E. Gern MD Associate Professor of Pediatrics, Department of Pediatrics, University of Wisconsin Medical School, Madison, Wisconsin, USA Anita Tartell Gewurz MD Professor, Department of Immunology/Microbiology, Internal Medicine, and Pediatrics, Rush Medical College, Senior Attending Physician, Immunology/Microbiology, Internal Medicine and Pediatrics, Rush-Presbyterian St. Luke's Medical Center, Voluntary Attending Physician, Department of Pediatrics, Cook County Hospital, Chicago, Illinois, USA Henry Gewurz MD Professor and Chairman,

Department of Immunology/Microbiology, Professor, Pediatrics and Medicine, Rush Medical College, Senior Attending Physician and Chairman, Immunology/Microbiology, Rush-Presbyterian St. Luke's Medical Center, Chicago, Illinois, USA Gerald J. Gleich MD Professor of Dermatology and Medicine, University of Utah Health Sciences Center, Salt Lake City, Utah, USA Edward J. Goetzl MD Professor of Medicine and Immunology, University of California—San Francisco, Moffitt-Long Hospitals, San Francisco, California, USA David B.K. Golden MD Associate Professor of Medicine, The Johns Hopkins University School of Medicine, Division of Allergy and Clinical Immunology, Director, Allergy Division, Department of Medicine, Sinai Hospital, Baltimore, Maryland, USA Frank M. Graziano MD, PhD Professor of Medicine, University of Wisconsin Medical School, University of Wisconsin Hospital and Clinics,

Madison, Wisconsin, USA Paul A. Greenberger MD Professor of Medicine, Division of Allergy-Immunology, Department of Medicine, Northwestern University Medical School, Attending Physician, Northwestern Memorial Hospital, Chicago, Illinois, USA Sudhir Gupta MD, PhD, MACP, FRCP (C) Professor of Medicine, Pathology, Neurology, and Microbiology and Molecular Genetics, Chief, Basic and Clinical Immunology, University of California—Irvine, Staff Physician, Department of Medicine, University of California, Irvine Medical Center, Orange, California, USA Qutayba Hamid MD, PhD James McGill Professor of Medicine, Meakins-Christie Laboratories, McGill University, Staff Physician, Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada Robert G. Hamilton PhD, D.ABMLI Professor of Medicine and Pathology, Department of Medicine, Division of Allergy and Clinical Immunology,

Johns Hopkins University School of Medicine, Director, Johns Hopkins Dermatology, Allergy, and Clinical Immunology Reference Laboratory, Baltimore, Maryland, USA Christopher Haslett BSc, MB, ChB, MRCP(UK), FRCP, FMEDSci, FRS Professor, MRC Centre for Inflammation Research, University of Edinburgh Medical School, Professor, Respiratory Medicine, New Royal Infirmary of Edinburgh, Edinburgh, United Kingdom Susan L. Hefle PhD Assistant Professor and Co-Director, Ford Allergy Research and Resource Program, University of Nebraska, Lincoln, Nebraska William R. Henderson Jr. MD Professor of Medicine and Head, Allergy Section, Department of Medicine, University of Washington, Director, Asthma and Allergy Clinics, Department of Medicine, University of Washington Medical Center, Seattle, Washington, USA

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C. Garren Hester BSc Research Specialist, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA Clement P. Hoffman MD Assistant Clinical Professor, Department of Reproductive Medicine, University of California—San Diego School of Medicine, Staff Physician, Department of Obstetrics and Gynecology, Kaiser Foundation Hospital, San Diego, California, USA Stephen T. Holgate MD, DSc, FRCP Professor, Respiratory Cell and Molecular Biology, University of Southampton School of Medicine, Southampton, Hampshire, United Kingdom Julian M. Hopkin MB, MD, MSc, MRCP Professor, Experimental Medicine Unit, University of Wales—Swansea, Swansea, United Kingdom Peter H. Howarth MD Reader in Medicine, Respiratory Cell and Molecular Biology Department, Southampton University, Consultant Physician and Head of Medical Specialties,

Allergy and Respiratory Medicine, Southampton General Hospital, Southampton, United Kingdom Thomas F. Huff PhD Vice Provost for Life Sciences and Professor of Microbiology and Immunology, Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, Virginia, USA Charles G. Irvin PhD Professor, Associate Chairman of Research Affairs, Director, Vermont Lung Center, University of Vermont, Department of Medicine-Pulmonary and Critical Care Division, Fletcher Allen Health Care, Burlington, Vermont, USA David B. Jacoby MD Associate Professor of Medicine, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Elizabeth F. Jaffe MD, PhD Timberlane Allergy and Asthma Associates, Clinical Assistant Professor, Department of Pediatrics, University of Vermont School of Medicine, Burlington, Vermont, USA

Henk M. Jansen PROFESSOR OF MEDICINE Professor, Department of Pulmonology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands Nizar N. Jarjour MD Associate Professor, Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Wisconsin, Madison, Wisconsin, USA Peter Jeffery MSc, PhD, DSc (Medicine), FRCPath Professor of Lung Pathology, Department of Gene Therapy, Imperial College, London, London, United Kingdom Allen P. Kaplan MD Professor of Medicine, Medical University of South Carolina, University Hospital of the Medical University of South Carolina, Charleston, South Carolina, USA Martien L. Kapsenberg PhD Professor, Histology and Cell Biology, Academic Medical Center of the University of Amsterdam, Amsterdam, The Netherlands

Arnon M. Katz MD, FRCPC Instructor, Department of Dermatology, University of Toronto, Department of Medicine, Division of Dermatology, Sunnybrook and Women's College Health Science Centre, Toronto, Ontario, Canada Arthur Kavanaugh MD Associate Professor, Department of Internal Medicine, University of California, San Diego, San Diego, California, USA H. William Kelly PharmD Professor of Pediatrics (Research), Department of Pediatrics, University of New Mexico Health Sciences Center, Professor, Clinical Research, Children's Hospital of New Mexico, Albuquerque, New Mexico, USA John M. Kelso MD Head, Allergy Division, Naval Medical Center, Clinical Associate Professor, Department of Pediatrics and Internal Medicine, University of California, San Diego, San Diego, California, USA

Hirohito Kita MD Professor of Medicine, Associate Professor of Immunology, Mayo Medical and Graduate Schools of Medicine, Consultant, Mayo Clinic and Foundation, Rochester, Minnesota, USA Kenneth S. Knox MD Assistant Professor of Medicine, Division of Pulmonary, Allergy, Critical Care, and Occupational Medicine, Indiana University Medical Center, Indianapolis, Indiana, USA Mario Kontolemos BSc Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada

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Aili L. Lazaar MD Assistant Professor of Medicine, Medicine/Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania School of Medicine, Attending Physician, Medicine/Pulmonary, Allergy, and Critical Care Physician, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA Kristin M. Leiferman MD

Professor of Dermatology, University of Utah Health Sciences Center, Salt Lake City, Utah, USA Robert F. Lemanske Jr. MD Professor of Pediatrics and Medicine, University of Wisconsin Medical School and Hospital, Madison, Wisconsin, USA Donald Y.M. Leung MD, PhD Professor of Pediatrics, University of Colorado Health Sciences Center, Head, Division of Pediatric Allergy-Immunology, National Jewish Medical and Research Center, Denver, Colorado, USA James T. Li MD Professor of Medicine, Allergic Diseases and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, USA Lawrence M. Lichtenstein MD, PhD Professor of Medicine, Department of Medicine, Division of Allergy and Clinical Immunology, Director, The Johns Hopkins Asthma and Allergy Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Phillip L. Lieberman MD

Clinical Professor, Departments of Medicine and Pediatrics, University of Tennessee College of Medicine, Memphis, Tennessee, USA Thomas F. Lint PhD Professor, Department of Immunology/Microbiology, Rush Medical College, Chicago, Illinois, USA Richard F. Lockey MD Professor of Medicine, Pediatrics and Public Health, Director, Division of Allergy and Immunology, Joy McCann Culverhouse Professor in Allergy and Internal Medicine University of South Florida College of Medicine, Physician, Department of Internal Medicine, Division of Allergy and Immunology, James A. Haley Veterans' Hospital, H. Lee Moffitt Cancer Hospital, Tampa General Hospital, All Children's Hospital—St. Petersburg, Tampa, St. Petersburg, Florida, USA Matthew J. Lodewick MD Allergy and Immunology Fellow, Department of Allergy and Immunology, University of Washington, Children's Hospital Regional Medical Center, Seattle, Washington, USA

Donald W. MacGlashan Jr. MD, PhD Professor of Medicine, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University, Baltimore, Maryland, USA Eric Macy MD Assistant Clinical Professor, Department of Medicine, University of California—San Diego School of Medicine, Partner, SCPMG, Department of Allergy, Kaiser Permanente, San Diego, San Diego, California Jean-Luc Malo MD Professor of Medicine, Université de Montréal, Chest Physician, Department of Medicine, Hôpital du Sacré-cœur de Montréal, Montréal, Quebec, Canada Adam J. Mamelak MD Dermatology Research Fellow, Department of Dermatology, The Johns Hopkins University and Hospital, Baltimore, Maryland, USA Ellen M. Mandel MD Associate Professor,

Departments of Otolaryngology and Pediatrics, University of Pittsburgh School of Medicine, Research Pediatrician, Pediatric Otolaryngology, Children's Hospital, Pittsburgh, Pennsylvania, USA Fernando D. Martinez MD Swift-McNear Professor of Pediatrics, Director, Respiratory Sciences Center, Department of Pediatrics and Respiratory Sciences Center, University of Arizona, Tucson, Arizona, USA E.R. McFadden Jr. MD Argyle J. Beams Professor of Medicine, Director, Center for Academic Clinical Research, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA Michael H. Mellon MD Associate Clinical Professor, Department of Pediatrics, University of California—San Diego School of Medicine, Staff Partner Physician, Department of Allergy, Kaiser Permanente, San Diego, California, USA Dean D. Metcalfe MD Chief,

Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA Mark H. Moss MD Assistant Professor, Departments of Medicine and Pediatrics, University of Wisconsin Medical School, Hospital, and Clinics, Madison, Wisconsin, USA

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Hedwig S. Murphy MD, PhD Assistant Professor, Department of Pathology, University of Michigan, Staff Pathologist, Pathology and Laboratory Medicine, Veterans Administration Medical Center, Ann Arbor, Michigan, USA P. RéGine Mydlarski MD, FRCPC Diplomate of the American Board of Dermatology, Lecturer, Division of Dermatology, Department of Medicine, University of Toronto, Dermatology Staff, Department of Medicine, Sunnybrook and Women's College Health Sciences Centre, Toronto, Ontario, Canada

Harold S. Nelson MD Professor of Medicine, University of Colorado Health Sciences Center, Senior Staff Physician, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado, USA Sultan Niazi MD Fellow, Department of Medicine, Division of Critical Care, Pulmonary, Allergic and Immunologic Diseases, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Paul M. O'Byrne MD Professor and Chair, Department of Medicine, McMaster University Health Sciences Center, Firestone Regional Chest and Allergy Unit, St. Joseph's Hospital, Hamilton, Ontario, Canada Kathleen M. O'Neil MD Clinical Associate Professor, Department of Pediatrics, State University of New York—Buffalo School of Medicine and Biomedical Sciences, Director, Section of Pediatric Rheumatology, Children's Hospital of Buffalo, Buffalo, New York, USA

Thomas G. O'Riordan MD, MRCPI Associate Professor of Clinical Medicine, Division of Pulmonary and Critical Care Medicine, State University of New York at Stony Brook, Stony Brook, New York, USA Dennis R. Ownby MD Professor, Pediatrics and Medicine, Medical College of Georgia, Augusta, Georgia, USA Reynold A. Panettieri Jr. MD Associate Professor of Medicine, Department of Medicine, Pulmonary, Allergy and Critical Care Division, University of Pennsylvania School of Medicine, Chief, Asthma Section, Department of Medicine, Pulmonary, Allergy and Critical Care Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA Mary E. Paul MD Assistant Professor, Department of Pediatrics, Baylor College of Medicine, Department of Pediatrics, Allergy and Immunology, Texas Children's Hospital, Houston, Texas, USA David B. Peden MD, MS

Associate Professor of Pediatrics and Associate Director, The Center for Environmental Medicine and Lung Biology, University of North Carolina, Chapel Hill, Asthma and Allergy Program Director, North Carolina Children's Hospital, University of North Carolina Hospitals, Chapel Hill, North Carolina, USA Stephen P. Peters MD, PhD Professor of Medicine, Wake Forest University Health Sciences Center, Center for Human Genomics, Winston-Salem, North Carolina, USA Vincent Piette MD Pulmonologist, Department of Allergology and Respiratory Medicine, Université Catholique de Louvain, Mont-Godinne CHU-Belgium, Physician and Pulmonologist, Allergology and Respiratory Medicine, Clinique-Notre-Dame de Grâce, Gosselies, Belgium Mark R. Pittelkow MD Consultant, Department of Dermatology, Professor of Dermatology and Biochemistry and Molecular Biology, Mayo Clinic and Medical School, Rochester, Minnesota, USA Douglas A. Plager PhD Department of Dermatology, Mayo Clinic and Foundation, Rochester, Minnesota, USA

Thomas A.E. Platts-Mills MD, PhD Professor of Medicine and Director, University of Virginia Asthma and Allergic Diseases Center, Head, Division of Asthma, Allergy, and Immunology, Department of Internal Medicine, University of Virginia, Charlottesville, Virginia, USA Dirkje S. Postma MD Director of Groningen Research Institute for Asthma and Chronic Obstructive Pulmonary Disease, Professor Doctor of Pulmonology, Groningen University Hospital, Groningen, The Netherlands David Proud PhD Professor of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada

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Hengameh Heidarian Raissy PharmD Fellow, Pediatric Pharmacotherapy, College of Pharmacy, University of New Mexico Health Sciences Center, Clinical Pharmacist, Pediatric Pulmonary Center, Department of Pediatrics, Children's Hospital of New Mexico, Albuquerque, New Mexico, USA

Karalasingam Rajakulasingam MD, MRCP Doctor, Respiratory Medicine, Homerton Hospital, Hon. Senior Lecturer, St. Bart's and The Royal London School of Medicine, London, United Kingdom Anuradha Ray PhD Associate Professor, Department of Internal Medicine, Pulmonary and Critical Care Section, Yale University School of Medicine, New Haven, Connecticut, USA Clive Robinson BSc, PhD, ILTM Reader in Respiratory Cell Science, Department of Basic Medical Sciences, Section of Pharmacology and Clinical Pharmacology, St. Georges' Hospital Medical School, London, United Kingdom Lanny J. Rosenwasser MD Marjorie and Stephen Raphael Chair in Asthma Research, Professor, Allergy-Immunology, National Jewish Medical and Research Center, Professor of Medicine and Immunology, University of Colorado Health Sciences Center, Denver, Colorado, USA Adriano Giorgio Rossi BSc (Hons), PhD

Physician, Respiratory Medicine Unit, Centre for Inflammation Research, University of Edinburgh Medical School, Edinburgh, United Kingdom John J. Ryan PhD Assistant Professor, Department of Biology, Virginia Commonwealth University, Richmond, Virginia, USA Jonathan M. Samet MD Professor and Chairman, Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland, USA Anthony P. Sampson PhD Senior Lecturer, Department of Respiratory Cell and Molecular Biology, University of Southampton School of Medicine, Southampton, United Kingdom Hugh A. Sampson MD Professor of Pediatrics and Biomedical Sciences, Department of Pediatrics, Mount Sinai School of Medicine, Chief, Pediatric Allergy and Immunology, Director, General Clinical Research Center, Mount Sinai Hospital,

New York, New York, USA Daniel N. Sauder MD, FRCPC, FACP Noxell Professor and Chairman, Department of Dermatology, Johns Hopkins University, Professor of Dermatology and Medicine, Johns Hopkins Hospital, Baltimore, Maryland, USA Michael Schatz MD, MS Clinical Professor, Department of Medicine, University of California—San Diego School of Medicine, Chief of Service, Department of Allergy, Kaiser Permanente Medical Center, San Diego, California, USA R. Robert Schellenberg MD, FRCPC Professor, Department of Medicine, University of British Columbia, Medical Staff, St. Paul's Hospital, Vancouver, British Columbia, Canada Robert P. Schleimer PhD Professor of Medicine, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

John T. Schroeder PhD Assistant Professor, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland, USA John C. Selner MD Clinical Professor of Pediatrics, University of Colorado Health Sciences Center, Denver, Colorado, USA Chun Y. Seow PhD Assistant Professor, Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada William T. Shearer MD, PhD Professor of Pediatrics and Immunology, Department of Pediatrics, Baylor College of Medicine, Chief of the Allergy and Immunology Service, Department of Allergy and Immunology, Texas Children's Hospital, Houston, Texas, USA Taro Shirakawa MD, PhD Professor, Department of Health Promotion and Human Behavior, Kyoto University Graduate School of Public Health,

Kyoto, Japan Janis K. Shute PhD Reader in Pharmacology, School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom

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Ronald A. Simon MD Head, Division of Allergy, Asthma and Immunology, Department of Medicine, Molecular and Experimental Medicine, Scripps Clinic and the Scripps Research Institute, Head, Division of Allergy, Asthma, and Immunology, Department of Medicine, Green Hospital of the Scripps Clinic, La Jolla, California, USA F. Estelle R. Simons MD, FRCPC Professor and Head, Section of Allergy and Clinical Immunology, Department of Pediatrics and Child Health, University of Manitoba, Head, Section of Allergy and Clinical Immunology, Children's Hospital, Health Sciences Centre, Winnipeg, Manitoba, Canada Reuben P. Siraganian MD, PhD

Head, Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, Bethesda, Maryland, USA David P. Skoner MD Director, Division of Allergy, Asthma, and Immunology, Department of Pediatrics, Vice Chairman of Clinical Research, Allegheny General Hospital, Pittsburgh, Pennsylvania, USA Jay E. Slater MD Children's National Medical Center, Washington, DC, USA, Chief, Laboratory of Immunobiochemists, DBPAP/OVRR/CBER, US Food and Drug Administration, Bethesda, Maryland, USA Gerald C. Smaldone MD, PhD Chief, Pulmonary/Critical Care Medicine, Professor of Medicine, Physiology, and Biophysics, Department of Medicine, State University of New York at Stony Brook, Stony Brook, New York, USA Joseph D. Spahn MD Associate Professor of Pediatrics, Department of Pediatrics,

University of Colorado Health Sciences Center, Staff Physician, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, USA Donald D. Stevenson MD Senior Consultant, Division of Allergy and Immunology, Department of Medicine, Molecular and Experimental Medicine, The Scripps Clinic and Scripps Research Institute, Staff member, Allergy, Asthma and Immunology, Department of Medicine, Green Hospital of the Scripps Clinic, La Jolla, California, USA Geoffrey Alexander Stewart PhD Professor, Department of Microbiology, School of Biomedical and Chemical Science, University of Western Australia, Perth, Western Australia Stanley J. Szefler MD Professor of Pediatrics and Pharmacology, University of Colorado Health Sciences Center, Helen Wohlberg and Herman Lambert Chair in Pharmacokinetics, Director, Clinical Research Unit—Pediatric Section, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, USA

Erika Avila Tang MD Doctoral Candidate, Department of Epidemiology, The Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA Steve L. Taylor PhD Professor and Co-Director, Food Allergy Research and Resource Program, University of Nebraska, Lincoln, Nebraska, USA Abba I. Terr MD Clinical Professor, Department of Medicine, University of California—San Francisco, San Francisco, California, USA Meri K. Tulic PhD Postdoctoral Fellow, Department of Medicine, Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada Homer L. Twigg III MD Associate Professor of Medicine, Division of Pulmonary, Allergy, Critical Care, and Occupational Medicine, Indiana University Medical Center, Indianapolis, Indiana, USA Bradley J. Undem PhD

Professor of Medicine, Department of Medicine, Division of Allergy and Clinical Immunology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA Paul Van Cauwenberge MD, PhD Professor of Medicine, Ear, Nose, and Throat Department, University Hospital, Ghent, Ghent, Belgium James Varani PhD Professor of Pathology, University of Michigan, Ann Arbor, Michigan, USA Donata Vercelli MD Associate Professor, Department of Cell Biology and Anatomy, Associate Research Scientist, The Respiratory Sciences Center, University of Arizona, Tucson, Arizona, USA Antonio Maurizio Vignola MD, PhD Professor of Respiratory Medicine, Department of Respiratory Diseases, University of Palermo, Ospedale “V. Cervello”, Palermo, Italy

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Erika Von Mutius MD, MSc Department of Pulmonology and Allergology, Ludwig-Maximilians, Universitat Munchen, Department of Asthma and Allergy, University Children's Hospital, Munich, Germany Scott S. Wagers MD Research Faculty, Vermont Lung Center, University of Vermont, Fellow, Pulmonary and Critical Care Medicine, Fletcher Allen Healthcare, Burlington, Vermont, USA Carol Ward BSc (Hons), PhD Doctor, Respiratory Medicine Unit, Centre for Inflammation Research, University of Edinburgh School of Medicine, Edinburgh, United Kingdom Peter A. Ward MD Professor and Chairman, Department of Pathology, University of Michigan and the University of Michigan Hospitals, Ann Arbor, Michigan, USA

Peter F. Weller MD, FACP Professor of Medicine, Department of Medicine, Harvard Medical School, Chief, Allergy and Inflammation Division, Co-Chief, Infectious Diseases Division, Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA Sally E. Wenzel MD Associate Professor of Medicine, University of Colorado Health Sciences Center, Associate Faculty Member, National Jewish Medical and Research Center, Denver, Colorado, USA Denise G. Wiesch PhD Formerly with the Department of Epidemiology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland, USA, Scientific Review Administrator, Epidemiology and Disease Control, Center for Scientific Review, National Institutes of Health, Bethesda, Maryland, USA Jerry Winkelstein MD Professor of Pediatrics, Medicine and Pathology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Director,

Division of Allergy and Immunology, Department of Pediatrics, The Johns Hopkins Hospital, Baltimore, Maryland, USA Leman Yel MD Assistant Clinical Professor, Division of Basic and Clinical Immunology, Department of Medicine, University of California—Irvine, Irvine, California, USA, University of California—Irvine Medical Center, Orange, California, USA Moira Chan Yeung MD Professor of Medicine, University of British Columbia, Active Staff, Vancouver General Hospital, Vancouver, British Columbia, Canada John W. Yunginger MD Professor of Pediatrics and Medicine, Mayo Medical School, Consultant in Pediatrics and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, USA Michael C. Zacharisen MD Assistant Professor of Pediatrics and Medicine (Allergy and Immunology), Departments of Pediatrics and Medicine, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

James G. Zangrilli MD Assistant Professor of Medicine, Division of Critical Care, Pulmonary, Allergic and Immunologic Diseases, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, USA Robert S. Zeiger MD, PhD Director of Allergy Research, Allergy Department, Kaiser Permanente Medical Center School of Medicine, San Diego, California, USA, Clinical Professor of Pediatrics, University of California—San Diego, La Jolla, California, USA Bruce L. Zuraw MD Associate Professor, Molecular and Experimental Medicine, The Scripps Research Institute, Member and Director of ARI Fellowship Program, Division of Allergy, Asthma, and Immunology, Green Hospital of the Scripps Clinic, La Jolla, California, USA Burton Zweiman MD Professor of Medicine and Neurology, University of Pennsylvania, Allergy and Immunology Division, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, USA

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To Elliott Middleton, Jr.

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Elliott Middleton, Jr. 1925–1999 In Tribute

We remember with affection and gratitude the life of Elliott Middleton, Jr., the founding editor of this textbook. Elliott was born in Glenridge, New Jersey. He attended college at Princeton and medical school at Columbia University College of Physicians and Surgeons. As a medical resident at Presbyterian Hospital in New York, he spent elective time with Michael Heidelberger, the foremost immunochemist of the era. Elliott continued training as a research fellow in immunology and allergy, first at the National Institutes of Health and later at the Robert A. Cooke Institute of Allergy at Roosevelt Hospital in New York. From 1956 to 1969, as was typical at the time, he combined a private practice in Montclair, New Jersey, with academic appointments at Columbia and Roosevelt universities, where he remained in close contact with Drs. Robert Cooke and William Sherman. In 1969 he became director of clinical services and research at the Children's Asthma Research Institute and Hospital (CARIH) in Denver, Colorado, (where immunoglobulin E was discovered by the Ishizakas in 1966) and associate clinical professor of medicine at the University of Colorado. Here he continued his research on the immunopharmacology of asthma. In 1976 he was appointed professor of medicine and pediatrics at State University of New York at Buffalo, succeeding Dr. Carl Arbesman. In Buffalo he became interested in the antiinflammatory effects of plant flavonoids, published many research papers on the subject, and was subsequently recognized as a world expert. An invited review on the subject published posthumously contained more than 1000 references. In 1995 he retired to Chebeague Island off the coast of Portland, Maine, where he died on March 7, 1999. Elliott served his allergy and immunology colleagues in many ways. He was president of the American Academy of Allergy in 1972, and editor of Journal of Allergy and Clinical Immunology from 1983 to 1988. He was honored by the academy in 1991 with its Distinguished Service Award. He was a director of the subspecialty Board of Allergy of the American Board of Internal Medicine at the time it merged with the subspecialty Board of American Board of Pediatrics to become the American Board of Allergy and Immunology. Elliott was well known both as a researcher and clinician. Less well known are his accomplishments as an educator. He was a gifted lecturer and teacher, and he

profoundly influenced fellows who trained under him in both Denver and Buffalo. Many of his trainees have become renowned in their own right. Indeed, this dedication to education was his chief motive in the organization of the contents of this textbook and the selection of its title—Allergy: Principles and Practice. Not the least, Elliott was a true gentleman who showed warmth and wit and who was beloved by his patients, colleagues, and staff. Elliott's vision of this book began in the mid 1970s during conversations with the staff of C.V. Mosby Company. His goal was: to produce a truly comprehensive book about allergy that focused not only on exciting developments in immunology but also provided in-depth coverage of equally pertinent new information on physiology and pharmacology, two areas of importance to the student of allergy. He realized from the beginning that this undertaking exceeded the capacity of a single person, and he recruited us as co-editors and, more importantly, 100 experts to write what

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ultimately became a definitive 63-chapter source of reference monographs for medical specialists in the field, as well as a textbook defining the area of knowledge for trainees. He divided the work into two volumes, basic science and clinical science, saying: The chapters dealing with immunology, physiology and pharmacology appear at the beginning in the basic science section of the book to provide the necessary conceptual framework for the clinical science section, which deals with the variety of clinical states that fall within the purview of allergy and the allergist. Allergy: Principles and Practice was first published in 1978 and was warmly received by the specialty. That combined with the rapid expansion of new knowledge made it apparent within only a few years of publication that updated editions would be needed. Elliott continued to serve as executive editor for four more editions, the fifth having been published in 1998. With his passing, the next generation of editors will continue the tradition of excellence he established, and new editions will be his legacy. It was a privilege to have known and worked with him over the years and on the previous five editions of this pioneering textbook. Elliot F. Ellis Charles E. Reed Editors Emeriti

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Special Features

Cellular and molecular biology and genetics continue to expand exponentially our understanding of the immunologic and inflammatory mechanisms of allergic diseases, including immunodeficiency states. This new knowledge explosion at the molecular level, as exciting as it has been over the past decade, may be just beginning. The promise of this revolution is for more precise and effective treatment of human disease. All serious students and practitioners of allergy/immunology will want to follow these developments closely. The information explosion has been so profound that a new, arcane language has become necessary to describe allergy/immunology. Like all foreign languages it can be difficult to learn, especially for adults who have not grown up speaking it every day. But learn it we must. To provide an easily accessible glossary of the origin and function of numerous cytokines, chemokines, and the leukocyte CD markers, this sixth edition retains a detailed list of inflammatory cytokines on the inside covers and provides tables of other groups of important molecules in the appendices. The editors intend that these tables will facilitate correlation of the basic science chapters with the clinical subjects and will provide a convenient source of definitions as our readers follow current journals. Although these tables are not a substitute for textbooks of cellular and molecular immunology and inflammation, we hope that these tools will facilitate communication and understanding between clinicians and basic scientists. These new and expanded features amplify our commitment to the original vision of Middleton, Reed, and Ellis for this compendium. In the sixth edition we believe that you will find the latest scientific knowledge, including newly defined pathways in the pathogenesis of allergic diseases and immunodeficiency. From this new knowledge will come novel therapeutic agents directed at newly discovered targets for treatment. Our abiding faith is that a better understanding of the details of disease pathogenesis promises improved clinical outcomes in the future.

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Preface

The sixth edition of Middleton's Allergy Principles & Practice is the first to be undertaken without the active participation and leadership of our three founding editors. About 30 years ago, Elliott Middleton, Jr., recruited his colleagues Charles E. Reed and Elliot F. Ellis to be co-editors with him of a new authoritative textbook in allergy/immunology. Enthusiasm for this new venture can clearly be seen in the preface for the first edition, which has been reprinted in each subsequent edition. This senior triumvirate within American allergy/immunology guided development of the text through five editions. Over the past 25 years, Middleton's

Allergy Principles & Practice has grown in both distribution and acclaim to become the most widely owned and referenced authoritative textbook for the discipline of allergy/immunology. Three additional co-editors (John W. Yunginger; N. Franklin Adkinson, Jr.; and William W. Busse) joined the project team along the way. The original editorial vision has not changed: to select editors who were experienced practitioners and investigators in the field and whose knowledge in aggregate would be sufficient to pursue and then critically evaluate invited contributions from authorities in all aspects of the discipline, from molecular medicine to genetics, epidemiology, and therapeutics. The selection of three new editors for the current edition continues the tradition of excellence, and at the same time adds a global perspective with editors from Canada and the United Kingdom. The new co-editors for the sixth edition have made numerous significant changes to the text as outlined below, while holding firm to the legacy of Drs. Middleton, Reed, and Ellis. We affectionately and respectfully dedicate this sixth iteration of Middleton's Allergy Principles & Practice to our senior colleagues and founding fathers, Elliott Middleton, Jr.; Charles E. Reed; and Elliot F. Ellis. They chartered the course of this pioneering textbook for a quarter of a century, evolved the text into a wellrespected authoritative reference compendium, and mentored their junior colleagues in essential editorial skills, ranging from layout and graphic design to gently coaching the best product from highly respected contributors. For our part we are grateful for the privilege of having worked closely with these senior colleagues, who became close friends as well as mentors. Our long-term readers may add their appreciation to the original three editors for documenting faithfully the progress of our medical discipline, and in the process nurturing the next round of discoveries. First among the trio was the soft-spoken, witty, and yet wise Elliott Middleton, who died in March 1999. Because of his visionary leadership and stature, Principles & Practice has become widely known as the “Middleton textbook.” Drs. Reed and Ellis have prepared the special tribute to Dr. Middleton, and it precedes this preface. The current editors have continued to focus the content of Middleton's Allergy Principles & Practice on the basic science components of allergic and inflammatory diseases, as well as the practical knowledge and resources needed for clinical diagnosis and management of allergic and immunodeficiency disorders. The scientific underpinnings of the discipline continue to expand at an astonishing rate. In the sixth edition, 10 new chapters provide new perspectives in signal transduction events, antigen processing pathways, biology of airway smooth muscle cells, apoptosis of inflammatory cells, animal models of asthma, immunopathology of airway inflammation, outdoor allergen aerobiology, molecular biology of allergens, nasal provocation testing, and therapy with immunomodulators. In keeping with the policy established for earlier editions, we have selected new authors for 40 of the 94 chapters in this new edition. In addition, with numerous contributions by authors from around the world, this edition has a new international flavor. The focus continues to be on human immunobiology and medicine, while not ignoring pertinent animal experimentation. To assist in reading and understanding the increasingly complex biology of inflammation, we have retained and updated reference charts of cytokines in allergic diseases, cluster of differentiation molecular definitions, and human chemokines for easy reference. Classification of adhesion molecules is now found in Chapter 9 . We have also expanded an appendix of relevant website addresses, where readers can find research updates of the molecular components of allergy and inflammation. We as editors and you as readers are deeply indebted to the 161 contributors to these volumes, each of whom undertook a sometimes monumental task to achieve a comprehensive review that is balanced in viewpoint and evidence based. Many of the authors worked extensively with editors to achieve the right scope and depth for their chapters. As editors

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we are very grateful for all of the goodwill and patient cooperation that made this collaborative venture possible. In addition, there are a few other people without whose skills and persistence this sixth edition would never have been completed. We acknowledge with gratitude excellent editorial assistance from various publishing professionals, including the very helpful oversight of several senior developmental editors at Mosby, W.B. Saunders, Harcourt, and now Elsevier. These include Jennifer Shreiner and Catherine Carroll, who saw us through to the finish line. Finally, each of us thanks our capable local assistants for tireless support during the editorial process. N. Franklin Adkinson Jr. Managing Editor John W. Yunginger William W. Busse Bruce S. Bochner Stephen T. Holgate F. Estelle R. Simons

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Preface to the first edition

Allergy, once a confusing subject for clinician and researcher alike, has emerged as a medical science in which immunology, physiology, and pharmacology interface uniquely. Our present state of knowledge is the culmination of the efforts of many workers over many decades of research in the clinic and laboratory. We want to acknowledge our incalculable debt to these investigators, both basic scientists and clinicians, who taught us not only fact but more importantly concepts and scientific method. Several textbooks on allergy are already in existence. Why another one? We pondered this question for some time before embarking on what turned out to be, expectedly, a rather formidable task. It was our opinion that a truly comprehensive book about allergy should focus strongly not only on the exciting developments of the past decade or two in immunology but also provide in-depth coverage of equally pertinent new information on physiology and pharmacology, two areas of critical importance to the student of allergy. We have made no attempt to cover all of the subject matter considered to fall under the general rubric of clinical immunology and so do not include sections dealing with rheumatology, other connective tissue disorders, immunohematology, or tumor immunology, for example, since these subjects are well covered elsewhere. The chapters dealing with immunology, pharmacology, and physiology appear at the beginning in the basic science section of the book to provide the necessary conceptual framework for the clinical science section, which deals with the variety of clinical states that fall within the purview of allergy and the allergist. The value

of the clinical descriptions is vastly enhanced by a careful reading of the earlier chapters. We were most fortunate in securing a truly outstanding “star-studded” cast of contributors who managed to find time in their already overcrowded schedules to help us write the book. We thank them all for their efforts and are grateful for the patient indulgence of a few who put up with some predictable editorial fussing meant to achieve proper balance and avoid excessive overlap. Most of the chapters can be read as free-standing articles or monographs on that particular subject. This has led to a certain irreducible amount of duplication. By and large, there is consistency among chapters in which comparable material has been presented by different authors, but the reader will find occasional areas of controversy, a natural state of affairs in a rapidly growing field. It is our opinion that some chapters in this book represent the most comprehensive summaries of the subject matter to be found in print. Thus Allergy: Principles and Practice serves not only as a textbook but as a reference book. Indeed, this was our intent, but original estimates for the length of the book were necessarily revised upward as it became clear that much excellent material could not properly be left out. The final product then turns out to be a book we hope will be useful to all students of allergy: practitioners, clinical investigators, other researchers, allergy trainees, and medical students. The generous and unstinting help of many people in addition to the contributors made this book possible. Without the competent and devoted secretarial assistance of Marci Dame, Evelyn Beimers, Bonnie Barcy, Carol Speery, and Candace Anderson, the task could not have been accomplished. We thank our wives and families for their forbearance, while we were sequestered away from home for day and night weekend sessions during the planning and editing phases. From the beginning their support has been essential to the successful completion of our job. A number of colleagues, too numerous to name, provided help in critical reading of manuscripts. To these and others who were helpful in a variety of ways, we offer thanks. We are saddened that two contributors died during the preparation of the book. Jane Harnett is the senior author of the chapter dealing with aspirin idiosyncrasy. Dr. Harnett compiled much of the information for the chapter and worked on the manuscript under extremely difficult circumstances up to within only a few days of her untimely death. She is remembered fondly and with respect by all those with whom she worked. Robert P. Orange, one of the most brilliant and creative investigators of his generation, died suddenly during the preparation of the book. No one can guess what additional important discoveries Dr. Orange would have made had he not died so prematurely. We would like to record here our personal sorrow at the loss of these fine physicians. We hope that their representation in this textbook will help keep memories of them alive. Elliott Middleton Jr. Charles E. Reed Elliot F. Ellis

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Color Plates Plate 1 Comparison of the three-dimensional structures of monomeric subunits of CCL5, or regulated on activation, normal T cells expressed and secreted (RANTES), and interleukin-8 (IL-8), as determined by nuclear magnetic resonance spectroscopy. The N-termini of the proteins are not well resolved in the structures. (For text mention see Chapter 11 , p.159.) (Modified from Wells T, Powers CA, Lusti-Narasimhan M, et al: J Leukoc Biol 59:53, 1996.)

Plate 2 Simplified representation of the major components of the epithelial adhesion complex in adjoining epithelal cells. A, Tight junction (TJ) occludes entrance to the paracellular space, showing the relationship of the transmembrane adhesion proteins (occludin and caludins) with components of the TJ plaque and the actin cytoskeleton. Other intracellular components of TJs and the transmembrane junctional adhesion molecules (JAMs) have been omitted for clarity. ZO, Zona occludens proteins. B, Adherens junction, showing the probable arrangement of the extracellular domains of E-cadherin, with the calcium-binding adhesion domain EC1 shown in red. Intracellular attachments to the actin cytoskeleton through catenins and attachment proteins are also shown. C, Desmosomal adhesion, showing the adhesion proteins (desmocolllins and desmogleins) and their attachment to intermediate filaments (IF through plakoglobin (PG) and desmoplaktin (DP). Also shown is the probably IF-dependent relationship to hemidesmosomal adhesion to the biomatrix, such as laminin-5 (Lam5), through integrins such as α6 β4 . (For text mention see Chapter 36 , p.606.)

Plate 3 A, Effect of house-dust mite serine peptidase allergens on epithelial tight junctions (TJs) (green fluorescence) and desmosomes (red fluorescence). Cell images were acquired by 2-photon molecular excitation microscopy after fluorescent antibody labeling of appropriate proteins from the interepithelial junctions and are shown as three-dimensional isosurface reconstructions to illustrate changes in a spatially meaningful way. Note that 1 hour (1h) after exposure to the allergens, small discontinuities become evident as TJ rings break down. This is accompanied by an increase in epithelial permeability (not illustrated). Note that 3 hours (3h) after allergen exposure, loss of TJs is total, and the desmosomal plaque protein desmoplakin shows an increased staining intensity. B, Representative immunoblots reveal proteolytic processing of a transmembrane TJ protein (occludin) and a plaque protein (zona occludens-1, ZO-1) after exposure of bronchial epithelial cells to house-dust mite serine peptidase allergens. With occludin, proteolytic processing is initiated by direct cleavage of ZO-1 arises through activation of intracellular processing pathways after perturbation by the peptidase allergins. (For text mention see Chapter 36 , p.607.)

Plate 4 Structure of human skin. A, Schematic representation of skin cross-section. The two major layers of human skin, the epidermis and the dermis, overlie subcutaneous fat and muscle. Arterioles (red) venules (blue), and lymph vessels (yellow-green) of the dermis form a lower and an upper vascular plexus. Capillary loops extend toward the epidermis from the upper plexus of blood vessels into the dermal papillae—approximately one loop per dermal papilla. Sensory and autonomic nerves (yellow fibers) are also arranged in a lower and an upper plexus, at the junction of the dermis and subcutaneous fat and in the upper dermis, respectively (not explicitly shown). Specialized sensory structures, including Meissner's (M), Pacinian (P), and Ruffini's (R) corpuscles and free nerve endings, arise from these nerve plexuses and extend into the epidermis. Sweat glands and hair follicles with their associated sebaceous glands are also integral components of the skin. B, Skin histology, showing epidermis (E), dermis (D), hair follicle (HF), and sebaceous gland (SG). Hematoxylin and eosin stain, × 50. C, Skin histology, showing keratinocyte (K), clear cell (CC; representing a melanocyte, Langerhans' cell, or Merkel cell) of the epidermis, dermal-epidermal junction (DEJ), dermal fibroblast (F), lymphocyte (L), and blood vessel (BV). Hematoxylin and eosin stain, × 250. Other cellular and structural components of the skin require special staining or immunomarkers for identification. (For text mention see Chapter 41 , p.671.)

Plate 4 Structure of human skin. A, Schematic representation of skin cross-section. The two major layers of human skin, the epidermis and the dermis, overlie subcutaneous fat and muscle. Arterioles (red) venules (blue), and lymph vessels (yellow-green) of the dermis form a lower and an upper vascular plexus. Capillary loops extend toward the epidermis from the upper plexus of blood vessels into the dermal papillae—approximately one loop per dermal papilla. Sensory and autonomic nerves (yellow fibers) are also arranged in a lower and an upper plexus, at the junction of the dermis and subcutaneous fat and in the upper dermis, respectively (not explicitly shown). Specialized sensory structures, including Meissner's (M), Pacinian (P), and Ruffini's (R) corpuscles and free nerve endings, arise

from these nerve plexuses and extend into the epidermis. Sweat glands and hair follicles with their associated sebaceous glands are also integral components of the skin. B, Skin histology, showing epidermis (E), dermis (D), hair follicle (HF), and sebaceous gland (SG). Hematoxylin and eosin stain, × 50. C, Skin histology, showing keratinocyte (K), clear cell (CC; representing a melanocyte, Langerhans' cell, or Merkel cell) of the epidermis, dermal-epidermal junction (DEJ), dermal fibroblast (F), lymphocyte (L), and blood vessel (BV). Hematoxylin and eosin stain, × 250. Other cellular and structural components of the skin require special staining or immunomarkers for identification. (For text mention see Chapter 41 , p.671.)

Plate 4 Structure of human skin. A, Schematic representation of skin cross-section. The two major layers of human skin, the epidermis and the dermis, overlie subcutaneous fat and muscle. Arterioles (red) venules (blue), and lymph vessels (yellow-green) of the dermis form a lower and an upper vascular plexus. Capillary loops extend toward the epidermis from the upper plexus of blood vessels into the dermal papillae—approximately one loop per dermal papilla. Sensory and autonomic nerves (yellow fibers) are also arranged in a lower and an upper plexus, at the junction of the dermis and subcutaneous fat and in the upper dermis, respectively (not explicitly shown). Specialized sensory structures, including Meissner's (M), Pacinian (P), and Ruffini's (R) corpuscles and free nerve endings, arise from these nerve plexuses and extend into the epidermis. Sweat glands and hair follicles with their associated sebaceous glands are also integral components of the skin. B, Skin histology, showing epidermis (E), dermis (D), hair follicle (HF), and sebaceous gland (SG). Hematoxylin and eosin stain, × 50. C, Skin histology, showing keratinocyte (K), clear cell (CC; representing a melanocyte, Langerhans' cell, or Merkel cell) of the epidermis, dermal-epidermal junction (DEJ), dermal fibroblast (F), lymphocyte (L), and blood vessel (BV). Hematoxylin and eosin stain, × 250. Other cellular and structural components of the skin require special staining or immunomarkers for identification. (For text mention see Chapter 41 , p.671.)

Plate 5 Cellular and vascular organization of normal human skin. Keratinocytes are the most abundant epidermal cells, and they are typically categorized from the skin surface to the dermal-epidermal junction (DEJ) as cornified, granular, spinous, and basal kertinocytes. Suprabasal Langerhans' cells (LC) and basal level

melanocytes (Mel), T cells (TC), and Merkel cells (Me) are also widely distributed in normal epidermis. Sensory nerve (SN) endings innervate all vital layers of the epidermis. The DEJ separates the epidermis from the papillary dermis, which extends down to the upper vascular plexus. Capillary loops (CL), approximately one loop per dermal papilla, project upward toward the epidermis. The postcapillary venule (PCV), important in inflammatory cel extravasation into the skin, is shown partially in cut-edge section and in an elarged cross-section (inset). Endothelial cells (EC) form the inner surface of the postcapillary venule, and they are surrounded by a lamelar basement emembrane. Pericytes (P) reside within layers of the venule basement membrane, and veil cells (VC) and mast cells (MC) often contact the outer surface of the venule basement membrane. Lymph vessels (LV) and nerves, including autonomic nerves (AN) controlling cutaneous involuntary muscle contraction, track with blood vessels. The remainder of the dermis down to the subcutaneous fat is referred to as the reticular dermis. Fibroblasts (F), mast cells (MC), and macrophages (MØ) are relatively more abundant in the papillary dermis than in the reticular dermis. Perivascular dermal T cells (TC) and dermal dendritic cells (DDC; e.g., dermal dendrocytes) also normally reside in the papillary dermis. The lower vascular plexus lies just above the subcutaneous fat layer, 2 to 4 mm below the surface of the skin. (For text mention see Chapter 41 , p.671.)

Plate 6 Dermal-epidermal junction. Desmosomes function to attach and laterally stabilize juxtaposed basal keratinocytes. Proteins important in desmosome formation include intermediate filaments (e.g., keratins), desmoplakins, desmogleins, desmocollins, and plakoglobin. Basal keratinocytes attach to the lamina densa (basement membrane) via hemidesmosomes, which are composed of bullous pemphigoid antigen 1 (BPAg1), plectin, BPAg2, and α6β4-integrin. Hemidesmosomeassociated BPAg2 and α6β4-integrin extend down to interact with laminin 5, which extends up from the lamina densa into the lamina lucida. As such, these are the primary components of anchoring filaments. Laminin 6 also is present in the lamina lucida. The lamina densa is composed of type IV collagen, laminin 1, enactin (i. e., nidogen), and heparan sulfate proteoglycan. Anchoring fibrils attach to the dermal side of the lamina densa and interweave with type I and type III collagen fibrils before attaching to anchoring plaques within the dermis or reattaching to the lamina densa. Note that exact locations, interactions, and identities of all dermalepidermal junction proteins are still being defined. (For text mention see Chapter 41 , p.673.)

Plate 7 Cutaneous innervation. Nerve fibers (yellow-green), which are stained with antibody recognizing a general nerve marker, protein gene product 9.5 (PGP9.5), often are closely associated with blood vessels (orange-red), which are stained with antibody against type IV collagen of the vessels' basement membrane. Fine nerve fibers also reside just beneath and traverse into the epidermis, as marked by the dermal-epidermal junction basement membrane, which also stains orange-red with type IV collagen antibody. (For text mention see Chapter 41 , p.675.) (Courtesy WR Kennedy, University of Minnesota.

Plate 8 Cutaneous immunology. Cutaneous immunity is divided into constiutive innate immunity, inducible innate immunity, and acquired immunity. On disruption of the skin barrier (upper left), an inflammatory response ensues, marked by the early release of primary cytokines such as interleukin 1α (IL-1α) and tumor necrosis

factor alpha (TNF-α). These cytokines activate a broad variety of cell types, including fibroblasts (F), mast cells (MC), macrophages (MØ), and endothelial cells lining the postcapillary venule, andadditional cytokines, chemokines, and mediators are released. Vasodilation and increased vasopermeability, characteristic of an inflammatory response, is the result. Subsequent inducible events such as increased antimicrobial polypeptide expression and complement activation contribute to the early neutralization of invading pathogens. The pattern of endothelial adhesion molecule expression—such as P-selectin (P), E-selectin (E), intercellular ahdesion molecule-1 (I), vascular cell adhesion molecule-1 (V), and surface-presented chemokines (C)—also responds to favor extravasation of neutrophils (N), monocytes (M), and, eventually, effector T cels (TC). Pathogen-recognition receptors constitutively expressed on a wide variety of cell types (red and green cell surfaceshapes, shown hereonly on neutrophils and a single macrophage and Langerhans' cell) also contribute to pathogen clearance and help direct the acquired immune response. After activation and antigen (Ag) uptake, epidermal Langerhans' cells can migrate to a lymph vessel and then to a draining lymph node and present Ag-derived peptides to naïve T cells. The resulting Ag-specific cutaneous lymphocyte-associated antigen (CLA)—positive effector T cells can then home back to the site of cutaneous inflammation and extravaste into the skin. Subsequent Ag presentation in the skin can activate these T cells to perform their programmed function (e.g., cytolysis, cytokine production). Plasma cell-—derived Ag-specific antibody also develops during the acquired immune response and infiltrates the inflammatory site to neutralize and mark an invading pathogen for destruction. Clearance of and heightened immunity against the invading pathogen, followed by resolution of the inflammatory response with concurrent wound healing, is the desired outcome; however, development of disease can also occur. SN, Sensory nerve. (For text mention see Chapter 41 , p.676.)

Plate 9 Masson's trichrome-stained 5-•m-thick cross-section of large, nonasthmatic airway. A, Low-magnification view shows epithelium (E), connective tissue

(CT), and smooth muscle (SM) layers. Aqua-staining collagen can be appreciated below epithelial basement membrane (S, subepithelial region [lamina reticularis]) as well as in smooth muscle layer. B, Higher magnification of smooth muscle bundle shows red-brown-staining muscle cells (red arrows) with elongated, darkbrown-staining nuclei (black arrow) and abundant aqua-staining collagen (yellow arrows) between myocytes. (For text mention see Chapter 43 , p.719.) (From Thomson RJ, Bramley AM, Schellenberg RR: Am J Respir Crit Care Med 154:749, 1996.)

Plate 10 Representation of normal (left) versus asthmatic airway (right), showing encroachment of the airway lumen by intraluminal secretions and thickened airway wall. (For text mention see Chapter 67 , p.1209)

Plate 11 A, Light micrograph of sputum showing a Curschmann's spiral (arrow) and Creola bodies (arrowheads) in a case of asthma (approximate scale, 20 •m). B,

Charcot-Leyden crystals (arrows) and eosinophils (arrowheads) in sputum in a case of asthma (approximate scale, 20•m; eosin stain). (For text mention see Chapter 67 , p.1211.)

Plate 11 A, Light micrograph of sputum showing a Curschmann's spiral (arrow) and Creola bodies (arrowheads) in a case of asthma (approximate scale, 20 •m). B, Charcot-Leyden crystals (arrows) and eosinophils (arrowheads) in sputum in a case of asthma (approximate scale, 20•m; eosin stain). (For text mention see Chapter 67 , p.1211.)

Plate 12 A, Gross appearance of airway plug (P) in bronchus with adjacent arteries (A) in a case of fatal asthma (approximate scale, 400 •m). B, Light micrograph of section through bronchial plud (P) that consists of exudates, mucus, and cellular elements in a case of ftal asthma. There is marked inflammatory cell infiltrate (arrows), enlargement of the mass of bronchial smooth muscle (arrowheads), and vasodilation of the bronchial vessels (V) (scale, 180 •m; hematoxylin-eosin [HE] stain). C, Immunohistologic staining of bronchial plug showing concentric lamellae in which there are EG2+ (i.e., activated) eosinophils (scale, 50 •m). (For text mention see Chapter 67 , p.1211.)

Plate 12 A, Gross appearance of airway plug (P) in bronchus with adjacent arteries (A) in a case of fatal asthma (approximate scale, 400 •m). B, Light micrograph of section through bronchial plud (P) that consists of exudates, mucus, and cellular elements in a case of ftal asthma. There is marked inflammatory cell infiltrate (arrows), enlargement of the mass of bronchial smooth muscle (arrowheads), and vasodilation of the bronchial vessels (V) (scale, 180 •m; hematoxylin-eosin [HE] stain). C, Immunohistologic staining of bronchial plug showing concentric lamellae in which there are EG2+ (i.e., activated) eosinophils (scale, 50 •m). (For text mention see Chapter 67 , p.1211.)

Plate 12 A, Gross appearance of airway plug (P) in bronchus with adjacent arteries (A) in a case of fatal asthma (approximate scale, 400 •m). B, Light micrograph of section through bronchial plud (P) that consists of exudates, mucus, and cellular elements in a case of ftal asthma. There is marked inflammatory cell infiltrate (arrows), enlargement of the mass of bronchial smooth muscle (arrowheads), and vasodilation of the bronchial vessels (V) (scale, 180 •m; hematoxylin-eosin [HE] stain). C, Immunohistologic staining of bronchial plug showing concentric lamellae in which there are EG2+ (i.e., activated) eosinophils (scale, 50 •m). (For text mention see Chapter 67 , p.1211.)

Plate 13 A, Light micrograph of section through normal bronchial mucosa from a motor vehicle accident death, showing a lumen (L) free of exudates, intact surface epithelium, normal amounts of bronchial smooth muscle (B), and surrounding alveoli (A). B, In contrast to A, light micrograph of mucosa from an asthma death shows loss of surface epithelium (arrow), hyaline and homogenous thickening of reticular basement membrane (R), and increases in subepithelial inflammatory cells (arrowheads) and bronchial smooth muscle mass. (scale, 80 •m; HE stain.) (For text mention see Chapter 67 , p.1211.)

Plate 13 A, Light micrograph of section through normal bronchial mucosa from a motor vehicle accident death, showing a lumen (L) free of exudates, intact surface epithelium, normal amounts of bronchial smooth muscle (B), and surrounding alveoli (A). B, In contrast to A, light micrograph of mucosa from an asthma death shows loss of surface epithelium (arrow), hyaline and homogenous thickening of reticular basement membrane (R), and increases in subepithelial inflammatory cells (arrowheads) and bronchial smooth muscle mass. (scale, 80 •m; HE stain.) (For text mention see Chapter 67 , p.1211.)

Plate 14 EG2 immunopositive (i.e., activated) subepithelial eosinophils in an area of bronchus from an astham death. The eosinophils are stained blue and lie immediately beneath the reticular basement membrane (arrows) (scale, 80 •m). (For text mention see Chapter 67 , p.1213.)

Plate 15 In situ hybridization of a bronchial biopsy to demonstrate interleukin-5 (IL-5) gene expression in a case of acute severe asthma. Each cell expressing the gene is densely labeled. Arrows indicate position of the epithelium (scale, 5 •m). (For text mention see Chapter 67 , p.1217.) (Courtesy Dechun Li, MD.)

Plate 16 Demonstration of adenoidal tissue partially obstructing the right posterior choana in a 9-year-old boy with recurrence of nasal obstruction 6 months after adenoidectomy. (For text mention see Chapter 77 , p.1415.) (From Selner JC, Dolen WK, Spofford B, Koepke JW, et al: Rhinolaryngoscopy, ed 2, Denver, 1989, Allergy Respiratory Institute of Colorado.)

Plate 17 Laryngocoeles causing muffled voice. (For text mention see Chapter 77 , p.1416.) (From Selner JC, Dolen WK, Spofford B, Koepke JW, et al: Rhinolaryngoscopy, ed 2, Denver, 1989, Allergy Respiratory Institute of Colorado.)

Plate 18 Vocal cord papillomas in a 67-year-old woman with hoarseness and coexistent allergic rhinitis. (For text mention see Chapter 77 , p.1416.) (From Selner JC, Dolen WK, Spofford B, Koepke JW, et al: Rhinolaryngoscopy, ed 2, Denver, 1989, Allergy Respiratory Institute of Colorado.)

Plate 19 Acute atopic dermatitis with excoriated lesions. (For text mention see Chapter 86 , p.1561.)

Plate 20 Chronic lichenified atopic dermatitis. (For text mention see Chapter 86 , p.1561.)

Plate 21 Severe keratoconjunctivitis with corneal scarring. (For text mention see Chapter 86 , p.1562.)

Plate 22 Staphylococcal superinfection in atopic dermatitis. (For text mention see Chapter 86 , p.1562.)

Plate 23 Acute vesicular allergic contact dermatitis from adhesive present in Elastoplast dressing. (For text mention see Chapter 87 , p.1588.)

Plate 24 Allergic contact dermatitis caused by perfume. (For text mention see Chapter 87 , p.1588.)

Plate 25 Allergic contact dermatitis caused by an ankle bracelet. (For text mention see Chapter 87 , p.1588.)

Plate 26 Skin biopsy of acute contact dermatitis demonstrating spongiosis (edema) of the epidermis with vesicle formation and a superficial perivascular inflammatory infiltrate (× 100). (For text mention see Chapter 87 , p.1590.)

Plate 27 High-power view of perivascular lymphocytic infiltrate and exocytosis of lymphocytes into the dermis (× 400). (For text mention see Chapter 87 , p.1590.)

Plate 28 Allergic contact dermatitis from plant (poison ivy); note streaky linear pattern. (For text mention see Chapter 87 , p.1593.)

Section A - Immunology

1

Chapter 1 - The Immune System: An Overview

William T. Shearer Thomas A. Fleisher

The practice of allergy at the level of pathogenesis and therapy has its roots in the science called immunology, the field that codifies the components and describes the functions of a human host defense system designed to protect against chemical substances, microorganisms, and cancer. This host defense system comprises specific cellular and numerous protein components that interact in a highly complex manner and that, in essence, act to preserve self and to neutralize or destroy nonself. Over the process of evolution, this system has developed into a remarkably efficient mechanism for protecting human life. However, as part of, or as a consequence of, this preservation system, immune responses have evolved that prove inadequate or detrimental, leading to human disease. Allergy is a clear example of the detrimental side of the immunologic system, an overreaction in certain individuals by a specific defense pathway that responds inappropriately to environmental encounters and results in annoying and sometimes debilitating secondary effects. Allergy is the study and treatment of human hypersensitivity reactions directed at nonself molecules termed allergens; this contrasts with these same hypersensitivity mechanisms providing protection when responding to parasitic infection. Thus, allergy is an integral part of the manifestation of immunity, and physicians who treat allergic disease must understand the scope and breadth of the science of immunology. The notion that allergists practice in a vacuum of scientific design and treatment is incorrect. Rather, the allergist, armed with the knowledge of underlying immunopathogenetic principles of allergic disease and the capability of exploiting the immunologic basis of allergy, will be able to relieve patient symptoms with scientifically based therapeutic measures. This chapter summarizes current knowledge of the origins of the immune system, the interworkings among the constituent parts of the immune network, and the varied consequences, including allergy, of the host immune response. This chapter serves as a reference point for all subsequent chapters, which discuss the clinical presentations, treatments, and management strategies of allergic disease.

FEATURES OF THE IMMUNE SYSTEM

Over the past 200 years, and particularly the last 25 years, the resistance mechanisms against communicable diseases have been documented and explained, even visualized. The basic property of the immune system is that it can distinguish nonself from self, a power that promotes survival and exists in a delicate balance between tolerance to self and response/rejection of nonself. Autoimmunity defines a state in which tolerance to self is lost. This implies that responses against self do not normally occur and that if they do occur with sufficient magnitude and duration, the outcome is harmful to the host. However, not all responses to self are harmful, but rather the capacity to recognize self in the context of specific cell surface molecules encoded by the major histocompatibility complex (MHC) and in antiidiotypic responses is important for the control and normal functioning of the intact immune system. Implicit in the recognition of nonself is the resultant capability for the rejection of nonself by the immune system. Although this power of the immune system to recognize and reject nonself can under unusual circumstances initiate self-destructive autoimmunity, by and large the immune system has preserved vertebrate species from the microbial and parasitic forces that seem programmed to destroy them. The extraordinary measures that are necessary to circumvent the forces of immune rejection, as required in transplantation and autoimmunity medicine, illustrate the complex strengths of these primal survival mechanisms. These measures involve powerful immunosuppressive drugs, irradiation, and antilymphocyte antibodies that are necessary to restrain the more serious rejection episodes, leaving the patient immunologically suppressed, particularly vulnerable to opportunistic infection, and other complications. Immune Memory A remarkable aspect of the immune system is its property of memory, which provides protection against harmful microbial agents despite reexposure being separated by prolonged periods, even decades. Immunologic memory is made possible by the clonal expansion of lymphocytes in response to antigen stimulation. From the time the human immune system first begins to differentiate in fetal life, uniquely reacting lymphocytes are created by the random recombination of segments of deoxyribonucleic acid (DNA) that code for antigen receptors expressed on the lymphocyte cell membrane. Through the expression of these receptors, each of the lymphocytes has the ability to bind to and become activated by a specific non-self antigen, either natural or artificial. Interaction with antigen not only activates the lymphocytes, but also results in the

2

generation of long-lived antigen-specific memory cell clones. Thus in the future, when the same antigen enters the body, there is an immediate recognition by these memory cells. Both cellular and humoral responses to the antigen are produced quickly, which offers protection in the case of an infectious agent or neoplastic cell, and another round (boosting) of memory cell generation ensues. This process of expansion of clonal populations of uniquely reacting lymphocytes first explained the B lymphocyte origin of antibody diversity and applies to cellular (T lymphocytes) immune responses as well.[

1] [2]

Surveillance Because of the early documentation of immune memory, a theory of immune surveillance arose that purported that the immune system was in a perpetual state of vigilance, screening and rejecting any nonself entity that appeared in the body. According to this theory, bacteria, viruses, and cancer cells are being regularly countered by this surveillance mechanism. However, criticism of this theory originates from the observation that, despite protection offered by the system, fatal malignancies frequently arise.[

3] [4]

Nevertheless, the phenomenal explosion of the acquired immunodeficiency syndrome (AIDS) epidemic has demonstrated that the

immune system, particularly the CD4+ T lymphocyte, plays a crucial role in preventing the acquisition of fatal opportunistic infections and the early appearance of at least certain malignancies, including Kaposi's sarcoma and lymphoid cancers.[

5]

Tolerance (Central vs. Peripheral) Tolerance is the process by which the immune system is programmed to eliminate foreign substances such as microbes, toxins, and neoplastic tissues but to accept 6 7

self-antigens.[ ] [ ] This process depends on the expression of unique B and T cell antigen receptors, involving somatic gene recombination events during cell development. Lymphocyte receptor genes recombine to give antigen specificity. When the lymphocyte that expresses rearranged receptor genes is stimulated by binding of the appropriate specific antigen to the receptor, the cell propagates, signaling the initial step toward elimination of the antigen or the cells bearing the foreign antigen. Immunologic self-tolerance is not complete at birth but is actively acquired and maintained during life. Several mechanisms may be operating. One mechanism is central clonal deletion, resulting in the elimination of self-reactive immature T lymphocytes in the thymus and self-reactive B lymphocytes in bone marrow. The process of central tolerance occurs in lymphoid tissue and is not complete because not all self-antigens are expressed in these organs. Peripheral tolerance appears to be maintained by several mechanisms, including clonal deletion, anergy, suppression, and clonal ignorance. Clonal anergy results in T cells incapable of responding to the specific antigen, whereas deletion results in the elimination of autoreactive T lymphocytes. Other nonlymphoid cells may induce suppression of self-antigen– reactive T cells, whereas naive T cells may ignore some self-antigens. Biologic Amplification System Although the memory capabilities of the immune system are conveyed by lymphocytes, a combination of cellular and humoral components of the immune and inflammatory systems are necessary for appropriate host defense. This combination of elements in the immune response is considered part of a biologic amplification system that greatly augments the capability of the immune response. Thus, both soluble molecules (e.g., activated complement proteins, interleukins, chemokines, other cytokines) and cellular components (e.g., polymorphonuclear leukocytes, monocytes, macrophages, eosinophils, basophils, mast cells; the “innate” immune 8 9

response) greatly amplify the recognition and rejection power of lymphocytes. [ ] [ ] Viewed from this perspective, many portions of the inflammatory and immune response become integrated into an elegant and multifaceted system that distinguishes self from nonself. Perhaps the clearest example of the cells and mediators of the allergic response participating in host defense is the protective role that eosinophils and 10

immunoglobulin E (IgE) play in parasitic infection. [ ] Eosinophils facilitate destruction of larvae by the release of cytotoxic proteins (e.g., major basic protein) when the eosinophil Fc receptor (FcR) FcαR (CD89) or FcγRII (CD32) binds immunoglobulin A (IgA), IgG, or IgE specifically bound to the helminths. All these antiparasitic responses involving mast cells, eosinophils, and immunoglobulin are exquisitely dependent on T cell control. Here, too, other components of the immune system, such as macrophages, neutrophils, and complement components, amplify the cytotoxic effects of the immediate hypersensitivity system. The combined power of these various components of host defense enables individuals to survive the myriad of insults from biologic and chemical agents.

CONSTITUENTS AND DEVELOPMENT OF THE IMMUNE SYSTEM

All the cells of the immune system derive from the bone marrow; in fact, all the inflammatory and ancillary cells that work in concert with the primary cells of the immune system derive from the pluripotent stem cell ( Figure 1-1 ). This pluripotential stem cell gives rise to a lymphoid stem cell and a myeloid stem cell. The lymphoid stem cell differentiates into three types of cells—the T lymphocyte, the B lymphocyte, and the non-T, non-B natural killer (NK) cell progenitor—and contributes to the development of subsets of dendritic cells. The myeloid stem cell gives rise to dendritic cells, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages, as well as megakaryocytes and erythrocytes. Differentiation of both these committed stem cells is critically dependent on an array of cytokine and cell-cell interactions. An ever-increasing number of biologically important surface membrane proteins are being characterized on cells of the immune system. Many of these molecules have been assigned a sequential number based on the cluster of differentiation (CD) nomenclature. T Cell Development: Role of Thymus Lymphoid stem cells leave the bone marrow through the bloodstream. Some cells enter the thymus gland and, by a poorly understood process, remain in the thymus gland, where precursor cells ultimately develop into mature T cells, emerging with distinct genome, surface antigen, and functional characteristics ( Figure 1-2 ).[ [12]

11]

When the lymphoid stem cell

3

Figure 1-1 Derivation of the cells of the immune system and hematopoietic system. Pluripotent stem cells are derived from the yolk sac and ultimately reside in the bone marrow. All lymphocytes are derived from the lymphoid stem cell and differentiate into three types of lymphocytes: (1) mature T cells after passage through the thymus gland, (2) large granular lymphocyte cells that possess natural killer (NK) function, and (3) B cells that can differentiate into plasma cells capable of secreting antibodies. Monocytes and macrophages are derived from myeloid stem cells; in addition, neutrophils, eosinophils, erythrocytes, megakaryocytes, mast cells, basophils, and dendritic cells are also derived from this stem cell. The differentiation of stem cells into mature specialized cells is under the control of numerous cytokines and cell-secreted factors.

Figure 1-2 Ontogeny and lineage relationships of maturing T cells expressing TCRαβ. Most thymocytes express both CD4 and CD8. T cell antigen receptor (TCR) expression commences in this double-positive stage, beginning with low numbers of receptors on each cell and increasing as maturation proceeds. Single-positive, that is, CD4+ or CD8+ TCR-αβ–expressing, mature cells are selected from this population. A small population of double-negative T cells bearing TCR-γδ also leave the thymus (not shown).

TABLE 1-1 -- Accessory Molecules (Receptor-Ligand Pairs) that Stabilize the Binding of T Lymphocytes to Antigen-Presenting Cells (APCs) T Cell

APC

T cell receptor

HLA and peptide

CD4

HLA-DR, HLA-DQ, HLA-DP

CD8

HLA-A, HLA-B, HLA-C

CD11a (LFA-1α), CD18 (LFA-1β)

CD54 (ICAM-1), ICAM-2, ICAM-3

CD2 (LFA-2)

CD58 (LFA-3)

CD40L

CD40

CD28

CD80 (B7)

LFA, Leukocyte function-associated antigen; ICAM, intercellular adhesion molecule. Essential to the proliferation of antigen-activated T cells is their expression of the CD25 (IL-2R α chain, p55) which combines with the β chain (p75, CD122) and γ chain (CD132) to form the high-affinity IL-2R. Signaling through this receptor initiates the production of IL-2, resulting in autocrine cell growth. A number of accessory glycoprotein adhesion molecules stabilize the binding of T cells to the APC during the recognition phase or to the target cell in the effector phase, as well as providing for a co-stimulatory signal required for T cell activation ( Table 1-1 ). Major Histocompatibility Complex and Immune Response The biologic basis for antigen recognition in the context of MHC molecules is to allow distinction between self and nonself. In humans the MHC gene complex is 14

located on chromosome 6 and comprises genes that code for human leukocyte antigen (HLA).[ ] Class I MHC molecules (HLA-A, HLA-B, HLA-C) are composed of a 44-kD variant chain that is noncovalently associated with the 12-kD non-MHC invariant chain, β2 -microglobulin. Class II MHC molecules (HLA-DR, HLADQ, HLA-DP) are composed of a 34-kD α-variant chain, noncovalently associated with a 29-kD β-variant chain. The biologic role of the MHC molecules is to display antigenic peptides that enables appropriate TCRs to bind them. In general, class I MHC molecules present endogenously derived antigenic peptide after antigen processing, such as viral epitopes, to CD8+ T cells,[

15]

whereas class II MHC molecules present exogenously derived antigenic peptide, such as soluble

16 cells.[ ]

bacterial protein-derived antigenic peptide, to CD4+ T Both class I and class II MHC gene products exhibit simple mendelian inheritance with co-dominant expression. Thus, single cells from any individual typically express pairs of the MHC gene products corresponding to the maternal and paternal alleles. 17

Class I MHC molecules have three external domains, and their crystalline structure has been resolved.[ ] The antigen-binding site resides within a groove formed by the first and second (α1 and α2 ) external domains of the class I MHC molecule, and the appropriate TCR on CD8+ T cells recognizes the antigen in association with these epitopes; the α3 domain has been implicated in the interaction with CD8 ( Figure 1-3 ).

Figure 1-3 Floor of HLA class I molecule on antigen-presenting cell (APC), as seen by a CD8+ T cell antigen receptor (TCR) complex. The floor (groove) of the HLA class I molecule on an APC is formed by the β-pleated sheets of the peptide backbone. Amino acids specific for each TCR complex anchor the two protein complexes in the presence of antigen. The TCR binding groove of HLA class II molecules binds to the CD4+ of T cells in a similar manner (see also Figure 1-4 ).

Figure 1-4 Activation of CD4+ T cells, with binding of T cell antigen receptor (TCR) to antigen-presenting cell (first signal) and accessory molecules (second signal). Cross-linking of TCR causes aggregation with the CD3 complex containing epsilon, δ, and ζ chains, together with the three dimers and activation of phosphorylation and differentiation. ZAP, Zeta-associated protein; MHC, major histocompatibility complex; MAPK, mitogenic-associated proliferation kinase; NFAT, nuclear factor of activated T cells; IL, interleukin; Lck, Fyn, tyrosine kinases.

ZAP, Zeta-associated protein; MHC, major histocompatibility complex; MAPK, mitogenic-associated proliferation kinase; NFAT, nuclear factor of activated T cells; IL, interleukin; Lck, Fyn, tyrosine kinases.

6

These intricate cell activation events are of considerable importance to the clinical practice of allergy and immunology because their discovery and elucidation is helping explain mechanisms underlying efficacious treatments given to patients for decades. Glucocorticoids, for example, inhibit early T cell gene activation events by the induction of proteins that bind to DNA sequences in the region of promoter response elements, whereas cyclosporin A and tacrolimus (FK506) inhibit a serine/ threonine-specific protein phosphatase called calcineurin that blocks specific gene transcription. B Cell Development The B cell matures in the bone marrow, but during fetal life this occurs in the liver. During maturation of the B cell, a series of DNA rearrangements of immunoglobulin heavy-chain genes and light-chain genes occurs for production of membrane-bound and secreted immunoglobulin molecules ( Table 1-2 ). As the pre-B cell matures, it acquires •-chain gene rearrangement together with surrogate invariant light chains (λ5 and V pre-B) necessary for effective transport of • to the cell surface expressed as the pre–B cell receptor. Association of the pre–B cell receptor with Igα and Igβ provides a means for signal transduction that facilitates continued maturation of the pre–B cell kappa (κ) or lambda (λ) gene rearrangements, ultimately resulting in the expression of a complete IgM molecule on the cell surface. Mature B cells coexpress surface IgM and IgD after heavy-chain mRNA splicing. All these maturation processes are antigen independent. Subsequent differentiation of the IgM+ /IgD+ mature B cells released into the periphery is antigen driven. Thus, activation of mature B cells into immunoglobulin-secreting B cells or long-lived memory B cells and final differentiation into plasma cells depend on antigen interaction. Isotype switching involves the further rearrangement of 24

immunoglobulin heavy-chain genes and DNA splicing and is a process under T cell control.[ ] The switching mechanisms involve at least two factors: (1) T cell to B cell contact (T cell receptor/B cell antigen presentation and B cell CD40/activated T cell CD40L) and (2) secretion of interleukin molecules, which are thought to make accessible the 5′ switch regions of the heavy-chain DNA sequence so that the γ, α, or epsilon gene can be transcribed, leading to production of IgG, IgA, or IgE. Concomitant with the immunoglobulin gene rearrangements and expression of surface immunoglobulin by developing B cells is the appearance of certain B cell markers that are useful in identifying stages of maturation and differentiation. B cell markers include the variable expression of terminal deoxynucleotidyl transferase (TdT); class II antigens; common acute lymphoblastic leukemia antigen (CALLA or CD10); the B cell-specific molecules CD19 and CD20; various membrane immunoglobulin isotypes, such as IgM; CD21, the complement receptor 2 (CR-2), which specifically binds the C3d fragment of complement component 3; CD23, the low-affinity Fc receptor for IgE; CD25, the IL-2 receptor α chain, and PC-1, the antigen that distinguishes plasma cells. Thus the phenotypic expression of markers can distinguish the stage of B cell differentiation ( Table 1-2 ). Similar to events in the thymus with the maturation and differentiation of T cells, the maturation and differentiation of B cells is thought to be under the control of cytokines (see inside cover). For example, interleukin-1 (IL-1) and IL-2 promote B cell activation and growth; IL-4 induces switching to the IgE isotype; IL-5 enhances eosinophils and B cell growth and terminal differentiation; IL-6 increases the rate of secretion of Ig by B cells; and IL-7 promotes proliferation of pre–B cells. TABLE 1-2 -- Correlation of Surface Markers and Immunoglobulin Production in Development of B Lymphocytes Characteristic

Stem

Pre-B Cell

Immature B Cell

Mature B Cell

Activated B Cell

Antibody-Secreting B (Plasma) Cell

Antigen dependency

−

−

−

−

+

+

Compartment

BM

BM

BM + PB

BM + PB

PB

PB

Intracellular proteins

TdT

RAG-1 RAG-2

Heavy-chain genes/ ISO type switch

Germline

V→J

VDJ

VDJ

Isotype switch

Isotype switch

Light-chain genes

Germline

Germline

V→J

VJ

VJ

VJ

Surface markers

MHC-II

MHC-II

CD45R

CD45R

CD45R

PC-1

CD10

•

MHC-II

MHC-II

MHC-II

CD20

CD19

CD19

IgM

IgM, IgD

IgM

CD38

CD38

CD20

CD19

CD19

CD19

CD38

CD20

CD20

CD20

CD40

CD21

CD21

CD21

CD23

CD23

CD25

CD40

CD40

CD40

Immunoglobulin (Ig) production

None

Cytoplasmic • Membrane •

Membrane IgM (κ/λ) Membrane IgD, IgM

Low-rate Ig (G, A, M, D, E)

High-rate Ig (G, A, M, D, E)

Modified from Janeway CA Jr, Travers P: The development of B cells in immunobiology in health and disease, ed 2, New York, 1996, Garland Publishing. BM, Bone marrow; PB, peripheral blood; TdT, terminal deoxynucleotidyl transferase; RAG, recombinase activating gene; V, variable; J, joining; D, diversity (gene segments); MHC-II, major histocompatibility complex class II.

7

The cytokine interferon gamma (IFN-γ) can exert a positive or negative influence on the effects of IL-6 on B cells. B Cell Antigen Receptor Gene Rearrangement and B Cell Activation Diversity of antibody specificity for antigens is made possible by rearrangements of immunoglobulin genes. Millions of specific immunoglobulin molecules can be

produced by the known rearrangements of heavy-chain and light-chain DNA coding sequences. Heavy-chain genes, located on chromosome 14, and light-chain genes, located on either chromosome 2 (κ light chains) or on chromosome 22 (λ light chains), must rearrange for the production of immunoglobulin molecules using intracellular processes that are similar to those for TCR. The heavy-chain variable region is encoded by VDJ gene segments, which are then juxtaposed to specific C regions for transcription of a complete RNA message. The light-chain variable region is encoded by VJ gene segments, which are juxtaposed to their respective C segment. Unique to B cell development is the phenomenon of heavy-chain isotype switching. Using the same mechanisms of DNA rearrangement, transcription, and RNA splicing, the cell can convert immunoglobulin production from IgM to IgA1, IgA2, IgE, or any of the four IgG subclasses, preserving the heavy-chain variable regional sequences and entire light-chain sequences and thus antigen specificity (see Chapter 9 ). Antigen activation of B lymphocytes is initiated by ligation of membrane-bound immunoglobulin, which has cytoplasmic tails consisting of only three amino acids from each heavy chain, that is, inadequate for direct physical coupling with cytoplasmic intermediaries for signal transduction. A complex of molecules 25 26

noncovalently associated with membrane immunoglobulin have cytoplasmic tails that can be phosphorylated and serve for signal transduction.[ ] [ ] These molecules include an IgM-specific 32-kD α chain and an IgD-specific 33-kD α chain, each of which is disulfide linked with a 37-kD β and a 34-kD γ chain. This membrane immunoglobulin-associated complex is considered to be analogous to the CD3 molecule of the TCR complex. 23 24

B cell activation events occur by many of the same signal transduction pathways described for T cells.[ ] [ ] Binding of the B cell antigen receptor by anti-IgM induces the rapid hydrolysis of phosphatidylinositol bisphosphate, production of IP3 and DG, activation of PKC, and eventual production of immunoglobulin. The intervening gene activation events are beginning to be explored, and findings similar to those seen in activated T cells are being discovered. In studies involving human B cell lines stimulated by the binding of platelet-activating factor (PAF) to B cell membrane receptors, in addition to the release of IP3 and DG from the lipid bilayer, arachidonic acid is released from phospholipids by phospholipase A2 or DG lipase and is subsequently converted to 5-hydroxyeicosatetraenoic acid (527 28

HETE) by activated 5-lipoxygenase. These secondary messengers are associated with the activation of the protooncogenes c-fos and c-jun. [ ] [ ] In activated human peripheral blood B cells, stimulated by HIV gp120, cyclic adenosine monophosphate (cAMP) metabolism was stimulated, cell growth occurred, the B cell 29

receptor was down-regulated, and immunoglobulin production was increased.[ ] Thus, justification can be made for B cell activation events that link cell membrane receptor binding, signal transduction pathways, intracellular cyclic nucleotide metabolism, secondary fatty acid metabolite production, activation of protooncogenes, and immunoglobulin secretion. Natural Killer Cells 30

A non-T, non-B cell lineage of lymphocytes has been defined both phenotypically and functionally.[ ] Lacking rearranged immunoglobulin or TCR genes, these cells do not express surface immunoglobulin or the TCR complex. Functionally, they may possess natural cytotoxic activity against tumor cells (NK activity), can mediate antibody-dependent cell-mediated cytotoxicity (ADCC) by activation through their IgG Fc receptors and release of cytolysins, or participate in T cell– mediated responses. NK cells have the morphologic appearance of large granular lymphocytes (LGLs) and phenotypically are CD3− /TCR− , CD16+ , CD56+ , and 31

CD57+ . Uniquely among lymphocytes, NK cells also express the IL-2R β chain (p75, CD122) and γ chain (CD132) in a resting state.[ ] Because this heterodimer has an intermediate affinity for IL-2 and a cytoplasmic extension capable of participating in signal transduction, resting LGL can be activated directly by IL-2 derived from antigen-activated T cells. A greater understanding of NK cell differentiation and function will be critical to a more complete understanding of their immune potential.

NK cell recognition of target cells has become more clear with the identification of two receptor classes: killer activator receptors (KARs), which bind to common 32

cell surface antigens, and killer inhibitor receptors (KIRs), which bind to MHC-I molecules.[ ] KIRs provide an inhibitory signal in response to cells expressing selected (less polymorphic) host MHC molecules with nonhost cells. The absence of this signal by KIRs facilitates cytotoxicity mediated by soluble mediators, including perforin and granzyme. Myeloid Stem Cell Lineage Leukocytes Differentiation of myeloid stem cell lineage leukocytes occurs under the influence of stromal cells and cytokines with both pleiotrophic and lineage-specific effects (see Figure 1-1 and inside cover). The pleiotrophic cytokines include granulocyte-macrophage colony-stimulating factor (GM-CSF) and several interleukins, especially IL-3. Lineage-specific cytokines include CSF for terminal differentiation of granulocytes (G-CSF) and of monocytes and macrophages (M-CSF). IL-5 is a specific terminal growth factor for eosinophils. GM-CSF plus IL-3 supports the differentiation of basophils; and stem cell factor, which binds to c-kit (CD 117), can induce mast cell differentiation from multipotent hematopoietic stem cells.[

33]

Although not programmed with immune memory, the circulating peripheral blood monocyte, tissue macrophage, and dendritic cell are essential components of specific immune responses because they process and present antigens in a highly structured fashion to T cells. These cells contain a number of important receptors that facilitate their interactions with antigens. These receptors are specific for the Fc region of IgG molecules and the third complement component. Monocytes and macrophages can express both class I and class II MHC molecules and thus present antigenic epitopes to TCR on either CD8+ or CD4+ T cells during antigen recognition. The contributions of the monocyte/macrophage system to the

8

general inflammatory response and the specific immune response are diverse, including functioning as phagocytic cells for intracellular pathways and as cytotoxic effector cells, particularly as effectors of ADCC. In addition, these cells produce multiple cytokines, including IL-1, IL-12, and tumor necrosis factor (TNF), that are central to inflammatory immune responses and that induce an extraordinary diversity of effects on hematopoietic and nonhematopoietic cells and tissues. Complement 34

Serum complement was first described more than 125 years ago as the fraction of serum that could be inactivated by heat.[ ] Even today, after the description of more than 25 component proteins, the complexity of nomenclature often obscures the vital role complement plays in natural immunity ( Figure 1-5 ). Most complement factors are produced in the liver, with smaller contributions made by monocytes and fibroblasts. Complement is a distinct humoral immune system that contributes to host protection against infection. It acts in concert with the other humoral system, antibody, to promote both physical clearance of pathogens and nonspecific inflammation. Coating a foreign membrane with complement proteins (opsonization) allows specific cellular components to ingest the pathogen. Anaphylatoxins and chemotactic factors, derived from complement proteins (e.g., C5a), contribute to nonspecific inflammation. Anaphylatoxins promote the release of histamine from basophils, promoting easy entry of inflammatory cells from the blood stream to the tissue

Figure 1-5 Complement cascade. Comparison of classic, mannan-binding lectin, and alternative pathways of complement. A major function of all pathways is generation of C3b, an important opsonin, and production of the membrane attack complex (MAC). These common goals are achieved by different routes of initiation and differing mechanisms for conversion of C3 and C5. The alternative pathway is initiated by any mechanism that increases the rate of C3b production or reduces the rate of C3b breakdown, such as the presence of microorganisms. The mannan-binding pathway is activated by serum mannan-binding lectins attaching to microbes with mannan residues on cell walls. The classic pathway initiates with the interaction of preexisting antibody (Ab) and C1q to facilitate the subsequent cleavage of C4. The alternative pathway C3 convertase forms through the interaction of C3b, factor B (B), and factor D (D). The resulting C3bBb, stabilized by properdin (P), converts C3 to C3b, which can act as an opsonin or contribute to the C5 convertase (C3bBb3b). The classic pathway uses C4b2a to convert C3 to C3b, which subsequently participates in the C5 convertase (C4b2a3b). Production of C5b by either C5 convertase allows progression to the lytic pathway, where C6, C7, C8, and C9 participate in the generation of the MAC. In the course of both pathways, cleavage of C3 and C5 generates C3a and C5a, which possess anaphylatoxin activity.

TABLE 1-3 -- Effects of Cytokines on Precursor Cells Involved in Immediate Hypersensitivity (IgE-Derived) Reactions Activity

IL-3

IL-4

IL-5

IL-9

IL-13

SCF

1. B cell proliferation

—

↑

—

↑

↑

—

••IgE production

—

↑

—

↑

↑

—

••MHC class II

—

↑

—

↑

↑

—

— ••CD23 (FcepsilonRII)

↑

—

↑

↑

—

— ••B cell differentiation

↑

—

↑

↑

—

2. Mast cell maturation

↑

↑

—

↑

↑

—

3. Eosinophil maturation

↑

↑

↑

↑

↑

—

4. Basophil maturation

↑

↑

↑

↑

↑

↑

IgE, Immunoglobulin E; IL, interleukin; SCF, stem cell factor; MHC, major histocompatibility complex.

10

Box 1-1. Mechanisms of Immunopathologic Reactions 1. 2. 3. 4. 5. 6. 7.

Inactivation/activation antibody reactions Cytotoxic or cytolytic antibody reactions Immune complex reactions Allergic reactions T cell cytotoxic reactions Delayed hypersensitivity reactions Granulomatous reactions

protein messages such as cytokines (e.g., TGF-β, IL-10), as well as antiidiotypic signals and regulatory T cells. These circuits allow for managing the magnitude and duration of an immune response and appear to depend, at least in part, on a process referred to as “programmed cell death” (apoptosis). The two general pathways of apoptosis are a passive process of cell death initiated by the removal of artificial growth factors and an active pathway that involves the interaction between specialized receptors and their specific ligands. Apoptosis is a mechanism for controlling a specific immune response, and it also is a central process in the elimination of autoreactive lymphocytes. Recent evidence suggests that complex regulatory processes are involved in apoptosis that differ between activated and nonactivated cells.[

40]

In addition, apoptosis appears to be a mechanism for controlling other inflammatory cells involved in the immune response and an effector

pathway for cytolytic lymphocytes. [

41]

Chemokines Chemokines are generally small-molecular-weight polypeptides that are capable of regulation of numerous cell functions (e.g., chemotaxis, adhesion, degranulation), except proliferation. At a functional level, chemokines are classified as being proinflammatory (e.g., IL-8, MIP-1α, eotaxin, MIP-1β) or developmental/homeostatic 42 43

(e.g., SDF-1, β-defensins, lymphotactin).[ ] [ ] The chemokines are further classified with regard to the pairs of cysteine amino acids that bind their molecules internally to maintain their folded structure, such as CC (double-cysteine molecules), CXC (cysteine residues separated by a single amino acid), C (single-cysteine molecule), and CX3C (cysteine residues separated by three amino acids). All chemokine receptors share a 7-transmembrane structure, a feature shared with G-protein type receptors. Chemokines signal through their corresponding chemokine receptors (e.g., CC) and produce changes in target cell response, either initiating new biologic responses in the case of the proinflammatory chemokines or maintaining an ongoing response in the case of developmental/homeostatic chemokines (see Appendix A). One unexpected discovery was that certain microbial organisms utilize chemokine receptors to enter target cells and carry out their life cycles. The best known of these is human immunodeficiency virus type 1 (HIV-1), which enters monocytes and macrophages by the cognate recognition of the CCR5 receptor and enters CD4 44 45

T helper cells by a similar mechanism involving the SDF-1 receptor CXCR4 and CD4.[ ] [ ] The specificity of these chemokine receptor entry systems was established when it was demonstrated that the soluble chemokines macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and RANTES (regulated on activation, normal T cells expressed and secreted) inhibited the binding of the HIV virion to target monocytes. Also, SDF-1 inhibited HIV binding to CD4 T cells.

The extraordinary importance of the role of chemokine receptors in human disease was further demonstrated when studies showed that gene deletions or mutations either rendered humans resistant to HIV infection or altered the course of HIV disease progression. Moreover, some viruses have incorporated the genetic code for chemokine receptors that they use for target cell entry into their own genome. These interesting findings raise the possibility that designer chemokine or soluble chemokine receptor molecules may be capable of being employed as specific immunotherapy for viral infections. Regulation of IgE Production by T Cells: Th1 vs. Th2 and Cytokine Profiles To link the immune response and immune regulation directly to immediate hypersensitivity, it is instructive to consider the immune regulation of IgE production. This is exquisitely sensitive to T cell control, as initially documented in the description of T cell deficiency, where IgE levels in patients were extremely elevated and returned to normal after T cell reconstitution following bone marrow transplantation. The hyper-IgE syndrome, a condition of recurrent bacterial infections, chronic eczema, and early malignancy, is characterized by defective T cell regulation and IgE 46

levels up to 100,000 IU/ml of serum.[ ] The precise molecular defect of the hyper-IgE syndrome is not known but likely involves at least a functional deficiency in T cell regulatory mechanisms and possibly defective T cell production of IFN-γ. 47

Evidence has been presented to suggest that certain T helper (Th) cell subsets may play an immunoregulatory role in cytokine regulation of immune responses.[ ] At a conceptual level the T helper 1 (Th1) cells are associated with cytotoxic or delayed-type hypersensitivity (DTH) immune responses involving IFN-γ and IL-12, whereas Th2 inflammatory immune responses involve IgE and eosinophils with production of IL-4 and IL-5. Th1 cells generate an immunologic response that provides an effective defense against viral infections and other intracellular pathogens and depends on T cell-monocyte/dendritic cell interactions. Th2 cells exert their effect on immunologic responses to parasites with augmentation of inflammation from IgE production and eosinophilic infiltration. In the study of the immunoregulation of allergic responses, CD4+ T cell clones specific for dust mite allergens were isolated from atopic and nonatopic individuals. Clones from the atopic persons secreted IL-2 and IL-4, but not IFN-γ and supported in vitro production of IgE. Clones from nonatopic persons secreted IL-2 and IFN-γ but little IL-4 47

and suppressed in vitro IgE production.[ ] Thus a functional compartmentalization of T cells seemed to produce varied amounts of IL-4 vs. IFN-γ, which has a profound effect on IgE production by B cells. This human system has been compared with the murine immunoregulatory system for IgE production, in which Th1 cells secrete IFN-γ, IL-2, and TNF-β and turn off IgE synthesis, whereas Th2 cells secrete IL-4, IL-5, and IL-6 and turn on IgE synthesis by B cells.

11

Immunotherapy of allergic disease has been shown to induce T cells capable of turning off IgE responses to allergen that likely represent a switch from Th2 to Th1 48

allergen-specific T cells.[ ] Undoubtedly, all the steps of immune sensitization described previously, beginning with presentation of antigen to T cells by APCs and ending with plasma cell secretion of antibody, apply to the IgE response to allergens. Moreover, the demonstration that IL-4 directs switching to IgE production provides the impetus to search for antagonist cytokines or molecules capable of down-regulating IL-4 production or expressing IL-4 receptors. Because a significant portion of the clinical practice of allergists actually deals with the regulation of IgE responses, it is appropriate for allergists to understand the fundamental pathways of IgE biosynthesis and regulation.

IMMUNOPATHOLOGY: STUDY OF UNEXPECTED IMMUNE RESPONSES Immunopathology is the study of untoward reactions produced by immune mechanisms that primarily exist for protection. For many years the classification system of Gell and Coombs enjoyed wide acceptance as a guide to understanding complex immunologic reactions that regularly produce clinical illness. The allergist and clinical immunologist caring for patients with these hypersensitivity diseases are, in fact, observing and treating the results of the unexpected excesses of the immune response, some of which are autoimmune in nature. The original Gell and Coombs classification lists four types of hypersensitivity (immunopathologic) reactions: I, immediate (IgE mediated); II, cytotoxic (IgG/IgM mediated); III, immune (IgG/IgM immune complex mediated); and IV, delayed (T cell mediated). The authors 49

endorse an alternate classification system proposed by Sell that categorizes immunopathologic reactions on the basis of seven mechanisms (see Box 1-1 ).[ ] In attempting to classify the type of immunopathologic reactions that some patients experience, several of these mechanisms may be applied. For example, patients who are allergic to drugs (e.g., penicillin) may display symptoms compatible with several of these mechanisms. Over the years the word “allergy” has been used to describe many of these reactions. The division of untoward immunologic reactions into specific categories, although having merit for purposes of classification, has led many clinicians into explaining allergic reactions by oversimplified mechanisms. In reality, allergic reactions typically involve components of the immune and inflammatory responses that are common to all immune reactions. A brief consideration of each of the seven general types of immunologic reactions illustrates the integration of immunology in the expression of allergic reactions. Mechanism 1: Immune-Mediated Inactivation/Activation of Biologically Active Molecules Antibody to a hormone, hormone receptor, blood-clotting factor, growth factor, enzyme, or drug may cause disease or treatment failure by inactivating the vital biologic function of these molecules. In addition, antibodies to receptors on cells may activate the secretory function of the cell. The disease caused by activation or inactivation depends on the function of the biologically active molecule or cell involved. In mechanism 1 immunopathologic reactions, two nondestructive and paradoxical antibody-mediated reactions are characterized by (1) target cell stimulation and (2) negative signaling or ligand blockade. Antibody to the thyrotropin receptor on thyroid epithelial cells is an example of an autoantibody that can act equivalent to the 50

ligand for the hormone receptor, yielding a positive signal to the cell.[ ] Autoantibodies to the insulin receptor in type IIB insulin-resistant diabetes mellitus can cause opposite effects on blood glucose levels over time in the same individual, depending on the relative levels of insulin receptor–stimulating or insulin receptor– blocking antibodies. Mechanism 2: Antibody-Mediated Cytolytic Reactions These immune reactions involve IgG and IgM and to a lesser extent IgA, directed to cell surface antigens on erythrocytes, neutrophils, platelets, and epithelial cells of glandular or mucosal surfaces or to antigens on tissues (e.g., basement membranes). The sensitizing antigens in these cases can be natural cell surface antigens, modified cell surface antigens, or haptens attached to cell surfaces. There are three categories of mechanism 2 immunopathologic reactions. The first occurs by opsonization, which is facilitated by complement activation; the second induces complement-mediated lysis; the third is cytotoxicity mediated by ADCC. These mechanisms are well known to afford protection against infections and eradication of malignant cells but can also result in damage to self-antigens in various tissues.

An example of the first category is the phagocytic cell destruction of antibody-coated platelets (opsonization) leading to thrombocytopenia. The platelet antigens to which autoantibodies bind are glycoprotein Iib (CD41) and glycoprotein IIIa (CD42). The second-category reaction is illustrated when penicillin binds to the surfaces 51

of erythrocytes, creating a nonself antigen composed of penicillin-modified erythrocyte cell surfaces.[ ] These new antigens elicit an immune response. Antipenicilloyl antibodies, initially IgM and later IgG, fix to erythrocyte surfaces and concomitantly activate complement, leading to the lysis of the cell with penetration of the terminal hydrophobic complement MAC (C5-9). Clinically this condition is known as penicillin-induced autoimmune hemolytic anemia. Other clinical examples of this reaction include quinidine-induced autoimmune thrombocytopenia and methyldopa-induced autoimmune hemolytic anemia. Because of the biologic amplification mechanism inherent in the activation process of complement, the thousands of activated complement components generated from a few target cells under immune attack by antibody can damage non-antibody-bound target cells, the “innocent bystander effect.” 52

Several classic forms of autoimmunity involve mechanism 2 immunopathologic reactions.[ ] Certain forms of thyroiditis involve both antibody and complementmediated cytotoxic reactions and ADCC (third category) attack of antithyroid antibody–coated thyroid glandular cells. The stimulus of this form of autoimmunity is not known but is thought to result from viral infections that alter the state of tolerance defined early in life when T lymphocytes are programmed in the

12

thymus gland not to react to self-antigens. Another example of a third-category reaction is the ADCC reaction produced when eosinophils bind through FcαR receptors to IgA-bound helminths and release cytolytic major basic protein. Mechanism 3: Immune Complex Reactions IgG and IgM antibodies, activated complement, and neutrophils are participants in immune complex–mediated reactions, again as the result of vigorous immune responses in genetically predisposed individuals on exposure to certain antigens, with a resultant immune injury. Knowledge of this immunologic disease became 53 54

widespread in the early 1900s, when physicians began using hyperimmune animal sera, usually equine derived, to treat bacterial infections.[ ] [ ] As many as 25% of patients treated with animal sera become seriously ill or even died. Even with the rudimentary understanding of the immune system at that time, physicians ascribed the clinical findings of inflammation of the reticuloendothelial system, skin, kidneys, and joints to an antibody process. With succeeding decades, it became clear that immune complexes of antibody and antigen, activated complement components, and chemotaxis of neutrophils were important participants in this hypersensitivity reaction. Clinical investigators described the appearance of dual sensitization whereby the IgG (and/or IgM) and IgE immune responses were stimulated. Highly sensitized patients would sustain the immune complex-mediated reaction 10 to 14 days after exposure to antigen, and this delayed reaction would have been heralded by a classic immediate allergic reaction. Mechanism 4: Allergic Reactions The immediate hypersensitivity reaction with release of mast cell or basophil mediators creating immediate and delayed (4 to 8 hours) responses to sensitizing allergens appears to be the result of host defense mechanisms to parasites. Anaphylactic responses require allergen-specific IgE antibody to attach to high-affinity

IgE receptors on mast cell or basophil surfaces, providing the means for triggering the cascade of cellular events after allergen binding, as with anaphylaxis to penicillins or with allergic rhinitis to ragweed pollen. Anaphylactoid responses are caused by IgE-independent mechanisms of mast cell or basophil degranulation, such as those associated with high-osmolar radiocontrast material. Although not directly involved in the immediate reaction, eosinophils are drawn to the site of the reaction by specific chemotactic factors generated during mechanism 4 responses. Eosinophils contain cationic proteins, the best characterized being major basic protein, which have the potential to contribute to ongoing inflammatory reactions by producing cellular injury[ hypersensitivity reaction by chemotactic factors.

55]

(see Chapter 19 ). In addition to eosinophils, it is likely that neutrophils are drawn to the site of an immediate

Therefore, at least four cell types (mast cell, basophil, eosinophil, neutrophil) may participate in the full expression of anaphylactic reactions. Each of these cells is programmed to secrete significant quantities of mediators, which contributes to the general state of inflammation. These mediators include histamine, prostaglandins, leukotrienes, and PAF. In the case of chronic allergic reactions such as asthma, mononuclear cell infiltrates containing T cells are prominently found at the site of inflammation, such as the lung (see Chapter 30 ).[

56]

Investigators increasingly are discovering that the early-phase, late-phase, and chronic manifestations of asthma involve a stepwise participation of many, if not all, components of the immune and inflammatory responses. Th cells, monocytes, eosinophils, cytokines, chemokines, and arachidonic acid metabolites all contribute to the clinical expressions of asthma. In addition, the expression of adhesive glycoproteins on bronchial endothelial cells are up-regulated by cytokines and play a role 57

in late-phase allergic reactions in model lung systems.[ ] IL-1 and TNF-α up-regulate the expression of intercellular adhesion molecule-1 (ICAM-1; see Table 1-1 ), endothelial-leukocyte adhesion molecule-1 (ELAM-1), and vascular cell adhesion molecule-1 (VCAM-1) in the endothelial cell surfaces, which in turn recruit and 58 59

produce adherence of neutrophils, eosinophils, and basophils.[ ] [ ] Monoclonal antibodies to ICAM-1 and ELAM-1 reduced the adhesiveness of all cell types to IL1-up-regulated adhesion molecules, and anti-VCAM-1 antibody reduced adherence of basophils and eosinophils. These new facts have changed the clinical management of allergic disease, with a progressive increase in the use of drugs that modify inflammatory responses. Drugs such as corticosteroids and sodium cromoglycate are now being used to blunt the excesses of the inflammatory responses underlying all forms of asthma. Therapies focused on the use of antagonists of cytokines, chemokines, and their receptors important in both IgE production and eosinophil, basophil, and mast cell growth are currently under study. The sum total of immune events that characterize the immediate hypersensitivity reactions describe a multicomponent response, with activated IgE-sensitized mast cells or basophils by allergen binding playing the leading role. Undoubtedly, most if not all components of the immediate hypersensitivity reaction are important in host defense. Because of genetic susceptibility of the host leading to an exaggerated IgE response to allergens, a significant proportion of the population experiences exaggerated allergic reactions. Mechanism 5: Cell-Mediated Cytotoxicity The mechanism 5 category of the immunopathogenic reactions is caused by T cells or NK cells. An example of such reactions is the T cell infiltration of blood vessels and alveoli in chronic asthma. Mechanism 5 immune responses are mediated by CD4+ T cells, CD8+ T cells, and NK cells. [

60]

CD8+ T cell cytolytic

responses to viruses and alloantigens are clinical examples of such immune reactions. NK cells play an important role in immune surveillance against certain viruses and tumor cells. Mechanism 6: Delayed Hypersensitivity The typical mechanism 6 immune response is the delayed hypersensitivity reaction, known to be caused by sensitized T cells, in particular the CD4+ (helper) cell population. A representative clinical example of reaction is contact dermatitis resulting from poison ivy Rhus antigen. This form of hypersensitivity reaction is typically considered an allergic reaction but actually is CD4+ T cell-mediated Th1 type of response

13

(rather than Th2 cells). Although the most apparent focus is on delayed hypersensitivity reactions involving the skin and sensitizing antigens, reactions involving other organ systems and target antigens are known. There is also an increasing awareness of the participation of other immune and inflammatory cells and cytokines in delayed hypersensitivity reactions. In the skin, for example, the HLA-DR+ Langerhans' cell presents antigen to T cells, and IFN-γ, IL-1, and TNF-α all modulate the intensity of the immune response. This type of immune response appears to play a central role in certain autoimmune diseases, such as rheumatoid arthritis, and serves as the theoretic basis for new therapeutic approaches directed at blocking inflammatory cytokines. Mechanism 7: Granulomatous Reactions Granulomas are focal collections of inflammatory cells in tissues, including macrophages, histiocytes, epithelioid cells, and giant cells, as well as lymphocytes and 49

plasma cells surrounded by varying amounts of fibrous tissue.[ ] The characteristic epithelioid cell is derived from a macrophage and has a prominent eosinophilic amorphous cytoplasm and a large, oval, pale-staining nucleus with a sharp, thin nuclear membrane and large nuclei. This characteristic pathologic appearance has been recognized for more than 150 years. These cells have been called epithelioid because of their resemblance to epithelial cells. Granulomas may progress from high cellular reactions to fibrous scars. Granulomatous reactions are cellular responses to irritating, persistent, and poorly soluble substances. These reactions are characteristically initiated by sensitized lymphocytes reacting with antigen but may also occur in response to poorly catabolizable antigen-antibody complexes that persist locally. Not all granulomas have their origin in an immune response. Common granulomatous reactions are those surrounding insoluble suture material or around urate deposits in gouty lesions. Similar lesions may be induced by other foreign bodies. Antibody-antigen complexes may provide a stimulus for granuloma formation and inflammation if the complex is insoluble and poorly digestible. Clinical conditions involving granulomatous reactions include tuberculosis, leprosy, parasitic diseases, berylliosis, and asbestosis.

CONCLUSION The total inflammatory response includes elements comprising nonspecific and specific cellular and humoral immunity and a growing network of interacting soluble mediators, cytokines, and chemokines. The “classic” allergic diseases use selected components of the inflammatory response in effector systems that produce clinical symptoms in patients. The various chapters of this textbook are directed at selected portions of these inflammatory responses and corresponding clinical conditions. For example, chapters on allergic rhinitis, asthma, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, sinusitis/otitis/recurrent infections, anaphylaxis, urticaria, atopic dermatitis, and food allergy all attempt to convey information on clinically diagnosed conditions and describe how portions of the inflammatory response are excessive, misguided, or deficient in producing clinical illness. As molecular biology uncovers more common molecules, mediators, and signal pathways in the seemingly separate components of the inflammatory response, it becomes easier to place the allergic response in the spectrum of inflammation. Systems that once were thought to operate only within the context of classic allergy have now been shown to exert effects in many effector systems. For example, major basic protein is thought to produce significant inflammatory effects in atopic eczema, chronic asthma, graft-versus-host disease, and immune surveillance against tumors. The interleukin family of peptide regulators exerts effects on mast cells, IgE production, T cells, and B cells. The integrin supergene family of surface accessory glycoproteins transcends cell type and displays an economy of DNA inheritance in molecular recognition, cellular adhesion, signal transduction, and cell activation. This overview provides a sampling of the new areas of research in allergy and clinical immunology that are covered in detail in subsequent chapters. With these advances come unprecedented opportunities to develop and apply new approaches to the diagnosis and management of allergic and immunologic disorders.

REFERENCES Features of the Immune System 1. Delves PJ, Roitt IM: The immune system (in two parts), N Engl J Med 343:37–49, 108–117, 2000. 2. Parkin J, Cohen B: An overview of the immune system, Lancet 357:1777–1789, 2001. 3. Pardoll DM: Cancer vaccines, Nat Med 4:525–531, 1998. 4. Caligaris-Cappio F: How immunology is reshaping clinical disciplines: the example of haematology, Lancet 358:49–55, 2001. 5. Wilson CC, Walker BD: Acquired immunodeficiency syndrome. In Rich RR, Fleisher TA, Shearer WT, et al, editors: Clinical immunology: principles and practice, ed 2, St Louis, 2001, Mosby, pp 1–28. 6. Kamradt T, Mitchison NA: Tolerance and autoimmunity, N Engl J Med 344:655–664, 2001. 7. Goodnow CC: Pathways for self-tolerance and the treatment of autoimmune diseases, Lancet 357:2115–2121, 2001.

8. Matzinger P: An innate sense of danger, Semin Immunol 10:399–415, 1998. 9. Medzhitov R, Janeway C Jr: Innate immunity, N Engl J Med 343:338–344, 2000. 10. Galli SJ, Maurer M, Lantz CS: Mast cells as sentinels of innate immunity, Curr Opin Immunol 11:53–59, 1999. Constituents and Development of the Immune System 11. Kruisbeek AM: Introduction: regulation of T cell development by the thymic microenvironment, Semin Immunol 11:1–2, 1999. 12. Jamieson BD, Douek DC, Killian S, et al: Generation of functional thymocytes in the human adult, Immunity 10:569–575, 1999. 13. Garcia KC, Teyton L, Wilson IA: Structural basis of T cell recognition, Ann Rev Immunol 17:369–397, 1999. 14. Klein J, Sato A: The HLA system (in two parts), N Engl J Med 343:702–709, 782–786, 2000. 15. Salter RD, Benjamin RJ, Wesley PK, et al: A binding site for the T-cell co-receptor CD8 on the α3 domain of HLA-A2, Nature 345:41, 1990. 16. Chapman HA: Endosomal proteolysis and MHC class II function, Curr Opin Immunol 10:93–102, 1998. 17. Bjorkman PJ, Saper MA, Samraoui B, et al: Structure of the human class I histocompatibility antigen, HCA-A2, Nature 329:506–512, 1987. 18. Brown JH, Jardetzky TS, Gorga JC, et al: The three-dimensional structure of the human class Ii histocompatibility antigen HLA-DRI, Nature 364:33, 1993. 19. Stern LJ, Brown JH, Jardetzky TS, et al: Crystal structure of the human class Ii MHC protein HLA-DR1 complexed with an influenza virus peptide, Nature 368:215, 1994. 20. Khalil I, d'Auriol L, Gobet M, et al: A combination of HLA-DQβ Asp67-negative and HLA-DQα Arg 52 confers susceptibility to insulin-dependent diabetes mellitus, J Clin Invest 85:1315, 1990. 21. Arstila TP, Casrouge A, Baron V, et al: A direct estimate of the human alpha/beta T cell receptor diversity, Science 286:958–961, 1999. 22. DeFranco AL, Weiss A: Lymphocyte activation and effector function, Curr Opin Immunol 10:243–367, 1998. 23. Healy JI, Goodnow CC: Positive versus negative signaling by lymphocyte antigen receptors, Annu Rev Immunol 16:645–670, 1998. 24. Casellas R, Nussenzweig A, Wuerffel R, et al: Ku80 is required for immunoglobulin isotype switching, EMBO J 17:2404–2411, 1998.

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25. Kurosaki T: Genetic analysis of B cell antigen receptor signaling, Annu Rev Immunol 17:555–592, 1999. 26. Scharenberg AM, Kinet JP: The emerging field of receptor-mediated inhibitory signaling: SHP or SHIP, Cell 87:961–964, 1996. 27. Schulam PG, Kuruvilla A, Putcha G, et al: Platelet activating factor induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line, J Immunol 146:1642–1648, 1991. 28. Kuruvilla A, Pielop C, Shearer WT: Platelet-activating factor induces the tyrosine phosphorylation and activation of phospholipase C-γ1,fyn, and lyn kinases, and phosphatidylinositol 3-kinase in a human B cell line, J Immunol 153:5433–5442, 1994. 29. Patke C, Shearer WT: gp120- and TNF-α-induced modulation of human B cell function: proliferation, cyclic AMP generation, Ig production, and B cell receptor expression, J Allergy Clin Immunol 105:975–982, 2000. 30. Biron CA, Nguyen KB, Pien GC, et al: Natural killer cells in antiviral defense: function and regulation by innate cytokines, Annu Rev Immunol 17:189–220, 1999. 31. Raulet DH, Held W: Natural killer cell receptors: the offs and ons of NK cell recognition, Cell 82:697–700, 1995. 32. Moretta A, Biassoni R, Bottino C, et al: Major histocompatibility complex class I-specific receptors on human natural killer and T lymphocytes, Immunol Rev 155:105–117, 1997. 33. Mekori YA, Oh CK, Metcalfe DD: IL-3-dependent murine mast cells undergo apoptosis on removal of IL-3, J Immunol 151:3775, 1993. 34. Holers VM: Complement. In Rich RR, Fleisher TA, Shearer WT, et al, editors: Clinical immunology: principles and practice, ed 2, St Louis, 2001, Mosby, pp 1– 18. 35. Walport M: Complement (in two parts), N Engl J Med 344:1058–1066, 1140–1144, 2001. Cellular Interaction and Immunoregulation 36. Oppenheim JJ, Feldman M, editors: Cytokine reference: a compendium of cytokines and other mediators of host defense, London, 2000, Academic Press. 37. McKenzie GJ, Fallon PG, Emson CL, et al: Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper cell type 2-mediated responses, J Exp Med 189:1565–1572, 1999. 38. Nelms K, Keegan AD, Zamorano J, et al: The IL-4 receptor: signaling mechanisms and biologic functions, Annu Rev Immunol 17:701–738, 1999. 39. Fitch FW, McKisic MD, Lancki DW, et al: Differential regulation of murine T lymphocyte subsets, Annu Rev Immunol 11:29, 1993. 40. Wickremasinghe RG, Hoffbrand AV: Biochemical and genetic control of apoptosis: relevance to normal hematopoiesis and hematological malignancies, Blood 93:3587–3600, 1999.

41. Scaffidi C, Kirchhoff S, Krammer PH, et al: Apoptosis signaling in lymphocytes, Curr Opin Immunol 11:277–285, 1999. 42. Zlotnik A, Yoshie O: Chemokines: a new classification system and their role in immunity, Immunity 12:121–127, 2000. 43. Von Andrian UH, Mackay CR: T cell function and migration: two sides of the same coin, N Engl J Med 343:1020–1034, 2000. 44. Berger EA, Murphy PM, Farber JM: Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease, Annu Rev Immunol 17:657–700, 1999. 45. Loetscher P, Moser B, Baggiolini M: Chemokines and their receptors in lymphocyte traffic and HIV infection, Adv Immunol 74:127–180, 2000. 46. Gorin LJ, Jeha SC, Sullivan MP, et al: Burkitt's lymphoma developing in a 7-year-old boy with hyper-IgE syndrome, J Allergy Clin Immunol 83:5–10, 1989. 47. Kay AB: Allergy and allergic diseases (in two parts), N Engl J Med 344:30–37, 109–113, 2001. 48. Frew AJ: Immunotherapy for allergic disease. In Rich RR, Fleisher TA, Shearer WT, et al, editors: Clinical immunology: principles and practice, ed 2, St Louis, 2001, Mosby, pp 1–10. Immunopathology: Study of Unexpected Immune Responses 49. Sell S: Immunopathology. In Rich RR, Fleisher TA, Schwartz BD, et al, editors: Clinical immunology: principles and practice, St Louis, 1996, Mosby, pp 449– 477. 50. Baker JR Jr: Autoimmune endocrine disease, JAMA 278:1931–1937, 1997. 51. Smith LA: Autoimmune hemolytic anemias: characteristics and classification, Clin Lab Sci 12:110–114, 1999. 52. McIntosh RS, Asghar MS, Weetman AP: The antibody response in human autoimmune thyroid disease, Clin Sci 92:529–541, 1997. 53. Kojis FG: Serum sickness and anaphylaxis: analysis of 6,211 patients treated with horse serum for various infections, Am J Dis Child 64:93–143, 1942. 54. Lawley TJ, Bielory L, Gascon P, et al: A prospective clinical and immunologic analysis of patients with serum sickness, N Engl J Med 311:1407–1413, 1984. 55. Gleich GJ, Adolphson CR, Leiferman KM: The biology of the eosinophilic leukocyte, Annu Rev Med 44: 85, 1993. 56. Busse W, Lemanske RF Jr: Asthma, N Engl J Med 344:350–362, 2001. 57. Leung DYM, Pober JS, Cotran RS: Expression of endothelial-leukocyte adhesion molecule-1 in elicited late-phase allergic reactions, J Clin Invest 87:1805–1809, 1991. 58. Bochner BS: Cellular adhesion and its antagonism, J Allergy Clin Immunol 100:581–585, 1997. 59. Wardlaw AJ: Molecular basis for selective eosinophil trafficking in asthma: a multistep paradigm, J Allergy Clin Immunol 104:917–926, 1999.

60. Biron CA, Nguyen KB, Pien GC, et al: Natural killer cells in antiviral defense: function and regulation by innate cytokines, Annu Rev Immunol 17:189–220, 1999.

15

Chapter 2 - Molecular Biology and Genetic Engineering

Sudhir Gupta Leman Yel

Until a decade ago, the function of a gene was studied by examining the effect of its mutation in the whole organism and, more recently, in cultured cells. However, the technology of gene cloning, in vitro mutagenesis, gene expression in heterologous cells, development of transgenic and knockout animals, genomics, and proteomics have revolutionized genetic analysis in human biology. The technology of molecular biology has allowed investigators to manipulate cells and organisms in a highly specific manner, permitting them to discern the effect of a designed change in a single protein on the cells or an organism. The consequences of recombinant technology are far-reaching in that it is possible to synthesize proteins in large quantities to study structure and functions. Furthermore, these molecules may be used as drugs or vaccines. New molecular biology techniques have made the Human Genome Project a reality, with multiple long-term therapeutic implications, including gene therapy.

ANATOMY OF THE GENE The gene, the basic unit of heredity, is carried by the chromosome and stored in the nucleus. Genes are made up of deoxyribonucleic acid (DNA), which is the genetic material for all cellular organisms. DNA contains three main components: the phosphate (PO4 ) groups, five-carbon sugars, and nitrogen-containing bases called purines, comprising adenine (A) and guanine (G), and pyrimidines, comprising thymine (T) and cytosine (C). These basic subunits are called nucleotides. Nucleic acids are polymers of repeating subunits of nucleotides. The carbon atoms are numbered 1′ to 5′ proceeding clockwise from the oxygen atom ( Figure 2-1 ). The phosphate group is attached to the 5′ carbon atom of the sugar, and the base is attached to the 1′ carbon atom. There is an additional free hydroxyl group (−OH) attached to the 3′ carbon atom. The presence of 5′ phosphate and 3′ hydroxyl groups allows DNA and ribonucleic acid (RNA) to form long chains of nucleotides. The 5′ phosphate of one nucleotide interacts with the 3′ hydroxyl group of another nucleotide, and a covalent bond (phosphodiester bond) is formed between the two molecules. This two-unit nucleotide still has a free 5′ phosphate at one end and a 3′ hydroxyl group at the other so that it can interact with other nucleotides at each end. In this manner, a long chain of nucleotides can be joined together to form a DNA or RNA molecule. All four nucleotides are not in equal amounts, but the

amount of adenine present in a DNA molecule is always equal to the amount of thymine, and the amount of guanine always equals the amount of cytosine (A = T and G = C). The three-dimensional structure was not known until early in the 1950s, when British chemist Rosalind Franklin, working in the laboratory of Maurice Wilkins, performed x-ray crystallography of DNA fibers. The diffractional pattern suggested that the DNA molecule had the shape of a helix or corkscrew, with a diameter of 1

2•nm and a complete helical turn every 3.4•nm. James Watson and Francis Crick[ ] built models of nucleotides and tried to assemble the nucleotides into a molecule. After exploring various possibilities, they proposed a “double helix” structure of the DNA molecule, in which the bases of two strands pointed inward toward one another (base pairing). They proposed that the base pairing was always between purines (large) pointing toward pyrimidines (small), thus keeping the diameter of the molecule at a constant 2•nm. The double helix is stabilized by a hydrogen bond between the bases in a base pair; adenine makes double hydrogen bonds with thymine, and guanine will form three hydrogen bonds with cytosine ( Figure 2-1 ).

RNA AND PROTEIN SYNTHESIS All eukaryotic cells use DNA to direct protein synthesis. Proteins are made in the cytoplasm on the ribosome. These polypeptide-making factories contain more than 50 different proteins, as well as RNA. RNA is similar to DNA, and its presence in ribosomes suggests its important role in protein synthesis ( Figure 2-2 ). RNA differs from DNA in two ways: RNA contains ribose as sugar rather than the deoxyribose in DNA, and RNA contains the pyrimidine uracil (U) instead of thymine 2

3

(T).[ ] In addition, RNA does not have a regular helical structure. The class of RNA present in ribosomes is called ribosomal RNA (rRNA).[ ] rRNA and ribosomal 4 5

proteins provide sites where polypeptides are assembled. Transfer RNA (tRNA) transports the amino acids to the ribosome for the synthesis of polypeptide.[ ] [ ] There are more than 40 different tRNA molecules in human cells. tRNA is smaller than rRNA and is present in free form in the cytoplasm. Messenger RNA (mRNA) comprises long strands of RNA molecules that are copied from DNA. mRNA travels to the ribosome to direct the assembly of polypeptides. RNA is synthesized on a DNA template by a process of DNA transcription in which RNA polymerase enzymes make an RNA copy of a DNA sequence. RNA polymerases are

16

Figure 2-1 Structure of DNA. Left, Model of Watson-Crick DNA double helix. A, Adenine, T, thymine; G, guanine; C, cytosine. Right, Chain-linked deoxyribose and phosphate residues forming the sugar-phosphate bond. (Courtesy Baback Roshanravan.)

(Courtesy Baback Roshanravan.) formed from multiple polypeptide chains with a molecular weight of 500,000 D.[

6] [7]

In eukaryotic cells there are three different types of RNA polymerases. RNA

polymerase II transcribes the gene whose RNAs will be translated into proteins. RNA polymerase I makes the large rRNA precursor (45S rRNA) containing the major rRNAs. RNA polymerase III makes very small, stable RNAs, including tRNA and the small 5S rRNA. In mammalian cells there are approximately 20,000 to 40,000 molecules of each of the RNA polymerases. Transcription The first phase of gene expression is the production of an mRNA copy of the gene. As in all other RNAs, mRNA is formed on a DNA template by a process of 6 7 8 9

transcription.[ ] [ ] [ ] [ ] Transcription is initiated when RNA polymerase binds to a specific DNA sequence, called the promoter, located at the 5′ end of the DNA, which contains the start site for RNA synthesis and signals where RNA synthesis should start. After binding to the promoter, the RNA polymerase opens up an adjacent area of the double helix to expose the nucleotides on a small stretch of DNA on each strand. One of the two exposed DNA strands serves as a template for complementary base pairing with RNA nucleotide. Therefore, G, C, T, and A in the DNA would signal the addition of C, G, A, and U, respectively, to the RNA. The RNA polymerase then moves stepwise along the DNA helix, exposing the next region of DNA for complementary base pairing (from the 5′ to the 3′ end) until the polymerase encounters another area of special sequences in the DNA, the stop (terminal) signal, where polymerase disengages from the DNA and releases the newly assembled single-stranded RNA chain and both the DNA templates. The RNA chain that is complementary to the DNA from which it was copied is called the primary RNA transcript. The primary RNA transcript is approximately 70 to 10,000 nucleotides long because only a selected portion of a DNA is used to produce an RNA molecule. Primary RNA transcripts (originally called “heterogenous nuclear RNA”) vary greatly in size because of the presence of long noncoding intron sequences. This is in contrast to mature, more uniform, small-size RNA sequences that are needed for encoding proteins. The primary RNA transcript is then capped by the addition of a methylated G nucleotide to its 5′ end (5′ cap). The 5′ cap plays an important role in protecting growing RNA transcript from degradation and later in the initiation of protein synthesis. The 3′ end of primary RNA transcript is cleaved at a specific site, and a poly-A tail (100 to 200 residues of adenylic acid) is added by poly-A polymerase. The poly-A tail facilitates the export of mature mRNA from the nucleus, influences the stability of some mRNAs in the cytoplasm, and serves as a recognition signal for the ribosome, which is required for translation of mRNA. For mRNA to move out of the nucleus, primary modified RNA transcripts undergo one or more RNA splicing events. The noncoding sequences (introns) are removed by ribonucleoprotein complex (the spliceosome), and the coding sequences (exons) on either side of the introns are joined together. These events result in a small, relatively stable, mature mRNA, which represents approximately 3% of the quantity of cellular RNA. Translation In the second phase of gene expression the information contained in mRNA is used for the synthesis of polypeptides by a process of translation. During the course of protein synthesis the translational machinery moves from the 5′ to the 3′ direction along an mRNA, and the mRNA sequence is read as a block of three nucleotides at 10

a time, termed a codon ( Table 2-1 ).[ ] Because RNA is made up of four types of nucleotides, there are 64 possible sequences composed of three nucleotides. Three of the 64 sequences do not code for amino acids but instead signal the termination of the polypeptide chain. These noncoding sequences are called stop codons. The remaining 61 codons specify only 20 amino acids; therefore most of the amino acids (with the exception of methionine and tryptophan, which have only one codon each) are represented by multiple codons, and the genetic code is considered to be degenerate.

11 12 13 14 15

Translation is mediated by tRNA, also termed adapter molecule, which has two important properties. [ ] [ ] [ ] [ ] [ ] First, tRNA is able to represent only one amino acid to which it is covalently bound. Second, tRNA contains a trinucleotide sequence, the anticodon, which is complementary to the codon in mRNA representing its amino acid. The anticodon enables the tRNA to recognize the codon through complementary base pairing. The events in protein synthesis are catalyzed on the ribosome,

17

Figure 2-2 Steps in protein synthesis. ADP, adenosine diphosphate; ATP, adenosine triphosphate; tRNA, transfer RNA; mRNA, messenger RNA. (Courtesy Baback Roshanravan.)

(Courtesy Baback Roshanravan.) which consists of two subunits; each subunit consists of several proteins associated with a long RNA (rRNA). A ribosome contains three binding sites for RNA molecules: one for mRNA and two for tRNAs. The site for tRNA that holds the growing end of the polypeptide chain is called the P-site (peptidyl-tRNA-binding site), whereas the site that holds the incoming tRNA molecules charged with an amino acid is termed the A-site (aminoacyl-tRNA-binding site). To accomplish the sequential synthesis of a protein, the ribosome moves along the mRNA one codon at a time. A ribosome attaches to mRNA at or near the 5′ end of a coding region;

moving along the RNA toward the 3′ end, it translates each triplet codon into an amino acid en route. The process of polypeptide chain elongation on a ribosome is a repeat of cycles with three distinct steps. During step 1 an aminoacyl-tRNA molecule binds to a ribosomal A-site by base pairing with the codon on mRNA exposed on A-site. In step 2 the carboxyl end of the polypeptide chain is uncoupled from the tRNA molecule bound to the P-site and joined by a peptide bond to the amino acid linked to the tRNA at the A-site. This reaction is catalyzed by peptidyl transferase. During final step 3, as the ribosome moves along the mRNA, the new peptidyl-tRNA in the A-site is translocated to the P-site. At the same time, the free tRNA molecule that was generated in the P-site in step 2 is released from the ribosome to reenter the pool of cytoplasmic tRNA. After completion of step 3, the unoccupied A-site on the ribosome is ready to take another tRNA linked to the next amino acid, which starts the cycle again. The stop codons (UAA, UAG, and UGA) are responsible for the termination of the translational process. Cytoplasmic proteins, the eukaryotic releasing factor (eRF), bind directly to any stop codon that reaches the A-site on the ribosome, resulting in the alteration of the activity of peptidyl transferase. This results in the addition of a water molecule instead of an amino acid to the peptidyl-tRNA. This frees the −COOH end of the growing polypeptide chain from its attachment to a tRNA molecule. Because only this attachment holds the growing polypeptide to the ribosome, the completed protein is released into the cytoplasm. The ribosome releases mRNA and dissociates into its two subunits, which are ready to assemble on another mRNA to begin the synthesis of a new protein. In summary, the termination reaction involves (1) release of the completed polypeptide from the last tRNA, (2) expulsion of the tRNA from the ribosome, and (3) dissociation of the ribosome from mRNA. 11 12

Additional factors are required at each stage of protein synthesis, [ ] [ ] characterized by their cyclic association with, and dissociation from, the ribosome. During the initiation phase of protein synthesis, the two subunits of ribosomes are

18

TABLE 2-1 -- The Genetic Code, with 20 Specified Amino Acids First Position (5′ End)

U

C

*

Third Position (3′ End)

Second Position U

C

A

G

Phe

Ser

Tyr

Cys

U

Phe

Ser

Tyr

Cys

C

Leu

Ser

Stop

Stop

A

Leu

Ser

Stop

Trp

G

Leu

Pro

His

Arg

U

Leu

Pro

His

Arg

C

A

G

Leu

Pro

Gln

Arg

A

Leu

Pro

Gln

Arg

G

Ile

Thr

Asn

Ser

U

Ile

Thr

Asn

Ser

C

Ile

Thr

Lys

Arg

A

Met

Thr

Lys

Arg

G

Val

Ala

Asp

Gly

U

Val

Ala

Asp

Gly

C

Val

Ala

Glu

Gly

A

Val

Ala

Glu

Gly

G

U, Uracil; C, cytosine; A, adenine; G, guanine; Phe, phenylalanine; Ser, serine; Tyr, tyrosine; Cys, cysteine; Leu, leucine; Stop, stop codon; Trp, tryptophan; Pro, proline; His, histidine; Arg, arginine; Gln, glutamine; Ile, isoleucine; Thr, threonine; Asn, asparagine; Lys, lysine; Met, methionine; Val, valine; Ala, alanine; Asp, aspartic acid; Gly, glycine; Glu, glutamic acid. * Codons are given as they appear in messenger RNA (mRNA).

brought together at the precise location on the mRNA where the polypeptide chain is to begin. An RNA sequence can be translated in any one of three reading frames, each specifying a completely different polypeptide chain. The sequence of the mRNA determines which of the three reading frames are read, which determines how the ribosome assembles. The initiation process involves a number of steps catalyzed by a set of proteins, the initiation factors (IFs). To start a new protein chain, the ribosome must bind an aminoacyl-tRNA molecule in its P-site (normally occupied by peptidyl-tRNA molecule), a special step performed by initiator tRNA, which provides the amino acid methionine that starts a protein chain. The initiator tRNA must be loaded onto a small ribosomal unit, with the help of eukaryotic initiation factor-2 (eIF-2), before this subunit can bind to a mRNA molecule. This process allows the subunit to find the start codon (AUG), as well as allowing the small ribosomal subunit to bind to its larger subunit.

DNA REPAIR Individual survival depends on genetic stability. Thousands of random changes are created in human cell DNA every day by heat energy and metabolic accidents; 16 17

however, most of these spontaneous changes are temporary because they are immediately corrected by a mechanism termed DNA repair. [ ] [ ] Only rarely are these changes in the DNA permanent, termed mutation. The process of DNA repair depends on the presence of a separate copy of the genetic information in each

strand of the double helix of the DNA. The DNA repair process takes place in three steps. In step 1 the damaged portion of the DNA strand is recognized and removed by DNA repair nucleases, leaving a small gap in the DNA helix. In step 2, another DNA polymerase makes a complementary copy from the undamaged strand of the double helix, binds to the 3′ −OH end of the cut DNA, and fills in the gap. In step 3 the break or “nick” that is left in the damaged strand when the DNA polymerase has filled the gap is finally sealed by DNA ligase. DNA polymerase and DNA ligase are also important in DNA replication. In addition to maintaining the integrity of DNA sequences by DNA repair, an accurate duplication of DNA is a prerequisite for all cell divisions.

DNA REPLICATION DNA replication is semiconservative because the original DNA duplex is not conserved after one round of replication; instead, each strand of the duplex becomes part of another duplex. DNA replication in mammalian cells occurs at a polymerization rate of about 50 nucleotides per second. The speed and accuracy with which the replication process takes place is regulated by a group of enzymes constituting a “replication machine.” The basis for the great accuracy of DNA replication is complementarity. [

18]

DNA templating is a process in which the nucleotide sequence of a DNA strand or a segment of DNA strand is copied by complementary base pairing in 19 20

complementary nucleic acid sequence.[ ] [ ] During this process, two strands of DNA helix are separated so that the hydrogen bond donor and acceptor groups on each base become exposed for base pairing. The DNA double helix is opened and untwisted ahead of the replication fork, by DNA helicase and single-stranded DNA21 22

binding proteins. [ ] [ ] This results in the separation of the template strand from its complementary strand, which is a requirement for DNA polymerases to copy the DNA. DNA helicases, when bound to single strands of DNA, hydrolyze adenosine triphosphate (ATP). Using the principle that hydrolysis of ATP can change the shape of a protein, DNA helicases move rapidly along the DNA single strand; where they encounter a region of double helix, they continue to move along their strand, thereby prying the helix apart. Single-strand DNA-binding proteins (helix-destabilizing proteins) bind to exposed DNA strands without covering the bases, allowing them to remain available for templating. These proteins also help open the DNA helix by stabilizing the unwound, single-stranded confirmation. Several classes of eukaryotic DNA polymerase have been identified. DNA polymerase α is a nuclear replicase; however, it synthesizes only one of the daughter strands. DNA polymerase δ is also involved in replication and probably synthesizes the other daughter strand. DNA polymerases β and epsilon are probably involved in DNA repair reactions, and DNA polymerase γ is responsible for replication of mitochondrial DNA. 23 24 25

The actual process of replication occurs at the DNA replication fork, [ ] [ ] [ ] which is asymmetric ( Figure 2-3 ). The replication of DNA always proceeds in the 5′→3′ direction on a growing DNA strand. Because the two parent strands are antiparallel, new strands are synthesized in opposite directions along the parent templates at each replication fork. Therefore the new strand must be elongated by different mechanisms. Two DNA polymerases molecules work at the DNA fork, one (polymerase δ) on the leading strand (a strand that elongates toward the replication fork) and the other (polymerase α) on the lagging strand (a strand that is elongated away from the

19

Figure 2-3 Structure of DNA replication fork. (Courtesy Baback Roshanravan.)

(Courtesy Baback Roshanravan.) fork). Once the replication fork is established, the DNA polymerase at the leading strand is continuously presented with a base-pair chain onto which it adds a new nucleotide at the 3′ end in a continuous manner; therefore the DNA daughter strand is synthesized continuously. In contrast, the lagging strand is synthesized discontinuously in a series of short segments called Okazaki fragments, which in eukaryotic cells are about 100 to 200 nucleotides long. Each Okazaki fragment is synthesized by DNA polymerase at the lagging strand in the 5′→3′ direction beginning at the replication fork and moving away from it. DNA polymerase requires only approximately 4 seconds to complete each short DNA fragment, after which it starts synthesizing a completely new DNA fragment at a site distant from the template strand. To achieve this, DNA primase, using ribonucleoside triphosphates, synthesizes short RNA primers (approximately 10 nucleotides long). RNA primers are made at intervals on the lagging strand, where they are elongated by DNA polymerase to synthesize Okazaki fragments. Synthesis of each Okazaki fragment ends when the DNA polymerase reaches the RNA primer attached to the 5′ end of the previous fragment. To produce a continuous chain of DNA from the

many DNA fragments made on the lagging strand, old RNA primers are removed and replaced with DNA. The 3′ end of the new fragment is joined to the 5′ end of the previous DNA fragment by DNA ligase, completing DNA replication. Because the synthesis of the leading strand is continuous, whereas that of the lagging strand is discontinuous, DNA replication is semicontinuous. How does the newly synthesized DNA strand become a double helix without tangling? It is estimated that every 10 base pairs replicated at the DNA replication fork correspond to one complete turn about the axis of the parental double helix. For a replication fork to move along the entire length of a chromosome, the fork must rotate rapidly, requiring a large amount of energy. Instead, a swivel is formed in the DNA helix by a group of proteins, the DNA topoisomerases, covalently binding 26 27

to a DNA phosphate, thereby breaking a phosphodiester bond in a DNA strand.[ ] [ ] Topoisomerase I causes a single-strand break (“nick”). The phosphodiester bond in the strand acts as a swivel point around which two sections of DNA helix on either side of the nick can rotate. Consequently, DNA replication can occur with the rotation of only a short length of helix. Topoisomerase II forms a covalent bond to both strands of the DNA helix at the same time, resulting in a transient doublestrand break in the DNA helix. Topoisomerase II enzymes are activated where two double helixes cross over each other. When topoisomerase binds to such a crossing site, (1) breakage of one double helix creates a DNA “gate,” (2) the second nearby double helix passes through the gate, and (3) the break reseals and dissociates from the DNA, thus preventing potential tangling that would otherwise occur during DNA replication.

CONTROL OF GENE EXPRESSION Control of gene expression is essential for directing development and maintaining homeostasis and can be regulated at several levels: transcriptional, RNA 28 29 30 31 32]

processing, RNA transport, mRNA degradation, translational, and posttranslation by protein phosphorylation. [ ] [ ] [ ] [ ] [ gene regulation is at the transcription level, which is affected by binding of proteins to regulatory sequences within the DNA.

The most common and important

Transcriptional Control To transcribe a gene, RNA polymerase binds to the promoter region, a specific sequence of nucleotides on the gene that informs the RNA polymerase where to begin transcribing. Other protein-binding nucleotide sequences on DNA regulate transcription by affecting the binding of RNA polymerase to the promoter. The interaction of proteins to the regulatory sequence either inhibits transcription by interfering with RNA polymerase binding to the promoter region or stimulates it by facilitating polymerase binding to the promoter. To initiate transcription, an assembly of a set of proteins, the transcriptional factors, on the promoter is required for the stabilization of binding of RNA polymerase to the promoter. The assembly begins some 25 nucleotides upstream from the transcription start site, where a transcription factor (basal factor) comprised of several subunits binds to a short TATA sequence ( Figure 2-4 ). Other transcriptional factors (coactivators) link the basal transcriptional factors with the regulatory proteins, the activators. This completes the formation of a full transcription complex that is able to engage RNA polymerase. The transcription complex then phosphorylates the bound RNA polymerase, disengaging it from the complex so that it is free to start transcription. Any factor that reduces the availability of a particular transcriptional factor, or blocks its assembly into the transcription complex, will likely inhibit transcription. Regulatory proteins bind to the edges of base pairs exposed in the major grooves of DNA. Most of these regulatory proteins contain structural motifs, such as zinc finger or leucine zipper. The regulatory proteins are composed of two distinct

20

Figure 2-4 Structure of human transcription complex, consisting of four types of proteins: basal factors, coactivators, activators, and repressors. (Courtesy Baback Roshanravan.)

(Courtesy Baback Roshanravan.) domains, the DNA-binding domain and the regulatory domain. The DNA-binding domain physically attaches the protein to DNA at a specific site, using one of the structural motifs. The regulatory domain interacts with other regulatory proteins. These two domains of regulatory proteins provide them with an advantage, allowing a regulatory protein to bind to a specific DNA sequence on one site of a chromosome and to exert its regulatory effect over a promoter at another site. The distant sites to which regulatory proteins bind are termed enhancers. The activator regulatory proteins bind to DNA through specific enhancer sequences. Interaction of specific basal transcriptional factors with particular activator proteins is necessary for the proper positioning of RNA polymerase. The rate of transcription is regulated by the availability of these activator regulatory proteins. The repressor regulatory protein, through its regulatory domain, binds to a “silencer” sequence, located adjacent to or overlapping an enhancing sequence. As a result, the corresponding activator protein will no longer be able to bind to the enhancer sequences and will be unavailable to interact with the transcription complex, repressing transcription.

One question remaining unanswered is how a regulatory protein can affect a promoter when these proteins bind to DNA at enhancer/repressor sites located far from the promoter. The current hypothesis is that the DNA loops around so that the enhancer is positioned near the promoter. This brings the regulatory domain of the protein attached to the enhancer into direct contact with the transcription factor associated with the RNA polymerase attached to the promoter. Posttranscriptional Control Although gene regulation typically occurs at the level of transcription, there are several posttranscriptional steps at which gene expression can be regulated, including RNA splicing, translational repressor proteins, and selective degradation of mRNA transcripts. Most eukaryotic genes comprise short coding sequences (exons) embedded within long stretches of noncoding sequences (introns). The initial mRNA copied from a gene by RNA polymerase, the primary transcript, is a copy of the entire gene including introns and exons. Before the primary transcript is translated, the introns (comprised of approximately 90% of the primary transcript) are removed by enzymes in a process of RNA splicing or RNA processing. This is a point where gene expression can be controlled because the exon can be spliced in different ways to allow different polypeptides to be assembled from the same gene. Another step in posttranscriptional regulation of gene expression is the level of transport of processed mRNA script from the nucleus to the cytoplasm. The processed mRNA script is transported across the nuclear membrane through a nuclear pore. This active process of transport requires recognition of poly-A tail (a chain of adenine residues at the 3′ end) of processed transcript by receptors lining the interior of the nuclear pore. Although no direct evidence indicates that the gene expression is regulated at this point, it remains a possibility. Because the translation of processed mRNA in the ribosome involves transcription factors, gene expression can be regulated by modification of one or more of these transcriptional factors. Translation repressor proteins shut down translation by binding to the beginning of the transcript so that it cannot be attached to the ribosome. Different mRNA transcripts have different half-lives. Transcripts contain sequences near their 3′ end that make them subject to enzymatic degradation. A sequence of A and U nucleotides near the 3′ end of poly-A tail of transcript promotes removal of the tail, destabilizing the mRNA. Other mRNA transcripts contain sequences near their 3′ end that are recognition sites for endonucleases, causing these transcripts to be digested quickly.

DNA REARRANGEMENT (GENETIC RECOMBINATION) 33] [34] [35] [36] [37] [38] [39] [40]

To adapt to an ever-changing environment, DNA undergoes rearrangement, which is caused by genetic recombination. [

21

The mechanisms of genetic recombination allow large sections of DNA helix to move from one chromosome to another. There are two classes of genetic recombinations: general, or homologous, and site specific.

In homologous recombination an exchange of genetic material takes place between two pairs of homologous DNA sequences located on two copies of the same 36 37

chromosome.[ ] [ ] This exchange involves breaking of two homologous DNA double helixes and joining of the two broken ends, by base pairing, to their opposite partners (crossover) to create two intact DNA molecules, each composed of parts of the original DNA molecule. The exchange of genetic material can occur anywhere in the homologous DNA sequences of two DNA helixes; however, the mechanism of homologous recombination ensures that two regions of DNA double helix undergo an exchange reaction provided they have extensive sequence homology. The homologous recombination does not normally change the rearrangement of the genes in a chromosome. In contrast, site-specific recombination alters the relative positions of the nucleotide sequence in a chromosome because DNA homology between the recombining DNA molecule is not required, and the pairing reaction depends on a recombination enzyme-mediated recognition of specific nucleotide sequences present on one or both recombining DNA molecules.[

33] [38] [39] [40]

There are two types of site-specific recombinations, conservative and transpositional. The conservative site38

specific recombination was first demonstrated in bacteriophage λ.[ ] This applies to many viruses. DNA sequences in the virus encode for integrase; in bacteriophage λ it is termed λ integrase. When a virus (in this case, bacteriophage λ) enters a cell, λ integrase is synthesized. Several molecules of integrase protein bind to a specific DNA sequence of the circular bacteriophage chromosome (mobile genetic element). This DNA-integrase complex binds to a related but different specific sequence on the bacterial chromosome (target chromosome), bringing the bacterial and bacteriophage chromosomes close together. Integrase then cuts the DNA section in both the bacteria and the bacteriophage and, by using a short region of sequence homology, reseals the reaction. The integrase then dissociates from the DNA and is ready to be used for the next recombination reaction. In transpositional site-specific recombination, mobile DNA sequences encode integrases that insert their DNA into target chromosome by a mechanism different from that described for bacteriophage λ. Similar to λ integrase, each of these integrases recognizes a specific DNA sequence in the mobile genetic element that must be integrated into the target chromosome. However, these integrases do not require specific DNA sequences in the target chromosome. Instead, both cut ends of the linear DNA sequence of mobile genetic element catalyze a direct attack on the target DNA, leaving two short single-stranded gaps in the recombinant DNA molecule, one at the 3′ end and the other at the 5′ end of the mobile genetic element. These gaps are then filled by DNA polymerase, and thus the entire process of recombination is completed. In summary, in conservative site-specific DNA recombination, integrase encoded by viral DNA (mobile genetic element) is involved in the entire process of recombination, that is, cutting of specific DNA sequences of both the mobile genetic material and the target DNA (cell) and resealing them. On the other hand, in transpositional site-specific recombination, integrase is involved in cutting of the specific DNA sequences of the mobile genetic element only.

RECOMBINANT DNA TECHNOLOGY Recombinant DNA technology has revolutionized the field of cell biology and led to the discovery of a large number of new genes and proteins. By allowing the study of the regulatory regions of genes, this technique has provided an important tool to understand and decipher various complex mechanisms of gene regulation in eukaryotic cells. In addition, recombinant DNA technology has been instrumental in the study of conservation of many proteins during evolution and in the determination of the functions of proteins and of individual domains within proteins. Recombinant DNA technology comprises a number of techniques, of which the most significant are the following: • Fragmentation, separation, sequencing, and recognition of DNA molecules • Nucleic acid hybridization • Gene cloning • Gene isolation

• Gene mapping • DNA engineering • Genomics and proteomics • RNA interference/RNA silencing

FRAGMENTATION, SEPARATION, SEQUENCING, AND IDENTIFICATION OF DNA DNA Fragmentation Cell DNA can be cleaved at specific sites by restriction nucleases to yield DNA fragments that are separated by gel electrophoresis and can be subsequently 41 42

sequenced.[ ] [ ] The restriction nucleases are bacterial enzymes that protect bacteria from viruses by degrading viral DNA. Each restriction nuclease cuts the double-helical DNA into fragments of DNA (restriction fragments) that are strictly defined by their property of recognizing a specific sequence of four to eight nucleotides. More than 100 restriction nucleases have been purified from various bacteria, most of which recognize different nucleotide sequences. Most of these restriction nucleases are now commercially available ( Table 2-2 ). Certain restriction nucleases produce staggered cuts, leaving short single-stranded tails at the two ends (cohesive ends) of each DNA fragment ( Figure 2-5 ). Any two DNA fragments can be joined together, provided both DNA fragments have the same cohesive ends (either generated by the same restriction nuclease or with another restriction nuclease, provided the DNA fragment has the same cohesive ends). DNA molecules produced in this manner by splicing together two or more DNA fragments are known as recombinant DNA molecules. As mentioned, each restriction nuclease yields a series of restriction fragments. A restriction map of a particular region of the gene can be generated by comparing the sizes of restriction fragments obtained by the treatment of DNA from a particular genetic region with a combination of restriction nucleases. Because different short DNA sequences are recognized by different restriction nucleases, these sequences serve as markers, and the restriction map reveals their arrangement in the region of the gene. By using a restriction map, it is possible to study the conservation of a region of chromosome that codes for a particular gene during evolution, that is, whether the coding region has remained unchanged during evolution. Restriction maps are also used in DNA cloning and DNA

22

TABLE 2-2 -- Selected Restriction Endonucleases and Their Recognition Sequences and Cleavage Sites (*) Cleavage Sites Enzymes 5′ Tetranucleotides

3′

TaqI

T

*

C

G

A

Msp1

C

*

C

G

G

Pentanucleotides EcoRII

*

C

C

T (A)

G

G

HinfI

G

*

A

N

T

C

Hexanucleotides BamH1

G

*

G

A

T

C

C

EcoRI

G

*

A

A

T

T

C

HindIII

A

*

A

G

C

T

T

PstI

C

T

G

C

A

*

G

SmaI

C

C

C

*

G

G

G

SphI

G

C

A

T

G

*

C

C

*

T

N

A

G

Heptanucleotides MstII

C

G

T, Thymine; C, cytosine; G, guanine; A, adenine; N, any base. engineering by identifying the gene of interest on a restriction fragment and therefore facilitating its isolation for DNA cloning and DNA engineering. Separation of DNA 43 44 45

Gel electrophoresis techniques separate DNA molecules by size. [ ] [ ] [ ] Polyacrylamide gel with a small pore size is used to separate single-stranded DNA fragments less than 500 nucleotides long (size range 10 to 500 nucleotides) that differ in size by as little as a single nucleotide. Agarose gel with a

Figure 2-5 Cleavage sites for commonly used restriction nucleases. (Courtesy Baback Roshanravan.)

Figure 2-6 Polymerase chain reaction, a cyclic process in which the number of DNA targets doubles with each cycle. (Courtesy Baback Roshanravan.)

(Courtesy Baback Roshanravan.)

part of the gene of interest, are synthesized. One of the primers is complementary to the sense strand, and the other is complementary to the antisense strand. The primers are mixed with genomic DNA or complementary DNA. The mixture is heated to 95° C to denature the double-stranded DNA and allowed to cool, during which oligonucleotide primers anneal to their complementary sequences. Then a special DNA polymerase (Taq) derived from a bacterium (Thermus aquaticus) is added to the mixture, and the temperature is raised to 72° C. The advantage of Taq is that it is not denatured at 95° C and is active at 72° C. During this reaction, DNA replicates with oligonucleotides as primers. This process of denaturing the DNA, reannealing the oligonucleotide primers, and replicating the DNA is repeated 30 times, using an automated thermal cycler, resulting in an exponential amplification of the gene. The DNA product of the PCR reaction can be inserted into a vector, cloned, and sequenced. Quantitative Real-Time PCR.

The amount of specific DNA product at the end of the PCR run does not correlate with the number of target copies present in the original specimen. However, some applications in medicine and research require quantification of the number of specific targets in the specimen. This has led to development of quantitative PCR techniques. Recent advances in technology allow detection of the increment per cycle of a specifically generated PCR product in “real-time mode.” Quantitative real63 64 65

time PCR is based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product. [ ] [ ] [ ] A probe (e.g., Taq) is designed to anneal to the target sequence between the traditional forward and reverse primers. The probe is labeled at the 5′ end with a reporter fluorochrome (usually 6carboxyfluorescein [6-FAM]), and a quencher fluorochrome (6-carboxy-tetramethyl-rhodamine [TAMRA]) is added at the 3′ end. As long as both fluorochromes are on the probe, the quencher molecule stops all fluorescence by the reporter. As Taq polymerase extends the primer, the intrinsic 5′→3′ nuclease activity of Taq

25

Figure 2-7 Quantitative real-time polymerase chain reaction (PCR). A, Primers are extended as in traditional PCR reaction. Probe labeled with reporter fluorochrome (R) and quencher fluorochrome (Q) anneals to the complementary gene sequence between the two primers. Fluorescent signal is generated when R is cleaved from the probe by Taq polymerase on extension of the primer. B, Amplification window showing the fluorescence obtained in each amplification cycle for each reaction. Threshold cycle (Ct) shows the cycle number at which fluorescence intensities are above background noise.

Figure 2-8 Complementary DNA (cDNA) microarray schema. Total RNA from both the test and reference sample is fluorescently labeled with Cy3- or Cy5-dUTP using reverse transcription. Fluorescent targets are pooled and hybridized to the clones on the array. Fluorescence is measured and data are calculated from a single experiment as a normalized ratio of Cy3/Cy5 in which significant deviations from 1 show increased (>1) or decreased ( PGE1 >> PGF2 > PGD2

SM, GI tract, uterus, bladder, pulmonary artery, trachea

Iloprost, SC19220, sulprostone

EP2

PGE2 = PGE1 >>> PGF2 , PGD2

SM, lymphocytes, lung, heart

Butaprost, M&B28767, misoprostol

EP3

PGE2 = PGE1 > PGF2 , PGD2

SM, adipocytes, autonomic nerves, CNS, EC, kidney

Enprostil, sulprostone, misoprostol, M&B28767

EP4

PGE2 = PGE1 >> PGF2 , PGD2

SM, EC, mast cells, sensory nerves, lung, kidney

Misoprostol, M&B28767, AH22921 and AH23848

DP

PGD2 >> PGE2 , PGF2 , PGI2

SM, CNS, platelets

BW245C, ZK 110841, RS 93520

FP

PGF2 > PGD2 > PGE2

Corpus luteum, SM, kidney, lung

Fluprostenol, closprostenol

IP

PGI2 > PGE1 > PGD2 , PGE2

Arterial SM, platelets, sensory nerves

Carbacyclin, cicaprost, iloprost, octimibate

TP

TxA2 >> PGD2

Venous SM, platelets, lung, kidney, EC U44069 and U46619, AH 23848, E45, ICI 192605

BLT1

*

LTB4

Granulocytes, macrophages, lymphocytes

SC 53228, Ro 25-4094, CGS 25019C, CP 105,696

BLT2

†

LTB4 > 12-HETE = 15-HETE

Many tissues, especially spleen, liver, ovary, leukocytes

ONO 4057

CysLT1

LTD4 >> LTC4 > LTE4

SM, epithelial cells, EC, macrophages, eosinophils

SKF 104353, MK-571, ICI 204219, Ro 24-5913

CysLT2

LTD4 = LTC4 >> LTE4

SM, lung macrophages, cardiac Purkinje cells, adrenal medulla, brain

None

PG, Prostaglandin; Tx, thromboxane; LT, leukotriene; HETE, hydroxyeicosatetraenoic acid; GI, gastrointestinal; CNS, central nervous system; SM, smooth muscle; EC, endothelial cells; Cys, cysteinyl. * High affinity; Kd = 1.1•nM. † Low affinity; Kd = 23•nM.

respect to LT-binding properties and signal transduction pathways, and analyzed for cell and tissue distribution. Their names correspond with those for the receptors for COX-derived mediators ( Table 14-4 ). Cultured smooth muscle cells, smooth muscle of lung tissue and gastrointestinal tissues, and mononuclear phagocytes express both cysteinyl leukotriene (CysLT) 1 122] [123] [124]

and 2 GPCRs for peptidoleukotrienes, whereas other cells bear predominantly CysLT1 or CysLT2.[

Granulocytes and some sets of mononuclear

125] [126] [127] [128]

leukocytes express both the BLT1 and BLT2 GPCRs for LTB4, whereas most other cells bear one or the other LTB4 receptor preferentially.[

Both

CysLT GPCRs bind peptidoleukotrienes with similarly high affinity, but with different specificities. In contrast, BLT1 binds LTB4 with twentyfold higher affinity than BLT2, and each has a distinct specificity for native LTs and pharmacological antagonists. Although the LTC4 receptor of some types of leukocytes and of 129 130

smooth muscle cells[ ] [ ] appears to consist of intracellular as well as plasma membrane proteins, the structural and functional relationships between the two sets have not been delineated definitively. The cloning of GPCRs specific for TxA2 , PAF, each PG and each major type of LT has provided structural bases for discovery 121] [131] [132]

of several clinically useful types of antiallergic and antiinflammatory compounds.[ development and clinical application of selective pharmacologic agents.

Thus, this family of mediators offers unique possibilities for the

The pathways of further metabolism and inactivation of eicosanoids are considered in this section because most are subject to uptake by cells and intracellular enzymatic conversions. The exceptions are TxA2 and PGI2 , which are inherently unstable and decay spontaneously within minutes in extracellular fluids to the respective TxB2 and 6-keto-PGF1α products, lacking significant biologic activity. In contrast, the rate-limiting step in the metabolism of PGD2 , PGE2 , PGF2α , and LTs of the 5-LO pathway is uptake by cells that have the capacity to biodegrade the mediators or, in the case of leukocytes, the release from cells of the inactivating enzymes. PGs of the E and F series are removed efficiently from the circulation by endothelial cells, especially in the pulmonary capillaries, and in many tissues these PGs are converted sequentially to the corresponding 15-keto-PG by a 15-hydroxy-PG dehydrogenase and then to the inactive 13,14-dihydro-15-keto-PG.[

133]

The intracellular mechanisms for the metabolic inactivation of peptidoleukotrienes are more varied than those for LTB4 . A constitutive sequence of enzymes in neutrophils, hepatocytes, and possibly other cells converts LTB4 by ω-oxygenation to 20-OH-LTB4 and then to 20-COOH-LTB4 . [ potent as a PMN chemotactic and activating factor than LTB4 , whereas 20-COOH-LTB4 is inactive.[

135]

134]

The 20-OH-LTB4 is less

LTC4 is metabolized by three separate pathways that are

expressed in different types of cells. First, PMNs, macrophages, and some other cells convert LTC4 to LTD4 and then to LTE4 by peptidolysis of 6S-glutathione. Second, a peroxidase–hypochlorous acid reaction in activated neutrophils, eosinophils, and monocytes transforms LTC4 , LTD4 , and LTE4 to sulfoxides and then to a sulfone that is less potent than the parent LT.[

136] [137]

An LTC4 sulfoxide also is transformed to the 12(R)- and 12(S)-6-trans isomers of LTB4 ,

223

which represent major products of LTC4 degradation by the leukocytes. [

136] [137]

Third, the 15-hydroxy derivatives of LTC4 and probably LTD4 and LTE4 are

products of 15-LO-predominant cells and exhibit significantly less activity than the index mediators. The extent to which the conversion of LTE4 to the less potent contractile and vasoactive factor LTF4 contributes to the overall biodegradative processes for peptidoleukotrienes remains to be established.

138]

PAF activates its target cells by binding to different classes of specific receptors. One class of receptors has a molecular weight of 39,000•kD, with Kd = 5.6•nM.[ [139]

This PAF receptor is a member of the family of receptors that transduce signals to cells by interacting with G proteins. Analyses of the biochemical pathways for cellular activation by PAF and of the effects of PAF-binding antagonists reveal the presence of a second class of PAF receptors that modulate monocyte140

macrophage activation and may be important to PAF's immunomodulatory actions.[ ] LPA and S1P increase in concentration in pulmonary airway fluids after segmental antigen challenge of asthma patients and, at those concentrations, evoke airway smooth muscle contraction, proliferation, and secretion of inflammatory 114 115 116 117

cytokines.[ ] [ ] [ ] [ ] Airway smooth muscle cells and epithelial cells, lung macrophages, and the inflammatory cells typical of asthma all express EdgRs specific for LPA and S1P. The roles and relative importance of this mediator system will be revealed when pharmacologic agonists and antagonists are developed that permit selective activation and blockade of each EdgR in allergic and inflammatory reactions.

PATHOGENETIC ROLES OF EICOSANOIDS AND OTHER LIPID MEDIATORS Products of both the cyclooxygenation and lipoxygenation of AA and PAF are detected at elevated concentrations in fluids and tissues affected by a wide range of hypersensitivity and inflammatory diseases, as well as other states with abnormally altered vascular, epithelial cell, or endothelial cell function or extensive tissue 141 142

injury.[ ] [ ] In most cases, AA-derived mediators are quantified by high-performance liquid chromatography (HPLC), radioimmunoassay (RIA), or both techniques in sequence, with appropriate corrections for losses during extraction and purification. In plasma, TxA2 and PGI2 are quantified radioimmunochemically as the respective metabolites, TxB2 and 6-keto-PGF1α , whereas monoHETEs, some PGs, and LTB4 are detected transiently in low concentrations as the native compounds. In contrast, the peptidoleukotrienes are best assessed in terms of the quantities of LTE4 in urine or in terms of N-acetyl-LTE4 —a terminal metabolite of LTC4 , LTD4 , and LTE4 —in bile as cumulative reflections of the plasma concentrations of the mediators in the interval preceding the time of collection.[ The findings of PGs and Txs in plasma, other fluids, and tissue extracts of patients with diverse diseases have been reviewed thoroughly,[ focuses principally on the LO products ( Table 14-5 ).

8] [10]

142] [143]

so this section

Four general types of eicosanoid abnormalities are postulated to be important in the pathogenesis of human allergic and inflammatory diseases or to develop after TABLE 14-5 -- Leukotrienes (LTs) and 5-Hydroxyeicosatetraenoic Acid (HETE) in Human Disease Disease

Source

Mediators

Rhinitis, conjunctivitis

Tears, nasal secretions

LTC4 /LTD4 /LTE4

Cutaneous allergy

Lesional fluid

LTC4 , LTB4 , 12-HETE

Asthma

Sputum, BAL fluid

LTC4 /LTD4 /LTE4 , LTB4

Allergic States

Urine

LTE4

Pulmonary fibrosis

Lung tissue homogenates

LTB4 , LTC4

Cystic fibrosis

Sputum

LTD4 , LTB4

Urine

LTE4

Acute respiratory distress syndrome

Lung edema fluid

LTD4 /LTE4 , LTB4

Psoriasis

Epidermis, lesional fluid

LTC4 /LTD4 , LTB4 , 12-HETE

Eosinophilic dermatitis

Exudate

LTC4 /LTD4

Spondyloarthritis, rheumatoid arthritis

Synovial fluid

LTB4

Urine

LTE4

Gout

Synovial fluid

LTB4

Inflammatory bowel disease

Mucosa

LTB4

Familial Mediterranean fever

Plasma

MonoHETEs

Neonatal hypoxemia, pulmonary hypertension

BAL fluid

LTC4 /LTD4

Severe trauma

Bile

N-acetyl-LTE4

Urine

LTE4

Other Inflammatory Diseases

Other Conditions

BAL, Bronchoalveolar lavage.

224

exposures to antiallergy or antiinflammatory drugs. First, C6 peptidoleukotrienes and LTB4 increase in concentration in tissue fluids and are excreted at higher

levels, either as the initial mediators or more often as their metabolites, in the course of allergic reactions. The results of many studies indicate that such increases are attributable predominantly to immunologically stimulated production by mast cells, mononuclear phagocytes, granulocytes, and epithelial cells. Second, defects in metabolism of some COX-derived and LO-derived mediators may alone evoke or more likely amplify the allergically induced elevations of eicosanoid concentrations in tissues and consequent pathology. Third, relative deficiencies or excesses of COX-1, COX-2, or 5-LO or abnormal interactions among the products of these different pathways may alter inflammatory responses. Fourth, oxygenase-active drugs may alter the quantities or types of eicosanoid products by mechanisms related to the third type of abnormality and thereby elicit or amplify inflammatory reactions of allergies. The first and second types of abnormality are characterized by transient or sustained elevations of the levels of one or more eicosanoid mediators resulting from overproduction (type one), or decreased biodegradation (type two), or a combination of these mechanisms. Although there are no known specific defects in the 144 145

biodegradation of eicosanoids, individuals affected by peroxisome deficiencies show reduced production of PGs and diminished degradation of LTs.[ ] [ ] The relative quantities of each of the LTs found at any reaction site also reflect properties of the involved tissue and the time after exposure to the eliciting stimulus. Introduction of pollen antigen into the skin of sensitized humans results in the local generation and accumulation of LTC4 , with only minimal conversion to LTD4 and LTE4 in 3 to 4 hours, and occasionally of LTB4 . [

146]

12-HETE, the predominant product of human keratinocytes, also is found in substantial amounts in fluids

of some cutaneous allergic lesions. In contrast, the LTC4 generated by allergically challenged eyes and inflamed airways is converted largely to LTD4 and LTE4 . [147]

The eicosanoid products found in inflammatory responses depend largely on the composition of the leukocytic infiltrate and to a lesser extent on the involved tissue. Various forms of acute arthritis and inflammatory bowel diseases, in which the neutrophil predominates, typically show high concentrations of LTB4 , whereas dermatitides rich in eosinophils, which are a major source of LTC4 , have high concentrations of LTC4 and LTD4 ( Table 14-5 ). Psoriasis represents an especially complex circumstance because extracts of epidermis and fluid from adherent chambers contain LTB4 from neutrophils, 12-HETE from keratinocytes, and LTC4 and LTD4 from indeterminate sources.[

148] [149]

The plasma of patients with attacks of familial Mediterranean fever have elevated concentrations of monoHETEs in

relation to disease activity, although the sites of synthesis of the HETEs have not been identified.[

150]

Elevated concentrations of C6 peptidoleukotrienes and LTB4 in

some subjects have been detected in sputum or pulmonary secretions in numerous diseases of pulmonary parenchyma, airways, or circulation. The highest concentrations of LTD4 and LTE4 are found in the airway edema fluid of patients with adult respiratory distress syndrome in relation to and often preceding the 147 151

clinical recognition of altered endothelial-epithelial permeability.[ ] [ ] In contrast, the airway free fluid in high-pressure pulmonary edema without altered permeability or inflammation has little LTD4 or LTE4 . The plasma concentrations of C6 peptidoleukotrienes are apparently elevated transiently after severe systemic trauma, but this event was recorded solely in terms of a rise in the biliary concentration of the metabolic product N-acetyl-LTE4 . [

143]

Quantification of PAF release into blood and tissues during disease states has been limited by available assays. PAF is routinely quantitated by bioassay after partial 100 103

purification by thin-layer chromatography (TLC) or HPLC.[ ] [ ] RIAs and enzyme-linked immunosorbent assays (ELISAs) for PAF have been developed and have improved quantitation in clinical and experimental studies. An additional problem is rapid PAF metabolism to products that are not quantitated by available assays. Increased quantities of PAF are detected in diseases associated with inflammation or vascular injury. Increased airway concentrations of PAF are detected in

asthma, chronic obstructive lung disease, and hypoxia.[

107]

PAF is released into the circulation after allergen-induced bronchospasm and after cold challenge of

patients with cold-induced urticaria. Local accumulation of PAF is noted after intradermal administration of antigen to atopic patients.[ PAF are recovered from inflamed joints of patients with rheumatoid arthritis and from inflamed rheumatoid tissues incubated ex vivo.[ systemically in animal models of anaphylaxis, serum sickness, nephritis, and

152]

Increased quantities of

107] [153]

PAF also is released

107 sepsis.[ ]

The third type of abnormality in eicosanoids, which is postulated to contribute to allergic and other forms of inflammation, is a disease-induced imbalance between the COX and LO pathways of AA metabolism. One experimental example is provided by the model of mice lacking any 5-LO activity as a result of inactivation of 154 155

the gene.[ ] [ ] In these mice, some inflammatory reactions are reduced, and the COX pathways assume a much greater role in mediating inflammation. Whereas inflammation elicited by AA and some other stimuli in normal mice was unaffected by pretreatment with COX inhibitors, inflammation of equal intensity in 5-LO– deficient mice was almost eliminated by COX inhibitors. Thus a relative deficiency in either pathway may contribute to greater allergic sensitivity or susceptibility to drug-induced allergic-like reactions, as well as to unusual allergy-related responses to oxygenase-directed medications. Many of the hypersensitivity reactions to aspirin, which are manifested by bronchospasm and nasoocular inflammation, are examples of this fourth type of abnormality in eicosanoid generation observed after nonsteroidal antiinflammatory drug (NSAID) treatment of some adult patients with asthma and less often with other allergic diseases. The mechanisms underlying such adverse reactions to aspirin have not been elucidated fully. One contributing factor is the exaggerated increase in the ratio of 5-LO to COX products of blood 156 157

leukocytes and presumably primary target organs at the time of exposure to aspirin, relative to the increases typical of aspirin-nonsensitive subjects.[ ] [ ] Prevention of aspirin hypersensitivity reactions by CysLT receptor antagonists implies a pathogenetic role for LTC4 / LTD4 /LTE4 . Qualitative alterations in the oxygenation of AA by aspirin also have been identified recently and include stimulation of mixtures of neutrophils and endothelial cells to synthesize 15R-epimers of lipoxins capable of inhibiting some functions of neutrophils. [

158]

Any roles for these and other distinctive

225

aspirin-evoked metabolites in aspirin hypersensitivity remain to be established.

REGULATION OF GENERATION AND FUNCTIONS OF LIPID MEDIATORS A mechanism-based framework can be proposed for analyzing the potential sites at which pharmacologic modification alters the activities of mediators derived from the 5-lipoxygenation or cyclooxygenation of membrane fatty acids (see Figure 14-2 ). Specific levels in the biochemical pathways may be amenable to pharmacologic agents that either enhance or diminish the effect of lipid mediators on their target tissues. The first level involves altering the rate at which membrane phospholipids and triglycerides are broken down by a cytoplasmic or another cellular phospholipase. One of the postulated mechanisms of action of corticosteroids is 159]

inhibition of eicosanoid synthesis by inducing the generation of one or more PLA2 -inhibitory proteins.[

Free fatty acid release also may be under negative-

feedback control by several of the monoHETEs. For example, in platelets, 5-HETE, 12-HETE, and 15-HETE all inhibit the activity of PLA2 .[

92]

The second level of regulation focuses on substitution of other unsaturated fatty acids for AA in cellular phospholipids. Dietary alteration or supplementation that provides unsaturated fatty acids structurally similar to AA, such as γ-linolenic acid and eicosapentaenoic acid (EPA, which predominates in marine fish), leads to absorption, incorporation into membrane phospholipids, release from stimulated cells, and oxygenation of the alternative fatty acids with AA. Dietary supplementation with EPA (20:5), which contains one more double bond than AA (20:4), results in displacement of AA and oxygenation of the substitute EPA by 160 161

stimulated cells.[ ] [ ] Additional mechanisms of EPA inhibition of PG-, Tx-, and LT-mediated reactions include suppression of phospholipase-dependent release of AA, reduction in the oxygenation of AA, and the generation from EPA of mediators corresponding to and competing with those derived from AA but with much lower potency. For example, LTB5 and TxA3 are less potent than the respective AA-derived LTB4 and TxA2 . The ingestion of supplements of 3 to 4•g/day of unpurified EPA or purified ethyl ester of EPA for 6 to 8 weeks by normal subjects or asthmatic patients altered the AA oxygenation pathways and the functions of 160 161

both PMNs and T lymphocytes.[ ] [ ] EPA decreased significantly the generation of PGs and LTs from AA by PMNs and mononuclear leukocytes. Concurrently, EPA suppressed PMN but not monocyte chemotaxis in vitro and enhanced the responses of T lymphocytes to mitogens without modifying the number in each major subset.[

160] [161]

The effects of EPA on inflammatory responses in vivo are more complex when examined in animal models but generally appear to be inhibitory. When rats were fed supplements of unpurified EPA before induction of immune inflammation in an air pouch, the local production of LTB4 and PGE2 were diminished in the chronic 162

phase but not the acute phase relative to the levels in vegetable oil–supplemented rats.[ ] In contrast, the infiltration of mononuclear phagocytes was increased by EPA in the chronic phase. EPA did not influence the volume or character of edema fluid in the immune air pouch or carrageenan-induced paw inflammation in the 162]

same group of rats. [ 163 mice,[ ]

Similar leukocyte-specific effects of dietary supplements of EPA delay the onset and reduce the severity of collagen-induced arthritis in

decrease the levels of autoantibodies and the incidences of autoimmune diseases in MRL-lpr and MRL-mp-lpr/lpr mice,[

development of amyloidosis in azocasein-treated

164] [165]

and retard the

166 mice.[ ]

Dietary supplementation with EPA in several human inflammatory diseases has decreased substantially the generation of PGs and LTs but generally achieved only slight or no therapeutic benefit. Although neutrophil function and production of LTB4 were impaired by EPA in patients with mild asthma, no improvement in airway reactivity or clinical manifestations were detected.[

167]

The increase in nasal mucosal blood flow and the intranasal accumulation of eosinophils elicited by 168]

antigen challenge in allergic rhinitis were blunted by EPA, but these were not accompanied by significant alleviation of symptomatic responses.[

In psoriasis,

169 patients.[ ]

EPA again altered neutrophil functions in vitro but resulted in only modest improvement in the erythema and scaling in 80% of Courses of EPA, which attained similar reductions in neutrophil oxygenation of AA, led to modest improvement in a small number of symptoms and signs of rheumatoid arthritis, including 170] [171]

the duration of morning stiffness, time to onset of fatigue, and number of tender joints, but regression was rapid after cessation of EPA.[

The third level of regulation of PG and LT generation involves separate inhibition of 5-LO, COX-1, and COX-2 enzymatic activity. Some of the LO products derived from EPA and eicosatrienoic acid, as well as from AA, appear to inhibit 5-lipoxygenation by a feedback mechanism. New generations of 5-LO inhibitors have been 172

developed but are handicapped by short duration of action and hepatotoxicity. [ ] In addition, certain flavonoids, in particular cirsiliol, reduce the release of LTs by sensitized guinea pig lung tissue fragments challenged with antigen. COX-1 and COX-2 activity both can be inhibited by most NSAIDs, leading to a reduction in 173]

prostaglandin, thromboxane, and prostacyclin production in specific tissues.[ preferentially with valine

7 174 509.[ ] [ ]

Selective inhibitors of COX-2 have been developed, some of which interact

Selective COX-2 inhibitors have the potential benefit of antiinflammatory effects equal to COX-1 inhibitors with far fewer

gastrointestinal side effects. One other major advantage of selective COX-2 inhibitors is their apparent relative safety in NSAID-sensitive patients, but this aspect of their profile requires additional confirmation. In addition, polyunsaturated acids such as docosahexaenoic acid also inhibit PG, but not LT, biosynthesis by competitive inhibition of the conversion of AA to PGs. [

175]

Potent, orally administered 5-LO inhibitors, such as A-64077 (zileuton), MK-886, and BAYx1005, have effectively suppressed airway inflammation, symptoms, and 176

functional abnormalities in patients with upper and lower respiratory hypersensitivity diseases.[ ] The benefits of 5-LO inhibitors have been significant in natural asthma and asthma evoked by exercise or by inhalational challenge with cold, dry air or antigens. One of the earliest randomized double-blind studies examined the 177

effects of a single oral dose of zileuton in asthma induced by inhalation of cold, dry air. [ ] The amount of cold, dry air required to reduce the 1-second forced expiratory volume (FEV1 ) by 10% and suppress the minute ventilation significantly in 13 patients was increased approximately 50% by zileuton. The published results of applying at least two other

226

equally potent 5-LO inhibitors in asthma elicited by physical or antigenic challenges have shown similar suppression of bronchoconstriction, symptoms, and 176 178 179 180

pulmonary inflammatory responses.[ ] [ ] [ ] [ ] The attenuation of antigen-evoked and exercise-evoked attacks of asthma by 5-LO inhibitors is uniformly partial compared with the almost complete abrogation of attacks of similar severity in sensitive patients challenged with aspirin. Several 5-LO inhibitors also have been of significant and sustained benefit for adults with mild to moderate chronic asthma based on clinical and pulmonary functional criteria ( Table 14-6 ).[

176] [180]

A few studies have addressed the possible effects of oral 5-LO inhibitors in antigen-induced allergic rhinitis. In one typical short-term analysis, a single oral dose of zileuton before intranasal antigen challenge of eight patients in a randomized double-blind protocol significantly reduced both the nasal congestion and the nasal wash fluid concentrations of 5-HETE and LTB4 relative to corresponding values after placebo.[

181]

However, there were no associated changes in sneezing or nasal

fluid concentrations of histamine or PGD2 . Two different selective 5-LO inhibitors have been applied topically in psoriasis to reduce the local tissue content of LTB4 and improve the skin lesions clinically. A 2% ointment TABLE 14-6 -- Leukotriene-Modifying Drugs in Evoked and Natural Asthma Drugs

Mechanism

Study Population and Design Benefits

Adverse Effects

5-LO inhibitor

Adults: exercise-induced asthma; cold, dry air challenge

None observed

Evoked Attacks Zileuton

Improvement in symptoms and pulmonary function

*

Adults: antigen challenge

Improvement in pulmonary function and pulmonary airway inflammation

None observed

BAYx1005

5-LO inhibitor

Adults: antigen challenge

Improvement in symptoms and in pulmonary function

None observed

ICI204219

CysLT1 antagonist

Adults: antigen challenge

Improvement in pulmonary function

None observed

MK-571

CysLT1 antagonist

Adults: exercise-induced asthma, exercise challenge

Improvement in symptoms and pulmonary function

None observed

LY171883

CysLT1 antagonist

Adults: cold, dry air challenge

Improvement in symptoms and pulmonary function

None observed

Zileuton

5-LO inhibitor

Adults: moderate chronic asthma; no inhaled corticosteroids

Improvement in symptoms and pulmonary function

Dyspepsia

ICI204219

CysLT1 antagonist

Adults: moderate asthma

Improvement in chronic asthma symptoms and pulmonary function

None observed

MK-571

CysLT1 antagonist

Adults: mild to moderate chronic asthma

Improvement in pulmonary function

None observed

Natural Chronic Disease

LO, Lipoxygenase; CysLT, cysteinyl leukotriene. * Hepatotoxicity has limited the use of most 5-LO inhibitors in clinical practice.

of the 5-LO inhibitor lonapalene (RS 43179) was applied twice daily without occlusion for 28 days to one area and vehicle alone to a symmetric area of equivalently severe psoriasis in a double-blind study of 10 adults.[

182]

Skin chamber fluid samples from lonapalene-treated sites had significantly lower concentrations of LTB4 ,

but not AA or 12-HETE, after only 4 days. Clinical improvement followed at 14 and 28 days. Identical clinical improvement of psoriasis in 44 patients was observed 183

in a study with similar design of a 2% ointment of the 5-LO inhibitor R 68151, as contrasted with less or no benefit for vehicle alone in 44 patients.[ ] R 68151 resulted in an improvement of 46% for scaling and 34% for erythema, as contrasted with 6% and 3%, respectively, in the vehicle control-treated lesions. In treatment of asthma, side effects initially were limited to mild gastrointestinal symptoms. However, some severe hepatic reactions and subsequent liver damage have been observed. The need for multiple daily doses also has decreased the patient acceptance of 5-LO inhibitors relative to the once-daily effectiveness of LT antagonists.

The fourth level of potential pharmacologic intervention involves selective inhibition of oxygenase-associated proteins, which are required for delivery of optimal concentrations of

227

AA and activation of the oxygenases (see Figure 14-2 ). The fifth level of pharmacologic control is the respective enzymes that convert PGH2 to each distinctive PG or to TxA2 and LTA4 to LTB 4 (LTA4 hydrolase inhibitors) or LTC4 (glutathione-S-transferase inhibitors).[

184]

On the sixth level is the possibility of blocking the

effect of specific mediators with antagonists that interfere with the occupancy of receptors and prevent the transduction of specific signals in target tissues. The agents at this level are the most selective to be designed because each blocks predominantly one LT or PG without affecting other mediators. Several CysLT1 receptor antagonists of distinctive chemical classes exhibit potencies differing by up to two orders of magnitude, unlike the similarly potent 5-LO inhibitors. CysLT1 receptors suppress levels of allergic inflammation in animal models of asthma and effectively reduce the manifestations of asthma evoked by physical and antigenic 185] [186] [187]

challenges in patients[

(see Table 14-6 ). The attenuation of evoked attacks of asthma by LT receptor antagonists has been partial, just as it has been for

the 5-LO inhibitors, but is equivalent to that afforded by long-acting β2 -agonists and the duration of effectiveness far exceeds that of 5-LO inhibitors.[

188] [189]

188 189 190

CysLT1 receptor antagonists also have been of significant and sustained benefit for most adults with mild to moderate chronic asthma.[ ] [ ] [ ] In different studies of the benefits of CysLT1 antagonists combined with inhaled corticosteroids, the benefits for asthmatic patients have been less or more than the combination 189] [190] [191]

of long-acting β2 -agonists and inhaled corticosteroids, but this point requires further studies in different subsets of asthma patients. [

Some leukotriene receptor antagonists may suppress allergic and inflammatory reactions outside the lower respiratory tract. Limited studies have been completed of CysLT1 receptor antagonists in human allergic skin diseases. Administration of the CysLT1 receptor antagonist ICI204219 orally to 18 normal male subjects did not alter significantly the mean threshold intradermal dose of LTD4 required to elicit a wheal-and-flare reaction in the group, but it did increase the dose more than 1 log for 28% of the subjects without a similar change in their responses to intradermal histamine.[ are still in progress.

192]

Similar studies of CysLT1 receptor antagonists in allergic rhinitis

Possible mechanisms for modulating PAF's actions can be analyzed with the same framework used for mediators derived from 5-lipoxygenation or cyclooxygenation of AA. PAF's biosynthetic pathway is relatively simple, and only a few unique enzymes can be manipulated selectively.[

100]

Activation of PLA2 is the initial step in

PAF synthesis. Activity of this enzyme and PAF synthesis are decreased nonspecifically by corticosteroids, but selective antagonists of PAF biosynthesis have not yet been developed. Although PAF is not derived from fatty acids, its synthesis is closely linked to fatty acid metabolism. Substitution of EPA or related polyunsaturated fatty acids for AA decreases PAF synthesis. [ been identified.

193]

At the third and fourth levels of regulation, no specific inhibitors of PAF acetyltransferase have

193]

A potentially fruitful area has been the development of specific antagonists of PAF binding to its receptors.[

Numerous potent and specific antagonists that block

PAF binding and cell activation selectively have been identified, but none has proved effective in controlled trials for allergic diseases. Some of these antagonists are derived from traditional Chinese medicines used to treat allergic and inflammatory disorders. The most hopeful approach had been administration of stable and bioavailable PAF acetylhydrolase, which efficiently degrades PAF and is currently being tested in several clinical trials, although early results do not seem hopeful. [100]

SUMMARY The constellation of lipids with potential roles in allergic diseases constitute the largest set of newly generated and unstored mediators. The range of structures encompasses complex fatty acids and phospholipids. They are all bound extensively by plasma and tissue proteins. Each acts on cells through one or more G protein– coupled receptors. A combination of these mediators can reproduce all aspects of hypersensitivity reactions. Many of the new, effective antiallergy and antiasthma drugs target the generation or cellular receptors of one or more such lipid mediators. Most allergy-like reactions to aspirin, NSAIDs, and related chemicals result from an imbalance in generation between two or more lipid mediators.

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175. Corey EJ, Shih C, Cashman JR: Docosahexaenoic acid is a strong inhibitor of prostaglandin but not leukotriene biosynthesis, Proc Natl Acad Sci USA 80: 3581, 1983. 176. Drazen JM: Leukotrienes. In Leff AR, editor: Pulmonary and critical care pharmacology and therapeutics, New York, 1996, McGraw-Hill. 177. Israel E, Dermarkarian R, Rosenberg M, et al: The effects of a 5-lipoxygenase inhibitor on asthma induced by cold, dry air, N Engl J Med 323:1740, 1990. 178. Hui KP, Taylor IK, Taylor GW, et al: Effect of a 5-lipoxygenase inhibitor on leukotriene generation and airway responses after allergen challenge in asthmatic patients, Thorax 46:184, 1991. 179. Muller-Peddinghaus R, Kohlsdorfer C, Theisen-Popp P, et al: Bay x1005, a new inhibitor of leukotriene synthesis: in vivo inflammation pharmacology and pharmacokinetics, J Pharmacol Exp Ther 267:51, 1993. 180. Israel E, Rubin P, Kemp JP, et al: The effect of inhibition of 5-lipoxygenase by zileuton in mild-to-moderate asthma, Ann Intern Med 119:1059, 1993. 181. Knapp HR: Reduced allergen-induced nasal congestion and leukotriene synthesis with an orally active 5-lipoxygenase inhibitor, N Engl J Med 323:1745, 1990. 182. Black AK, Camp RD, Mallet AI, et al: Pharmacological and clinical effects of lonaalene (RS 43179), a 5-lipoxygenase inhibitor, in psoriasis, J Invest Dermatol 95:50, 1990. 183. Degreef H, Dockx P, DeDoncker P, et al: A double-blind vehicle-controlled study of R 68 151 in psoriasis: a topical 5-lipoxygenase inhibitor, J Am Acad Dermatol 22:751, 1990. 184. Bach MK, Brashler JR, Peck RE, et al: Leukotriene C synthetase, a special glutathione S-transferase: properties of the enzyme and inhibitor studies with special reference to the mode of action of U-60,257, a selective inhibitor of leukotriene synthesis, J Clin Immunol 74:353, 1984. 185. Taylor IK, O'Shaughnessy KM, Fuller RW, et al: Effect of cysteinyl-leukotriene receptor antagonist ICI 204,219 on allergen-induced bronchoconstriction and airway hyperreactivity in atopic subjects, Lancet 337:690, 1991. 186. Manning PJ, Watson RM, Margolskee DJ, et al: Inhibition of exercise-induced bronchoconstriction by MK-571, a potent leukotriene D4 -receptor antagonist, N Engl J Med 323:1736, 1990. 187. Ihaku D, Cameron L, Suzuki M, et al: Montelukast, a leukotriene receptor antagonist, inhibits the late airway response to antigen, airway eosinophilia, and IL-5expressing cells in brown Norway rats, J Allergy Clin Immunol 104:1147, 1999. 188. Gaddy JN, Margolskee DJ, Bush RK, et al: Bronchodilation with a potent and selective leukotriene D4 (LTD4 ) receptor antagonist (MK-571) in patients with asthma, Am Rev Respir Dis 146:358, 1992. 189. Spector SL, Smith LJ, Glass M, et al: Effects of 6 weeks of therapy with oral doses of ICI 204,219, a leukotriene D4 receptor antagonist, in subjects with bronchial asthma, Am J Respir Crit Care Med 150:618, 1994.

190. Coreno A, Skowronski M, Kotaru C, et al: Comparative effects of long-acting β2 -agonists, leukotriene receptor antagonists, and a 5-lipoxygenase inhibitor on exercise-induced asthma, J Allergy Clin Immunol 106:500, 2000. 191. Nelson HS, Busse WW, Kerwin E, et al: Fluticasone propionate/salmeterol combination provides more effective asthma control than low-dose inhaled corticosteroid plus montelukast, J Allergy Clin Immunol 106:1088, 2000. 192. Bernstein JA, Greenberger PA, Patterson R, et al: The effect of the oral leukotriene antagonist, ICI 204,219, on leukotriene D4 and histamine-induced cutaneous vascular reactions in man, J Allergy Clin Immunol 87:93, 1991. 193. Braquet P, editor: Handbook of PAF and PAF antagonists, Boca Raton, Fla, 1991, CRC Press.

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Chapter 15 - Neuronal Control of Airway Function in Allergy

Bradley J. Undem Brendan J. Canning

Both the immune system and the nervous system are critical to host defense. With respect to the airways, the immune system uses cellular and humoral mechanisms to protect the peripheral air spaces from invasion and colonization by microorganisms. The nervous system protects the airways by orchestrating reflexes such as sneezing, coughing, mucus secretion, and bronchospasm. The difference in mechanisms of defense means that these two systems act in a complementary, nonredundant manner. The two systems (in addition to the endocrine system), however, can also be viewed as integrated systems. Immune tissues are directly innervated by the autonomic nervous system, providing the pathways by which the brain can influence immune function. Stimulation of nerves can also indirectly augment immune function by stimulating inflammation in so-called neurogenic inflammatory reactions. On the other hand, mediators released during immune reactions can act on the nervous system to modulate its activity. These nerve-immune interactions can be beneficial to the host. For example, in experimental animals, chemical 1

denervation of airway C-fiber sensory nerves has been found to decrease substantially the host's ability to clear Mycoplasma infection of the airways.[ ] Deleterious nerve-immune interactions can also occur in allergy when the immune response is inappropriate. In this case the immune response triggered by allergen exposure can recruit the nervous system in a way that is not beneficial to the host but rather causes or exacerbates the symptoms of allergic disease: irritation, pruritus, sneezing, coughing, hypersecretion, reversible bronchospasm, and dyspnea.

This chapter provides an overview of the neurophysiology of the airway wall, followed by a review of the literature on the mechanisms by which this neurophysiology is altered during allergic reactions. Although this discussion focuses on the airways, the fundamental principles are likely to extend to any organ in which allergic reactions take place.

AIRWAY INNERVATION Extrinsic Innervation Vagus Nerves

The airways and lungs are innervated bilaterally by the vagus nerves (cranial nerve X). The vagi are mixed nerves, with the majority of vagal fibers being afferent (or 2

sensory) in nature.[ ] Vagal afferent nerve fibers have their cell bodies in one of two ganglia: the jugular (superior vagal) or nodose (inferior vagal) ganglia.[

3] [4]

5

These ganglia are of distinct embryonic origin, which has important influence over the physiologic properties of the nerves they project to the viscera.[ ] The remaining vagal nerve fibers are preganglionic parasympathetic nerve fibers innervating parasympathetic ganglia and motor nerve fibers innervating the striated muscle of the larynx, upper airways, and esophagus. Vagal afferent nerve fibers terminate in integrative centers in the brainstem, primarily the nucleus tractus solitarius (nTS). The parasympathetic nerves and the vagal motor nerve fibers arise from discrete brainstem nuclei, including the dorsal motor nucleus of the vagus nerve (dmnX) and the nucleus ambiguus (nA). Although these brainstem structures have viscerotopic organization, considerable overlap among the sites of afferent nerve subtype termination and of efferent projection is apparent. This overlap contributes in part to the nonselective clustering of autonomic reflexes (e.g., effects on 6] [7]

heart rate, respiratory pattern, and airway caliber) initiated by selective activation of specific afferent nerve subtypes.[ Spinal Nerves

3

The majority of postganglionic sympathetic nerves projecting to the airways arise bilaterally from the superior cervical ganglia and the stellate ganglia.[ ] Although physiologic and morphologic evidence indicates spinal afferent innervation, the functional role of these nerves is poorly understood. The superior laryngeal nerves, recurrent laryngeal nerves, and the bronchial branches of the vagus nerves carry the vagal and spinal nerve fibers projecting to the airways. Both afferent and efferent vagal nerves project bilaterally, although ipsilateral innervation is much more extensive. No evidence shows contralateral projections of postganglionic sympathetic nerves. Intrinsic Innervation 8 9 10

Afferent and efferent nerve fibers occupy multiple nerve plexuses in the airway wall from the larynx to the terminal bronchioles.[ ] [ ] [ ] Afferent nerve fibers are found just beneath and between epithelial cells in an epithelial nerve plexus. The epithelial plexus is composed primarily of afferent nerve endings, but efferent 11]

innervation of the epithelium has been described.[

Both afferent and efferent nerves are found in the plexus of the lamina propria, where most effectors of the

airways (airway smooth muscle, mucus glands, arterioles) are located ( Figure 15-1 ).[

12]

Airway ganglia occupy a serosal nerve plexus of the extrapulmonary

airways, a plexus that merges with the lamina propria plexus in the intrapulmonary airways.[ the airway wall. Parasympathetic ganglia

9] [10] [13] [14] [15]

Occasionally, ganglia may also be found elsewhere in

232

containing as few as one neuron to more than 100 neurons are randomly and sparsely dispersed in the serosal nerve plexus and are associated primarily with the extrapulmonary airways. Ganglia associated with the intrapulmonary airways are typically localized to branch points in the bronchial tree. No ganglia are found in or adjacent to the bronchioles. 16

Except for afferent nerve endings terminating in neuro-epithelial bodies of the epithelium,[ ] most airway afferent nerves do not assume structures characteristic of muscle spindle sensory nerves, or touch-sensitive sensory nerves of the somatic system. Rather, airway afferent nerves form apparently nonspecialized (based on appearance) receptive fields in the epithelium and in and around various structures of the airway wall. Swellings associated with airway afferent nerve terminals in the epithelium contain synaptic vesicles with neurotransmitters that may be released during axonal reflexes. Afferent nerve fibers may also innervate other effector 17] [18]

tissues in the airway wall, including glands,[

3

airway smooth muscle,[ ] blood vessels,[

19]

and airway parasympathetic ganglia.[

Postganglionic autonomic nerves innervate structures throughout the airway wall, including glands,[ adjacent airway para-sympathetic the bronchioles.[

13] [23] [25]

14 24 ganglia.[ ] [ ]

22]

19]

blood vessels,[

20] [21]

airway smooth muscle,[

23]

and perhaps

Morphologic analyses reveal little change in nerve fiber densities in the smooth muscle from the large bronchi to

The neurochemistry of these nerve fibers, however, may differ considerably in the large and small airways.

Figure 15-1 Fluorescence photomicrograph of confocal microscope image stacks showing dense neural network in biopsy specimen from asthmatic airway. (Nerves stained with protein gene product 9.5; ×calibration bar: 50••m.) (From Goldie RG, Fernandes L, Rigby P: Curr Opin Pharmacol 2:273, 2002.)

(From Goldie RG, Fernandes L, Rigby P: Curr Opin Pharmacol 2:273, 2002.) Airway Parasympathetic Ganglia

Parasympathetic nerve fibers are found throughout the airways. Parasympathetic ganglia, however, are few in number and contain only a handful of neurons. Airway 14]

parasympathetic tone is thus determined by the actions of relatively few ganglia neurons in fewer still ganglia.[

Airway ganglia neurons are not simply relays between the central nervous system (CNS) and the effector tissues of the airway wall. Rather, airway ganglia neurons subserve an impor-tant integrative role ( Figure 15-2 ). This function is facilitated by the complex morphology of the ganglia neurons and by the many biophysical

properties of the neurons that facilitates integration of synaptic input.[

14] [15] [26] [27]

Figure 15-2 A, Drawing of single neuron injected with Neurobiotin and processed for peroxidase histochemistry; asterisk (*) indicates axon (calibration bar = 20• •m). B, Fast excitatory postsynaptic potentials (fEPSPs) in human bronchial ganglia. Overlay of traces shows responses by human bronchial ganglia neuron to 10 consecutive peribronchial nerve stimulations (shock artifact at vertical arrow; stimulus = 1.0•msec, 20•V, 0.5•Hz). fEPSPs are subthreshold for action potential generation and graded in amplitude (arrowheads). C, Single stimulus (shock artifact at vertical arrow) to preganglionic nerve trunk elicits three temporally distinct fEPSPs (arrowheads), indicating convergence of preganglionic axons. (From Kajekar R, Rohde HK, Myers AC: Am J Respir Crit Care Med 164:1927, 2001.)

(From Kajekar R, Rohde HK, Myers AC: Am J Respir Crit Care Med 164:1927, 2001.)

233

Synaptic transmission between preganglionic and postganglionic parasympathetic nerves in the bronchi is mediated primarily, if not exclusively, by acetylcholine 15 28

acting on nicotinic receptors.[ ] [ ] When activated, nicotinic receptors on the airway ganglia neuronal dendrite initiate depolarizations known as fast excitatory postsynaptic potentials (fEPSPs). In the airway ganglia, most fEPSPs are subthreshold for action potential formation. Summation of several fEPSPs may be necessary to reach threshold for action potential generation ( Figure 15-2 ). The filtering capacity of airway ganglia neurons can be modulated by a number of mechanisms, either through modulatory effects of noncholinergic neurotransmitters or through alterations in the excitability of the ganglia neurons. Airway ganglia neurons are innervated by preganglionic nerve fibers carried by the vagus nerves. Neurons are often innervated by several convergent preganglionic fibers, which in turn may diverge extensively in the airways and innervate multiple airway ganglia. This divergence may facilitate coordination of airway reflexes.[ Airway ganglia neurons may also innervate adjacent ganglia neurons.[

24]

14]

Collaterals of afferent nerves containing neuropeptides are also found in airway ganglia and

may regulate synaptic transmission in the ganglia through peripheral reflexes.[

29] [30] [31]

Reflex Regulation of Airways Afferent Nerve Subtypes

Multiple afferent nerve subtypes innervate the airways. These nerve subtypes can be subclassified based on their neurochemistry, responsiveness to physical and 2 32 33

chemical stimuli, myelination, conduction velocity, sites of termination in the CNS, and ganglionic origin.[ ] [ ] [ ] Airway low-threshold mechanoreceptors have at least two subtypes: rapidly adapting receptors (RARs) respond to the dynamic physical effects and slowly adapting receptors (SARs) to the sustained physical effects of lung inflation. Some airway mechanoreceptors can also be activated indirectly by bronchoconstrictors, such as histamine, acetylcholine, and leukotrienes. [34] [35]

When activated, airway mechanoreceptors may initiate alterations in autonomic nerve activity and cough and may play an integral role in controlling 2 32 33

36

respiratory rate and tidal volume.[ ] [ ] [ ] Not surprisingly, therefore, many airway mechanoreceptors are active during the respiratory cycle ( Figure 15-3 ).[ ] This continuous activity of airway mechanoreceptors may be of fundamental importance to the maintenance of baseline autonomic tone, respiratory pattern, and possibly, how subsequently evoked reflexes proceed.[

37]

Afferent nerves that are similar to the nociceptors of the somatic nervous system also innervate the airways. These nociceptors, most of which are unmyelinated C 38

fibers, are generally unresponsive to mechanical stimuli and are thus essentially quiescent during tidal breathing[ ] ( Figure 15-3 ). These nociceptors, however, are activated by inflammatory mediators such as bradykinin and 5-hydroxytryptamine (5-HT, serotonin) and may also be activated by low pH, hypertonic saline, or the 2 5 39 40 41

vanilloid capsaicin.[ ] [ ] [ ] [ ] [ ] Capsaicin activates airway nociceptors by opening the ion channel and receptor VR-1 (vanilloid Receptor-1).[ an important role in regulating signaling by nociceptors. Endogenous ligands for VR-1 include

42]

VR-1 may play

Figure 15-3 Representative experimental records illustrating three basic phenotypes of afferent nerves in the lungs through the action potential (AP) discharge in response to capsaicin (Cap) injection, tidal breathing, and hyperinflation of rat lungs. A, Pulmonary C fiber arising from an ending in right upper lobe of anesthetized, open-chest rat (conduction velocity of fiber = 1.05•m/sec). Note that C fiber responds to capsaicin, but not to mechanical effects of tidal breathing (top) or even hyperinflation (bottom). B, Fiber with rapidly adapting receptor (RAR) located in right upper lobe (conduction velocity = 21.4•m/sec). Note that RAR does not respond to capsaicin, but does respond with AP discharge during tidal breathing. The fiber adapts to the mechanical stimulus of prolonged hyperinflation. C, Slowly adapting receptor (SAR) located in right lower lobe (conduction velocity = 23.5•m/sec). Note that SAR does not respond to capsaicin, but does respond during tidal breathing and in a nonadapting manner to prolonged hyperinflation. Upper panels, Capsaicin (1•g/kg in 0.2-ml solution) was first slowly injected into the catheter (dead space 0.3•ml) and then flushed into the right atrium (arrow) as a bolus with saline (0.4•ml). Lower panels, Hyperinflation was generated by maintaining a constant tracheal pressure (Pt ) at 30•cm H2 O for 10 seconds while the respirator was turned off. ABP, Arterial blood pressure. (From Ho CY, Gu Q, Lin YS, et al: Respir Physiol 127:113, 2001.)

(From Ho CY, Gu Q, Lin YS, et al: Respir Physiol 127:113, 2001.)

234

12-lipoxygenase (12-LO) and 15-LO products and the endo-genous cannabinoid anandamide. [

43] [44]

When activated, airway nociceptors initiate alterations in

autonomic nerve activity and cough and have unique effects on respiratory pattern. Autonomic Nerve Subtypes

Both sympathetic and parasympathetic nerves innervate the airways. Sympathetic nerves primarily innervate the bronchial vasculature, whereas airway parasympathetic nerves inner-vate the vasculature but also the glands and airway smooth muscle.[

45] [46]

For almost a century, autonomic control of the airways was viewed as a balance between the opposing actions of the sympathetic and parasympathetic nerves. It was further assumed that the actions of the parasympathetic nervous system were mediated by acetylcholine, whereas the sympathetic nerves used norepinephrine to regulate airway function. Despite the accuracy of some aspects of this model and its great predictive value, it has become apparent that autonomic control is much more complex than being described simply as the opposing actions of acetylcholine and norepinephrine. Multiple neurotransmitters have been localized to the autonomic nerves innervating the airways. These neurotransmitters have multiple effects on the end organs in the airways, and their role as true neurotransmitters and neuromodulators has been confirmed.[

45] [46]

Autonomic Regulation of Airway Smooth Muscle Tone

Postganglionic parasympathetic nerves innervate airway smooth muscle from the trachea to the terminal bronchioles. When activated, airway parasympathetic cholinergic nerves initiate marked contractions of airway smooth muscle throughout the airway tree. Sympathetic innervation of human airway smooth muscle is either sparse or nonexistent. A comparable paucity of sympathetic innervation of the intrapulmonary airways in other species has been noted. Indeed, even though human airway smooth muscle expresses abundant beta adrenoceptors (primarily β2 -adrenoceptors), direct functional evidence of functional sympathetic (adrenergic) innervation of human airway smooth muscle is lacking. It seems likely, therefore, that hormonal catecholamines are the primary ligand for the βadrenoceptors expressed on human airway smooth muscle. The only functional relaxant innervation of airway smooth muscle in many species, including humans, is provided by the parasympathetic nervous system. Parasympathetic nerve-mediated relaxation of airway smooth muscle may be mediated by vasoactive intestinal peptide (VIP), pituitary adenylate cyclase–activating peptide (PACAP), and polypeptide with histidine at N-terminal and methionine at C-terminal (PHM, human form of PHI, with isoleucine at C-terminal), as well as the gaseous transmitter nitric oxide (NO, synthesized from arginine by neuronal NO synthase). These nonadrenergic, noncholinergic (NANC) relaxant responses can 46] [47]

be evoked in airways from the trachea to the small bronchi.[

It was long assumed that the neurotransmitters mediating NANC relaxation of airway smooth muscle were co-released with acetylcholine from a single population of postganglionic parasympathetic nerves. It was further hypothesized that the function of these neurotransmitters was to serve as a break on the parasympathetic nerves, preventing excessive constriction during periods of elevated parasympathetic nerve activity. Studies in guinea pigs, however, reveal that at least in this species, noncholinergic parasympathetic neurotransmitters are not necessarily co-released with acetylcholine from postganglionic parasympathetic nerves. Rather, data support the hypothesis that entirely distinct parasympathetic pathway regulates noncholinergic nerve activity in the airways.[ subtypes and reflexes also differentially regulate these distinct parasympathetic

48 50 51 52 pathways.[ ] [ ] [ ] [ ]

48] [49]

Preganglionic nerve

Morphologic and physiologic studies in ferrets and guinea 10] [48] [53] [54]

pigs indicate that the parasympathetic ganglia mediating cholinergic and noncholinergic responses in the airways are also distinct.[

Distinct

50] [51]

parasympathetic pathways also likely regulate cholinergic and noncholinergic responses in cats.[ ganglion neurons have also been identified in human airways.

Separate cholinergic and noncholinergic parasympathetic

[13]

46

Many stimuli initiate reflex alterations in airway parasympathetic nerve activity.[ ] The homeostatic role of these alterations is not readily apparent, but these may serve to optimize the efficiency of gas exchange and may facilitate clearance mechanisms during cough by regulating airflow velocity. Bronchoconstrictors such as 52] [55] [56]

histamine, prostaglandin D2 (PGD2 ), leukotrienes, and even methacholine, for example, initiate both cough and reflex bronchospasm.[ stimulants such as capsaicin, bradykinin, hypertonic saline, and acidic solutions also initiate reflex bronchospasm.[

44] [52] [55] [56] [57]

Nociceptor

Other stimuli initiating reflex 44] [48] [52]

bronchospasm include chemoreceptor stimulation, esophageal afferent nerve stimulation, and upper airway afferent nerve stimulation.[

46] [50] [52] [56] [57] [58]

Stimuli initiating reflex bronchodilation include stimulants that activate airway mechanoreceptors and airway nociceptors.[ chemoreceptor stimulation is without apparent effect on noncholinergic parasympathetic nerve activity.[

50] [52]

By contrast,

Activation of skeletal muscle afferent nerves, as might

occur during exercise, also initiates reflex bronchodilation, primarily through withdrawal of baseline cholinergic tone.[

59] [60]

Reflexes initiating alterations in airway sympathetic nerve activity are not well described. Clearly, however, adrenoceptors and thus the endogenous catecholamines play an important role in regulating airway caliber and airway responsiveness. The relative contribution of hormonal and neuronal sources of the catecholamines is not clear. In humans the circulating catecholamines are likely more important in regulating airway function than the sparse sympathetic adrenergic innervation of the airways. Autonomic Regulation of Glands

Airway glands are regulated primarily by the parasympathetic nervous system. Acetylcholine (ACh) is the primary neurotransmitter regulating airway glandular secretion, but other peptide neurotransmitters may also play a role in mucus secretion. Sympathetic nerves play little or no role in mucus secretion. Neurotransmitters 22] [61]

associated with sympathetic nerves have subtle if any effect on secretion but may play a role in regulating parasympathetic nerve activity. [

Reflexes initiating parasympathetic nerve–dependent mucus secretion are induced by many of the same stimuli that initiate reflex bronchospasm. Therefore the postganglionic parasympathetic nerve–regulating mucus secretion and smooth muscle tone in the airways may be derived from the same subpopulations of airway parasympathetic nerves. Evidence suggests,

235

however, that the neurochemistry of the postganglionic parasympathetic nerves innervating glands may differ from those innervating airway smooth muscle. The trophic influence of these end organs on the phenotype of the autonomic nerves has not been studied.

Autonomic Regulation of Bronchial Vasculature 19 62 63 64

Sympathetic and parasympathetic nerves regulate bronchial vascular tone.[ ] [ ] [ ] [ ] Sympathetic nerves mediate vasoconstric-tion through the actions of norepinephrine and neuropeptide Y, whereas parasympathetic nerves mediate vasodilatation through the actions of ACh, NO, and perhaps peptides (e.g., VIP). Reflex regulation of bronchial vascular tone is poorly described, in large part due to the difficulty in studying the bronchial vasculature. Airway nociceptor 65]

stimulation, however, is known to initiate parasympathetic reflex dilation of the bronchial vasculature.[ Axon Reflexes

Activation of some sensory nerves, primarily nociceptors, causes the release of proinflammatory transmitters, such as substance P and perhaps adenosine 66

triphosphate (ATP), from their peripheral endings that act on the tissue of innervation, thereby initiating neurogenic inflammation.[ ] This peripheral release of neurotransmitters from sensory nerve collaterals and the resulting end-organ effects are called the axon reflex. Afferent nerves innervating the airway mucosa of most species, including humans, express the anatomic attributes of the sensory nerves mediating axon reflexes in somatic tissues. Many of these afferent nerve endings contain potent proinflammatory peptides such as substance P, neurokinin A, and calcitonin gene-related peptide (CGRP). When administered exogenously, these putative neurotransmitters have profound effects in the airways, initiating bronchospasm, mucus secretion, vasodilation, plasma exudation, and inflammatory cell 67]

recruitment.[

These observations naturally led to the hypothesis that axonal reflexes contribute to the pathogenesis of inflammatory airway disease.

Axon reflexes have been well defined in the airways of rats and guinea pigs, and evidence indicates that axon reflexes may regulate human upper airway responses to bradykinin and capsaicin. The role of axon reflexes in the lower airways is less clear. Morphologic studies of the afferent innervation of the human airway mucosa reveal a dense plexus of afferent nerves innervating the epithelium but a general sparseness of neurokinin-containing nerve fibers. Capsaicin evokes contractions of isolated human airway smooth muscle preparations and mucus secretion from airway mucosal explants, but these effects are unlikely to be mediated by neurokinins. [68] [69] [70]

Bronchospasm initiated by putative C-fiber stimulants in human subjects and in species other than rats or guinea pigs are caused primarily by CNS-

dependent parasympathetic reflexes.[

71] [72]

Cough

Cough reflexes play an essential role in the clearance of inhaled pathogens, aeroallergens, irritants, particulate matter, secretions, and aspirate and thus protect the 73

airway mucosa from damage. However, cough may also be “dry,” or nonproductive.[ ] This irritating type of cough is a common symptom of patients with allergic inflammation of the airways. Vagal afferent nerves regulate the cough reflex. In general, stimuli that initiate reflex bronchospasm also initiate cough. The afferent nerves mediating cough are likely both the airway mechanoreceptors, which are activated by inhaled particulate, accumulated mucus, and bronchospasm, and the 55]

airway nociceptors, which are activated by bradykinin and capsaicin.[

AIRWAY NEUROPHYSIOLOGY AND THE ALLERGIC REACTION

The symptoms of allergic disease are largely the result of altered neuronal activity. Whether the symptom is pruritus or “itchy airways” resulting in excessive coughing, eye irritation, gastrointestinal irritation, runny nose, or the rapid and reversible bronchospasm that leads to dyspneic sensations, the nervous system serves as the principal transducer between immunologic aspects of allergic inflammation and the symptomatology of immediate hypersensitivity. This may occur as the result of allergen-induced mediator production and release from resident cells interacting with receptors on sensory and autonomic nerves. Relatively little is known about the specific pharmacology of allergen-nerve interactions, but the mediators likely include histamine, arachidonic acid metabolites, and neuro-trophins, as well as chemokines and cytokines.[

2] [74] [75] [76]

As outlined earlier, the peripheral nervous system functions in terms of reflex arcs. Although an increase in airway reflex activity is self-evident to those coughing and sneezing during their allergy season, it is difficult to measure hyperreflexia experimentally in humans. Studies in the human nose have provided a clear-cut example of up-regulation in reflex physiology by allergy. In these studies a sensory nerve irritant (bradykinin) was applied to one nostril of a subject before and during their allergy season. When subjects were not experiencing allergies, bradykinin had very little effect on airway physiology. Applying bradykinin to the nose of the same subjects during their allergy season, however, led to excessive sneezing and reflex secretions ( Figure 15-4 ). [ 78] [79] [80]

other laboratories and may be observed with other types of sensory nerve stimulants.[ when cough is used as the outcome

77]

This observation has been repeated in

This same type of hyperreflexia can be seen in the lower airways

81 variable.[ ]

The reflex can be conceptually subdivided into four components ( Figure 15-5 ). First, the reflex begins with stimulation of afferent or sensory nerves that innervate all tissues in the body. Second, action potentials (APs) travel along the afferent axon until they reach the central terminals, where APs evoke the release of neurotransmitters at the synapse with secondary neurons in the CNS. Synaptic activation of secondary neurons is then transmitted to other centers in the CNS, leading to sensations, respiratory reflexes, or increases or decreases in the activity of preganglionic autonomic fibers. Third, APs in preganglionic fibers reach the terminals of these fibers, where they evoke the release of ACh at the synapse of autonomic ganglion neurons. Fourth, synaptic activation of postganglionic autonomic neurons leads to autonomic transmitter release at effector cells (e.g., smooth muscle, vasculature). The allergic reaction appears particularly adept at influencing reflex physiology in the airways by modifying each of the four steps in the pathway of reflex action. Primary Afferent Nerves in Airways Most, if not all, airway afferent nerves are sensitive to mechanical perturbation. Allergic reactions can lead indirectly to

236

Figure 15-4 Effect of nasal provocation with bradykinin in nine patients with seasonal allergy challenged in season (white circles) and out of season (black circles). Left, Ipsilateral responses; Right, contralateral responses; Dil, diluent. Significant increase in contralateral secretion weights was seen in subjects challenged in season (P < 0.01) but not out of season. (From Riccio MM, Proud D: J Allergy Clin Immunol 97:1252, 1996.)

(From Riccio MM, Proud D: J Allergy Clin Immunol 97:1252, 1996.) mechanosensitive afferent nerve activation by releasing mediators that cause bronchial smooth muscle contraction. For example, histamine leads to activation of 33]

afferent RARs and SARs by a mechanism that can be inhibited by bronchodilators.[ challenge in rabbit

82 airways.[ ]

This finding may explain the AP discharge in RARs observed after allergen

In

Figure 15-5 Airway central reflex pathway, showing points along the pathway that may be modulated by allergic inflammation. (1) Mediators released during an allergic reaction may activate primary afferent nerves, leading to action potential (AP) discharge. Alternatively, mediators may increase excitability of afferent nerve endings such that the threshold for other stimuli (e.g., mechanical stimulation) is decreased. (2) Mediators released during the allergic reaction may interact with nerve terminals whereby signals are sent to the cell bodies in the sensory ganglia, leading to changes in gene expression, as when allergen inhalation increases expression of the preprotachykinin gene in vagal sensory ganglia. (3) Allergic inflammation of airways can lead to increases in excitability of secondary neurons in the brain stem, where changes in accommodation have been observed. Changes in amount and type of transmitter released from the central terminals of afferent nerves may also lead to augmented synaptic transmission (central sensitization). (4) Allergic inflammation has been shown to increase synaptic transmission in the bronchial ganglia, thereby decreasing the capacity of the ganglia to act as filters of preganglionic input. This, in theory, would lead to a generalized increase in parasympathetic tone in the airways. (5) Allergic inflammation has been associated with increases in the amount of acetylcholine released per AP, and with decreases in the amount or efficacy of the nonadrenergic, noncholinergic (NANC) parasympathetic transmitters vasoactive intestinal peptide (VIP) and nitric oxide (NO).

Figure 15-6 Mechanical sensitivity of an afferent rapidly adapting receptor (RAR)–like fiber in isolated guinea pig trachea before and after allergen challenge. Mechanical sensitivity was determined using von Frey filaments and expressed in millinewtons (mN) before and 15 minutes after allergen challenge in trachea isolated from actively sensitized guinea pigs. Allergen (ovalbumin, OVA) exposure did not evoke action potential discharge in RAR-like fibers (not shown) but did cause on average a significant fourfold decrease in amount of force required to activate the mechanical receptive field. (From Riccio MM, Myers AC, Undem BJ: J Physiol 491:499, 1996.)

(From Riccio MM, Myers AC, Undem BJ: J Physiol 491:499, 1996.) Central Nervous System Integration An increase in the activity of primary afferent nerves will result in an increase in the activity of the secondary neurons to which the primary nerves project. Because most vagal afferent nerves project to neurons in the nucleus tractus solitarius (nTS), allergen provocation in visceral tissues will likely lead to an increase in the 98

activity of neurons within the nTS. For example, intestinal anaphylaxis in rats leads to stimulation of numerous neurons in the rat nTS.[ ] Investigators studying the mechanism underlying various pain syndromes observe that peripheral inflammation not only leads to synaptic activation of secondary neurons, but can also lead to 99

increases in efficacy of synaptic transmission such that neurotransmission is magnified within the spinal cord. This process is referred to as central sensitization. [ ] Given the similarities between visceral hyperreflexia and somatosensory hyperalgesia, allergic inflammation may also lead to central sensitization. Evidence shows 44]

synergistic interactions between airway afferent nerves mediating reflex bronchospasm.[

Likewise, in an allergic monkey model, sensitization to and repeated

inhalation exposure with house-dust mite allergen led to profound changes in the electrophysiologic properties of secondary neurons in brainstem (nTS) neurons.[ The nTS neurons from control monkeys responded to prolonged suprathreshold current pulses with about 20 APs, whereas nTS neurons isolated from allergen-

100]

exposed monkeys responded to the depolarizing current pulse with more than 100 APs ( Figure 15-7 ). These data support the hypothesis that airway inflammation can alter afferent input into the CNS such that plastic changes occur in the basic electrophysiologic properties of neurons within the nTS. Because nociceptors and RARs mediate similar reflex effects in the airways, these afferent nerve subtypes might act synergistically to initiate these reflexes. Such synergistic interactions are facilitated by the likely convergence of the central nerve terminals of these afferent nerves in key integrative sites in the brainstem, such as the commissural nucleus of the solitary tract.[ 99 101 tissues.[ ] [ ]

6] [7]

These interactions would be analogous to the processes initiating hyperalgesia and pain sensations in somatic

The parallels between hyperalgesia and airway hyperresponsiveness (AHR) seem obvious. Such interactions between afferent nerve subtypes may

explain in part how extrapulmonary disorders such as gastroesophageal reflux disease,[ pulmonary symptoms such as cough and reflex bronchospasm and perhaps AHR.

102]

103]

allergic rhinitis,[

104]

and upper respiratory tract infections[

initiate

Autonomic Ganglionic Neurotransmission By increasing primary afferent nerve trafficking and increasing synaptic transmission in the CNS, allergic inflammation will likely lead to an increase in activity of autonomic preganglionic nerves. In addition, however, experimental evidence supports the hypothesis that allergic inflammation may also increase autonomic tone, by increasing the efficacy of synaptic transmission within autonomic ganglia. This process of ganglionic sensitization is analogous to the previously discussed central sensitization.[

14]

238

Figure 15-7 Current-clamp recordings of neurons in nucleus tractus solitarius (nTS) of isolated monkey brain stem from control animals and animals chronically exposed to inhalation of house-dust mite allergen. Membrane potential is recorded in response to 500-msec injections of increasing amounts of current (40 to 100• pA). Note that neurons in nTS of allergen-exposed animal responded to the current injection with many more action potentials than observed in control monkeys. In other words, chronic inhalation of allergen led to increased excitability of neurons in the central nervous system. (From Chen CY, Bonham AC, Schelegle ES, et al: J Allergy Clin Immunol 108:557, 2001.)

(From Chen CY, Bonham AC, Schelegle ES, et al: J Allergy Clin Immunol 108:557, 2001.) The parasympathetic division of the autonomic nervous system supplies most of the autonomic innervation of the airways. Airway parasympathetic ganglia filter or 14 105

integrate input from the CNS.[ ] [ ] Any process that increases the efficacy of synaptic transmission, resulting in increases in EPSP amplitude, will result in a decrease in filtering and a generalized increase in airway parasympathetic tone. Allergen challenge to bronchi isolated from actively sensitized guinea pigs increases 106

the excitability of bronchial ganglion neurons. [ ] This response occurs through several mechanisms and may involve many different mediators. Histamine released from mast cells in or near the ganglia results in decreases in potassium conductance and a membrane depolarization, whereas PGD2 challenge inhibits 107 108

accommodation of airway ganglia neurons such that the same stimulus results in many more APs.[ ] [ ] In preliminary unpublished experiments, we have also noted that when mast cell–activating anti–immunoglobulin E (IgE) is added to human bronchial preparations, the membrane potentials of bronchial parasympathetic ganglion neurons depolarize in a manner similar to that observed in guinea pigs. As discussed earlier, the parasympathetic innervation of the airways can be subdivided into cholinergic and noncholinergic pathways. The noncholinergic branch is responsible for the relaxant innervation of airway smooth muscle and likely also regulates mucus and vascular physiology. No studies have systematically compared the effect of allergen challenge on synaptic efficacy in the cholinergic versus noncholinergic parasympathetic ganglia. However, work in the sympathetic system suggests that regardless of the nature of the autonomic ganglia, mediators associated with allergen challenge will lead to an increase in synaptic efficacy and a generalized increase in autonomic tone. For example, exposing the superior cervical, mesenteric, or myenteric ganglia to a sensitizing antigen results in ganglionic 109 110 111

mast cell activation and a pronounced increase in synaptic neurotransmission.[ ] [ ] [ ] Autonomic synaptic efficacy is affected by very low concentrations of antigen. The antigen-induced effect is persistent; a brief (5-minute) antigen challenge of an isolated superior cervical ganglion potentiates synaptic transmission for longer than 3 hours (as long as the experiment lasted). This process has been termed antigen-induced long-term potentiation.[ Postganglionic Neuroeffector Transmission

112]

The final place in an autonomic reflex loop that can be modulated is at the postganglionic neuroeffector junction. This junction is the site where APs arising from the ganglionic synapse invade the postganglionic varicosities, causing neurotransmitter release, which then affects the effector cells. In the airways, allergen challenge is associated with an elevation in the amount of ACh released from the airway postganglionic nerve varicosities.[ with postganglionic nerve fibers to enhance transmitter

45 release.[ ]

113] [114]

Several mediators have been found to interact

In addition to conventional autacoid effects, allergen-induced eosinophilic inflammation may

augment ACh release by inhibiting the function of cholinergic muscarinic M2 receptors.[

113] [114] [115]

The prejunctional M2 receptors serve as negative-feedback 116]

“autoreceptors,” such that when the released ACh acts on these receptors, signals are produced that lead to an inhibition of further ACh release.[

challenge inhibits this process, apparently through an inhibitory effect of eosinophil-derived major basic protein (MBP) on M2 receptor function.

Allergen

[117]

Few studies have examined the effect of allergen challenge on the release of noncholinergic transmitters from postganglionic parasympathetic nerve endings. 118 119

Allergen challenge has been shown to attenuate subsequently evoked noncholinergic relaxant responses in animals.[ ] [ ] The mechanism for this effect of allergen challenge is unclear. Elevated levels of superoxide, perhaps derived from infiltrating eosinophils, may attenuate relaxant responses mediated by neuronal NO. [119] [120]

97]

Alternatively, mast cell tryptase or other peptidases may attenuate relaxant responses mediated by VIP and other relaxant neuropeptides.[ bronchoprotective effects of

The

239

deep inspiration have been associated with NO formation, although the NO source is unknown.[ NO may be inhibited in asthmatic perhaps NO synthase in the

123 124 patients.[ ] [ ]

121] [122]

Interestingly, the bronchoprotective effect of endogenous

Some studies indicate that the inflammation associated with asthma may decrease VIP levels in the airways or

125 126 lung.[ ] [ ]

CONCLUSIONS Many of the signs and symptoms of patients with allergic disease are the result of inappropriate neural function. Depend-ing on the site of allergen exposure, these manifestations may include cutaneous reactions and excessive itching, gastrointestinal disturbances, sneezing, rhinorrhea, coughing, bronchospasm, and sensations of breathlessness. The molecular mechanisms by which allergen exposure affects the nervous system have not been elucidated in detail. The experimental evidence from human studies and animal models, however, indicates that allergic inflammation may affect reflex physiology at multiple sites (see Figure 15-5 ). The effects include changes in excitability of primary afferent terminals, changes in gene expression in sensory ganglion neurons, increases in synaptic transmission within the CNS and within autonomic ganglia, and changes in transmitter secretion at the level of the nerve-effector junction. Future research into the mediators and mechanisms of allergen-induced neuromodulation will not only increase our basic understanding of the pathophysiology of allergic disease, but may also suggest novel therapeutic strategies.

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86:723, 1985. 117. Evans CM, Fryer AD, Jacoby DB, et al: Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs, J Clin Invest 100: 2254, 1997. 118. Miura M, Ichinose M, Kimura K, et al: Dysfunction of nonadrenergic non-cholinergic inhibitory system after antigen inhalation in actively sensitized cat airways, Am Rev Respir Dis 145:70, 1992. 119. Miura M, Yamauchi H, Ichinose M, et al: Impairment of neural nitric oxide-mediated relaxation after antigen exposure in guinea pig airways in vitro, Am J Respir Crit Care Med 156:217, 1997. 120. Ricciardolo FL, Timmers MC, Geppetti P, et al: Allergen-induced impairment of bronchoprotective nitric oxide synthesis in asthma, J Allergy Clin Immunol 108:198, 2001. 121. Skloot G, Permutt S, Togias A: Airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration, J Clin Invest 96:2393, 1995.

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122. Silkoff PE, Sylvester JT, Zamel N, Permutt S: Airway nitric oxide diffusion in asthma: role in pulmonary function and bronchial responsiveness, Am J Respir Crit Care Med 161:1218, 2000. 123. Ricciardolo FL, Geppetti P, Mistretta A, et al: Randomised double-blind placebo-controlled study of the effect of inhibition of nitric oxide synthesis in bradykinin-induced asthma, Lancet 348:374, 1996. 124. Ricciardolo FL, Di Maria GU, Mistretta A, et al: Impairment of bronchoprotection by nitric oxide in severe asthma, Lancet 350:1297, 1997. 125. Ollerenshaw S, Jarvis D, Woolcock A, et al: Absence of immunoreactive vasoactive intestinal polypeptide in tissue from the lungs of patients with asthma, N Engl J Med 320:1244, 1989. 126. Samb A, Pretolani M, Dinh-Xuan AT, et al: Decreased pulmonary and tracheal smooth muscle expression and activity of type 1 nitric oxide synthase (nNOS) after ovalbumin immunization and multiple aerosol challenge in guinea pigs, Am J Respir Crit Care Med 164:149, 2001.

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243

Chapter 16 - Biochemical Events in Basophil/Mast Cell Activation and Mediator Release

Reuben P. Siraganian

Although mast cells and basophils were first described by Paul Ehrlich more than 100 years ago, many years passed before it was recognized that these cells play critical roles in immediate hypersensitivity reactions and that they contain histamine and other inflammatory mediators. Mast cells and basophils have many features in common. Both cell types have surface receptors (e.g., FcepsilonRI) that bind immunoglobulin E (IgE) with high affinity, and both have prominent cytoplasmic granules containing inflammatory mediators. These cells are stimulated to secrete mediators when the IgE on their surface reacts with multivalent antigen. However, there are also many differences between basophils and mast cells (see Chapters 13 , 20 , and 21 ). The stimulation of basophils or mast cells is initiated by the interaction of secretagogues with cell surface receptors. This results in a series of biochemical events that culminate in the release of biologically active mediators that mediate allergic reactions. The mediators released from basophils or mast cells are either pre-formed and stored in secretory granules or newly generated. Pre-formed mediators include the biogenic amines, such as histamine and serotonin, which on a molar basis are the major components of the secretory granules. Human basophils and mast cells contain only histamine; however, serotonin is present in the mast cells of some species (e.g., rodents). The amount of histamine in mast cells is much greater than that in basophils. In the granule, histamine is associated with the carboxyl groups of proteoglycans and other proteins by ionic binding. During secretion the interior of the granule is exposed to the extracellular ions, and histamine is released by a cation exchange mechanism from these binding sites. Other granular contents include the neutral proteases, proteoglycans, several acid hydrolases (e.g., βhexosaminidase, β-glucuronidase), and chemotactic molecules. Several mediators are newly generated when mast cells or basophils are activated by an appropriate stimulus. During the release process, arachidonic acid (AA) is released from cellular phospholipids because of the activation of phospholipase enzymes. The metabolism of this AA can then proceed along different pathways depending on the enzymes present. If it is metabolized along the cyclooxygenase pathway, it results in the formation of the prostaglandins, whereas the lipoxygenase pathway leads to the formation of the leukotrienes. Platelet activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is generated from alkyl phospholipids after cell activation and has many potent biologic activities (see Chapter 14 ). 1

Activation of mast cells and basophils also results in an increase in messenger ribonucleic acid (mRNA) expression and the release of several cytokines. [ ] These factors include interleukin-1 (IL-1), IL-3, IL-4, IL-5, IL-6, IL-8, interferon gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF),

macrophage inflammatory protein, or peptide (MIP)-1α and MIP-1β, T cell activation gene 3, and tumor necrosis factor alpha (TNF-α). Thus, besides being inflammatory mediators, mast cells and basophils are also a source of factors that regulate the growth and function of other cells. Interestingly, the binding of IgE to FcepsilonRI with or without aggregation also results in the stimulation of mast cell proliferation, which could be caused by the autocrine activity of factors released 2 3 4

from these cells. [ ] [ ] [ ] This chapter describes the current knowledge of the secretion of the pre-formed and newly generated mediators. Although most of the early steps in the activation of cells for the generation of different classes of mediators are similar, differences probably exist in the regulation of the later steps. Because basophils and mast cells are sparsely distributed in blood or tissues, progress in the understanding of the biology of these cells can be largely attributed to the development of techniques to obtain large numbers of these cells. The earliest experiments with human materials used peripheral blood leukocytes or organ fragments in which only a small fraction of the cells were basophils or mast cells. Methods were then developed to purify basophils or mast cells from either 5] [6]

peripheral blood or lung fragments. Even with these techniques, however, the number of cells obtained is small, and few biochemical studies can be performed.[ [7]

The development of cell culture systems has been very important for basophil/mast cell studies. Rat basophilic leukemia (RBL-2H3) cells can be activated for 8 9

histamine release and therefore have been used extensively in biochemical studies to characterize the molecules important for secretion.[ ] [ ] Cell lines have also been established from a human basophilic leukemia patient and from human mast cell tumors. However, these lines cannot be activated for FcepsilonRI-mediated secretion. Unlike these cell lines, which were established from tumors or after viral transformation, the use of growth factors has allowed the growth of untransformed mouse and human basophils and mast cells. The culture of mouse bone marrow

244

10]

or spleen cells with IL-3 stimulates pluripotential stem cells to grow and differentiate into mast cells.[ basophils have been grown from cord blood or bone marrow by more complicated

11 12 procedures.[ ] [ ]

Using growth factors, cells similar to human mast cells or

Although these methods for culturing nontransformed cells are

more labor intensive and yield low numbers of cells, they have become useful for biochemical studies. [

13]

FCepsilonRI: THE HIGH-AFFINITY RECEPTOR FOR IMMUNOGLOBULIN E Many cells have membrane receptors that bind the Fc portion of immunoglobulin molecules. There are two types of receptors that bind IgE. The high-affinity IgE receptor (FcepsilonRI) present on mast cells and basophils is characterized by having a very high affinity for binding of monomeric IgE. Although originally thought to be present only on mast cells and basophils, recent evidence suggests that FcepsilonRI is also expressed on monocytes, eosinophils, peripheral blood dendritic 14

cells, and Langerhans' cells in the skin.[ ] In contrast, other cells, including lymphocytes, platelets, and macrophages, have receptors that bind IgE with much lower affinity and accordingly are called the low-affinity IgE receptor (FcepsilonRII). Although FcepsilonRII has striking homology to animal lectins such as the asialoglycoprotein receptor, it binds to the protein core of IgE, and its binding does not require the presence of carbohydrate on the molecule.[ Immunoglobulin E Receptor Binding

15]

The binding of IgE to its receptor on mast cells follows the kinetics of a simple reversible bimolecular reaction:

In such reactions the molar concentration of the IgE, R (receptor), and R-IgE determines the equilibrium constant. The forward rate constant (k1 ) is 105 M−1 · sec−1 . The reaction has a very small first-order reverse rate constant (k−1 ) of 10−5 · sec−1 . Thus the affinity (Ka ) of the receptor for IgE is high, about 1010 M−1 . The dissociation rate of IgE from cells is therefore very slow, with variable estimates of a half-life for cell-bound IgE of 20 to more than 100 hours. In a biologic setting, however, the effective half-life of IgE is probably even longer because the IgE that dissociates from cells is functionally normal and can rebind to the same or other cells: thus, it has been estimated that the half-life of IgE in the skin is 13 days. Specificity

The binding of IgE with its high-affinity receptor is not effectively inhibited by immunoglobulins of other classes. However, there is some binding with much lower affinity of some immunoglobulin G (IgG) classes to the FcepsilonRI; for example, rat IgGa binds and can activate the rat FcepsilonRI.[

17]

There is also a species

specificity of IgE binding to receptors. In general, IgE from one species binds only to the receptors on the cells from the same or closely related species; for example, human IgE binds to human and monkey basophils or mast cells but not to murine mast cells. An exception to this rule is the binding of mouse IgE to human FcepsilonRI.[

18]

However, human IgE does not bind to the rat FcepsilonRI.[

16]

Structure The high-affinity IgE receptor (FcepsilonRI) on basophils and mast cells is a multimeric complex consisting of one IgE-binding alpha (α) chain, one beta (β) chain, 19

and two disulfide-linked gamma (γ) chains[ ] ( Box 16-1 ). The different receptor subunits are not covalently linked but have both tightly bound and covalently linked lipids that may be important in their association and membrane insertion. FcepsilonRI present on other cells, such as monocytes or Langerhans' cells, appears to be a αγ2 complex lacking the β subunit. The IgE-binding component is the 45-kD to 60-kD α chain, which contains carbohydrate and is exposed on the outer surface of the cell. A leader sequence is important for membrane insertion of the molecule. The extracellular portion of 180 amino acids has two, about 40–amino acid domains (D1 and D2) that have similarity to the Ig gene superfamily,

Box 16-1. Characteristics of High-Affinity Immunoglobulin E (IgE) Receptor (FcepsilonRI) Subunits

Alpha (α) subunit

Extracellular portion with two Ig domains (D1, D2), one transmembrane domain, and a short cytoplasmic tail IgE-binding component: interaction of IgE with D2

Beta (β) subunit

Four transmembrane domains; both ends in cytoplasm Has an ITAM sequence, which is tyrosine-phosphorylated after FcepsilonRI aggregation Binds signaling molecules: • Quiescent cells: Lyn, protein kinase C-δ • Activated cells: primarily Syk, potential binding site for phospholipase C-γ1, Shc, SHIP, and SHP-2

Signal transducer or signal amplification component for receptor Gamma (γ) subunit

Covalently linked homodimers, each with short extracellular domain and one transmembrane domain Has ITAM sequence, which is tyrosine-phosphorylated after FcepsilonRI aggregation Binds Syk after tyrosine phosphorylation Signaling component

ITAM, Immunoreceptor tyrosine-based activation motif; SHIP, SH2-containing inositol phosphatase; SHP-2, SH2-containing protein tyrosine phosphatase-2.

245

and there are multiple sites for N-linked glycosylation. The IgE-binding sites reside exclusively in the second (D2), membrane-proximal Ig domain of the α subunit.

[20] [21]

The transmembrane region has a charged amino acid together with the hydrophobic residues. The carboxy terminals of the rat and human receptors are surprisingly different, with the human subunit longer by 12 extra amino acids. This strongly indicates that the cytoplasmic domain is probably not critical for signaling by the receptor. The β subunit does not contain carbohydrate. Although by gel analysis the protein is 33•kD, the complementary deoxyribonucleic acid (cDNA) suggests a protein with 243 amino acids. The sequence analysis reveals no leader sequence and four putative transmembrane regions. Thus, both the amino and carboxy terminal portions of the molecule are in the cytoplasm, with small parts of the molecule that are extracellular. Interestingly, the β component is much more conserved between 19]

the rat, mouse, and human than is the α subunit.[

The γ component is a homodimer approximately 15•kD in size. Analysis of the cDNA sequence of the rat, mouse, and human indicate that they have similarities both at the nucleotide and amino acid level (86% identical amino acids). A leader sequence is followed by a transmembrane segment and a mature protein of 68 residues (about 8•kD). The cleavage of the leader sequence leaves a five–amino acid, short extracellular segment. Of the two cysteine residues, the residue near the start of the transmembrane region is involved in the formation of dimers. Unlike the α and β subunits, the γ subunit is more widely distributed in other cells besides basophils and mast cells. It is present in macrophages and natural killer (NK) cells and associates with FcγR. Sequence analysis of the γ subunit suggests that it belongs to the same family as the ζ component of the T cell receptor. Both molecules are dimers and have analogous exon organization. In NK cells there are also γ-ζ heterodimers and γ-γ homodimers that associate with both the FcγRIII and with the T cell receptor. FcepsilonRI has been expressed by gene transfer in cells that do not normally have this receptor. The genes for all three subunits of the rat FcepsilonRI must be transferred together to produce cell surface expression of the α chain and IgE binding. By contrast, the cell surface expression of human α requires only the presence 22

23 24

of the γ subunit.[ ] None of the cytoplasmic domains of the chains is critical for surface expression or for internalization after aggregation.[ ] [ ] The receptor expressed in monkey kidney Cos 7 cells results in normal IgE binding, but it is incapable of activating most of the biochemical events because these cells lack some of the early critical signaling molecules.[

25]

In other experiments a chimeric human α was expressed without β or γ by using the extracellular domain of α fused to 26

the gene of another transmembrane protein.[ ] These cells bind human IgE normally, proving that β and γ are not necessary for this interaction. The human α chain has also been introduced into RBL-2H3 cells by transfection and is expressed on the cell surface, and it can induce secretion. These models have been used for 27] [28] [29]

studies of the structure-function relationship of the receptor and other related surface molecules that may be important for cell activation.[ IgE-FcepsilonRIα Interaction

Many studies have shown that IgE binds to its receptor through the Fc portion of the molecule. The 95-kD Fc fragment obtained by papain digestion of IgE contains the Cepsilon2, Cepsilon3, and Cepsilon4 portions of the molecule, is biologically active, and binds to the receptor; in contrast the F(ab′)2 fragment obtained by pepsin digestion and lacking in Cepsilon2, Cepsilon3, and Cepsilon4, is inactive. The reduction of disulfide bonds in the IgE molecule also will abolish its capacity to bind to FcepsilonRI. The use of monoclonal antibodies and molecular biologic techniques defined the parts of IgE that are important for binding to the receptor and 30 31

regions of IgE that are hidden in the receptor when IgE is bound to FcepsilonRI.[ ] [ ] IgE produced in bacteria is nonglycosylated but retains its capacity to bind with high affinity to FcepsilonRI, suggesting that the carbohydrate on the IgE is not critical for binding. Fragments of IgE as small as a 76 amino acids from the 15]

junction of the Cepsilon2 and Cepsilon3 domains inhibit IgE binding, as determined by passive sensitization of mast cells or basophils.[

Results from many

32]

experiments suggest that the Cepsilon3 domain is sufficient for binding to FcepsilonRI.[

Recent understanding of the interaction of IgE with FcepsilonRI has come from the determination of the crystal structure of IgE, FcepsilonRIα, and the complex of 33 34 35 36

two molecules.[ ] [ ] [ ] [ ] The D1 and D2 immunoglobulin-like domains of the α subunit are folded at an acute angle in respect to each other and therefore interact with each other over a broad interface. Both the amino terminal region of D1 and the carboxy terminal region of D2 domain are directed toward the cell membrane, and the exposed surface of the domains is available for interaction with IgE. Mutational studies of FcepsilonRIα have shown that these interactions are in the D2 region of the molecule. This finding was confirmed by the crystal structure of part of the Fc portion of IgE bound to FcepsilonRIα. The interaction between the receptor and IgE involved the IgE-Cepsilon3 region of the molecule that interacts with the D2 domain of the receptor. Both Cepsilon3 domains from the two chains of IgE bind to two different sites on the same D2 loop of the receptor. The crystal structure of the IgE Cepsilon3-Cepsilon4 domains in solution is different from that bound to the receptor; in solution, IgE-Fc has a more compact and closed configuration that places the two Cepsilon3 domains in proximity, thereby decreasing an interdomain cleft and blocking the site that is involved in interaction with the receptor. Structural and biophysical studies suggest that there is a 1:1 stoichiometry of IgE with FcepsilonRIα. The crystal structure supports observations that the Cepsilon4 portion of the IgE is not hidden in the receptor and is available for binding by antibodies.[ rapid reorientation

16]

Binding of IgE to the receptor restricts its segmental flexibility, although the antigen-binding Fab segments can undergo a

16 37 motion.[ ] [ ]

Distribution The FcepsilonRI are distributed diffusely over the surface of the cell without any significant pool of receptors in the cytoplasm. The monomeric IgE-receptor 16

complex is freely mobile in the plane of the membrane, with a diffusion coefficient similar to other membrane proteins.[ ] IgE receptors are univalent (i.e., one IgE bound per receptor) and independently mobile, although internalization studies suggest that some interaction occurs between different IgE receptors. Several proteins are close to the receptor and may interact with the receptor components.[ association of the receptor with protein tyrosine

27 40 kinases.[ ] [ ]

28] [38] [39]

Furthermore, specialized gangliosides on mast cells may play a role in the

Such molecules may be critical for receptor-mediated signaling.

246

The presence of monomeric IgE bound to the FcepsilonRI decreases the rate of receptor turnover.[ 103 and 106 per cell on different cell

16]

The estimate of the number of FcepsilonRI has varied between

18 41 42 preparations.[ ] [ ] [ ]

It was reported some time ago that the number of FcepsilonRI on basophils correlates directly with the serum concentration of IgE.[ 45 46 47 vivo.[ ] [ ] [ ]

43] [44]

Recent studies have found

that IgE appears to regulate the expression level of FcepsilonRI on basophils and mast cells both in vitro and in For example, the baseline expression of FcepsilonRI on basophils and mast cells is dramatically reduced in genetically IgE-deficient mice, and this can be up-regulated by the in vitro culture of the cells with IgE. Allergic patients treated with monoclonal anti-IgE show a dramatic decrease in the total serum IgE level and a greater than 90% decrease in FcepsilonRI

number of basophils. This reduction in FcepsilonRI expression could be an important mechanism for the efficacy in the use of anti-IgE therapy in allergic disease (see Chapter 56 ). Internalization and Recycling The addition of antigen or anti-IgE to basophils or mast cells results in the cross-linking of IgE molecules with the formation of aggregates, with progression into patching and capping at one pole of the cell. This aggregation of IgE results in the immobilization of the receptor, which is caused by the interaction of the receptors with cytoskeletal elements. Eventually, internalization and degradation of these complexes occur.[ common phenomenon of many aggregated cell surface receptors.

48]

Such internalization of aggregated surface receptors is a

In RBL-2H3 cells the aggregation of IgE with multivalent antigen results in rapid internalization of the complexes; the half-life for this reaction is 3 to 5 minutes.[ [49]

16]

23 24

The cytoplasmic domains of the three subunits of FcepsilonRI are not required for aggregation-induced internalization of FcepsilonRI.[ ] [ ] Even when the internalization reaction has gone to completion, most of the IgE is left on the cell surface. Larger IgE aggregates are more efficiently internalized than small IgE dimers. During endocytosis of IgE-antigen aggregates, there is co-internalization of receptors that have bound monomeric IgE. Internalization of FcepsilonRI can 50

also be induced by activation of protein kinase C (PKC), although this enzyme does not appear to be directly involved in aggregated receptor internalization.[ ] Unlike histamine release, internalization does not require extracellular Ca2+ (Ca++ ) and therefore it must be an event that precedes or is independent of secretion. Internalization of aggregated receptors occurs through clathrin-coated pits and probably does not play a role in signaling the cell for secretion. Dynamin is known to play a role in clathrin-mediated endocytosis and is rapidly dephosphorylated after receptor aggregation.[ signal for targeting the aggregated receptors to the endocytic pathway.

51]

This dephosphorylation of dynamin may provide the

Aggregation The initial event in the stimulation of the basophil or mast cell is the interaction of antigen with IgE that is already bound to FcepsilonRI on the cell surface. The antigen then bridges two IgE molecules to initiate a series of biochemical events that eventually result in the secretion of mediators from the cells. Therefore the optimal conditions for the release of histamine depend on the concentration of antigen-specific IgE antibody on the cell surface, the concentration of the antigen, and the affinity of the IgE for the antigen.[ individuals.

52] [53] [54] [55]

In vitro, as little as 1•ng of ragweed antigen E (2.5 × 10−11 M) can activate basophils from ragweed-allergic

56

Studies with defined-length bivalent haptens have shown that bridging of two IgE molecules initiates the cell-triggering signal.[ ] Small bivalent haptens with a different antigenic site at each end will activate cells, suggesting that the bridging is between adjacent IgE molecules on the cell surface. Different-length rigid bivalent haptens can activate RBL-2H3 cells most effectively when the molecules are separated by 80 to 240 Å, whereas there is a poor response when they are less 57] [58]

than 50 Å apart.[

59]

Monoclonal anti-FcepsilonRI antibodies activate cells to release, again demonstrating that receptor bridging is the critical event.[ [60]

there appear to be orientational constraints, and not all aggregates are equally effective in signaling for secretion. are active in triggering mast cells and human basophils, although dimers are much less effective than triggering signal is qualitatively different from stimulation by trimers or oligomers.[

64]

However,

Pre-formed, chemically cross-linked IgE dimers

61 62 63 trimers.[ ] [ ] [ ]

With human basophils the dimeric

Basophils from some donors respond to pre-formed IgE dimers and larger

65

oligomers, whereas basophils from other donors require at least trimers to release mediators. [ ] This difference in response correlates with the capacity of the cells to release histamine, a parameter that has been called releasability (see later section). Therefore, dimers are the unit signal for activating cells of only some donors. Once bound to the cell, dimers can coalesce to form larger aggregates because of intrinsic changes in the receptor or the interaction of the IgE-receptor complex with 66]

the cytoskeleton. [

Activation of basophils or mast cells requires the bridging of a few IgE molecules on the cell surface. Studies suggest that bridging of 100 or fewer IgE molecules will completely activate the cell for histamine release. Because mast cells or basophils have approximately 105 receptors per cell, it is obvious that bridging of less 52 53 63

than 1% of the total IgE molecules on the cell surface results in degranulation.[ ] [ ] [ ] In rat mast cells the addition of secretagogues immobilized on beads induces localized degranulation; therefore, full secretion from the cell requires the bridging of IgE molecules on different sectors of the cell surface. The IgE receptors are diffusely distributed on the cell surface. The binding of multivalent antigen with IgE on the cell surface results in the interaction of the 67

receptors with the cytoskeleton.[ ] Clusters, aggregates, and patches form, with capping of these into one pole of the cell. The addition of wheat germ agglutinin to cells results in binding to the α subunit of FcepsilonRI, the association of the receptor with the insoluble cytoskeleton, and a decrease in receptor-mediated signaling. [68]

Thus the ability of receptors to be freely mobile in the membrane is important for signal transduction.

Functional Studies The receptor subunits lack any known enzymatic activity, and therefore FcepsilonRI must rely on associated molecules for transducing intracellular signals. The extracellular domain of the α chain of FcepsilonRI binds IgE, whereas its relatively short cytoplasmic domain probably does not play a role in cell signaling.

247

In contrast, the COOH-terminal cytoplasmic domains of both the β and the γ subunits are important in FcepsilonRI-mediated signal transduction. The cytoplasmic domains of the β and the γ subunits of FcepsilonRI contain a sequence of amino acids that is important for signal transduction that is called immunoreceptor tyrosinebased activation motif (ITAM). The ITAM is as follows:

where X refers to any amino acid, and the tyrosine (Tyr) residues become phosphorylated after receptor aggregation. This motif is present in the β and γ chains of 69

FcepsilonRI, in the subunits of the T and B cell receptor complexes.[ ] The ligand-binding domain of the receptors on all these cells lacks intrinsic enzymatic activity but associates with transducing subunits that contain this cytoplasmic motif that is critical for cell activation. Chimeric proteins with the cytoplasmic domain of these ITAM-containing subunits of the T cell receptor or β or γ subunit of FcepsilonRI linked to the extracellular and transmembrane domains of other proteins have been used for signal transduction studies.[

69] [70] [71] [72]

Aggregation of these chimeric molecules by antibodies to their extracellular domains results in cell

activation. For FcepsilonRI the γ component appears to be the essential receptor subunit for signaling cells for secretion. As evidence of this, aggregation of chimeric molecules prepared with FcepsilonRIγ or the T cell antigen receptor-ζ (TCR-ζ), but not with FcepsilonRIβ, induces secretion in RBL-2H3 cells.[ studies in T cells, the critical signaling probably results from the ITAM contained in the cytoplasmic domain of the γ subunit.

72]

By analogy to

STIMULI FOR BASOPHIL OR MAST CELL SECRETION Many different stimuli can activate basophils and mast cells ( Box 16-2 ). Both types of cells respond to physiologically relevant stimulation through the IgE receptor. However, differences still appear to exist in their response to other secretagogues; for example, compound 48/80 causes histamine release from human cutaneous mast cells but not from human lung mast cells or basophils. [ to the same stimulus.

41]

There are also differences in the response of mast cells or basophils from different species

Box 16-2. Stimuli for Mast Cell or Basophil Activation FcepsilonRI mediated • Cell-bound IgE: antigen, anti-IgE, lectins (e.g., concanavalin A) • Anti-FcepsilonRI IgG receptor C5a receptor Lymphokines, cytokines, chemokine receptors Formyl-methionine peptide receptors Eosinophil granule major basic protein Other compounds: basic compounds (e.g., compound 48/80, morphine, substance P)

Immunoglobulin E Receptor Mediated

A characteristic of mast cells and basophils is the presence of FcepsilonRI on their cell surface. As noted previously, IgE binds through the Fc portion of the molecule, whereas the Fab fragments are available for interacting with antigen. Activation of the cell then results from the cross-linking of the IgE molecules by 56

59

antigen.[ ] Antibodies to the FcepsilonRI can activate cells in the absence of IgE.[ ] Therefore, IgE functions to confer antigenic specificity to the FcepsilonRI. The sera of some patients with chronic urticaria have anti-FcepsilonRIα antibodies that may play a role in their disease. Basophils and mast cells also can be activated to release by antibodies that react with the IgE on the cell surface (e.g., antibody that recognizes the immunoglobulin light or heavy chain). Such reactions could occur in vivo with autoimmune antibodies that react with IgE on cells. Mitogens such as concanavalin A or 73

phytohemagglutinin can cross-link cell surface IgE by binding to the carbohydrates on the IgE molecules. [ ] Protein A from Staphylococcus aureus also can react with IgE and release histamine. Similarly, viral superantigens are capable of activating basophils and mast cells by nonspecifically interacting with IgE on the cell surface.[

74]

Histamine-releasing factor (HRF) was originally described as a mononuclear cell–derived factor that reacts with a specific type of IgE on the basophil surface and 75

releases histamine.[ ] The early studies suggested that only the IgE from some donors (referred to as IgE+) was effective, which could be caused by differences in glycosylation. However, more recent studies suggest that the effect of this molecule, which has now been cloned, results from binding of an unidentified receptor 76] [77]

present on cells.[

Immunoglobulin G Receptor Mediated Basophils and mast cells besides FcepsilonRI have FcγRIIB and FcγRIII that binds IgG.[

78] [79]

The binding of IgG to these FcγR is of much lower affinity than the 78] [80]

binding of IgE to the FcepsilonRI. The IgG bound to the receptor easily dissociates from the cell surface unless it is in the form of antigen-IgG complexes.[ 81] [82]

Basophils or mast cells, especially from rodents, can be activated through the FcγRIII to degranulate,[

although this is a less efficient and requires much more 17

antibody than does the IgE receptor. Some subclasses of rodent IgG antibodies can also stimulate mast cells by binding to FcepsilonRI.[ ] There is some evidence to suggest that IgG receptor–mediated degranulation occurs from human mast cells. Recent experiments indicate that coaggregation of FcγRIIB with FcepsilonRI can generate negative signals that down-regulate the activation signals induced by FcepsilonRI (see discussion on inhibitory signals). Alloantisera directed toward the major histocompatibility complex (MHC) can trigger mast cells. In mouse cells these IgG alloantisera bind to the mast cell surface by their Fab and trigger the cell through the Fc portion of the molecule, probably binding to FcγRIII. Complement Mediated The two major complement-derived anaphylatoxins, C5a and C3a, can release mediators from mast cells and basophils.[ for C5a, the release by C3a may be similar to other basic molecules and not receptor

83] [84]

Whereas there is a distinct receptor

248

85

mediated.[ ] On a molar basis, C5a is much more potent than C3a in stimulating cells for histamine release. Both mast cells and basophils have C3b receptors, but these do not cause the activation of cells to release. With human basophils the activation of the C3b receptor results in enhancement of IgE-mediated histamine release. Cytokines Several HRFs have been identified in the supernatants of T and B lymphocytes, mononuclear cells, platelets, and human lung macrophages.[

86]

Some of these

76 surface.[ ]

molecules might release histamine by reacting with IgE on the cell Chemokines related to interleukin-8 (IL-8) release histamine from human basophils include the following, in order of decreasing potency: monocyte chemotactic protein (MCP)-1, MCP-3, RANTES (regulated on activation, normal T cells expressed 86 87

and secreted), MIP-1 α, and IL-8.[ ] [ ] These chemokines probably act through several different receptors that are members of the seven-transmembrane family that activate guanosine triphosphate (GTP)–binding proteins. A few cytokines, such as the interferons, bind to cell surface receptors and modulate IgE-mediated histamine release.[

88] [89]

Such modulation of mediator release

could be important at sites of inflammation and during viral infections. For example, human basophils have interleukin-3 (IL-3) receptors, [ 3 results in enhancement of histamine secretion both by IgE and non-receptor-mediated

89 90 91 92 mechanisms.[ ] [ ] [ ] [ ]

90]

and the addition of IL-

IL-3 at high concentrations can also induce

histamine release from the cells of some donors. Interleukin-5 (IL-5) and GM-CSF act similarly and have a common receptor subunit. [

93]

Also, the addition of

91 94 release.[ ] [ ]

95] [96]

granulocyte or macrophage CSF, IL-1α, and IL-1β enhances Some of these cytokines can release histamine directly at high concentrations.[ The cytokines also can change the response spectrum of the mediators released on stimulation; for example, basophils stimulated with C5a do not release leukotrienes unless the cells have been pretreated with IL-3.[

97]

Similarly, platelet-activating factor (PAF) induces histamine release only after such priming.

Other Compounds Formyl-methionine–containing tripeptides are chemotactic for some cells and have similarities to chemotactic factors produced by bacteria. They bind to specific 98] [99]

receptors of the seven-transmembrane family and induce histamine release from human basophils.[ characteristics of piecemeal degranulation.

The morphology of this release has some of the

[100]

Approximately half the eosinophil granule protein consists of the arginine-rich major basic protein (MBP). MBP damages cells and activates basophils and mast cells for noncytotoxic histamine release.[

101]

Many other compounds activate mast cells or basophils for histamine release. Ionophores are lipophilic compounds that insert themselves into cell membranes and transport ions across the membrane. The Ca2+ ionophore A23187 triggers mediator release from basophils or mast cells in the presence of Ca2+ in the medium. Other

compounds that are of experimental interest are tumor promoters, for example, phorbol myristic acid, which releases histamine from human basophils without a 102 103

requirement for extracellular calcium.[ ] [ ] This compound binds and activates PKC (see discussion on the role of PtdIns in the release pathway). Other secretagogues include polycationic compounds such as polymyxin B, compound 48/80, polylysine, and polyarginine. Other compounds that cause mediator release are neuropeptides such as substance P, luteinizing hormone–releasing hormone (LHRH), morphine, codeine, tubocurarine, adenosine triphosphate (ATP), dextran, mellitin, chymotrypsin, and proteins released from neutrophils.[ various agents (see Chapters 20 and 22 ).

104]

Human basophils and subtypes of mast cells differ considerably in responsiveness to these

MODEL FOR BASOPHIL OR MAST CELL ACTIVATION This section discusses some general principles of the phosphorylation of proteins in signaling, the present model of signaling events in mast cells, and the changes in FcepsilonRI. These biochemical events and the molecules involved are then discussed in detail. Phosphorylation of Proteins in Signaling In many biologic systems, extracellular ligands bind to cell surface receptors and activate protein kinase enzymes, which then phosphorylate cellular proteins. Such phosphorylation of a protein, by resulting in conformational changes, can regulate enzymatic activity and also modify its interaction with other proteins. The phosphorylated proteins are then dephosphorylated by phosphatases, which limit the effect of the phosphorylated proteins. Such phosphorylation-dephosphorylation of proteins regulates many intracellular processes. 105 106

An important concept is that defined domains in proteins are critical for protein-protein interactions.[ ] [ ] Proteins are made up of modular units that belong to families of related structures. These modules allow for protein-protein interactions by interacting with other modules. These protein domains form compact units that maintain their structure in isolation and occur in many enzymes, including protein tyrosine kinases, protein phosphatases, and phospholipases ( Table 16-1 ). They are also present on adapter proteins, which have no enzymatic function but act to aggregate other proteins. A protein may have several different types of these modules, with or without having any enzymatic activity. The specificity of the interaction of these domains regulates the interaction of proteins and therefore the specificity of the signaling that occurs in the cell. These modules allow for the amplification of the signals at different steps after receptor aggregation. These modules also are critical for the localization and translocation of a signaling protein within a cell that affects the molecule with which that protein can interact. The translocation of a signaling protein to a different location within the cell could result in proximity to a different enzyme, which then could result in its phosphorylation and activation. The src homology-2 (SH2) domain is an approximately 100–amino acid residue motif that is present in many protein tyrosine kinases and other molecules. SH2 domains mediate the interaction with specific phosphorylated tyrosine residues in other proteins. The three-dimensional structures of SH2 domains of different proteins are very similar, and each binds to a distinct sequence of amino acids surrounding a phosphotyrosine residue. The three amino acids immediately C-terminal to the phosphotyrosine are critical for the specificity of

249

TABLE 16-1 -- Noncatalytic Conserved Domains in Molecular Interactions Domain

Interaction Partner

Characteristics

SH2

Phosphorylated tyrosine residues

Sequence of ∼100 amino acids Binding specificity determined mostly by three amino acids N-terminal to the phosphotyrosine

PTB

Phosphorylated tyrosine residues

Sequence of ∼200 amino acids Binding specificity determined by three amino acids COOH-terminal of the phosphotyrosine

SH3

Proline-rich sequence

Sequence of ∼60 amino acids

PH

Polyphosphoinositides

Sequence of ∼100 amino acids

binding. The src homology-3 (SH3) domain, in contrast, is slightly smaller (approximately 55 to 70 residues) but is present in a large number of proteins. SH3 selectively binds to proline-rich sequences in other proteins, with different SH3 domains binding to different proline-rich sequences. The phosphorylated tyrosinebinding (PTB) domain is present in adapter molecules (e.g., Shc) and interacts with proteins that are tyrosine-phosphorylated. Its binding specificity is determined by residues N-terminal to the phosphorylated tyrosine. The pleckstrin homology (PH) domain is found on many proteins, including protein Tyr and Ser/Thr kinases. PH was originally described in pleckstrin, a substrate of PKC, and is thought to be involved in binding of proteins to phosphoinositide lipids so that PH functions in bringing molecules to the membrane. The SH3 and PH domains are present on cytoskeletal proteins, where they may play a role in regulating cell shape. The initial observation in mast cells was that aggregation of the FcepsilonRI results in rapid protein tyrosine phosphorylations, the most prominent being a 72-kD protein.[

107]

This quickly led to the recognition that protein tyrosine kinases are critical for this signal transduction pathway.[

108] [109] [110] [111]

Sequence of Events In the IgE-mediated reaction, mast cells are stimulated to secrete mediators by antigen reacting with IgE molecules bound to FcepsilonRI on the cell surface ( Figure 16-1 and Box 16-3 ). Optimal conditions for the reaction depend on the concentration of IgE on the cell surface, the concentration of the antigen, and the affinity of the IgE for the antigen. The cross-linking by antigen of two IgE molecules on the cell surface initiates the cell-triggering signal. The aggregation of a very small fraction of the total surface IgE is required for cell activation, most likely with cross-linking of less than 100•IgE molecules representing 1% of the total receptors on the cell surface. The release of histamine and other mediators is a rapid process that is complete in less than 30 minutes. Mediator release is a secretory (exocytotic) event that is noncytotoxic and does not require serum. However, there is an absolute requirement for the presence of Ca2+ in the medium for the IgE receptor– mediated release reactions. The release reaction is temperature dependent, with optimal release at 37°C, except for skin mast cells, where release is optimal at 30°C.

Figure 16-1 Molecules involved in the early FcepsilonRI-mediated signaling pathways. Arrows indicate both the interaction of molecules and the products of these reactions. Arrows do not always indicate a direct interaction but may include intervening steps. (See Boxes 16-4 , 16-5 , 16-6 , 16-7 , 16-8 , 16-9 , and 16-10 .)

Box 16-3. Biochemical Events during IgE-Mediated Basophil or Mast Cell Secretion IgE binding to receptors on the cell surface Multivalent antigen interacting with cell bound IgE Aggregation of the IgE-FcepsilonRI complex Lyn protein tyrosine kinase phosphorylation of Tyr residues of ITAM of β and γ subunits of FcepsilonRI Recruitment to FcepsilonRI of protein tyrosine kinase Syk resulting in its activation Tyrosine phosphorylation of substrates (e.g., PLCγ, LAT, SLP-76, PI3K, Btk) Activation of PI3K, which generates PIP3 that recruits PLCγ and Btk to

the membrane, activates Akt Hydrolysis of PtdIns by PLCγ; generation of IP3 and DG • IP3 binds to IP3 receptors and releases Ca21 from intracellular sources • DG activates protein kinase C (PKC) Influx of extracellular Ca21 Phosphorylation of proteins on Ser and Thr residues by PKC Activation of Ras pathway to MAP kinase Activation of phospholipases A2 and D Release of arachidonic acid Activation of other enzymes (e.g., FAK, Pyk2 tyrosine kinase) Changes in cytoskeleton Morphologic changes Fusion of granular membrane to plasma membrane Opening of granule to extracellular space Release of granular contents

Protein tyrosine phosphorylation is an early and critical signal for FcepsilonRI-induced degranulation. The FcepsilonRI subunits lack any known enzymatic activity, and thus this receptor depends on associated molecules for signaling. The extracellular domain of the α chain of FcepsilonRI binds IgE, whereas signal transduction in the cell is mediated by the associated β and γ subunits. In the plasma membrane, FcepsilonRI associates predominantly, through its β subunit, with Lyn, a member of the src family of nonreceptor tyrosine kinases. Aggregation of the receptors results in the phosphorylation of the tyrosine residues in the ITAMs of both the β and γ subunits of the FcepsilonRI, probably by Lyn or another src-family protein tyrosine kinase (PTK). The two tyrosines in both the ITAM of the β and γ subunits that are phosphorylated then function as scaffolds for binding of additional cytoplasmic signaling molecules that have SH2 domains. There is increased Lyn binding to the β subunits, whereas the tyrosine phosphorylation of the γ subunit appears to be essential for propagating the signal for degranulation. The phosphorylated ITAMs recruit the cytoplasmic tyrosine kinase Syk, which binds mainly to the γ subunit of the receptor by its two SH2 domains. This results in the activation of Syk and its autophosphorylation and in propagation of downstream signaling events.

The activated Syk then either directly or indirectly tyrosine-phosphorylates a number of molecules, including the linker for activation of T cells (LAT), SH2containing leukocyte protein-76 (SLP-76), Vav (p95vav ), phospholipase C-γ1 (PLC-γ1), and PLC-γ2. Receptor aggregation also results in the activation of phosphatidylinositol 3-kinase (PI3K), which results in the formation of phosphatidylinositol 3,4,5-phosphate (PIP3). This recruits the tyrosine-phosphorylated PLCγ1 and PLC-γ2 to the membrane, which catalyze the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), resulting in the generation of inositol 1,4,5trisphosphate (IP3) and 1,2-diacylglycerol (DG). These second messengers release Ca2+ from internal stores and activate PKC, respectively. Both events are essential for FcepsilonRI-mediated secretion. After FcepsilonRI aggregation, free cytosolic intracellular calcium concentration ([Ca++ ]i ) increases because of the mobilization of Ca2+ resulting from the binding of IP3 to specific intracellular receptors. This initial rise in [Ca2+ ]i is followed by the sustained influx of Ca2+ from the extracellular medium. The increase in [Ca2+ ] i

regulates Ca2+ /calmodulins dependent events. One of these downstream enzymes is calcineurin, a calcium/calmodulin-dependent serine phosphatase.

Aggregation of FcepsilonRI also activates other cytoplasmic PTKs, including the Tec family of kinases. Bruton tyrosine kinase (Btk), for example, associates with and is phosphorylated by PKC. Btk plays an important role in the sustained influx of Ca2+ . Another family is the PTKs activated by adhesion, including focal adhesion kinase (FAK, pp125FAK ) and Pyk2. Both are tyrosine-phosphorylated after receptor aggregation and may play a role in the late steps of degranulation. The aggregation of FcepsilonRI also induces the tyrosine phosphorylation and activation of many other molecules, including Vav and SLP-76, which play a role in the generation of signals for Ca2+ influx. The early tyrosine phosphorylation events activate the small guanosine triphosphatases (GTPases) such as Rac, Ras, and Rho, resulting in the stimulation of the extracellular signal–regulating kinase (ERK), c-Jun NH2 -terminal kinase (JNK), and p38 mitogen-activated protein (MAP) kinase pathways. Other proteins in these pathways include the adapter protein Shc, which recruits Grb2, which in turn recruits SOS (the mammalian homologue of the Drosophila “son of sevenless”). SOS is constitutively associated with Grb2 in a complex that is cytosolic but becomes membrane associated after cell activation. SOS is a guanine nucleotide exchange factor that activates Ras to the GTP form by the promotion of guanine nucleotide exchange. Vav also may have similar guanine nucleotide exchange factor activity. Activation of Ras by the dissociation of GDP and the binding of GTP results in stimulation of downstream kinases. Ras is deactivated by GTPase-activating proteins (GAPs), which results in the hydrolysis of GTP. In mast cells, Ras activates the Raf pathway. Activated Ras binds Raf, a cytosolic serine/threonine kinase, and recruits it to the plasma membrane, resulting in its activation. Raf then activates MAP kinase kinase (MAPKK) by phosphorylating it on serine residues. The MAPKKs then activate MAP kinases by phosphorylating them on both tyrosine and threonine residues. The substrates of the MAP kinases include transcription factors such as c-Jun, c-Fos, and cytoplasmic phospholipase A2 (cPLA2 ). Phosphorylation of the transcription factors of the c-jun and c-fos families allows their nuclear migration and the induction of new genes. The Ras pathway thus leads to the release of AA and nuclear events such as

251

the induction of cytokine genes. The other two ras-related small GTPases, Rho and Rac, also appear to play a role in secretion.

The PKC family of enzymes phosphorylates proteins on serine or threonine residues and is essential transducers of signals for secretion. RBL-2H3 cells contain the α, β, δ, epsilon, and ζ isoforms of PKC. After FcepsilonRI aggregation, PKC translocates to the membrane fractions. The β or δ isozymes of PKC isozymes are required for optimal FcepsilonRI-mediated secretion, whereas the α and epsilon isozymes inhibit the receptor-induced hydrolysis of inositol phospholipids. Activated PKC phosphorylates several proteins, including myosin light chain and the γ subunit of FcepsilonRI. A number of morphologic changes occur during mast cell secretion. The bridging of receptors transforms the microvillous surface of basophils to a plicated appearance; the cells spread and become more adherent. These changes are secondary to the rise in [Ca2+ ]i , phosphorylation of cellular proteins, and activation of GTP-binding proteins and are probably related to the cytoskeletal reorganization. The cytoskeleton is also involved in the large-scale clustering and capping of aggregated FcepsilonRI. The degranulation of human mast cells results in swelling of the individual granules, a change in the electron-dense granular contents, and the formation of interconnected granules with the granule closest to the cell surface fusing with the plasma membrane and thus open to the extracellular medium. The activation of phospholipase A2 (PLA2 ) in stimulated cells results in the release of AA from cellular phospholipids. The activity of cytosolic PLA2 is regulated by phosphorylation by MAP kinase and PKC. The AA released is metabolized by either the cyclooxygenase (COX) or the lipoxygenase (LO) pathways. The products of the LO pathway are the leukotrienes (LTC4 , LTD4 , LTE4 , LTB4 ), whereas the COX pathway results in the formation of prostaglandin D2 (PGD2 ). Some thromboxane A2 (TXA2 ) and prostacyclin also are formed. FcepsilonRI Tyrosine Phosphorylation and Membrane Rafts 112 113

Aggregation of FcepsilonRI in RBL-2H3 cells results in the rapid tyrosine phosphorylation of the β and of the γ subunits of FcepsilonRI. [ ] [ ] Interestingly, only the β and γ subunits that are aggregated are phosphorylated on tyrosine. Mutations of the β subunit in which Tyr residues in the ITAM sequence are replaced with 72

phenylalanine (Phe) greatly decrease the in vivo and in vitro tyrosine phosphorylation of the γ subunit.[ ] This finding suggests that the tyrosine phosphorylation of the β subunit, presumably by Lyn, plays an important role in the tyrosine phosphorylation of FcepsilonRIγ. Furthermore, the tyrosine-phosphorylated ITAM of FcepsilonRIβ may promote the further binding of either the same or a different PTK that subsequently phosphorylates the γ subunit. The tyrosine phosphorylation of the β and γ subunits by the aggregation of FcepsilonRI renders both subunits accessible for interacting with SH2-containing molecules and allows the further propagation of the receptor-mediated signals.[

114]

Many observations suggest an important role for the tyrosine phosphorylation of the FcepsilonRIβ and FcepsilonRIγ in receptor-mediated signaling and secretion. First, tyrosine phosphorylation of both subunits is rapid. Second, when the Tyr residues in the ITAM motif of β are replaced with Phe, not only is the FcepsilonRIinduced tyrosine phosphorylation of β blocked, but tyrosine phosphorylation of FcepsilonRIγ and other intracellular proteins is decreased as well. Third, SH2 domains of Syk or Lyn bind to tyrosine-phosphorylated β and γ subunits. Fourth, aggregating the extracellular domain of chimeric proteins containing the cytoplasmic domain of the γ subunit induces signaling and secretion in RBL-2H3 cells, although it is unclear whether this chimeric molecule is itself tyrosinephosphorylated. Fifth, deletion of the C-terminal cytoplasmic tail of γ that contains an ITAM motif abrogates activation responses. Cell membranes often contain domains whose compositions differ from the rest of the membrane. These domains, called lipid rafts or glycolipid-enriched

microdomains (GEMs), are rich in sphingolipids, cholesterol, and certain membrane proteins that have a hydrophobic glycosylphosphatidylinositol anchor or an acyl chain. Important signaling molecules present in the lipid rafts include Lyn and the adapter molecule LAT. The co-localization of signaling molecules in rafts could provide a physical environment rich in kinases, adapter molecules, and intracellular effectors, thereby facilitating signaling. For example, the disruption of lipid rafts by cholesterol depletion results in inhibition of mast cell activation. FcepsilonRI appears to be predominantly located outside these membrane rafts in unstimulated cells. After antigen stimulation, some of the receptor translocates into the lipid rafts by a process that probably requires the activity of Lyn and cytoskeletal reorganization. Once within the rafts, FcepsilonRI would interact more efficiently with Lyn that is in the rafts, thereby resulting in ITAM tyrosine phosphorylation. This phosphorylation then recruits and activates Syk, through binding of the tandem SH2 domains to doubly phosphorylated ITAMs. The LAT that is also present in these rafts would be tyrosine-phosphorylated by Syk and then would probably recruit other molecules, such as PLC-γ, Vav, SLP-76, and PI3K. However, receptor phosphorylation may also occur outside these lipid rafts. [

115]

MOLECULES AND EVENTS IN CELL SIGNALING Protein Tyrosine Kinases Src Family

The src family of kinases is associated with the inner surface of the plasma membrane and appears to function in signal transduction, especially of transmembrane receptors that lack intrinsic enzymatic activity ( Box 16-4 ). These kinases contain a region that is unique for each member of the family, followed by SH3, SH2, kinase, and COOH-terminal regulatory domains. The enzymatic activity of these kinases is tightly regulated by intramolecular interactions. The COOH-terminal regulatory domain has tyrosine phosphorylation sites that can be phosphorylated by a specific tyrosine kinase, Csk or Ctk. The tyrosine phosphorylation of these regulatory sites by Csk results in intramolecular interactions with this regulatory region binding with the SH2 domain and the SH3 domain interacting with the linker region between the SH2 and kinase regions of the molecule. This results in suppression of enzymatic activity. Conversely, the dephosphorylation of the regulatory Tyr by a specific tyrosine phosphatase or the interaction of the SH3 domain with proline-rich regions of other molecules results in activation.

252

Box 16-4. Protein Tyrosine Kinases in Basophils and Mast Cells

Lyn

Member of Src family Unique, SH3, SH2, and regulatory domains Associated with plasma membrane and constitutively with β subunit of FcepsilonRI Increased association with the receptor after cell activation Mast cells from Lyn knockout mice signal normally for degranulation.

Src

Similar to Lyn in structure; also associated with plasma membrane Although not associated with FcepsilonRI, Src activated after receptor aggregation

Syk

Cytosolic protein, member of Syk/ZAP-70 family Binds by its two tandem SH2 domains, to ITAM of γ and β subunits of FcepsilonRI, which results in activation and tyrosine phosphorylation Essential for mast cell secretion

Btk

Cytosolic kinase member of the Tec family Has proline-rich region and SH3, SH2, and PH domains Tyrosinephosphorylated and activated after FcepsilonRI aggregation Translocates to plasma membrane and interacts with protein kinase C

Important for sustained influx of Ca2+ Itk

Another member of Tec family Phosphorylated on Tyr, Thr, and Ser after FcepsilonRI aggregation Associates with protein kinase C

FAK

Focal adhesion kinase; is cytosolic Activated and tyrosinephosphorylated by both integrin and FcepsilonRI aggregation Associates with other signaling molecules

Pyk2

Another member of FAK family Tyrosinephosphorylated by FcepsilonRI aggregation

Csk, Ctk

Contain SH2 and SH3 domains

Negatively regulate Src family kinases by phosphorylating regulatory tyrosine Csk function regulated by Cbp (see Box 16-7 )

Lyn.

The PTK Lyn has both myristate and palmitate fatty acids attached near its amino-terminal region, which results in its localization in membrane rafts ( Box 16-4 ). In nonstimulated cells, only a small fraction of FcepsilonRI receptors are associated with Lyn, but there is approximately a fourfold increase in associated Lyn after receptor aggregation. [

40] [116] [117] [118]

In nonactivated cells, this interaction is between the unique N-terminal region of Lyn and the C-terminal cytoplasmic 119

domain of the β chain, but not with the cytoplasmic domain of the other subunits of FcepsilonRI.[ ] When the receptors are aggregated, the Lyn associated with one receptor phosphorylates the subunits of the other receptor, with which it is now in close contact. This transphosphorylation reaction is similar to that described for PTK receptors. Once the tyrosines in the ITAM of the receptor subunits are phosphorylated, the SH2 domain of Lyn binds to tyrosine-phosphorylated β subunit, but 114

not the γ subunit of FcepsilonRI, which results in more Lyn association with the receptor.[ ] Although FcepsilonRI aggregation induces rapid tyrosine phosphorylation of Lyn and an increase in its kinase activity, no direct evidence indicates that this kinase is responsible for tyrosine phosphorylation of the β or γ subunits. Bone marrow–derived mast cells from Lyn-deficient mice still degranulate normally, although there is decrease in the FcepsilonRI-induced protein tyrosine phosphorylations and Ca2+ mobilization.[

120]

This finding suggests that other src-family kinases, such as c-Src, may functionally substitute for Lyn.

Src.

The aggregation of FcepsilonRI also results in the rapid tyrosine phosphorylation of pp60c–src , a tyrosine kinase that, as with Lyn, has SH2 and SH3 domains and is associated with the plasma membrane because of myristoylation. Tyrosine phosphorylation of pp60c–src is apparent within 30 seconds after FcepsilonRI aggregation and correlates with the increase in its tyrosine kinase activity. Unlike Lyn, however, this kinase does not associate with FcepsilonRI and is not in the membrane rafts. Syk Family 121 122

Syk and zeta-chain-associated protein-70 (ZAP-70) comprise a family of protein tyrosine kinases expressed in most hematopoietic cells.[ ] [ ] Syk is present in B cells, mast cells, immature T cells, and platelets, whereas ZAP-70 is expressed predominantly in T cells, NK cells, and possibly human basophils. These two kinases are characterized by a domain structure consisting of two tandem SH2 domains in the N-terminal half of the molecule separated by a linker region from a C-terminal

123

kinase domain. They contain many tyrosine residues that are phosphorylated after activation.[ ] These phosphorylations regulate their activity and also provide binding sites for the SH2 domain of other molecules. Among these phosphorylation sites are the two adjacent tyrosines in the activation loop that are important for regulating enzymatic activity. [ importance.

124] [125]

Syk plays a major role in phosphorylating its activation loop tyrosines, whereas other kinases, such as Lyn, are of lesser

110 126 127

Aggregation of FcepsilonRI results in tyrosine phosphorylation of Syk and its association with FcepsilonRI.[ ] [ ] [ ] Although some Syk may be associated with the receptor in the nonstimulated state, the predominant interaction is with receptors that are aggregated and therefore tyrosine-phosphorylated. The SH2 domains of Syk bind strongly to the tyrosine-phosphorylated (but not to nonphosphorylated) γ subunit and less efficiently to the β subunit of FcepsilonRI.[

114] [127]

[128]

This binding requires the two SH2 domains of Syk, suggesting that the binding occurs when the two tandem SH2 domains form a bidentate association with the two phosphorylated tyrosines of the same ITAM. The N-terminal SH2 domain binds to the C-terminal pYxxL of the ITAM, whereas the C-terminal SH2 domain binds to the N-terminal pYxxL. The binding of the diphosphorylated γ-subunit ITAM to Syk induces a conformational change and increase in its kinase activity.[ 110] [127] [128]

Syk is a cytosolic protein, a very small fraction of which is recruited to the FcepsilonRI in the membrane after receptor aggregation.[ some Syk is detectable in the membrane rafts, although membrane localization of Syk does not make it more efficient for

129]

In activated cells,

130 131 signaling.[ ] [ ]

The linker region of Syk, located between the second SH2 and the kinase domain, plays an important role in regulating

253

its function. An alternatively spliced variant of Syk, termed SykB, lacks a 23–amino acid sequence in the linker domain. Both full length and this shorter form of Syk are present in mast cells. Unlike Syk, SykB is inefficient at coupling stimulation of FcepsilonRI to the early and late events of cellular activation because of its 132

reduced capacity to bind phosphorylated ITAM.[ ] The linker region also has three tyrosines that once phosphorylated are postulated to be binding sites of molecules such as the negative regulator Cbl and the signaling molecules PLC-γ1 and Vav. The in vivo tyrosine phosphorylation of Syk is rapid and parallels the in vitro increase in its kinase activity. Chimeric proteins with the cytoplasmic domain of β or FcepsilonRIγ fused to the extracellular and transmembrane domains of other proteins have been used for signal transduction studies. Antibodies to their extracellular domains then aggregate the chimeric molecules. In such experiments the cytoplasmic domains of FcepsilonRIγ but not FcepsilonRIβ result in tyrosine phosphorylation of Syk and its activation, although the chimeric molecules that contain β are not themselves tyrosine-phosphorylated. This suggests that the γ subunit is critical for the activation of Syk and the propagation of signals that result in degranulation. 133 134

Syk is essential for signaling from FcepsilonRI, as demonstrated by Syk-deficient mast cells not degranulating after FcepsilonRI aggregation.[ ] [ ] The transfection of Syk into these cells restores the secretory function. Studies in these cells have shown that the FcepsilonRI-induced tyrosine phosphorylation of the following proteins is independent of Syk: FcepsilonRI β and γ subunits, SH2 domain–containing inositol (phosphate polyphosphate) 5-phosphatase (SHIP), and SH2-

135 136

containing protein tyrosine phosphatase-1 (SHP-1) and SHP-2.[ ] [ ] In contrast, the FcepsilonRI-induced tyrosine phosphorylation of a large number of proteins is downstream of Syk; these include PLC-γ1, PLC-γ2, LAT, Vav, Shc, MAP kinase, JNK kinase, FAK, Pyk2, paxillin, HS-1, platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), Cbl, rasGAP, SLP-76, and HS-1. These results do not necessarily imply that there is direct phosphorylation of these proteins by Syk, although some may be substrates of Syk. Tec Family Tyrosine Kinases

Bruton tyrosine kinase (Btk) and Itk/Emt are members of the Tec family of tyrosine kinases present in mast cells. Btk is also present in other hematopoietic cells, and mutations in this protein result in immunodeficiency. Btk is a 77-kD cytosolic protein that contains a unique Tec homology domain that also contains a proline-rich region and SH2, SH3, and PH domains. Both Btk and Itk are rapidly tyrosine-phosphorylated after aggregation of the FcepsilonRI.[

137] [138]

The activation of Btk by

139 140 phosphorylation.[ ] [ ]

FcepsilonRI aggregation is downstream of Syk and requires two steps: membrane localization followed by tyrosine Tec-family kinases like Btk are recruited to the membrane, especially to the raft microdomains, by the interaction between their PH domain and PIP3 that is generated by the activation of PI3K. Src-family tyrosine kinases such as Lyn then phosphorylate the membrane-recruited Btk, leading to its activation, followed by intramolecular autophosphorylation. Such interactions of Btk are responsible for the sustained phase of Ca2+ influx from outside of the cells, which is downstream of the activation 141] [142]

of PLC-γ.[

FcepsilonRI aggregation of mast cells from Btk-deficient mice results in only mild if any changes in release of histamine, but does result in 143] [144]

defective activation of the JNK pathway, which may have a role in the production of cytokines.[ membrane translocation and enzymatic activity.[

139] [145]

Btk associates with the βI isoform of PKC and regulates its

PKC-βI plays a role in regulating the JNK pathway, leading to the transcriptional activation of the IL-2 and

TNF-α genes. Btk is also a substrate for PKC, and PKC-mediated phosphorylation of Btk on Ser residues results in the down-regulation of Btk's enzymatic activity, 146

decrease in membrane association, and decrease in mast cell secretion.[ ] FcepsilonRI aggregation in PKC-depleted cells results in enhanced tyrosine phosphorylation of Btk, suggesting that PKC may modulate FcepsilonRI-induced phosphorylation of Btk. Focal Adhesion Kinase and Pyk2

FAK and Pyk2 are nonreceptor protein tyrosine kinases that accumulate at focal adhesion sites, also called adhesion plaques, which are areas where the plasma membrane of cells is attached to the extracellular matrix.[

147]

FAK and Pyk2 link signals from cell surface adhesion receptors such as integrins to downstream

[147] [148]

events. These kinases have a central catalytic domain, an amino-terminal region, and a carboxyl-terminal noncatalytic region. These cytosolic proteins lack SH2 and SH3 domains and membrane localization signals. FAK appears to function as an integration site for extracellular signals resulting from cell adhesion and from receptors such as FcepsilonRI. Although FAK does not have SH2 or SH3 domains, it interacts with many molecules. Thus the autophosphorylation of FAK 147] [148]

creates a high-affinity site for the binding of the SH2 domains of src-family kinases such as Src, Fyn, and Lyn.[

This binding of src-family kinases to FAK 147 148 149

leads to tyrosine phosphorylation of FAK and other proteins (e.g., paxillin, tensin, p130cas ) that accumulate with FAK at focal adhesion sites.[ ] [ ] [ ] FAK and Pyk2 also interact with several signaling molecules, including paxillin, Cas, the p85 subunit of PI3K, Grb2, Crk, Csk, and a 77-kD protein that is prominent in mast cells.[

150]

The aggregation of FcepsilonRI results in tyrosine phosphorylation of FAK and Pyk2 in RBL-2H3 cells.[

151] [152]

This receptor-induced phosphorylation of FAK and

151 153

Pyk2 is dramatically enhanced by adherence of the cells to fibronectin and this adherence also results in enhanced secretion.[ ] [ ] Therefore, adherence may regulate the extent of mast cell degranulation by modulating intracellular protein tyrosine phosphorylations. The receptor-induced tyrosine phosphorylation and activation of FAK and Pyk2 is downstream of Syk and of the rise in intracellular Ca2+ . Sites on FAK that are phosphorylated by adherence and in receptorstimulated cells are similar.[

154]

The protein tyrosine kinase FAK may play an important role during the late stages of signaling leading to degranulation. A monoclonal antibody (mAb) to an 40 155

FcepsilonRI-associated ganglioside induces tyrosine phosphorylation of Lyn, PLC-γ1, Syk, and the β and γ subunits of FcepsilonRI, but not of FAK[ ] [ ] This mAb does not induce significant degranulation, however, suggesting a role for FAK tyrosine phosphorylation in secretion from mast cells. Further evidence for an 156

important function of FAK in degranulation are studies with a FAK-deficient variant of the RBL-2H3 cells.[ ] FcepsilonRI-mediated, but not ionophore-mediated, secretion is defective in these variant cells with decreased levels of FAK. The stable transfection of FAK greatly enhances the FcepsilonRI-mediated secretion. This enhancement does not require

254

the catalytic activity or the autophosphorylation site of FAK, suggesting that FAK is functioning as an adapter or linker molecule. In fact, the NH2 domain of FAK, 154

lacking the enzymatic and COOH-terminal regions, is sufficient to reconstitute secretion. [ ] However, the catalytically inactive FAK and truncated forms of FAK expressed in cells are still tyrosine-phosphorylated after FcepsilonRI aggregation. Therefore, tyrosine-phosphorylated FAK may function as an adapter or linker molecule to transduce FcepsilonRI-initiated signals. Carboxy-Terminal Src Kinases

Carboxy-terminal Src tyrosine kinases phosphorylate the regulatory carboxy-terminal tyrosine in Src-family kinases such as Lyn, which then results in downregulation of the enzymatic activity of the Src kinase. The down-regulation is caused by intramolecular interaction in the Src kinase. There are two members of this family, Csk, and Ctk, both of which are present in RBL-2H3 cells and which may function to regulate the activity of Lyn. These molecules have an SH3 and a SH2 domain and associate with a number of molecules, including the adapter molecule called PAG/Cbp, protein tyrosine phosphatases, FAK, and paxillin. Phosphoprotein associated with GEMs/Csk-binding protein (PAG/Cbp) is a recently identified protein localized to membrane rafts that binds and regulates the function of these kinases. PAG/Cbp has many similarities to LAT; both molecules have short extracellular domains, are palmitylated and therefore in membrane rafts, and have a cytoplasmic domain with multiple Tyr residues. The phosphorylated PAG/Cbp binds the SH2 domain of Csk and recruits Csk to the membrane. The interaction with PAG/Cbp is important in regulating the function of Csk. At least in T cells, constitutive tyrosine phosphorylation of PAG/Cbp apparently keeps Csk at the membrane and the Src kinase in an inactive state. Cell activation results in dephosphorylation of PAG/Cbp with the release of Csk from the membrane, which allows for the dephosphorylation and activation of the Src kinase. In mast cells, Csk appears to regulate some of the functions of Lyn, and the overexpression of Csk or PAG/Cbp in RBL-2H3 cells inhibits signal transduction.[ Protein Tyrosine Phosphatases

157] [158]

The extent of the tyrosine phosphorylation of proteins depends on the dynamic balance between competing reactions: increased phosphorylation by protein tyrosine kinases versus dephosphorylation by protein tyrosine phosphatases (PTPs). The PTPs are a large family of molecules, some of which resemble receptors, as best characterized by CD45 (leukocyte common antigen), whereas others have no extracellular domains. The CD45 molecule is essential for activation of ZAP-70 in T cells, probably because it dephosphorylates and activates the src-related tyrosine kinase p56lck . Many different PTPs have been detected in mast cells ( Box 16-5 ), including CD45, a transmembrane PTP that regulates src-family kinases in hematopoietic cells. [159]

Evidence is contradictory as to whether CD45 plays any role in signal transduction from FcepsilonRI. Evidence for a role for CD45 comes from observations that the addition of mAbs to CD45 to human basophils blocked IgE-mediated histamine release but not that induced by formyl-methionyl (fMet) peptide, Ca2+ ionophore A23187, or phorbol myristate acetate Box 16-5. Protein Tyrosine Phosphatases (PTPs) in Basophils and Mast Cells

CD45

Transmembrane protein with two intracellular phosphatase domains May dephosphorylate Lyn at its regulatory site, resulting in activation Mast cells from CD45 knockout mice fail to secrete, although cell lines defective in CD45 secrete normally.

Hematopoietic PTP (HePTP)

Cytosolic PTP

Tyrphosphorylated after mast cell activation SH2-containing PTP-1 (SHP-1)

Cytosolic phosphatase Has two SH2 domains Limits signal transduction

SH2-containing PTP-2 (SHP-2)

Cytosolic phosphatase Has two SH2 domains

(PMA).[

160]

The binding of the anti-CD45 antibodies may induce changes in the protein tyrosine phosphatase activity of CD45 and a decrease in the intracellular 161

protein tyrosine phosphorylation. Mast cells have been derived from mice in which the CD45 was genetically inactivated. [ ] These cells secrete when activated with calcium ionophore but not when stimulated through FcepsilonRI. However, the exact nature of the defect in these cells and whether there is inhibition of the early tyrosine phosphorylation steps have not been determined. The contradictory evidence are observations that CD45 is essential for ZAP-70, but not for Syk, to 162]

reconstitute FcepsilonRI-initiated degranulation signals in RBL-2H3 cells.[

Several other PTPs are present in basophils and mast cells. Hematopoietic protein tyrosine phosphatase (HePTP) is present in RBL-2H3 cells and is tyrosine163

phosphorylated after cell activation.[ ] HePTP is also tyrosine-phosphorylated after stimulation of cells with calcium ionophore, suggesting that phosphorylation is a late event in degranulation. Other PTPs in basophils are SHP-1 (previously called PTP1C and SH-PTP1) and SHP-2 (previously called Syp, SH-PTP2, PTP1D, and PTP2C). Both SHP-1 and SHP-2 have two SH2 domains and bind tyrosine-phosphorylated proteins such as growth factor receptors, including the c-kit receptor. Both are tyrosine-phosphorylated after growth factor stimulation of cells. SHP-1 regulates signaling to JNK and the generation of cytokines such as TNF-α, but not the release of granule content.[

164]

A PTP also is associated with the high-affinity receptor that dephosphorylates both the tyrosine-phosphorylated β and γ subunits, but

165 Syk.[ ]

not Lyn or Therefore, this PTP may be involved in regulating the steady-state tyrosine phosphorylation level of the FcepsilonRI subunits. Also, PTP activity is increased after cell stimulation by either FcepsilonRI aggregation or with Ca2+ ionophores and is dependent on the increase [Ca2+ ]i , suggesting that it may play a role in degranulation.[ mast cells.

166]

These data suggest that protein tyrosine kinases and phosphatases play critical roles in the FcepsilonRI-mediated signaling in basophils and

255

Phosphoinositide 3-Kinase Phosphoinositide (phophatidylinositol) 3-kinases (PI3Ks) are lipid kinases that phosphorylate the D3 position of the inositol head group of the phosphoinositide lipid PtdIns(4,5)P2 , also called PIP2 , to produce PtdIns(3,4,5)P3 , also called PIP3 . This lipid product, PIP3 , is responsible for the recruitment to the membrane of proteins that contain PH domains; these include PLC-γ, Tec-family PTKs, SOS, and Vav. The binding of these PH-containing proteins to PIP3 results in their translocation to the plasma membrane, where activation can occur, and may also induce conformational changes that result in allosteric activation. Although there are multiple isoforms of PI3Ks, the isoform linked to FcepsilonRI activation is class 1, heterodimers consisting of a catalytic subunit with a molecular weight of 110•kD (p110) and an adapter regulatory subunit. There are three adapter subunits, p85α, p85β, and p55γ. The p85 subunit has an SH3 domain and two SH2 domains. The key to PI3K regulation is SH2-mediated recruitment of the catalytic subunit to the plasma membrane, where the catalytic unit then can phosphorylate its substrate and generate PIP3 . There appear to be alternative and redundant mechanisms to recruit PI3K to the membrane; in mast cells these may include adapters such as the transmembrane LAT protein or Gab2. Studies with inhibitors of PI3K, such as wortmannin, suggest that this molecule is important in the signaling cascade from FcepsilonRI. Wortmannin interacts with the p110 catalytic subunit of PI3K and inhibits both FcepsilonRI-mediated histamine secretion and formation of the lipid products of this enzyme, such as PIP3 . [ Similarly, the overexpression of a fragment of p85 in RBL-2H3 cells inhibits the receptor-induced increase in [Ca2+ ]i and degranulation.[

168]

167]

Therefore the

activation of PI3K plays a role in the pathway that leads to the influx of calcium. Microinjection of antibodies in RBL-2H3 cells suggest that it is the p110β and p110δ but not the p110α catalytic isoforms that regulate the sustained increase in [Ca2+ ]i .[ activation of PI3K and PKB.[

170]

169]

Experiments also suggest a role for Rac upstream or parallel to the

Bone marrow–derived mast cells (BMMCs) deficient in p85α, p55α, and p50α degranulate normally after FcepsilonRI

aggregation, but not when stimulated by the c-Kit receptor. [

171]

In vivo, mice that lack the p85α subunit are deficient in the number of mast cells in the peritoneum

and the gastrointestinal tract, but they have a normal number of mast cells in the skin.[

172]

The downstream effectors of PI3K activation include the Tec-family protein tyrosine kinases (Btk, Itk), GTPases, and Ser/Thr kinases. The activation of PI3K recruits the PH-containing proteins Btk and PLC, which are critical for the increase in intracellular calcium. The activation of the GTPases (e.g., Rac, Rho, Cdc42), which regulate the organization of the actin cytoskeleton, is probably downstream of Vav, another PH-containing protein. The important Ser/Thr kinase in this pathway is protein kinase B (PKB, also called Akt), which has a PH domain and therefore is also recruited to the membrane after cell activation. Another Ser/Thr kinase that is recruited by a PH domain to the membrane is phosphoinositide-dependent protein kinase-1 (PDK-1), which phosphorylates and activates PKB. Other proteins phosphorylated by PDK-1 include PKC and p70S6K (p70 ribosomal protein S6 kinase), which may control protein synthesis in cells. PKB (or Akt) influences several signaling pathways that lead to cell survival, proliferation, and growth. PKB phosphorylates glycogen synthase kinase-3 (GSK-3),

which results in its inactivation, thus regulating cell metabolism. Other important molecules regulated by PKB include the nuclear factors of activated T cells (NFATs), which are transcription factors important for the induction of cytokines. The nuclear import/export of these NFAT factors is controlled by a phosphorylation/dephosphorylation cycle; phosphorylated forms of NFAT are predominantly in the cytoplasm whereas the dephosphorylated form is in the nucleus. The dephosphorylation results from the calcium phosphatase calcineurin, whereas the phosphorylation occurs through kinases such as GSK-3. The inactivation of GSK-3 activity by PKB favors the increase in the nuclear concentration of NFATs. The activation of FcepsilonRI results in the activation of PKB, and this pathway regulates IL-2 and TNF-α production.[

173]

There are two major mechanisms for degrading the PIP3 and thereby reversing the effects of the activation of PI3K. PTEN (phosphates and tensin homologue deleted on chromosome 10) converts PtdIns(3,4,5)P3 to PtdIns(4,5)P2 , whereas SHIP converts PtdIns(3,4,5)P3 to PtdIns(3,4)P2 . Both PTEN and SHIP are present in mast cells, and as discussed in a later section, SHIP is an important negative regulator of mast cell activation. Phospholipase C-γ Activation and Hydrolysis of Phosphoinositides The activation of many different types of receptors results in the hydrolysis of myo-inositol-containing phospholipids caused by the activation of PLC, which then results in the generation of products that are intracellular second messengers. There are three myo-inositol-containing phosphatides: PtdIns, PtdIns(4)P (PIP), and PtdIns(4,5)P2 (PIP2 ). They account for less than 5% of the total phospholipid of the cell, with PtdIns accounting for more than 90% of the inositides. Receptor stimulation results in the hydrolysis of PtdIns with the release of water-soluble inositol phosphates and DG. These two then are important synergistic second messengers within the cell; the DG activates PKC, whereas the IP3 formed by hydrolysis of PIP2 releases Ca2+ from ATP-dependent Ca2+ stores in the endoplasmic reticulum. Hydrolysis of other PtdIns and the interconversion of the inositol phosphates and their degradation products also occur and might be secondary to the rise in intracellular Ca2+ . These enzymatic pathways result in the rapid conversion of IP3 to inositol 1,3,4,5-tetrakisphosphate and subsequently to inositol 1,3,4174]

trisphosphate.[

174

The FcepsilonRI stimulation of RBL-2H3 cells results in a rapid breakdown of the PtdIns.[ ] Although some of this PtdIns hydrolysis precedes the rise in intracellular Ca2+ , the major hydrolysis of PtdIns is a Ca2+ -dependent event. Once the hydrolysis of PtdIns has started, it is no longer dependent on external Ca2+ for continuation, although secretion of histamine is totally dependent on external Ca2+ . Some hydrolysis of PtdIns occurs after stimulation with the Ca2+ ionophore 174

A23187, which correlates with secretion. There is also hydrolysis of PtdIns in permeabilized cells and cytoplasts devoid of organelles. [ ] In permeabilized cells there is release of IP3 by either stimulation of the FcepsilonRI or the activation of G proteins by GTPγS. In these cells there is also formation of inositol(1,4)P2 , suggesting that both

256

PtdIns(4)P and PtdIns(4,5)P2 are substrates for the activated PLC. The increase in IP3 still requires other biochemical events to result in degranulation. [

175]

The hydrolysis of membrane inositol phospholipids after FcepsilonRI aggregation results from the activation of PLC-γ, whereas stimulation of the cells by seventransmembrane receptors for thrombin or chemokines activate other members of the PLC family of enzymes.[

176]

The two PLC-γ isoforms, PLC-γ1 and PLC-γ2, are

[176]

present in mast cells. Structurally these enzymes have two SH2 domains, an SH3 domain, and a PH domain. The SH2 domains allow their recruitment to tyrosine phosphorylated signaling molecules, and the PH domain docks the enzyme to the inner membrane by binding PIP3 . The activities of PLC-γ1 and PLC-γ2 are regulated by several mechanisms: direct tyrosine phosphorylation, SH2-mediated conformational changes of the enzyme, and physical recruitment to the membrane-localized receptor complex. Once at the membrane, PLC-γ functions to catalyze the hydrolysis of PtdIns(4,5)P2 , resulting in the generation of IP3 and DG. Both of these events are essential for FcepsilonRI-mediated secretion. PLC-γ1 and PLC-γ2 are rapidly phosphorylated on tyrosine and serine residues after 113] [177] [178] [179]

FcepsilonRI aggregation.[

The tyrosine phosphorylation of both these enzymes is downstream of Syk.[

translocation of the phospholipases to the plasma membrane, for the increased hydrolysis of membrane lipids.[ which by forming PIP3 allow for the translocation of PLC-γ to the

141 membrane.[ ]

180]

133]

This phosphorylation still requires

Such translocation requires Btk and or PI3K,

A 44-kD protein tyrosine kinase has been found to be associated with PLC-γ1 in

178

BMMCs.[ ] This kinase phosphorylates PLC-γ1 on tyrosine in vitro, and aggregation of FcepsilonRI activates the kinase, as determined by the increase in its in vitro autophosphorylation and the increase in phosphorylation of PLC-γ1. The activation parallels the increase in the in vivo phosphorylation of PLC-γ1 by FcepsilonRI aggregation. This 44-kD kinase may be a proteolytic fragment of Syk. Although structurally similar, experiments suggest that there are functional differences between these two PLC-γ isoforms. Disruption of the PLC-γ1 gene results in embryonic lethality; in contrast, PLC-γ2 is not lethal, although the PLC-γ2–deficient mice have a number of defects in signaling through immunoglobulin receptor 181

superfamily receptors. The BMMCs from these mice have defective degranulation, probably related to defective calcium mobilization. [ ] In RBL-2H3 cells there appears to be much more PLC-γ2 than PLC-γ1, and the distribution of these PLC isoforms differs, with some PLC-γ2 inherently associated with LAT in the plasma membrane, whereas PLC-γ1 is recruited to membrane ruffles only after FcepsilonRI aggregation.[ sensitive than PLC-γ2 to inhibition by wortmannin, a PI3K inhibitor.

179] [182]

The receptor-induced activation of PLC-γ1 is more

Adapters and Other Tyrosine-Phosphorylated Proteins A number of other enzymes and proteins that function as adapters are tyrosine-phosphorylated after receptor aggregation ( Boxes 16-6 and 16-7 ).

Box 16-6. Other Enzymes Involved in FcepsilonRI Signaling

PI3K

Phosphatidylinositol 3-kinase; lipid kinase with two subunits regulatory 85-kD (85α) enzyme with two SH2 domains, SH3 domain, and catalytic 110-kD subunit. Generates lipids (PIP3 ) that recruit PH-containing proteins (e.g., PLC-γ, Btk, Akt) to membrane

PLC-γ

Phospholipase C gamma; two isoforms, PLC-γ1 and PLC-γ2 Has two SH2 domains and an SH3 domain Recruited to plasma membrane, where isoforms hydrolyze PtdIns (e.g., PIP2 ) to inositol 1,4,5-trisphosphate (IP3 ) and diacylglycerol (DG) Important for Ca2+ mobilization

Vav (p95vav )

Exchange factor for Rac1; adapter protein Two SH3 domains, one SH2 domain, PH domain, acidic CDC24 homology regions, and a region homologous to the DG-binding site of PKC Binds to activated receptors and is itself tyrosine-phosphorylated after FcepsilonRI aggregation Guanine nucleotide exchange factor of Rac1

PKC

Protein kinase C; family of Ser/Thr kinases In permeabilized cells, PKCs β/δ critical for secretion and PKCs α/epsilon inhibit secretion

cPLA2

Cytoplasmic phospholipase A2 ; phosphorylated on Ser by ERK-1/2 Translocates to plasma membrane in activated cells Releases arachidonic acid

Scramblase

Tyrosine-phosphorylated after FcepsilonRI aggregation, moves phospholipids across the membrane

PKB (Akt)

Protein kinase B; Ser/Thr kinase with PH domain

Recruited to plasma membrane by PI3K activation Ser/Thr phosphorylated by PDK1 RasGAP

GTPase-activating protein (GAP) for Ras Contains two SH2 domains, SH3 domain, and PH domain Deactivates Ras

257

Box 16-7. Adapter Proteins in FcepsilonRI Signaling

LAT

Linker for activation of T cells; transmembrane protein present in membrane rafts Has many tyrosines in cytoplasmic domain that are phosphorylated downstream of Syk Decreased degranulation in cells defective in this protein

SLP-76

SH2-containing leukocyte protein; contains one SH2 domain, proline-rich region, and sites for tyrosine phosphorylation Associates with Grb2 Phosphorylated downstream of Syk; regulates Ca2+ influx Decreased degranulation in SLP-76–defective cells

Gab2

Has PH domain, proline-rich regions, and many Tyr residues Interacts with PI3K, SHP-2, PLC-γ, Shc, and Lyn; important for PI3K activation

Decreased signaling in mast cells from Gab2−/−mice Nck

Function unknown; has one SH2 domain and three SH3 domains

Paxillin

Cytoskeletal protein; accumulates at focal adhesion sites Associates with Lyn, Src, Csk, FAK, and Crk Binds to vinculin, another cytoskeletal protein, which interacts with other cytoskeletal proteins, including talin, tensin, and actin filaments

HS1 (SPY75)

Has SH3, nuclear localization, and DNA-binding domains Associates with Lyn May transmit signal to the nucleus

Crk

Adapter kinase; has one SH2 domain and two SH3 domains Associates with paxillin

Cbp

Csk-binding transmembrane protein, also called PAG (phosphoproteinassociated with GEMs) Localizes to membrane rafts, binds Csk (see Box 16-4 ) Overexpression of this protein inhibits signal transduction

183

Vav (p95vav ) is a cytosolic protein that is primarily expressed in cells of the hematopoietic lineage.[ ] Structurally it has several motifs similar to signaling molecules, including one SH2 domain, two SH3 domains, a PH domain, a Dbl domain, and a calponin homology domain. These domains are important for Vav's interaction with molecules such as Grb2, Lck, ZAP-70, and tubulin. The major tyrosine-phosphorylated protein that associates with Vav is SLP-76, a protein that also associates with Grb2. Vav functions, in a tyrosine phosphorylation–dependent manner, as a GDP-GTP exchange factor for the Rac/Rho family of small GTP-binding proteins, including Rac1 and Cdc42. Rho regulates the assembly of focal adhesion and actin stress fibers, whereas Rac regulates membrane ruffling. Rac1 also activates the JNK pathway and is important for actin reorganization for membrane ruffling. Cdc42 interacts with Wiskott-Aldrich syndrome protein (WASP) to 184

induce filopedia formation. Both Rac and Rho regulate mast cell secretion.[ ] The tyrosine phosphorylation of Vav may play an important role in regulating the function of these small GTP-binding proteins and thereby affect cell morphology, membrane ruffles, secretion of granules, and the generation of cytokines such as IL6. Vav is tyrosine-phosphorylated after activation of FcepsilonRI and c-Kit receptors and redistributes to the membrane rafts due to SH2 domain interactions.[ [186]

The tyrosine phosphorylation of Vav is downstream of Syk.[

p38 or

189 ERK-2.[ ]

In BMMC from

Vav−/−

degranulation is only slightly decreased.[

187] [188]

185]

Vav overexpression in RBL-2H3 cells results in constitutive activation of JNK-1 but not

mice there is inhibition of the tyrosine phosphorylation of PLC-γ, generation of IP3 , and calcium mobilization, although

190]

This suggests that Vav, by regulating PI3K, Rac1, or Cdc42, can control calcium influx.

191

SLP-76 (SH2-containing leukocyte protein-76) is another hematopoietic cell protein that is tyrosine-phosphorylated after FcepsilonRI aggregation.[ ] SLP-76 has an amino-terminal acidic region with several potential tyrosine phosphorylation sites that, when phosphorylated, binds Vav and Nck, a central proline-rich region that mediates interaction with Grb2 and Gads, and a single carboxy-terminal SH2 domain that interacts with the Fyn-binding protein FYB/SLAP-130. SLP-76 may also associate with several other proteins, including ZAP-70 and Lck. Its tyrosine phosphorylation is downstream of Syk but does not require extracellular Ca2+ . BMMCs from mice that lack SLP-76 are defective in FcepsilonRI-induced degranulation; the defect is downstream of Syk, with reduced tyrosine phosphorylation of PLC-γ1 192]

and calcium mobilization.[

Therefore, SLP-76 is required for the optimal tyrosine phosphorylation and activation of PLC-γ. A related molecule is Clnk (also

called BASH) that appears to function upstream of [Ca2+ ]i in mast cells.[

193]

LAT (linker for activation of T cells) is an integral membrane protein with a short extracellular domain and one transmembrane region. It is further anchored in the membrane by palmitoylation that targets LAT to the membrane rafts, which is essential for cell activation in T cells and presumably in mast cells. The intracellular domain of LAT contains many Tyr residues that are phosphorylated downstream of Syk after FcepsilonRI aggregation. Once phosphorylated, these sites bind

258

important signaling molecules, including Grb2, Gads, PLC-γ1, PI3K, SLP-76, and Cbl. There is dramatic reduction in the receptor-induced degranulation of BMMCs from LAT-deficient mice in parallel to a decrease in the tyrosine phosphorylation of SLP-76, PLC-γ1, and PLC-γ2 and in the mobilization of calcium. However, 194

there are no changes in the tyrosine phosphorylation of Syk and Vav, although the activation of ERK-2 and JNK are inhibited.[ ] These changes are similar to the defects seen in the absence of SLP-76 and suggest that both LAT and SLP-76 are involved in the optimal activation of the signals that are required for the influx of calcium. Therefore, PLC-γ can be recruited to the membrane both by binding through its PH domain to PIP2 and through its SH2 domain to LAT. Such binding appears to be essential for the optimal phosphorylation and activation of PLC-γ. Gab2 (Grb2-associated binder 2) is a 97-kD protein that contains an N-terminal PH domain followed by a long region with multiple tyrosine-containing motifs and 195]

two proline-rich domains.[

The tyrosine-containing motifs, once phosphorylated, provide binding sites for SH2-containing molecules, including p85α subunit of 196]

PI3K, PLC-γ, and SHP-2. The proline-rich domains of Gab2 are potential binding sites for SH3-containing proteins, such as Shc-family and Src-family PTKs.[ Therefore, Gab2 is a scaffolding molecule that can assemble multiple molecules involved in signaling pathways.

FcepsilonRI aggregation results in the tyrosine phosphorylation of Gab2 and translocation of a significant fraction of the Gab2 from the cytosol to the plasma membrane. In mast cells, Gab2 associates with several signaling molecules, including PI3K, SHP-2, Grb2, Lyn, and PLC-γ. FcepsilonRI-mediated signaling is defective in BMMCs from Gab2−/− mice, with a decrease in the receptor-induced activation of the PI3K pathway, a decrease in the generation of PtdIns(3,4,5)P3 , the tyrosine phosphorylation of PLC-γ1, and the formation of IP3 .[

197]

These cells also show a decrease in the receptor-induced increase in [Ca2+ ]i , especially of the

delayed response, with parallel decreases in degranulation and cytokine generation. These data suggest that Gab2 recruits and activates the PI3K, thus playing an

important role in degranulation. Overexpression of Gab2 in RBL-2H3 cells inhibits FcepsilonRI-induced signal transduction at an early step, which includes the 198]

tyrosine phosphorylation of the receptor subunits and the activation of Syk. [ FcepsilonRI-induced mast cell signaling and functions.

This suggests that Gab2 may have both positive and negative regulatory effects on

Paxillin is a cytoskeletal protein that accumulates at focal adhesion sites and has multiple sequence motifs, including binding sites for SH2 and SH3 domains. Paxillin forms complexes with several PTKs, including FAK, Lyn, and Csk. Paxillin also binds other cytoskeletal proteins (e.g., vinculin, α-actinin, talin) and, as with FAK, may help direct actin filament-membrane interactions. Paxillin also associates with adapter molecules (e.g., Crk) that have SH2-binding or SH3-binding sites. Tyrosine phosphorylation of paxillin is by FAK and other PTKs. In RBL-2H3 cells, FAK and paxillin become coordinately phosphorylated on tyrosine in 199

response to FcepsilonRI aggregation,[ ] and as with FAK, the FcepsilonRI-induced tyrosine phosphorylation of paxillin is also dependent on the expression of Syk. However, this does not indicate that Syk directly phosphorylates paxillin. The coordinate tyrosine phosphorylation of paxillin and FAK may be important in the 147] [200] [201]

development of actin stress fibers and the reorganization of F-actin[ and in the receptor-mediated morphologic changes.

and thus may play a role in signal transduction from receptors to the cytoskeleton

Other Molecules The aggregation of FcepsilonRI also induces the tyrosine phosphorylation of many other proteins; those identified include HS1 (also called SPY75), scramblase, and Nck. All these proteins are phosphorylated on tyrosine residues after FcepsilonRI cross-linking in RBL-2H3 cells or in BMMCs. Because several of these proteins have SH2 or SH3 domains and become phosphorylated on tyrosine, they can associate with other molecules during signal transduction and therefore may play a role in protein-protein interactions during FcepsilonRI signaling. HS1 has one SH3 domain close and putative nuclear localization signal and repetitive helix-turn-helix 111

202

motifs found in many DNA-binding proteins.[ ] Nck is composed almost exclusively of three SH3 domains and one SH2 domain.[ ] Through its interaction with SLP-76, Nck is involved in the activation of Pak1, which also interacts with WASP to regulate actin polymerization. Scramblase is an enzyme that moves phospholipids bidirectionally across the plasma membrane. FcepsilonRI aggregation results in transient externalization of the phosphatidylserine, a molecule that usually is on the inner leaflet of the membrane.[

203]

The phosphorylation of phospholipid scramblase may activate this enzyme to redistribute the lipids in the plasma

204 membrane.[ ]

Changes In Intracellular Calcium The essential role for Ca2+ in mast cell and basophil degranulation has been recognized since early in vitro experiments. First, Ca2+ has to be present in the medium for the release of histamine, and Ca2+ chelators inhibit this process. Second, different Ca2+ ionophores (e.g., A23187, ionomycin) transport Ca2+ into cells and initiate secretion. Third, the introduction of Ca2+ into the cells with micropipettes results in secretion. Fourth, measurements with isotopic Ca2+ demonstrate the uptake of this cation into the cell before the release of mediators. Fifth, measurement of [Ca2+ ]i with fluorescent dyes demonstrates an increase in [Ca2+ ]i after FcepsilonRI activation. 205]

Immediately after receptor cross-linking there is a rapid initial rise in [Ca2+ ]i that is independent of extracellular Ca2+ .[

This rise is caused by the release of Ca2+

from the endoplasmic reticulum inside the cell resulting from the IP3 binding to specific receptors. [

206] [207] [208]

This depletion of the Ca2+ stores in the 209 210

endoplasmic reticulum results in influx of Ca2+ from the medium due to a mechanism that has been called capacitative or “store-operated” Ca2+ entry.[ ] [ ] This Ca2+ influx occurs through low-conductance Ca2+ channels that have been characterized by electrophysiologic measurements and referred to as Ca2+ release– 211] [212] [213] [214]

activated Ca2+ (ICRAC ) channels.[

This current is very selective for Ca2+ ; cyclic adenosine monophosphate (cAMP), cyclic guanosine

monophosphate (cGMP), GTP, and several other molecules do not activate it. There is a marked difference in the activation of store-operated Ca2+ current ICRAC compared with the Ca2+ release: low concentrations of IP3 induce substantial Ca2+ release without any activation of ICRAC , whereas higher concentrations of IP3 activated Ca2+ influx.[

214]

This suggests that there are functionally distinct stores controlling Ca2+ release and influx: one set of stores

259

involved in Ca2+ release, and the other lower affinity store for Ca2+ entry. The buffering of Ca2+ by mitochondria regulates the concentration at which IP3 will activate the store-operated calcium channel.[ degranulation.[

210] [216]

215]

However, localized high concentrations of Ca2+ near the plasma membrane or the Ca2+ stores are not required for

Despite maximal activation of ICRAC , Ca2+ influx can be graded due to changes in membrane potential or as a result of inhibitory signals (e. 217

g., PKC) that are activated after receptor stimulation.[ ] The addition of the PKC activator PMA to RBL-2H3 in suspension completely inhibits the receptorstimulated Ca2+ influx. Adherent cells are much less sensitive to these effects than are cells in suspension, although there are still effects on the release of Ca2+ from intracellular stores.[

218] [219]

The molecular identity of the ICRAC channel is unknown, although some suggest that members of the TRP superfamily of molecules may function as these store220

operated calcium channels.[ ] It is unclear how the emptying of the Ca2+ stores in the endoplasmic reticulum communicates with the plasma membrane. According to one hypothesis, a diffusible signal is released from the endoplasmic reticulum that activates plasma membrane store-operated channels. Another theory states that there is direct interaction of the IP3 receptor with the Ca2+ channel protein (which may be a TRP-family molecule). Drugs that change the cytoskeleton have no effect on the coupling of the emptying of the endoplasmic stores to Ca2+ influx, suggesting that no direct interaction exists between the endoplasmic Ca2+ stores and the ICRAC channels.[

221]

Although it is assumed that the emptying of the calcium stores of the endoplasmic reticulum by IP3 then activates the influx of calcium,

ICRAC is present in mutant cells that lack all three IP3 receptor isoforms. Studies have defined some of the mechanisms that regulate ICRAC channel activity. PLC and PI3K activity are essential for the activation of ICRAC by a mechanism that is independent of the pathway of IP3 binding to its receptors in the endoplasmic reticulum.[

222]

The activation of this channel is also modulated by an ADP

ribosylation factor and by Rac/Cdc42, independent of the effect of these molecules in generating IP3. [

223] [224]

225]

Sphingosine blocks ICRAC , suggesting that the sphingomyelin pathway may play a role in regulating calcium influx. [

Similarly, increasing the intracellular level

of sphingosine inhibits FcepsilonRI-induced leukotriene synthesis and cytokine production by blocking the activation of the MAP kinase pathways, but not the release of granular contents.[

226]

However, the changes in ICRAC would suggest that degranulation should also have been inhibited. FcepsilonRI aggregation also

leads to sphingosine-kinase activation and conversion of sphingosine to sphingosine-1-phosphate. Inhibition of this pathway blocks the receptor-induced calcium 227]

influx, suggesting that sphingosine-1-phosphate may act as a second messenger to activate the calcium influx pathway.[

However, these results are complicated 226]

by the observations that mast cells not only have a receptor for sphingosine-1-phosphate, but also release this molecule during degranulation. [ sphingosine-1-phosphate to its receptor further degranulates mast cells. The increase in [Ca2+ ]i starts at the periphery of the cytoplasm and then spreads to the nucleus.[ cytoplasm.[

230]

228] [229]

The binding of

The rise in the nucleus is secondary to the rise in the

In studies with individual cells, FcepsilonRI stimulation results in rapid increases in [Ca2+ ]i , with different lag periods and heterogeneity in the

extent of the increase.[

231]

After FcepsilonRI aggregation there are oscillations in [Ca2+ ]i , especially in basophils.[

nM, and this influx is inhibited by compounds that increase intracellular cAMP.[

232]

In human basophils the rise is from 85 to 190•

233] [234]

Although an increase in [Ca2+ ]i is a prominent signal in mast cells or basophils, it alone is not sufficient to trigger exocytosis. For example, the Ca2+ ionophore 235

A23187 can induce an equivalent increase in intracellular Ca2+ to that induced after FcepsilonRI aggregation without resulting in secretion.[ ] Similarly, activation of PKC together with the increase of [Ca2+ ]i to these levels is an insufficient secretory signal. The microinjection of IP3 induces a rise in intracellular calcium, but there is no secretion unless a stable analog of GTP is also present. Similarly, addition of GTPγS alone induces slow secretion unless [Ca2+ ]i is also elevated. Therefore, multiple signals are essential for optimal degranulation. The Ca2+ binding protein calmodulin mediates many of the intracellular regulatory functions of Ca2+ . This protein has a molecular weight of 17,000 daltons and contains four Ca2+ -binding sites. The binding of Ca2+ to any one of the four binding sites results in a conformational change that brings about the association of this complex with enzymes. The Ca2+ -calmodulin complex regulates many enzyme systems, including PLA2 and microtubule assembly. A few inhibitors (e.g., trifluoperazine) bind to calmodulin and inhibit its action in isolated systems; still, when these inhibitors are added to cells, they probably affect several other systems. Protein Kinase C Protein kinase C is a superfamily of protein serine-threonine kinases whose members can be further divided into subfamilies based on structure and activation requirements: classic PKCs (α, βI , βII , γ) are regulated by calcium, DG, and phospholipids; novel PKCs (δ, epsilon, η, theta) are regulated by DG and phospholipids; and atypical PKCs (ζ and λ), are insensitive to both calcium and DG. Recently, two new members have been described (• also called PKD, and υ). Structurally the PKCs have membrane-targeting domains and in the case of PKD a PH domain. Therefore the increase in [Ca2+ ]i , and the formation of DG that

results from cell stimulation activate some of these PKC isozymes. PKC activation is accompanied by translocation of PKC isozymes to different cellular compartments due to interaction of each PKC with specific localizing protein. This allows the PKC to phosphorylate and regulate the function of different substrates. Phorbol esters directly bind and activate PKC. PKC is an essential transducer of signals for secretion in mast cells.[ esters stimulate secretion from basophils and mast (PKD), and ζ isoforms of PKC.[

239]

following

the PKC isozymes.[

238]

The direct activation of PKC by phorbol esters could be the mechanism by which phorbol

The RBL-2H3 cells contain the Ca2+ -dependent α and β and the Ca2+ -independent δ, epsilon •

In nonstimulated cells, most of the PKC is in the cytoplasm, but after FcepsilonRI activation the PKC isozymes, except for ζ,

translocate to various extents to the membrane. [ 236 experiments.[ ]

102 237 cells.[ ] [ ]

236]

238] [239]

PKC isozymes are probably necessary for optimal FcepsilonRI-mediated secretion, as shown by the

Permeabilized rat mast cells release histamine on the addition of Ca2+ or GTPγS but rapidly lose their capacity to respond as they lose all

The secretory response is

260

reconstituted by the addition of either the β or the δ isozyme of PKC, but the response still requires the presence of Ca2+ . In these permeabilized cells the addition of 237

α and epsilon isozymes of PKC inhibits the FcepsilonRI-induced PtdIns hydrolysis, probably by reducing the tyrosine phosphorylation of PLC-γ1. [ ] Similarly, overexpression of PKC-α and PKC-epsilon in RBL-2H3 inhibits receptor induced cPLA2 activation, whereas PKC-β enhances degranulation, cPLA2 activity, and cytokine production.[

240]

Degranulation and production of cytokines induced by FcepsilonRI activation or by calcium ionophore is decreased in mast cells derived

from PKC-β–deficient mice.[

241]

These results suggest that there are distinct roles for both Ca2+ and different PKC isozymes in the secretory pathway. 242 243

There is also interaction between the protein tyrosine kinases and PKC. PKC-δ and PKC-theta are tyrosine-phosphorylated after FcepsilonRI aggregation.[ ] [ ] Both also associate with Lyn, and PKC-δ interacts and phosphorylates Lyn on serine residues. This interaction depends on FcepsilonRI aggregation and may regulate Lyn activity.[

244]

PKC-δ associates with the β subunit of FcepsilonRI and after receptor stimulation phosphorylates the γ chain on Thr residues, which may play a 244

role in receptor internalization.[ ] In contrast, PKC-theta only translocates to the membrane after receptor stimulation. Overexpression of PKC-theta in RBL-2H3 cells results in a slight enhancement of degranulation and cytokine generation. PKC-•, also called PKD, is a more distantly related member of this family of Ser/Thr kinases. It is activated by FcepsilonRI aggregation downstream of the stimulation of the other classic and novel PKCs.[ 247 stimulation.[ ]

in membrane rafts) after of the PKC superfamily of kinases.

246]

In the mast cell, PKD is in the cytoplasm but is transiently associated with the plasma membrane (although not

These results suggest that in receptor-activated cells, there is a network of activation pathways between different members

Targets for PKCs include the MAP kinase pathways, the cytoskeleton, and transcription factors involved in the induction of cytokine genes. In RBL-2H3 cells the

237 248

activation of PKC by phorbol esters enhances the Ca2+ ionophore A23187–induced degranulation, but does not directly activate the cells for secretion.[ ] [ ] PKC also could play a role in modulating the secretory event; thus the treatment of RBL-2H3 cells with phorbol esters results in the inhibition of PtdIns hydrolysis but the 249]

enhancement of exocytosis.[

This effect is seen with RBL-2H3 cells in suspension but not when they are adherent to fibronectin.[

218]

There is also receptor-

[250]

induced phosphorylation on Ser of myosin light and heavy chains, which is caused by both the Ca2+ /calmodulin-dependent myosin light-chain kinase[ PKC. These phosphorylations are closely associated with degranulation.

251]

and

The role of PKC in degranulation of human basophils is unclear. PKCs βI , βII , and δ have been detected in human basophils and are directly activated to release histamine by the addition of phorbol esters, suggesting a role for the activation of PKC. Although FcepsilonRI aggregation results in a total rise in cellular PKC activity, no membrane translocation of PKC enzymes can be detected.[

252] [253]

Interestingly, inhibitors of PKC activity enhance the receptor-induced histamine

release. In contrast, there is a redistribution of cytoplasmic PKC to the membrane induced by fMet peptide-mediated cell stimulation.[

252] [254]

255]

The phosphorylations induced by PKC are reversed by Ser/Thr phosphatases present in cells. These phosphatases are inhibited by okadaic acid. [ blocks both histamine and LTC4 release from mast cells.

This inhibitor

Phospholipase D Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine, the major membrane phospholipid, to form phosphatidic acid and choline. The phosphatidic acid can then be metabolized to other important lipids, including lyso-phosphatidic acid, and dephosphorylated by phosphatidate phosphohydrolyase to form DG. The two mammalian PLD enzymes (PLD1 and PLD2 ) have been identified and are similar structurally; they have two domains (PX and PH) that bind to phosphoinositides, followed by the enzymatic region and an N-terminal domain, especially of PLD1 , that interacts with GTPases.[ 257]

cells PLD1 is localized to the secretory granules, whereas PLD2 is at the membrane.[

256]

Studies suggest that in mast

Receptor aggregation then recruits the PLD1 to the plasma membrane.

FcepsilonRI aggregation, calcium ionophore, PMA, and compound 48/80 activate the PLD pathway in mast cells, suggesting that PLD stimulation is downstream of the Syk-mediated rise in intracellular Ca2+ and the activation of PKC.[

258] [259] [260] [261] [262] [263] [264]

source (about 75%) of the DG formed while the rest results from the PLC hydrolysis of phase is caused by PtdIns hydrolysis, and the second phase results from the PLD

265 PtdIns.[ ]

266 pathway.[ ]

After receptor aggregation the PLD pathway is the major

There is a biphasic increase in the production of DG; the initial

Tyrosine kinase inhibitors block only the second wave of DG

formation, suggesting that there may also be tyrosine kinase–independent pathways for PLD activation. [

264]

PLD activity is regulated by at least four factors: the phospholipids PIP2 and PIP3 , PKC, and the Ras-related proteins Arf and Rho. [ 268 269

267]

In mast cells, brefeldin A, an

inhibitor of Arf, blocks the FcepsilonRI-induced and calcium ionophore A23187–induced activation of PLD.[ ] [ ] However, FcepsilonRI-induced secretion is more easily inhibited than the activation of PLD. The activation of PLD and secretion are also blocked by Clostridium difficile toxin B, which inhibits the Rho-family

270] [271]

proteins RhoA, Rac1, and Cdc42.[ [272] [273]

However, activation of PLD without a concomitant rise in intracellular Ca2+ does not result in degranulation.[

263] [268]

These results suggest that PLD plays a role in FcepsilonRI-mediated secretion.

Guanosine Triphosphate–Binding Proteins The GTP-binding proteins are a family of molecules that are the transducers of signals from many receptors to intracellular effectors. There are several classes of GTP-binding proteins (or GTPases), including the heterotrimeric GTP-binding proteins, or G proteins. Receptors of the seven-transmembrane domain family (e.g., muscarinic, adrenergic, or C5a receptors) use the heterotrimeric G proteins for signal transduction. Although the three components of the FcepsilonRI together have seven transmembrane-spanning domains, they do not have any amino acid similarity to the G protein–coupled receptor (GPCR) family. A second group of GTPases is the low-molecular-weight monomeric GTP-binding proteins that are related to Ras. Both types of GTPases are widely distributed in different tissues and are present in basophils and mast cells. Evidence suggests a role for G proteins in exocytosis from mast cells. In permeabilized rat mast cells, nonhydrolyzable

261

GTP analogs stimulate secretion of histamine. Similarly, microinjection of these GTP analogs into mast cells results in degranulation if there is Ca2+ in the medium. [274]

There appear to be two different sites for the action of GTP analogs within the cell; one site is postulated to be in the plasma membrane and upstream of PtdIns

hydrolysis, and a second site is distal to the action of Ca2+ and PKC.[ membrane fusion mechanism and may activate exocytosis.[

277]

275] [276]

This second GTP-binding protein, called GE by some, may be an integral part of the

In permeabilized cells, addition of Rac and Cdc42 allows secretion caused by Ca2+ alone, suggesting

278

that these may be the G proteins involved in secretion.[ ] Similarly, the dominant negative forms of these proteins inhibit the Ca2+ -induced and GTP-induced secretion of permeabilized cells. Therefore the signals generated by GTP, together with those induced by the increase in [Ca2+ ]i , result in exocytosis. Evidence also exists for a role of G proteins in the FcepsilonRI-mediated signal transduction leading to degranulation. In RBL-2H3 cells, stimulation of G proteins by either AlF4 − complexes or by GTPγS, a nonhydrolyzable GTP analog, induces both PtdIns hydrolysis and histamine release. [

279]

Furthermore, GDPβS, an 280

inhibitor of G protein activation, decreases both PtdIns hydrolysis and histamine secretion induced by either FcepsilonRI cross-linking or by GTPγS.[ ] Similarly, the depletion of GTP by mycophenolic acid inhibits the FcepsilonRI-mediated cell activation. By contrast, some evidence suggests that in BMMCs the PLC activation and PtdIns are independent of G proteins.[ FcepsilonRI to PtdIns.

281]

More recent evidence discussed later clearly indicates a role for small GTP-binding proteins in coupling of

Toxins that adenosine diphosphate (ADP)–ribosylate some G proteins have been used to inhibit histamine release. In most experimental systems, pertussis toxin,

which reacts with the high-molecular-weight Gi , does not inhibit the FcepsilonRI-mediated release.[

282] [283]

Still, pertussis toxin does inhibit the release induced by

other secretagogues. For example, in rat mast cells, pertussis toxin inhibits the compound 48/80-induced Ca2+ uptake and release of AA and histamine. This suggests that a pertussis toxin–sensitive G protein is involved in activating the Ca2+ channel with this secretagogue. In these cells, compound 48/80 appears to activate G proteins directly, and this response is blocked in permeabilized cells by the addition of a synthetic peptide corresponding to the COOH-terminal end of the sequence of Gαi3 .[

284] [285]

Similarly, in BMMCs, pertussis toxin inhibits the thrombin, but not the IgE-mediated degranulation.[

inhibits the release induced by fMet peptide and C5a but not that by IgE-antigen.[ FcepsilonRI, are coupled to pertussis toxin–sensitive G proteins.

286]

282]

With human basophils, pertussis toxin

Therefore the seven-transmembrane receptors for these secretagogues, but not

Role for Small GTP-Binding Proteins

The small G proteins of the Ras superfamily play a role in many cellular processes by acting as molecular switches, cycling between active (GTP-bound) and inactive (GDP-bound) states and are important in mast cell and basophil secretion. For example, the microinjection into rat mast cells of the ras oncogene results in 287

exocytosis.[ ] The Rho GTPases are activated by guanine nucleotide exchange factors (GEFs), which include molecules such as Vav. Among the Ras family of small GTP-binding proteins, members of the Ras, Rab, Rho, and Arf families are thought to play a role Box 16-8. Small GTP-Binding Proteins in Basophil and Mast Cell Secretion

Rab3

Several isoforms; may play a role in granule fusion

Ras

Activated by SOS Activates Raf/MAPK pathway, leading to ERK-1/2

Rho, Rac, Cdc42

Participate in changes in cytoskeleton Rho essential for stress fiber formation Rac essential for membrane ruffles

SOS, Mammalian homologue, “son of sevenless”; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulating kinase.

in secretion in mast cells. Previous sections discuss the role of Arf in regulating PLD, and the activation of the Ras pathway that leads to the MAP kinase pathways is

described later. This section discusses some of the other small GTP proteins involved in mast cell signaling ( Box 16-8 ). These small G proteins also regulate the cytoskeleton, as discussed later. 288

Rab3 isoforms are associated with secretory organelles, including synaptic and mast cell secretory granules.[ ] They interact with vesicle specific proteins and may play a role in the docking and regulation of exocytosis in cells. There are four isoforms (Rab3A, -B, -C, and -D); the predominant one expressed in mast cells is 203] [288] [289] [290]

Rab3D, although Rab3A and Rab3B have also been detected.[ fraction.

[290]

The majority of Rab3A is in the cytosol, whereas Rab3D is in the membrane

A novel member of the Rab family, Rab8B, has been cloned from RBL-2H3 cells, which when overexpressed in these cells induced outgrowths of the

plasma membrane.[ 292 secretion.[ ]

291]

The intracellular perfusion of mast cells with Rab3A or with a 16–amino acid peptide based on the amino-terminal sequence of Rab3 induces

However, even the extracellular addition of these peptides induces intracellular Ca2+ changes and secretion. The overexpression of wild type or a 203] [289] [293]

dominant negative form of Rab3A or Rab3D in RBL-2H3 cells inhibits secretion,[ 289 secretion.[ ]

although in one of the studies, Rab3D but not Rab3A inhibited

These results suggest that Rab3 plays a role in granule fusion and exocytosis.

The Rho family of small G-proteins includes Rho, Rac, and Cdc42. The Rho GTPase, by controlling the organization of the actin cytoskeleton, regulates cellular functions such as motility, morphology, and adhesion. Activated Rho induces the formation of actin stress fibers, activated Rac induces actin-dependent membrane ruffling, whereas activated Cdc42 induces protrusion of actin-rich microspikes from the cell surface. As discussed, Vav has guanine nucleotide exchange factor activity toward Rac1, whereas Rho/Cdc42 and Arf stimulate PLD activity. One of the downstream targets of the Rho family of GTPases is Rho kinase, which, by phosphorylating both myosin light-chain phosphatase and myosin light chain, regulates actin organization in the cells. The phosphorylation of both myosin heavy and 250] [251] [294]

light chains by PKC and by myosin light-chain kinase correlates with antigen or calcium ionophore/PMA-induced secretion in RBL-2H3 cells.[ Inhibition of either of the two kinases blocks phosphorylation as well as secretion, [

251]

although secretion

262

294]

correlates best with the PKC-induced phosphorylation.[ relationship of this to secretion is not clear.

Therefore, Rho kinase probably participates in the control of myosin in mast cells, although the

The present model for the regulated actin polymerization is by the GTP-bound Rho family of proteins to recruit and activate WASP-family proteins, which in turn recruit the Arp2/3 complex and stimulates its nucleation activity. The Arp2/3 complex then nucleates formation of actin filaments and cross-links them into a branching network. WASP-family proteins are downstream effectors of Rho-family proteins. The WASP family of proteins consists of WASP, N-WASP, and SCAR. To initiate actin polymerization, Cdc42 acts through WASP or N-WASP, and Rac acts through SCAR. WASP was originally identified as a protein that was altered in patients with Wiskott–Aldrich syndrome, a genetic immunodeficiency disorder characterized by reduced motility of lymphoid immune cells. After FcepsilonRI stimulation, activated Cdc42 recruits the WASP to the plasma membrane, where it associates with and becomes phosphorylated by Lyn and Btk. Both Rac and Rho appear to play a role in signaling from FcepsilonRI aggregation. Inhibitors of endogenous Rac and Rho reduce secretory responses of

permeabilized rat peritoneal mast cells, whereas constitutively active Rac or Rho mutant proteins enhance secretion and increase the level of F-actin.[ actin becomes rearranged into membrane ruffles and also associates with myosin in a cytoplasmic

296 meshwork.[ ]

184] [295]

The F-

Rho regulates actin and exocytosis by different

297

295

mechanisms.[ ] Although degranulation is independent of actin polymerization, cytoskeletal reorganization and secretion are still strongly correlated.[ ] There is further evidence to suggest a role for these molecules in signal transduction. First, in permeabilized mast cells, secretion is reconstituted by the addition of a complex 278] [298]

of Rac and RhoGDI or Cdc42, whereas the dominant negative forms inhibit secretion.[ rat mast cells by means of a patch pipette, inhibits the GTPγS-induced

299 release.[ ]

Second, RhoGDI, which inhibits Rho activity, when introduced into

Third, Clostridium botulinum C3 toxin, an inhibitor of Rho, blocks FcepsilonRI as 300]

well as calcium ionophore/GTPγS-induced secretion from permeabilized RBL-2H3 cells.[

The Clostridium difficile toxin B, which monoglucosylates and 270]

inactivates RhoA, Rac1, and Cdc42, inhibits FcepsilonRI-, GTPγS-, or PMA-induced activation of PLD in both intact and permeabilized RBL-2H3 cells.[

This

271 A23187,[ ]

toxin also inhibits secretion in intact cells stimulated by either FcepsilonRI aggregation or with the calcium ionophore probably due to inhibition of RhoA or Cdc42. Since C3 transferase, which ADP-ribosylates RhoA, B, and C, does not inhibit secretion, the results suggest that Cdc42 is the GTPase that 271]

participates in degranulation.[

Fourth, overexpression of a dominant negative mutant form of Cdc42 in RBL-2H3 cells decreases cell adhesion and inhibits 301]

FcepsilonRI-induced actin plaque formation, recruitment of vinculin to these adhesion sites, and receptor-induced degranulation, [ dominant active Cdc42 and Rac enhanced FcepsilonRI-stimulated 170 303 304 IP3.[ ] [ ] [ ]

302 secretion.[ ]

whereas expression of a

Cdc42 and Rac also function upstream of the calcium influx and the generation of

Fifth, BMMCs from Rac2-deficient mice have reduced FcepsilonRI-mediated degranulation with decreased activation of the PI3K pathway and

305 PKB.[ ]

Altogether these results suggest that the different small GTP binding proteins control distinct pathways that affect cytoskeletal change and are involved in degranulation. The small GTP-binding proteins probably also play an important role in the fusion of the granule to the plasma membrane, which results in the release of the granular contents. Mast cell degranulation that results from the granule fusing with the plasma membrane has many similarities to the vesicle fusion in neurons releasing neurotransmitters. In this exocytic process the first step is docking of the granule with the plasma membrane, followed by the formation of a protein complex that is involved in the fusion event. This leads to the integration of the granule and plasma membrane and the creation of a fusion pore. Such a process involves proteins that associate with the granular membrane and those associated with the plasma membrane and is thought to involve molecules that are called SNAREs: the v-SNAREs (vesicle SNAREs) present on vesicle membranes and t-SNAREs (target SNAREs) on the plasma membrane. The SNARE protein on the granule is VAMP (also called synaptobrevin), which interacts with the t-SNAREs, syntaxin 4, and SNAP-23. These events are regulated by the small Rab3D, which also associates with the granules, and by the calcium-sensing protein synaptotagmin.[

203] [306] [307] [308]

In mast cells, synaptotagmin type II is the most abundant isoform and is associated 308

with lysosomes rather than with secretory granules; it regulates the exocytosis when the cells are stimulated with Ca2+ ionophore or with antigen.[ ] The overexpression of synaptotagmin I in RBL-2H3 cells enhances degranulation, whereas that of type II inhibits lysosomal release with minimal effects on the release of 308 309

secretory granules.[ ] [ ] Overexpression of SNAP-23, which associates with the plasma membrane, results in enhanced degranulation.[ that the fusion events in mast cells are similar to vesicle release in neurons.

310]

These data suggest

Mitogen-Activated Protein Kinase Pathways Mitogen-activated protein kinases are a cascade of enzymes that are important in signaling pathways for cell proliferation, differentiation, and death. There are three major groups of MAP kinases: extracellular signal–regulated protein kinase (ERK), p38 MAP kinases, and c-Jun NH2 -terminal kinase (JNK), which are also known

as the stress-activated protein kinases. There are two members of the ERK family (ERK-1, ERK-2; sometimes referred as p44/p42 MAP kinases), several isoforms of JNK, and four p38 MAP-kinases (p38α, p38β, p38γ p38δ). Recently, ERK-5 has been described as a new MAP kinase that is distinct from these traditional pathways. These MAP kinase pathways are activated by many stimuli, such as cytokine and growth factor receptors and environmental stress. FcepsilonRI stimulation results in the activation and phosphorylation of the ERK, JNK, p38, and ERK-5. This activation is a late event in signaling and correlates with the extent of secretion. The activation of ERK-1/2 reaches a peak in 1 to 5 minutes, whereas that of JNK is slower. Not only FcepsilonRI activation, but also Ca2+ ionophores, cKit receptor, and activation of G protein–mediated receptors, result in activation of these MAP kinase pathways in mast cells. The MAP kinase pathways contain at least three protein kinases that work in series ( Figure 16-2 ). The MAP kinases are activated by dual phosphorylation at the motif Thr-Xaa-Tyr, which is different in each group of MAP kinases: ERK (Thr-Glu-Tyr), p38 (Thr-Gly-Tyr), and JNK (Thr-Pro-Tyr). The dual phosphorylation of Thr and Tyr residues is by a conserved protein kinase cascade. The ERK MAP kinases are

263

Figure 16-2 Mitogen-activated protein (MAP) kinase (MAPK) pathways. Prototypical cascade of enzymes is shown at left. MAPK is controlled by a MAPK kinase (MAPKK), which in turn is controlled by a MAPKK kinase (MKKK). In some cases this three-component system is regulated by a MAPK kinase kinase kinase (MKKKK). As indicated, these pathways are all downstream of small guanosine triphosphatase molecules. Arrows have not been added when some of these multiple interactions are not clearly defined. Activated MAPKs phosphorylate the indicated transcription factors.

Box 16-9. Components of Ras/Extracellular Signal–Regulating Kinase (ERK-1/2) Activation Pathway

Shc

Adaptor protein; contains PTB and SH2 domains Binds Grb2, which then binds SOS Tyrosinephosphorylated in nonstimulated RBL-2H3 cells

Associates with β subunit of FcepsilonRI Grb2

Adaptor protein with SH3-SH2SH3 structure Links receptors to Ras activation Binds several proteins, including Shc and SOS

SOS

Guanine nucleotide exchange factor Proline-rich region binds to SH3 domain of Grb2 Ras exchange factor; converts inactive RasGDP to active Ras-GTP

Ras

Small GTPase; binds Raf Activated by guanine nucleotideexchange factor (e.g., SOS) Inactivated by Ras-GAP

Raf

Ser/Thr kinase; binds to activated Ras Activated by recruitment to plasma membrane and by tyrosine phosphorylation

MAPKK

MAP kinase kinase; dualspecificity kinase Phosphorylates both Tyr and Thr residues MEK-1/MEK-2 activated by Ser phosphorylation by Raf

ERK-1/2

Ser/Thr kinase Activated by dual phosphorylation on Tyr and ser by MAPKK Substrates include cPLA2 and trancription factors

GTP form by the promotion of guanine nucleotide exchange. Thus, Shc binds to tyrosine-phosphorylated membrane receptors and recruits the adapter protein Grb2 and the associated SOS. Vav also has guanine nucleotide exchange factor activity and may activate Ras. Thus, two pathways, one mediated by Shc-Grb2-SOS and the other by Vav, could result in the activation of Ras.

Ras is a member of the small GTPase superfamily. In the inactive state, Ras has bound GDP and is activated by the dissociation of GDP and the binding of GTP, a step facilitated by a guanine nucleotide exchange factor such as SOS. This results in the activation of downstream kinases. Ras is then deactivated by GAP (GTPaseactivating proteins), which results in the hydrolysis of GTP. Ras can activate several effector pathways, which may involve the Raf, Jun, and p38 pathways. Both the Raf and the Jun pathways are activated in mast cells. Some inhibitor studies suggest that there may be Ras/Raf-independent pathways for FcepsilonRI-induced activation of ERK-1/2.[

321]

Raf is a 70- to 75-kD cytosolic protein Ser/Thr kinase. The activation of Raf by tyrosine kinase pathways requires Ras. Activated Ras binds Raf and thus recruits Raf to the plasma membrane, where another tyrosine kinase–mediated signal results in its activation. Tyrosine phosphorylation of Raf may play a role in its activation that leads eventually to the activation of ERK-1/2 (see Figure 16-2 ). Release of Arachidonic Acid 322 323

The activation of basophils or mast cells also results in the release of AA, an unsaturated fatty acid, from cellular phospholipids.[ ] [ ] AA is typically present in the sn-2 position on the glycerol backbone of phospholipids. The major membrane phospholipid is phosphatidylcholine, with lesser amounts of phosphatidylethanolamine, phosphatidylserine, and PtdIns. There are different pools of phospholipids in cells, and the source from which the AA is released depends on the stimulus. The release of AA requires the presence of extracellular Ca2+ and parallels the release of histamine but is slower than the increase in [Ca2+ ]i . [

322] [323] [324]

In

permeabilized cells, GTPγS induces PLA2 activation, indicating that G protein activation is upstream of cPLA2 . Although both IgE and Ca2+ ionophore stimulation of cells release AA, there are several differences in the release induced by these two secretagogues in RBL-2H3 cells. With the Ca2+ ionophore, more of the total 326

cellular AA is released and is derived predominantly from the cellular phosphatidylethanolamine.[ ] In contrast, with IgE-mediated release, the major source of the AA is phosphatidylinositol and phosphatidylserine. This suggests that different pools of phospholipids are accessible to the activated cPLA2 . Mast cells and basophils contain at least three different PLA2 enzymes.[ generation of the AA-derived eicosanoids.[

328]

326] [327]

The low-molecular-weight secreted form, called sPLA2 , does not participate in the

The major phospholipase in mast cells is the high-molecular-weight cytosolic PLA2 (cPLA2 ). The FcepsilonRI-

mediated release of AA is regulated by cPLA2 , which has a Ca2+ -dependent phospholipid-binding domain and a single serine-containing consensus site for phosphorylation by ERK-2. The increase in [Ca2+ ]i in stimulated cells results in the translocation of cPLA2 from the

265

cytosol to membranes, and phosphorylation on Ser activates the phospholipase activity of the molecule.[ Ras/Raf/MEK-1/ERK-2

315 pathway.[ ]

327] [329]

This phosphorylation is by MAP kinase through the

PKC may influence AA release, either directly or indirectly, through regulating ERK kinase.[

330]

from cPLA2 -deficient mice, there is enhanced secretion of granular content but a complete absence of the generation of eicosanoids.[ some experiments that suggest that an AA product from the LO pathway may play a role in cell secretion.[

When BMMCs are isolated

331]

This finding could explain

332]

The AA released by cell stimulation can be metabolized by either the COX or the LO pathway. As noted earlier, in mast cells the COX pathway leads predominantly to the formation of PGD2 with formation of some TXA2 and prostacyclin. In the COX pathway there are the COX-1 and COX-2 enzymes; although COX-1 is 333

expressed constitutively in mast cells, the expression level of COX-2 is regulated by cytokines such as c-Kit ligand, IL-3, and IL-10.[ ] The increased expression of COX-2 is blocked by glucocorticoids. The LO pathway includes 5-LO, 5-LO–activating protein, and leukotriene C4 synthase and results in the formation of the cysteinyl leukotriene C4 (see Chapter 14 ). Degranulation is not always accompanied by AA release. For example, with human basophils, several secretagogues (e.g., C5a, PMA) release histamine but not 334]

LTC4 , whereas with cutaneous mast cells, substance P and codeine release histamine but not PGD2 or LTC4 .[

With FcepsilonRI stimulation, human lung mast

cells release tenfold more LTC4 than do skin mast cells. In rats, mast cells isolated from connective tissue release predominantly PGD2 , whereas those from mucosal tissue release more LTC4 . Such data suggest that there are differences in these cells in the enzymes that are required for the further metabolism of the AA released during cell activation. Mast cells also synthesize PAF by the addition of an acetyl residue in the sn-2 position of the phospholipids cleaved by PLA2 . PAF is an important mediator of inflammation that has diverse physiologic actions (see Chapter 14 ). Cytoskeletal and Morphologic Changes Aggregation of FcepsilonRI induces cytoskeletal reorganization and transforms the microvillous surface of basophils to a plicated ruffled appearance; the cells also spread and become more adherent. These morphologic changes are secondary to the changes in phosphoinositides (especially PIP2 ), the rise in intracellular Ca2+ , phosphorylation of cellular proteins, and activation of small GTP-binding proteins. These changes are downstream of Syk and precede degranulation. The cytoskeleton is also involved in the large-scale clustering and capping of the aggregated FcepsilonRI. The large-scale clustering and capping of aggregated FcepsilonRI is caused by their interaction with the membrane skeleton, such as with filamentous actin.[

335]

The detergent insolubility of these cross-linked receptors

correlates inversely with secretion; at the highest antigen concentration there is maximal insolubility and minimal secretion.[ detergent solubility of the receptor proteins is not caused by changes in the

336]

However, the decrease in the

337 microfilaments.[ ]

Cytoskeletal reorganizations probably play an important role in granule migration and in regulation of the access of secretory vesicles to the plasma membrane. As discussed in previous sections, the small GTPases Rho and Rac are essential for the formation of stress fibers and membrane ruffles, respectively. The addition of

338 339

GTPγS to permeabilized mast cells results in reorganization of actin filaments. [ ] [ ] The content of filamentous (F) actin is reduced in the cortical region and increased in the cell interior. This appearance of actin filaments in the interior of the cell depends on Rac and Rho, with Rho responsible for actin polymerization and Rac for the entrapment of actin filaments in the interior of the cell.[

338]

Microtubules are labile filamentous protein polymers that are in a state of constant dynamic equilibrium with the pool of tubulin subunits. The assembly of the tubules is controlled by [Ca2+ ]i and other intracellular signals such as the activation of PKC. Experiments with compounds that disrupt microtubules have suggested that these structures may play a role in secretion. Colchicine and vinblastine bind to tubulin dimers and prevent their polymerization into microtubules although they may also have other effects in cells. Colchicine and vinblastine inhibit histamine release from both basophils and mast cells. Conversely, deuterium oxide (heavy water) stabilizes microtubules and enhances IgE-mediated histamine release. However, deuterium oxide also enhances PtdIns hydrolysis, suggesting that it also could have effects at early steps of the release process. Inhibitors of microtubular function block the release of AA without affecting the increase in [Ca2+ ]i . Therefore the coupling of the Ca2+ influx PLA activation requires the microtubular system. However, cytoskeletal-inhibiting agents do not block the generation of inositol 340]

phosphates, although they do inhibit later steps of cell activation, suggesting that microtubules are not involved in the early activation steps.[

Microfilaments are filamentous structures that are usually distributed close to the cell surface. The cytochalasins are fungal products that inhibit cellular movement due to their interaction with microfilaments, although they also inhibit membrane transport systems. Cytochalasin B inhibits the lateral diffusion of FcepsilonRI but enhances the receptor-mediated histamine release, whereas leukotriene formation is decreased. Disruption of the microfilaments in RBL-2H3 cells with cytochalasin 341

B increased histamine release but did not change the receptor-mediated PtdIns hydrolysis or Ca2+ influx.[ ] In permeabilized cells it also enhanced the GTPγSinduced release, suggesting that microtubules regulate a late step in the release process. These data with inhibitors suggest that microtubules and microfilaments are probably involved in mast cell secretion. 201

There is redistribution of cytoskeletal elements during activation of mast cells and basophils.[ ] In nonstimulated RBL-2H3 cells the actin is localized to the cell periphery just under the plasma membrane. After stimulation it is in the periphery, concentrated under the membrane ruffles that are prominent in activated cells. 200

Stimulated cells exhibit increased adhesive properties and form actin plaques that contain phosphorylated proteins.[ ] The intermediate filaments and microtubules are also redistributed throughout the cell body. Vimentin, an intermediate cytoskeletal protein, is normally distributed around granules. After stimulation, vimentin is 342]

phosphorylated on Ser, probably by PKC, and becomes diffusely distributed in the cytoplasm.[ Morphologic Study of Degranulation

Microscopic studies have detected changes that occur when mast cells and basophils are stimulated to degranulate.[

266

343]

When basophils are observed under phase-contrast microscopy, addition of antigen results in a loss of their oriented motility. Cells spread and extend pseudopodia in several directions. Some cells exhibiting this change do not go on to degranulate. Degranulating basophils develop small “vesicles” in their cytoplasm that rapidly increase in size and coalesce with a resulting decrease in the number of specific granules. By electron microscopy the surface of the RBL-2H3 cells shows many microvilli. Within seconds of the addition of antigen, the microvillous surface transforms to a plicated appearance; the cells spread with increased adhesion to the substrate. There is also increased fluid pinocytosis. These changes can occur in the absence of degranulation. 343 344

Transmission microscopic studies have described the changes that occur in basophils or mast cells of different species during secretion.[ ] [ ] The stimulation of human basophils with antigen to release histamine results in a series of characteristic morphologic changes. The granular membranes fuse with the plasma membrane around the circumference of the cell, resulting in the extrusion of the granular material to the outside. The diameters of the openings to the exterior are variable. Occasionally, interconnected chains of granules within the cell open to the exterior at a single point on the cell surface. The granule matrix is released as a whole to the outside, but the granule membrane is left behind. Frequently, membrane-free granular contents are seen attached to the cell exterior. In a fully degranulated basophil there is a complete loss of recognizable cytoplasmic granules. In contrast, a different morphologic picture of degranulation is seen in basophils infiltrating sites of delayed hypersensitivity reactions. The process has been termed piecemeal degranulation; the granules never fuse with the cell membrane and lose their matrix in a piecemeal manner over days. This type of degranulation probably does not occur in immediate hypersensitivity reactions. 345

The degranulation of human mast cells after IgE-receptor activation has similar morphologic characteristics.[ ] After stimulation a dimple forms in the plasma membrane in areas where the membrane is in contact with the secretory granule membrane. This area becomes clear of proteins, and electrophysiologic 346

measurements demonstrate the formation of a small fusion pore, which is stable for 10 to 15,000•msec.[ ] The earliest event observed is this formation of narrownecked pores joining the membranes of the granules with the plasma membrane. Swelling of the individual granules is accompanied by a change in the electron347

dense granular contents. However, the swelling of the secretory granule is not the driving force for membrane fusion.[ ] In contrast to basophils, the individual granular contents fuse with each other to form an interconnected chain filled with altered granule matrix. These channels then open to the exterior by fusion with the 348

plasma membrane. The granule fusion does not need to be an all-or-none event and can result from partial release.[ ] During the release process, prominent cytoplasmic filaments are observed close to the granules. The granule membrane is under tension, which results in a net transfer of membrane from the plasma membrane to the secretory granule when they are connected by fusion pores.[

349]

This tension also is the force that causes the expansion of the fusion pore.[

There are differences in the degranulation-induced morphologic changes observed in human skin compared with lung mast cells.[ reconstitution of granules in rat mast cells after a secretory event can take up to 4 weeks.

345]

350]

Interestingly, the full

Other Metabolic Events in Mast Cell Activation There is extensive literature on the inhibition of basophil or mast degranulation by different classes of inhibitors. Although these experiments may not directly reveal the enzymes involved, they do indicate that there are complex mechanisms for the regulation of cell activation and the release of inflammatory mediators. Requirement for Metabolic Energy

Energy is required for maintaining general cellular functions and for the secretion of mediators. Several inhibitors block energy utilization in mast cells and basophils, including anoxia in the absence of glucose, as well as cyanide, 2-deoxyglucose, and antimycin A. Use of such inhibitors suggests that energy for histamine

release may be from either aerobic or anaerobic glycolysis. Direct measurements with isolated cells suggest that oxidative phosphorylation (aerobic glycolysis) plays the dominant role during mediator release. The depletion of intracellular ATP by mitochondrial inhibitors blocks the increase in [Ca2+ ]i . Adenylate Cyclase and Formation of Cyclic Adenosine Monophosphate

Adenylate cyclase is a membrane-associated enzyme that is regulated by activated G proteins. It results in the formation of cAMP, which acts as an intracellular second messenger. In some cellular pathways, increased levels of cAMP provide a signal for cell activation. There is no clear evidence that a rise in cAMP plays any role in signal transduction in basophils and mast cells. Some, but not all, investigators have observed an increase in intracellular cAMP after FcepsilonRI stimulation of human and rat mast cells or basophils.[ the increase in the [Ca2+ ]i . [

351]

41]

When reported, the cAMP rise is an early, rapid response, which occurs in the absence of extracellular Ca2+ and before

A late rise in cAMP has also been observed, probably a secondary effect caused by the PGD2 released from cells. FcepsilonRI-

mediated stimulation of RBL-2H3 cells does not result in an increase in the intracellular cAMP levels,[ secretion.

352]

suggesting that a rise in cAMP is not essential for

The level of cAMP appears to modulate the secretion of mediators from mast cells and basophils. Several pharmacologic agents, such as β-adrenergic agents and prostaglandins, bind to specific cell surface receptors, activate adenylate cyclase, and increase intracellular cAMP levels. The intracellular levels of cAMP also can be raised by the addition of exogenous cAMP or its analog 8-bromo-cyclic AMP and by the addition of inhibitors of the phosphodiesterase enzyme involved in the breakdown of cAMP. These agents inhibit mediator release in a variety of in vitro systems, including human basophils and mast cells. Therefore the increased cAMP levels could be a normal turnoff signal for secretion in these cells. The intracellular effects of cAMP are mediated by the cAMP-dependent protein kinases. Increased intracellular cAMP levels activate cAMP-dependent protein kinases that phosphorylate their respective protein substrates on Ser/Thr residues. In rat mast cells, both type I and type II cAMP-dependent protein kinase activity is present in the cytoplasm and is activated after IgE-mediated cell triggering. The resulting phosphorylation of cellular proteins could then modulate the release process.

267

Activation of Proteases

Both protease inhibitors and substrates of proteolytic enzymes block secretion from mast cells and basophils. These inhibitors include serine protease inhibitors such as diisopropyl fluorophosphate and some trypsin and chymotrypsin substrates. These chemicals block the rise in [Ca2+ ]i , the cAMP increase, and cell secretion. In some experimental systems the inhibitors are only effective if present during cell stimulation; that is, if cells are incubated with the inhibitor and washed before the addition of the secretagogue, there is normal release. This suggests that the proteolytic enzyme is activated only after the start of the secretory process. The activation of this protease is Ca2+ independent and may be an early step after receptor aggregation. Also, a different protease may be involved in cell desensitization (see section on desensitization for the release reaction). The activation of this second protease occurs in the absence of Ca2+ and is sensitive to lower concentrations of the

inhibitors. Methylation Reactions

In methylation reactions, methyltransferase enzymes transfer methyl groups from the high-energy methyl donor, S-adenosylmethionine, to lipids, proteins, or nucleic acids. These reactions regulate chemotaxis and gene expression. Inhibitors of methylation reactions block FcepsilonRI-mediated secretion in a variety of mast cells and basophils.[

322]

There is also inhibition of the increased [Ca2+ ]i and of AA release.[

322] [323]

This inhibition is limited to the FcepsilonRI-mediated cell activation 352] [353]

but not to stimulation with either compound 48/80, the Ca2+ ionophore A23187, fMet peptides, or C5a.[ be at multiple sites in the

354 cell[ ]

The effect of the methyltransferase inhibitors could

; therefore the mechanism of their action is not clear.

Glucocorticoids

These inhibit mediator release in in vitro experiments. With human basophils this inhibition is limited to FcepsilonRI-mediated activation but not stimulation with 355]

other secretagogues (e.g., Ca2+ ionophore A23187, fMet peptides, PMA).[

In contrast, there is no inhibition of the IgE-mediated release of histamine or PGD2

from human mast cells, although there are decreases in the release of some COX products. [

356]

In both mouse mast cells and RBL-2H3 cells, dexamethasone inhibits

histamine and AA release, the increased influx of Ca2+ , and PtdIns hydrolysis, suggesting that it is acting at a very early step in signaling.[

357] [358] [359]

Secretion

360 factor.[ ]

and PtdIns hydrolysis are not blocked in permeabilized cells, suggesting that inhibition by steroids is caused by a cytosolic Inhibition requires the exposure of these cells to the steroid for several hours, probably due to a requirement for the synthesis of a PLA2 inhibitory protein. Although such an inhibitor can explain the decrease in AA release, it does not explain the inhibition of the Ca2+ entry into the cells nor the inhibition of the IgE receptor–mediated PitdIns hydrolysis. Besides effects on some of these early events that interfere with PtdIns hydrolysis and Ca2+ influx, steroids have effects at the steps downstream of calcium influx. Glucocortocoids inhibit the MAP kinase pathways, especially in the activation of ERK-2, because of effects on Raf-1 or increases in the level expression of the MAP kinase phosphatase-1. [

316] [361] [362]

The decrease in the activation of cPLA2 would result in inhibition of AA release.

RELEASABILITY There is wide variation in the extent of histamine release from the cells of both atopic and nonatopic individuals; this has been termed the releasability of the cells. The extent of stimulated histamine release from the cells of any one individual can vary from as low as no different from spontaneous release to values as high as 98 99 363

90% to 100% of total cellular histamine content. The releasability of the same cells can also vary with different secretagogues.[ ] [ ] [ ] Differences also exist in the extent of release from basophils compared with mast cells. The release by FcepsilonRI-mediated activation of the cells has drawn the most interest because it may relate to clinical allergy. There is good positive correlation between antigen-induced histamine release in vitro with basophils of allergic patients and the extent of 44 364

their symptoms. No correlation exists, however, between the number of IgE molecules present on the surface of basophils and releasibility.[ ] [ ] Variations have also been observed over time as to the releasability of the cells of the same donor. In comparing cells from monozygotic and dizygotic twins, good correlation was seen in IgE-induced histamine release only when the basophils were from monozygotic twins.[

365]

Recent evidence suggests that these differences in secretion are regulated by intracellular factors. Studies have shown that basophils of individuals who are high responders release histamine with chemically cross-linked IgE dimers; whereas the cells of the low responders release with chemically cross-linked trimers but very 65

poorly with dimers.[ ] The sensitivity of cells to different stimuli is set by differences in intracellular signaling mechanisms. It can be modified by factors such as adherence of the cells to other cells or to extracellular matrix (ECM) proteins, by cytokines, by steroids, or by dietary lipids incorporated into the membranes (see following discussions). Adhesion The adherence of cells to other cells and to the extracellular matrix regulates developmental, cellular, and immunologic processes. Such adherence is mediated by cell surface receptors. Adherence also activates intracellular signals that not only evoke new cellular responses but also modulate the response of cells to other 366 367 368 369

stimuli. Both basophils and mast cells have surface adherence receptors that can mediate binding to other cells and to ECM glycoproteins.[ ] [ ] [ ] [ ] This adherence has effects on cellular functions and may be important not only for regulating the migration of these cells into sites of inflammation but also for controlling the releasibility of these cells. Adherence of basophils and mast cells is mediated by several classes of cell adhesion molecules (CAMs), such as the integrins, many of which have been identified on these cells.[

367] [369]

There are changes in the expression of integrins in murine and human mast cells cultured with different growth factors, suggesting

modulation of integrin expression during mast cell differentiation.[

370] [371] [372]

Adherence of cells is also enhanced after stimulation, which may be caused by the

redistribution of surface adhesion receptors, their enhanced expression on the cell surface, or the modulation of their affinity to ligands.[

373] [374]

Adherence of cells not only may control their migration and localization at sites of inflammation but also may regulate their

268

proliferation and phenotype. Adherence of cells results in the aggregation of adhesion receptors and the transduction of intracellular signals, which includes reorganization of the cytoskeleton, changes in intracellular signaling, and changes in the degranulating capacity of the cells. These signals are mediated by intracellular changes induced by adhesion such as protein tyrosine phosphorylation, phosphatidylinositol hydrolysis, changes in intracellular pH or calcium, and the expression of new genes. Adherence of mast cells can transduce signals that affect cell growth, differentiation, and secretion. For example, culture of mature human 375 376

or rat mast cells on fibroblasts maintains their phenotype, viability, and secretory function.[ ] [ ] BMMCs that are derived by the culture of mouse bone marrow cells in IL-3 are immature progenitors of serosal and mucosal mast cells, although they are more similar to mucosal mast cells in morphology, histochemistry, biochemistry, and responses to secretagogues. When transferred to feeder layers of mouse 3T3 fibroblasts, BMMCs persist in the absence of IL-3, but if IL-3 is added to these co-cultures, these cells acquire a more serosal phenotype, with an increase in the content of mediators and a switch from synthesizing predominantly 377]

chondroitin sulfate proteoglycans to mostly heparin proteoglycans.[

Many of these effects are caused by the interaction of the fibroblast product, stem cell factor,

with its receptor (c-kit) on mast cells. [

378] [379]

378] [380]

Co-culture of mast cells with fibroblasts regulates secretory function.[

sulfidopeptide leukotrienes, whereas after co-culture with fibroblasts there is a shift to production of PGD2

381 382 .[ ] [ ]

82] [383]

results in an increase in surface expression of FcγRIII and enhanced degranulation by FcγR activation.[

When activated, BMMC produce

The co-culture of BMMCs with fibroblasts

RBL-2H3 cells lack responsiveness to compound

48/80 and substance P. However, when cultured for 2 weeks on fibroblasts, these cells release histamine in response to these compounds.[ of mast cells to fibroblasts modulates secretory function.

384]

Therefore, adherence

The adherence of mast cells results in changes in the cytoskeleton, cell spreading, the redistribution of secretory granules to the periphery of the cell, and the tyrosine phosphorylation of several proteins.[

153] [385] [386]

Adhesion may also change the activity of PKC.[

These proteins include FAK, Pyk2, PECAM-1 (CD31), and the cytoskeletal protein paxillin. [

218]

153] [368] [387] [388]

These changes induced by cell adherence may play an important role in modulating the intracellular signaling

that leads to degranulation. For example, there is enhanced secretion from RBL-2H3 cells that are adherent to surfaces coated with fibronectin. [

153]

FcepsilonRI aggregation of cells also results in enhanced cell adherence, indicating that it regulates CAMs. This regulation could be secondary to signals (e.g., tyrosine phosphorylation) that are induced by receptor aggregation. For example, FcepsilonRI aggregation results in the tyrosine phosphorylation of PECAM-1, to 388 389

390

which SHP-2 then binds.[ ] [ ] PECAM-1 functions in the transmigration of cells across the endothelium into sites of inflammation.[ ] Therefore the level of the tyrosine phosphorylation of PECAM-1 may modulate its interaction with other molecules, thereby regulating the migration of basophils into inflammatory sites. Cytokines Several lymphokines, such as IL-3, IL-5, IFN-γ, and granulocyte or macrophage colony-stimulating factor (G-CSF, M-CSF) enhance both FcepsilonRI and non86] [91]

receptor-mediated histamine release from human basophils.[ histamine release, suggesting a role for

391 CAMs.[ ]

Monoclonal antibodies to some but not all integrins inhibit the IL-3–enhanced anti-IgE–induced

Thus the enhancement of release with these cytokines may be caused by changes in cell adherence.

HETEROGENEITY Cells 378 380 392

The concept of heterogeneity in the population of mast cells developed from studies in rodents.[ ] [ ] [ ] Studies have found heterogeneity in human mast cells obtained from different sites in their content of proteases and cytokines (see Chapters 13 and 20 ). Different secretagogues (e.g., compound 48/80) may be used to demonstrate differences in the response of these different types of mast cells. However, all mast cells have FcepsilonRI and respond to IgE receptor or calcium ionophore stimulation with the release of mediators. As discussed, however, there are also differences in mediators generated by the different cell types. For example, human mast cells make PGD2 but also produce LTC4 and other leukotrienes. In contrast, human basophils do not generate PGD2 and produce only small amounts of the leukotrienes. Some of these differences could be caused by differences in the maturation state of the cells, which could regulate the presence or absence of receptors or enzymes.

Release Reactions An important question is whether the mechanism for mediator release is similar in the various cell systems studied, such as human mast cells compared with basophils or rat mast cells. Differences exist in the response of various cells to diverse nonphysiologic stimuli; for example, rat mast cells and human skin mast cells are stimulated with compound 48/80, whereas human basophils do not respond. Still, all systems respond to the physiologically relevant system of IgE plus antigen, although even with this system there are differences in the rate of release with different cells. For example, the rate of histamine release is faster with human mast cells than with human basophils. Many differences also exist in degranulation between mast cells and basophils. By electron microscopy, degranulation of mast cells is of the “compound” type, with granules fusing with each other, whereas in basophils, granules fuse directly with the plasma membrane. When tested with different secretagogues, several differences have been observed between mast cells and basophils. Basophils and skin mast cells respond to C5a and fMet peptides, whereas lung mast cells do not 393

release with these stimuli.[ ] Basophils also release when activated with PMA, whereas this has no effect on lung mast cells. Similarly, the LO product hydroperoxyeicosatetraenoic acid (hPETE) releases histamine from basophils but not from lung mast cells. There are also differences in mast cells isolated from different tissues; skin mast cells respond to compound 48/80, bradykinin, substance P, and morphine (similar to rat peritoneal mast cells).[

394]

However, lung,

[395] [396]

intestinal, and synovial mast cells do not respond to these compounds. Even cells from one tissue share some heterogeneity; human basophils separated by density show functional differences in response to FcepsilonRI-mediated and non-receptor-mediated

269

activation.[

397]

There is also density heterogeneity of human lung mast cells, with some differences in the quantitative but not qualitative response to secretagogues.

[398] [399]

Mast cells require higher concentrations of anti-IgE for activation than basophils. These variations could be caused by differences in the maturation of the cells, which could result in changes in the number or type of receptors expressed on cells. There are also differences in the capacity of pharmacologic agents to inhibit FcepsilonRI-mediated secretion from basophils compared with mast cells. Inhibitors of the COX pathway enhance release from basophils but not from mast cells. Histamine H2 agonists inhibit release from basophils but have no effect on mast cells, 41]

whereas PGD2 enhances the release from human basophils but has no effect on mast cells.[

Other differences include the effect of dexamethasone, which inhibits

mediator release from human basophils but not from human mast cells, and adenosine, which inhibits the response of basophils but slightly enhances mast cell release. Such differences in the effects of pharmacologic agents could be caused by the presence or absence of agonist-specific receptors on these cells. Differences have also been observed in the mediators released by mast cells compared with basophils. In contrast to mast cells, human basophils release very little, if any, of the COX-derived products of AA, such as PGD2 . However, both mast cells and basophils release LTC4 . These secondary mediators are generated by enzymes acting on released AA. The differences between basophils and mast cells could result from the presence or absence of the enzymes involved in the

production of these metabolites. Although differences exist between the basophil and mast cell, little evidence is available to suggest that the secretory process is fundamentally different in the two cell types.

INHIBITION OF RELEASE REACTION The cross-linking of IgE receptors on the basophil or mast cell results in the activation of biochemical reactions that not only result in degranulation but also function to regulate the extent of the release reaction. The extent of this degranulation of cells results from a balance between activation and deactivation signals. This section discusses the phenomenon called cell desensitization and the inhibitory molecules and signals that tightly control the activation of mast cells. Such inhibitory signals may result from transmembrane proteins that are activated from outside the cell and intracellular molecules that down-regulate the signal transduction process ( Box 16-10 ). Cell Desensitization Desensitization of basophils or mast cells refers to the decrease in secretion of mediators that is observed when a secretagogue is added under conditions that do not result in secretion and after a defined time the permissive conditions are restored, for example, adding antigen to cells in the absence of Ca2+ or at concentrations that 55 80 400 401

402 403

are supraoptimal or at nonoptimal temperatures.[ ] [ ] [ ] [ ] Cell desensitization has been studied most extensively with human basophils.[ ] [ ] When different secretagogues are used with human basophils, the desensitization is specific for the stimulus; for example, desensitization for IgE-mediated release does not 99 363

inactivate the cells for release with C5a or with the fMet peptides.[ ] [ ] The FcepsilonRI-mediated desensitization requires receptor aggregation and can be initiated by IgE dimers, although larger oligomers are more effective. The IgE-mediated desensitization can be either Box 16-10. Molecules that Generate Inhibitory Signals in Mast Cells

Cell Surface Molecules FcγRIIB

Transmembrane protein Low-affinity receptor for IgG coligation with FcepsilonRI inhibits signaling Cytoplasmic domain contains ITIM-binding SHIP.

gp49B1

Transmembrane protein Receptor for integrin; two ITIM domains in cytoplasmic domain bind SHP-1

MAFA

Mast cell function–associated antigen; transmembrane protein; lectin-type molecule Cytoplasmic domain has ITIM-like sequence that binds SHIP.

Aggregation of MAFA inhibits degranulation. Intracellular Molecules SHIP

SH2-containing inositol polyphosphate 5-phosphatase Dephosphorylates PIP3 in plasma membrane Cells from SHIP−/− mice have increased sensitivity to FcepsilonRI aggregation

Cbl

Ubiquitin ligase; ring finger protein Associates with many other proteins; forms a complex with Syk and PI3K Negatively regulates secretion May be involved in ubiquitination of proteins for degradation

PTEN

Lipid phosphatase and tensin homologue deleted on chromosome 10 Dephosphorylates PtdIns 3,4,5-P3 to PtdIns 4,5-P2, decreasing signals generated by PI3K

ITIM, Immunoreceptor tyrosine-based inhibition motif.

270

specific, that is, specific to the antigen used for the desensitization process, or nonspecific, in which case the cells do not respond to challenge with any antigen.[

404]

405

Specific desensitization occurs when there are fewer IgE molecules on basophils, whereas at high IgE levels there is nonspecific desensitization.[ ] Desensitization can occur in the absence of added Ca2+ , suggesting that it involves a step independent of the influx of Ca2+ . Further evidence for such an early step is the observation that rechallenge of desensitized cells by an IgE-dependent stimulus results in no Ca2+ influx. Desensitization is an active process, similar to the events that result in cell secretion, and is blocked by the addition of some inhibitors; thus with human basophils, but not with lung mast cells, the serine esterase inhibitor DFP decreased desensitization and enhanced release. There is a decrease in the receptor-mediated Ca2+ response of desensitized human mast cells, again suggesting that desensitization involves early steps of the release process.[

406]

407]

However, the changes in Ca2+ influx cannot alone explain desensitization [

desensitized by exposure to anti-IgE have an increased response on stimulation with PMA to activate

and basophils

408 PKC.[ ]

The mechanism of desensitization is uncertain. Cell desensitization is not caused by a loss from the cell surface of antigen-specific IgE from endocytosis or shedding

of IgE-antigen complexes.[

409] [410]

Although there is rapid endocytosis of IgE-antigen complexes from the cell surface, enough IgE-receptor complexes remain on 409

the cell surface to stimulate release. In desensitized cells there is the continued capacity of antigen to bind to the IgE on the cell surface.[ ] Similarly, nonspecific desensitization is not caused by the co-internalization of all cell surface IgE when the specific IgE is aggregated. Specific desensitization appears to be caused by an alteration in a very early biochemical event, whereas the nonspecific desensitization probably results from more distal events. Desensitization could be caused by the decay of an unstable intermediate formed by the aggregated receptors interacting with another protein. There appears to be differences in the down-regulation of the 402

pathways that lead to the release of granular contents (e.g. histamine), lipid products, and cytokines.[ ] Although most of the detectable intracellular events occur early after receptor aggregation, the generation of cytokines requires continued receptor cross-linking. PKC inhibitors have no effect on desensitization, but srcfamily kinase inhibitors block desensitization as well as secretion, suggesting that PKC has little role in down-regulating the IgE-mediated basophil response.[ Therefore, as with the activation-signaling cascade, the desensitization process depends on the activation of Src-family kinases.

411]

Inhibitory Cell Surface Molecules 412

Mast cells express a number of cell surface molecules that can regulate FcepsilonRI-induced signaling.[ ] These include FcγRIIB, gp49B1, mast cell function– associated antigen (MAFA), CD63, CD81, and a unique ganglioside recognized by mAb AA4. Some of these molecules (e.g., the ganglioside and MAFA) are close to FcepsilonRI. Inhibitory signals by these molecules optimally require their aggregation, and with gp49 and FcγRIIB, the molecules must be coligated with FcepsilonRI. Recent studies have suggested the biochemical mechanism for the inhibition by the molecules that require coligation with FcepsilonRI. The cytoplasmic domains of several of these molecules, including FcγRIIB, gp49b1, and MAFA, contain the immunoreceptor tyrosine-based inhibition motif (ITIM) with the amino acid 413]

sequence of I/VxYx2 L/V.[

In addition to initiating immunoreceptor signaling, Src-family protein tyrosine kinases such as Lyn can initiate inhibitory signals by

phosphorylating the Tyr in these ITIMs. This leads to the activation and recruitment of inhibitory molecules, such as SHP-1/-2 and SHIP. SHP-1 and SHP-2 can then dephosphorylate proteins such as the receptor subunits and decrease signal transduction, whereas SHIP, as described below, regulates signals that are important for calcium influx. FcγRIIB is a low-affinity receptor for IgG that functions in phagocytosis by binding multivalent antigen. FcγRIIB is expressed in mast cells and many other 414

hematopoietic cells and has a cytoplasmic domain with an ITIM.[ ] The inhibitory function of FcγRIIB has been clearly demonstrated in B cells, where it was shown that coligation of FcγRIIB with the B cell receptor (BCR) leads to inhibition of BCR-induced responses. The coaggregation of FcγRIIB with FcepsilonRI results in the inhibition of FcepsilonRI-mediated degranulation and generation of TNF-α. This co-aggregation of FcγRIIB with FcepsilonRI leads to the rapid tyrosine phosphorylation of the tyrosine in the ITIM of FcγRIIB by the FcepsilonRI-associated Lyn. The phosphorylated ITIM recruits SHIP, which catalyzes the removal of the 5-phosphate from PtdIns(3,4,5)P3 and inositol (1,3,4,5)P4 and the direct cleavage of PtdIns(3,4,5)P3 . This results in the inhibition of the membrane recruitment and activation of PLC-γ and Btk and therefore downstream signaling. However, the FcepsilonRI-induced tyrosine phosphorylation and activation of Syk are unaffected. FcγRIIB appears to regulate mast cell responses in vivo. For example, FcγRIIB-deficient mice exhibit increased vascular permeability in IgG-dependent passive

415

cutaneous anaphylaxis and higher mortality during systemic anaphylaxis compared with normal mice.[ ] This suggests that FcγRIIB may play a role in regulating FcepsilonRI-mediated mast cell activation even in the absence of the coligation of these two receptors. Coaggregation of FcγRIIB with FcepsilonRI may also occur under physiologic conditions, as when allergen is complexed with IgG. Such coaggregation could explain some of the beneficial effects of specific immunotherapy. Treatment of allergic patients with injections of increasing doses of allergen results in an allergen-specific IgG response. The formation of allergen-IgG complexes could then inhibit IgE-induced mast cell activation by coaggregating FcepsilonRI and FcγRIIB. gp49B1 is another transmembrane protein that has two cytosolic ITIMs. Besides mast cells, it is also expressed on macrophages and activated NK cells. The integrin αv β3 , which is expressed on endothelial cells, fibroblasts, and epithelial cells, is the ligand for gp49B1. The coligation of gp49B1 to FcepsilonRI inhibits the IgEinduced degranulation and the generation of leukotrines by a mechanism that requires the ITIM tyrosines of gp49B1 and the PTP SHP-1. A ligand for this protein, αv s3 , also may modulate mast cell responses. In vitro, there is decreased degranulation when the antigen is presented together with the recombinant integrin to BMMCs. [416]

In vivo, gp49B1-deficient mice are more sensitive to IgE-dependent passive cutaneous anaphylaxis; there is greater tissue swelling and mast cell degranulation and a lower threshold for antigen challenge, even though in vitro

271

417]

responses of mast cells isolated from these mice are normal.[

Therefore the interaction of gp49B1 with its ligand can modulate the mast cell response.

Some members of the paired immunoglobulin-like receptor (PIR) families are similar to gp49B1, with cytoplasmic ITIMs that recruit SHP-1. When expressed in RBL-2H3 cells, PIRs can inhibit FcepsilonRI-mediated signaling, although it is unclear whether they are present or have any function in mast cells. MAFA is a transmembrane protein with a cytoplasmic tail that contains a single ITIM-like sequence and an extracellular domain with significant homology to the carbohydrate-binding domain of a family of calcium-dependent lectins. The molecule binds mannose-containing saccharides. MAFA is close to or directly associated with FcepsilonRI on the cell surface. Aggregation of MAFA results in Ser and Tyr phosphorylation, which appears to have the potential to bind SHP-1, SHP-2, and 418

SHIP although the inhibitory effects appear to be caused by SHIP.[ ] When aggregated with antibodies, MAFA inhibits FcepsilonRI-induced cell activation. The original description of this molecule was on RBL-2H3 cells, but a homologous gene was identified in humans and is probably expressed in basophils and other cells. [419]

Intracellular Inhibitory Molecules Many of the molecules discussed, primarily in regard to their role in activation of cells, probably have duel functions in both initiating and inhibiting these pathways in the cells. For example, Src-family PTKs (e.g., Lyn) phosphorylate ITAMs and start the activation pathways, but at the same time, by phosphorylating ITIMs, these kinases can recruit inhibitory signaling molecules. Similarly, as described earlier, PTPs probably play an important role in regulating the level of the phosphorylation

of proteins, thereby starting and stopping signaling cascades. However, other intracellular molecules, including SHIP and c-Cbl, appear to act only to regulate FcepsilonRI-mediated signaling negatively in basophils and mast cells. SHIP (SH2-containing inositol phosphatase) is a cytoplasmic protein that has an N-terminal SH2 domain, a centrally located inositol 5-phosphatase domain, two 420

NPXY sequences that when phosphorylated can bind PTB domains, and a proline-rich sequences in the C-terminal region.[ ] As discussed, SHIP binds with its SH2 domain to the phosphorylated ITIM in several inhibitory molecules. SHIP also interacts through its SH2 and NPXY domains, with Shc and by its proline-rich region to Grb2. SHIP dephosphorylates PIP3 or inositol (1,3,4,5)P4 . This results in a decrease in the concentration of PIP3 , which is the product of PI3K. In the cell membrane PIP3 is important for recruiting to the membrane PH-containing molecules, such as Btk, PKB (Akt), PDK-1, Vav, and PLC-γ. These proteins are important for regulating the influx of calcium so that a decrease in their membrane recruitment and activation results in inhibition of the increase in intracellular calcium. Interestingly, SHIP is tyrosine-phosphorylated after FcepsilonRI stimulation even in Syk negative cells, and in vitro SHIP binds to a phosphorylated peptide 421

based on the ITAM of the β subunit of FcepsilonRI.[ ] The enzymatic activity of SHIP is not regulated by tyrosine phosphorylation, but its function in the cell depends on its localization. However, the tyrosine-phosphorylated SHIP can bind other proteins (e.g., Shc, p62dok), which may play a role in inhibiting the activation of the Ras pathway. SHIP appears to be essential in regulating the FcepsilonRI-induced cell activation for degranulation. BMMCs from SHIP-deficient mice, unlike cells from normal mice, have increased responses in PtdIns(3,4,5)P3 levels and calcium influx to both FcepsilonRI and c-Kit receptor signalling.[

422] [423]

The binding of only IgE

alone to these SHIP-deficient cells, without the need of aggregation, results in degranulation. This suggests that the level of SHIP in mast cells may regulate the extent of the activation of these cells. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a 43-kD protein that contains a phosphatase domain and has extensive homology at its Nterminus to both tensin, a cytoskeletal protein that interacts with actin filaments at focal adhesions, and auxilin, a protein involved in synaptic vesicle transport. Although initially thought to be a protein tyrosine phosphatase (PTP), it was later shown that PTEN's major enzymatic activity is to remove the 3-phosphate from PtdIns. Therefore, PTEN dephosphorylates PtdIns(3,4,5)P3 to PtdIns(4,5)P3 , decreasing the level of this important molecule and reversing the effect of PI3K. PTEN is expressed in RBL-2H3 cells, and by regulating the level of PtdIns (3,4,5)P3 in the cell, PTEN may control the activation of Btk, PKB, PKC, and the generation of IP3. Cbl (p120c–cbl ) is another cytoplasmic protein that may negatively regulate FcepsilonRI signaling. Cbl is the protein product of the c-cbl protooncogene, which structurally consists of an amino-terminal SH2 domain and a carboxy-terminal region containing a RING finger motif, a large proline-rich region, and a leucine zipper, which are motifs that mediate protein-protein interactions.[

424]

Cbl functions in down-regulating several receptor-coupled signaling pathways, including 425 426 427

immunoreceptor signaling. Cbl associates with Syk independent of the aggregation of FcepsilonRI and is tyrosine-phosphorylated downstream of Syk.[ ] [ ] [ ] Overexpression of Cbl in RBL-2H3 cells leads to inhibition of tyrosine phosphorylation and activation of Syk, resulting in suppression of mediator release. This suggests that Cbl may control the kinase activity of Syk and therefore the signaling pathways that result in degranulation. This could be a mechanism that involves the ubiquination of proteins, as Cbl can serve as a ubquitin protein ligase (E3), which by ubiquinating proteins results in their targeting for degradation. Similarly, the mutation of the tyrosine in the linker region of Syk that is the putative site for Cbl interaction results in enhanced signal transduction and increased histamine release.

CONCLUSIONS A better understanding of signaling in mast cells and basophils is crucial for advancing knowledge of immediate hypersensitivity reactions. Major advances have occurred in understanding the biochemistry and molecular biology of secretion in these cells. This knowledge could allow a more rational development of pharmacologic agents capable of abrogating these secretory events.

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Chapter 17 - Biology of Lymphocytes

Lauren Cohn Anuradha Ray

Lymphocytes are central to the development of immune responses, either in allergy or in host defense to pathogens. Although allergy is an inappropriate response to environmental antigens that leads to an injurious response, whereas host defense is an essential protective response, the same principles apply. The immune system is

made up of adaptive and innate elements of the immune response. A specific response that occurs as an adaptation to encountering a new antigen, such as an inflammatory response to contact with poison ivy, is one of the many types of adaptive immune responses that often confer immunity to a specific antigen for the life of the host. Adaptive immune responses depend on lymphocytes. Innate immune responses largely take advantage of the immediate and nonspecific responses of neutrophils and macrophages, although other inflammatory cells, including lymphocytes, have been shown to respond early to various foreign antigens. Innate immune responses appear to be less critical in the outcomes of allergic diseases compared with their protective role against pathogens. The presumed sites of exposure to allergens, including skin, respiratory tract, and gut, are under surveillance by cells of the innate limb of the immune response. The interaction of the innate response to allergens may have an important influence on the development of adaptive immune responses. This chapter focuses on T and B lymphocytes, how these cells respond to protein antigens such as allergens, and how T and B cells contribute to allergic inflammation in asthma.

T LYMPHOCYTES Adaptive immune responses are initiated by T cells bearing alpha-beta (α/β) T cell antigen receptors (TCRs), which make up a majority of T cells. TCR comprises two polypeptide chains and has an antigen-binding or variable region, a constant region, and a region that anchors TCR to the cell membrane (hinge, transmembrane, and cytoplasmic tail regions). During development in the thymus, T cells undergo TCR rearrangement, which creates unique antigen-binding domains and positive selection, which permits maturation of T cells that will recognize antigens in the context of self–major histocompatibility complex (MHC) antigens. Once released into the bloodstream, a mature T cell bears a single TCR with unique antigen specificity. An estimated 106 different TCRs exist in an individual. This extraordinary diversity of TCRs allows the individual to respond to a vast range of antigens throughout life. Two classes of α/β T lymphocytes, CD4 and CD8, are involved in adaptive immune responses and they are distinguished by the expression of this cell surface coreceptor, their interaction with different MHC molecules and by their different functions. CD4+ T cells are traditionally called T helper (Th) cells. They recognize antigen presented by class II MHC molecules, which are present only on antigen-presenting cells (APCs), including B cells, macrophages, and dendritic cells. Protein antigens are taken up by APCs and processed into peptides in endocytic vesicles, which are presented on the cell surface bound to class II MHC molecules. CD8 cytotoxic T (Tc) cells recognize antigen presented by class I MHC molecules. Class I MHC is present on the surface of all nucleated cells. CD8+ T cells are typically activated by pathogens, especially viruses, which may infect any cell. Therefore, CD8+ T cells are not induced in response to allergens, but, as discussed later, they may enhance airway inflammation in allergic diseases. CD4+ T Cell Activation When a mature CD4+ T cell is released from the thymus into the circulation, it is considered naive, having never come into contact with its specific antigen. The T cell circulates from the bloodstream into peripheral lymphoid tissue, entering the lymph node by way of specialized blood vessels, the high endothelial venules (HEVs). Naive T cells express L-selectin, which binds to vascular addressins expressed on the HEVs and in turn allows for specific homing into the lymphoid tissue. [1]

Secondary lymphoid tissue chemokine (SLC), produced by both the HEVs and cells within the lymphoid tissue, binds to its receptor (CCR7) on naive T cells and

promotes its recruitment into the lymph node.[

2]

In the peripheral lymphoid tissues (lymph nodes, spleen, mucosal lymphoid tissues), T cells come into contact with APCs that bear foreign antigens. Dendritic cells

3

(DCs) are the primary APCs that activate naive T cells.[ ] At mucosal sites, DCs form a dense network to catch antigens that penetrate the surfaces. DCs take up antigen when it binds specifically to DC surface receptors or by nonspecific macropinocytosis. B cells take up antigen by specific binding to cell surface

280

immunoglobulin, and macrophages engulf large particles and pathogens. All these APCs break down antigens into peptide fragments in endocytic vesicles, where the 3

peptides are loaded onto class II proteins and then transported to the cell surface.[ ] The act of processing antigen leads to maturation of immature DCs to mature DCs, which then leave the mucosal surface and migrate to local lymphoid tissues. In the peripheral lymphoid tissues, naive CD4+ T cells pass by APCs, sampling by cell contact, causing the TCR to interact with class II MHC and its bound peptide. If specific antigen is not encountered, the CD4+ T cell passes back into the bloodstream and continues recirculating until it contacts its antigen. When a CD4+ T cell contacts its specific antigen on an APC, it migrates no farther. For T cell activation to be initiated, two signals are required: (1) TCR recognition of MHC class II peptide and (2) a simultaneous co-stimulatory signal delivered by the same APC. If both these signals are received, the T cell goes in G1 phase of the cell cycle and begins to produce interleukin-2 (IL-2). IL-2 is an essential T cell autocrine growth factor that stimulates the T cell to progress though the cell cycle and undergo clonal expansion, giving rise to a population of effector cells with the identical TCR specificity to the parental cell ( Figure 17-1 ). The principal co-stimulatory molecules expressed on the surface of APCs are the B7 molecules, B7-1 (CD80) or B7-2

Figure 17-1 Two-signal mechanism of CD4 T cell activation. Antigen-presenting cell (APC) takes up a protein antigen and processes it into peptide fragments that are presented by class II major histocompatibility complex (MHC) molecules. The first signal required for CD4 T cell activation is recognition by the T cell antigen receptor (TCR) of the class II MHC–peptide complex. The second, co-stimulatory signal is an interaction of CD28 on the T cell with B7-1 or B7-2 on the APC. These signals stimulate interleukin-2 (IL-2) production and induce CD4 T cell proliferation. IL-2R, IL-2 receptor.

Figure 17-2 Generation of T helper types 1 and 2 (Th1, Th2) cells from a naive CD4+ T cell. A naive CD4+ T cell does not secrete cytokines and has low levels of GATA-3 and c-Maf expression. Differentiation along the Th1 or Th2 pathway is triggered by stimulation by antigen presented to the T cell receptor in the context of the major histocompatibility complex (MHC) by the appropriate antigen-presenting cell (APC) and a second signal imparted by ligation of co-stimulatory molecules B7-1/B7-2 and CD28. Dendritic cells (DCs) represent the key APCs for naive T cells and are classified into two subsets, mature (DC1) and immature (DC2). The mature DCs express high levels of MHC class II on their surface and produce interleukin-12 (IL-12), which drives Th1 differentiation. The immature DCs express low levels of MHC class II and produce interleukin-10 (IL-10), which favors Th2 differentiation. Furthermore, the cytokines present in the microenvironment, IL-4/ IL-10 versus IL-12, play a decisive role in orchestrating the differentiation along the Th1 or Th2 lineage.

Figure 17-3 Molecular mechanisms of differentiation of a naive CD4+ T cell into Th2 cells. A naive CD4+ T cell contains a condensed chromatin structure with extensive methylation. Stimulation by antigen together with engagement of the interleukin-4 receptor (IL-4R) results in activation of signal transducer and activator of transcription-6 (STAT-6, Stat6), which in turn causes specific demethylation around the IL-4/IL-5/IL-13 locus (similarly, antigen plus IL-12 causes demethylation around the IFN-γ locus). Chromatin remodeling is accompanied by induction of Th2-specific transcription factors (e.g., GATA-3, c-Maf), which bind to target sequences in the IL-4/IL-5/IL-13 locus. The chromatin, rendered accessible by demethylation and perhaps by binding of GATA-3, c-Maf, and other as yet undiscovered Th2-specific transcription factors, is next bound by more widely expressed and transiently induced transcription factors (e.g., AP-1, NF-κB, NF-Atc, C/ EBPβ). This may allow synergistic interactions between the tissue-specific and general transcription factors, resulting in the active transcription of the IL-4, IL-5, and IL-13 genes. Effector/memory cells are postulated to be in a state of suspended animation with an open chromatin structure and high levels of GATA-3 and c-Maf expression. Restimulation of these cells by antigen would result in transient induction of the general factors, leading to rapid induction of Th2 gene expression. TCR, T cell antigen receptor.

Figure 17-4 B cell activation requires CD4 T cell “help.” The B cell is stimulated to differentiate into an antibody-producing cell when it comes into contact with an activated CD4 T cell that has the same antigen specificity. Two signals are required for B cell activation: (1) T cell antigen receptor (TCR) recognition of the class II MHC–peptide complex and (2) interaction of CD40L on the activated T cell with CD40 on the B cell. Once these signals have been transmitted, B cells undergo proliferation, immunoglobulin (Ig) heavy-chain class-switching, and affinity maturation. IL-4–producing CD4 Th2 cells are potent inducers of B cell antibody production, stimulating both proliferation and heavy-chain class switching to immunoglobulin E (IgE).

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1999. 141. Huang TJ, MacAry PA, Eynott P, et al: Allergen-specific Th1 cells counteract efferent Th2 cell-dependent bronchial hyperresponsiveness and eosinophilic inflammation partly via IFN-γ, J Immunol 166:207, 2001.

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155. Warren HS, Kinnear BF, Phillips JH, et al: Production of IL-5 by human NK cells and regulation of IL-5 secretion by IL-4, IL-10, and IL-12, J Immunol 154:5144, 1995. 156. Walker C, Checkel J, Cammisuli S, et al: IL-5 production by NK cells contributes to eosinophil infiltration in a mouse model of allergic inflammation, J Immunol 161:1962, 1998. 157. Korsgren M, Persson CG, Sundler F, et al: Natural killer cells determine development of allergen-induced eosinophilic airway inflammation in mice, J Exp Med 189:553, 1999. 158. Mikus LD, Rosenthal LA, Sorkness RL, et al: Reduced interferon-gamma secretion by natural killer cells from rats susceptible to postviral chronic airway dysfunction, Am J Respir Cell Mol Biol 24:74, 2001. 159. Hayday AC: γ/δ Cells: a right time and a right place for a conserved third way of protection, Annu Rev Immunol 18:975, 2000. 160. Wen L, Barber DF, Pao W, et al: Primary γ/δ cell clones can be defined phenotypically and functionally as Th1/Th2 cells and illustrate the association of CD4 with Th2 differentiation, J Immunol 160:1965, 1998. 161. Krug N, Erpenbeck VJ, Balke K, et al: Cytokine profile of bronchoalveolar lavage-derived CD4+, CD8+, and γ-δ T cells in people with asthma after segmental allergen challenge, Am J Respir Cell Mol Biol 25:125, 2001. 162. Spinozzi F, Agea E, Bistoni O, et al: Local expansion of allergen-specific CD30+Th2-type γ/δ T cells in bronchial asthma, Mol Med 1:821, 1995. 163. Lahn M, Kanehiro A, Takeda K, et al: Negative regulation of airway responsiveness that is dependent on γ/δ T cells and independent of α/β T cells, Nat Med 5:1150, 1999. 164. Zuany-Amorim C, Ruffie C, Haile S, et al: Requirement for γ/δ T cells in allergic airway inflammation, Science 280:1265, 1998. 165. Gauchat JF, Henchoz S, Fattah D, et al: CD40 ligand is functionally expressed on human eosinophils, Eur J Immunol 25:863, 1995. 166. Gauchat JF, Henchoz S, Mazzei G, et al: Induction of human IgE synthesis in B cells by mast cells and basophils, Nature 365:340, 1993. 167. Muller KM, Rocken M, Joel D, et al: Mononuclear cell-bound CD23 is elevated in both atopic dermatitis and psoriasis, J Dermatol Sci 2:125, 1991. 168. Yu P, Kosco-Vilbois M, Richards M, et al: Negative feedback regulation of IgE synthesis by murine CD23, Nature 369:753, 1994. 169. Corry DB, Kheradmand F: Induction and regulation of the IgE response, Nature 402:B18, 1999. 170. Haczku A, Takeda K, Hamelmann E, et al: CD23 exhibits negative regulatory effects on allergic sensitization and airway hyperresponsiveness, Am J Respir Crit Care Med 161:952, 2000. 171. Coyle AJ, Wagner K, Bertrand C, et al: Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine

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Chapter 18 - Biology of Neutrophils

Antonio Maurizio Vignola Jean Bousquet

Neutrophils are known to play an important role in inflammatory responses by virtue of their ability to perform a series of effector functions that collectively represent a major mechanism of innate immunity against injury and infection. In recent years, however, it has become obvious that the contribution of neutrophils to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. Neutrophils can be induced to express a number of genes whose products lie at the core of inflammatory and immune responses. These mediators include not only Fc receptors, complement components, cationic antimicrobial proteins, and NADPH oxidase proteins, but also a variety of cytokines, growth factors, and chemokines.[

1]

Neutrophils are present in increased numbers at sites of inflammation and play an important role in the pathogenesis of many lung disorders (e.g., cystic fibrosis, chronic obstructive bronchitis, asthma) and diseases of the lung parenchyma (e.g., emphysema, adult respiratory distress syndrome). Neutrophilic inflammation may 2 3 4

be increased in asthmatic patients during exacerbations,[ ] [ ] [ ] but the mechanisms involved in their recruitment and activation remain largely unknown. This chapter examines the origins and immunologic fate of neutrophils, with a focus on their possible importance in causing asthma.

NEUTROPHIL MIGRATION AND ACTIVATION Neutrophils migrate from the blood vessel lumen into the lung interstitium and reach the airway lumen, where they may play an active role during infection and inflammation. Neutrophil maturation and migration from the bone marrow to the airways is a complex process driven by the release of a wide range of chemotactic factors and the expression of a several adhesion molecules. Mature neutrophils are incapable of cell division, and their sustained generation from the bone marrow (1011 cells per day in a normal adult) is the result of a highly controlled process of myelopoiesis. Precursor cells divide and differentiate from the pluripotent hematopoietic stem cell and proceed to committed stem cells, which provide lineage-restricted progenies. During maturation, neutrophils acquire their granule products, which equip the activated neutrophils to kill microorganisms and to participate to the development of an inflammatory response. 5

During migration and on arrival in the lung, neutrophils may contribute to tissue inflammation and injury.[ ] Tissue injury prevails when neutrophils accumulate in high numbers, when they receive inappropriate stimuli, or when the activity of their products is not adequately controlled. Neutrophils are equipped with a great variety of pre-formed compounds, which are stored in different types of neutrophil granules. These compounds include serine and metalloproteases, oxygen radicals, lipid mediators, and defensins. It is unlikely that any one of these families of toxins acts in isolation at inflammatory sites. For the purposes of description in this chapter, however, each is considered separately. Myeloid Development Neutrophils originate in bone marrow from stem cells that have the property of self-renewal. These stem cells have the capacity to develop into committed pluripotent progenitors. Studies of these cells in culture indicate that pluripotent progenitor cells are intermediate progenitors at one stage, with the capacity to differentiate further into monopotent progenitors, resulting in a single type of leukocyte. The first stage of differentiation of the mature neutrophil from the monopotent progenitors is recognized as the myeloblast. This cell develops into the promyeloblast. Subsequently, these cells mature into metamyelocytes and then into band forms, which are occasionally seen in the circulation at times of stress, such as severe infection. The final step of development is into the mature neutrophil. The duration of this maturation process is 10 to 15 days and is followed by the influx of neutrophils at certain sites ( Figure 18-1 ). This process depends on the production of cells in the bone marrow, detachment from the marrow microenvironment, and immigration into the blood, followed by the adherence to the endothelium and emigration into inflamed tissues. Many factors act cooperatively to influence each of these individual steps. Within the bone marrow, changes in the adherence of hematopoietic cells are likely to play an important role in neutrophil maturation. Adherence is mediated by weak but multivalent carbohydrate-protein interactions and by stronger protein-protein associations. Such a bone marrow–specific adhesion system seems to play an important role in the development and subsequent release of precursors from the bone marrow. Specific molecules play a crucial role in such a system. For example, hemonectin, a bone marrow glycoprotein, assists in the specific local attachment of immature granulocytes.[

6]

292

Figure 18-1 Identification of neutrophils in bronchial biopsy and induced sputum samples obtained from severely asthmatic subjects. Neutrophils are phenotypically identified by an immunostaining with a specific monoclonal antibody against neutrophil elastase. Immunoreactive neutrophils show a red staining.

and hydrogen peroxide (H2 O2 ).[

57]

The further reaction of these radicals produces much more toxic oxygen radicals, such as HOC1 , through the catalytic effect of 57]

myeloperoxidase in neutrophils, as well as the most potent hydroxyl radical (OH• ) through a reaction involving the reduction of ferrous in ferric iron.[

Release of oxygen radicals from neutrophils of other inflammatory cells apparently plays an important role in causing tissue damage at sites of inflammation and is likely associated with neutrophil mediated lung damage in emphysema and possibly with chronic bronchitis and asthma. Previous studies examining the role of oxygen radicals in the pathogenesis of airway diseases have implicated the xanthine–xanthine oxidase system in the pathogenesis of acetylcholine AHR in cats.[ Studies have demonstrated that generation is

295

58]

increased from neutrophils of asthmatic subjects compared with normal controls[ generation in patients with COPD.[

59]

and that AHR correlates with

60]

More recently the potential role of OH• in causing ozone-induced airway neutrophil influx and airway hyperresponsiveness in dogs has been studied. These studies demonstrated that pretreatment with the antioxidant allopurinol and deferoxamine significantly inhibited ozone-induced AHR.[ inhibitor of xanthine oxidase and thus prevents the formation of and H2 O2 [

62]

61]

Allopurinol is a competitive

; it is also a powerful oxidant scavenger with direct reducing activity against OH• . Deferoxamine is an iron-chelating agent that, by binding iron, 63

prevents the formation of iron-dependent OH• radicals.[ ] This inhibition occurred without a concomitant reduction in the magnitude of ozone-induced neutrophil infiltration into the airways. These studies suggest that OH• released from activated neutrophils is important in ozone-induced AHR. Lipid Mediators A variety of lipid mediators can be released from activated neutrophils. Those implicated in causing neutrophil migration (and possibly activation) into the airways are LTB4 and platelet-activating factor (PAF). Evidence also indicates that platelets are an important source of thromboxane A2 (TXA2 ) in the circulation when TXA is released from neutrophils at sites of tissue inflammation. Neutrophils are a source of both LTB4 and PAF, both of which have the capacity to cause neutrophil migration and activation.[

64]

Thus neutrophils have the capacity

to magnify a neutrophil inflammatory response once initiated. Interestingly, neutrophils also have the ability to metabolize these mediators and therefore potentially to turn off neutrophil migration. Both LTB4 and PAF have been implicated in causing airway disease, particularly asthma, because they are present in asthmatic airways or released after challenge of the airways with inflammatory stimuli, such as allergens. Inhalation of LTB4 has also been shown to cause neutrophil migration and airway hyperresponsiveness in canine, but not human, airways, whereas inhalation of PAF has been shown to cause AHR in human subjects in some studies. Selective antagonist for LTB4 are not yet available; however, studies with selective PAF antagonists on allergen-induced asthmatic responses have not yet supported an important role for PAF in causing these responses or for PAF antagonists in treating asthma. The importance of thromboxane in causing airway hyperresponsiveness in dogs has come from studies demonstrating that inhibition of thromboxane synthesis using OKY-046 prevented the development of AHR after LTB4 , ozone, allergen, and PAF challenge in dogs. Also, pretreatment with the thromboxane U46619 increased acetylcholine airway responsiveness in dogs and primates and transiently increased methacholine AHR in humans. However, studies using potent thromboxane receptor antagonists have not demonstrated inhibition of ozone-induced neutrophil migration and AHR in dogs or any effect of thromboxane synthase inhibitor on the development of allergen-induced AHR in human subjects.

Defensins 65 66 67

The human defensin family now comprises six members, four of which are known to be expressed in neutrophils.[ ] [ ] [ ] Human neutrophil defensins, or human neutrophil peptides (HNP-1 to HNP-4) are small (relative molecular mass 3.54•kD) cationic peptides that were first identified on basis of their antimicrobial activity. 68]

Mature defensins are stored in a dense subset of azurophilic granules that contain no or little elastase and myeloperoxidase[

and that constitute 30% to 50% of the

total protein content of the azurophilic granules. Neutrophil defensins display several activities that may be relevant to inflammation and the immune response.[

69]

NEUTROPHIL CLEARANCE AND DEATH Many of the neutrophils produced daily undergo apoptosis either before leaving the bone marrow or within inflamed tissues. Apoptosis is the most common form of physiologic cell death and a necessary process to maintain cell numbers in multicellular organisms. In many chronic inflammatory diseases, including asthma, 70

reduced cell death of different types of granulocytes is one important mechanism for cell accumulation.[ ] Granulocytes are constantly produced in large amounts in the bone marrow, and the same numbers die, under normal circumstances, within a defined period. Changing the rate of apoptosis rapidly changes cell numbers in such systems. Overexpression of interleukin-5 (IL-5) appears to be crucial for delaying eosinophil apoptosis in many allergic disorders, whereas overexpression of GM-CSF and granulocyte colony-stimulating factor (G-CSF) is associated with suppression of neutrophil apoptosis in bacterial and nonbacterial inflammation.[ Cytokine withdrawal leads to the induction of apoptosis both in vitro and in vivo.

71]

In contrast to the role of survival cytokines, little is known about the role of death factors and their receptors in the regulation of granulocyte apoptosis. Recent observations suggest a role for mitochondria in both eosinophil and neutrophil apoptosis, although the mechanisms that trigger mitochondria to release proapoptotic factors remain to be determined. Besides similarities, there are differences in the regulation of apoptosis between these granulocyte subtypes, including both 71 72

expression and function of Bcl-2 and caspase family members.[ ] [ ] Nuclear factor kappa B (NF-κB) activation has been shown to be a critical regulator of human granulocyte apoptosis in vitro, possibly through regulation of the production of proteins that protect neutrophils from the cytotoxic effects of various cytokines, including tumor necrosis factor alpha (TNF-α). [

73]

74

Apoptotic neutrophils can be removed by macrophages or by dendritic cells (DCs).[ ] With macrophages, neutrophils are degraded, whereas with DCs, one apoptotic process can lead to antigen presentation and autoimmunity. Antiapoptotic signals generated by growth factors and cytokines through integrin-mediated signaling can affect neutrophil survival after neutrophils leave the bone marrow during diapedesis and at the site of bacterial intrusion or inflammation. Macrophage clearance of apoptotic neutrophils occurs even for cells that still have their granules intact and therefore is a crucial step in helping resolve the inflammatory process. [75]

Corticosteroids can increase the ability of macrophages to exert the phagocytosis of apoptotic cells.[

76]

NEUTROPHILS IN ASTHMA Neutrophils are one of the first inflammatory cells to be recruited into the airways after exposure to infectious agents

296

77]

and to allergens.[

Although their role in the pathogenesis of asthma remains to be clarified, neutrophils have been described in the airways of both allergic and

nonallergic asthmatic patients, [

78] [79] [80]

even though their numbers in bronchial biopsies is usually low. Previous studies have also shown that neutrophils can be

recovered in the sputum of asthmatic patients,[

2] [81]

although in low numbers in BAL fluid.[

80] [82]

IL-8, a chemokine involved in neutrophil recruitment, and 83] [84]

neutrophil-derived mediators such as myeloperoxidase were not found to be increased in BAL of asthmatic patients. [ IL-8 and IL-5 have been shown during an acute exacerbation, with IL-8 concentrations also reduced at resolution.

In contrast, increased levels of sputum

[2]

Because neutrophils can be detected in some but not in all asthmatic subjects, these cells may play a role in the development of specific asthma subphenotypes. 85] [86]

Indeed, neutrophil numbers have been found to be increased in the airways during the late-phase reaction after an allergen challenge,[ died within hours after an asthma corticosteroid-dependent asthma[

87 88 exacerbation,[ ] [ ]

91] [92]

[89]

in nocturnal asthma,

in some patients with long-standing

90 asthma,[ ]

in some patients who

and in those with severe

(see Figure 18-1 ).

Acute Asthma Neutrophils are key players in acute or in fatal asthma. After bronchial allergen challenge an increased number of neutrophils has been described in the airways of 85

asthmatic subjects,[ ] suggesting the ability of neutrophils to respond to allergen stimulation. Although the mechanisms underlying neutrophil activation in acute asthma are still poorly understood, it has recently been suggested that their ability to express receptors for IgE might play an important role. Neutrophils isolated 93

from asthmatic subjects express the high-affinity receptor for IgE (FcepsilonRI), and its engagement leads to the release of IL-8.[ ] The lack of FcepsilonRI expression by neutrophils isolated from normal subjects indicates that the expression of this receptor is not constitutive but is modulated by factors released during inflammation. Previous in vivo and in vitro studies have shown that surface expression of many Fc receptors on human neutrophils is up-regulated or induced after treatment with cytokines. In particular, the T helper cell type 2 (Th2) cytokines, GM-CSF, and IL-4 have been shown to induce the expression of the low-affinity 94

95

receptor for IgE (CD23) and of CD11b/CD18 molecules.[ ] Because GM-CSF and IL-4 receptors are expressed by human neutrophils,[ ] neutrophils may become more prone to respond through an IgE-dependent mechanism under the effects of these Th2 cytokines. This may explain why the use of a specific anti-CD4+ 96

antibody inhibits not only allergen-induced recruitment of eosinophils, but also the corresponding recruitment of neutrophils in sensitized mouse airways.[ ] However, Th2-driven neutrophil recruitment may also occur independently of an IgE-mediated mechanism. In vivo experimental evidence shows that T lymphocyte– related cytokines, such as IL-17 and IL-9, can link the activation of T lymphocytes to the recruitment and activation of airway neutrophils. The IL-17–induced neutrophil recruitment is mediated through induced CXC chemokine release by steroid-sensitive mechanisms and is modulated by the release of endogenous tachykinins. These effects of IL-17 are potentiated by other proinflammatory cytokines, such as IL-1β and TNF-α. [ Neutrophil activation can also be modulated by IL-9[

98] [99]

97]

through the stimulation of the IL-9 cell surface receptor, whose expression is increased in asthma and can

lead to an increased release of IL-8. Interestingly, as with IgE receptors, IL-9 receptor expression also can be induced by GM-CSF and IL-4,[

98]

supporting the

concept that neutrophils are an important target of Th2 cytokines ( Figure 18-2 ). From a pathophysiologic standpoint, increased neutrophils numbers in acute asthma may lead to several consequences. The release of oxygen free radicals and proteases contributes to tissue damage and amplifies the inflammatory process. In addition, neutrophil elastase can reproduce many of the pathologic features of asthma or chronic bronchitis, including mucous gland hyperplasia,[

100]

excess mucus secretion,[

101]

epithelial damage,[ 104]

Neutrophil elastase can also promote neutrophil recruitment in the lung by inducing IL-8 production[ bronchoconstriction and AHR mediated by neutrophil accumulation and 5-LO

105 products.[ ]

41] [102]

103]

and connective tissue destruction.[

and may be involved in antigen-induced

Furthermore, elastase exposure has been found to increase directed

fibroblast migration through the extracellular matrix, a phenomenon that may play a role in the development of airway remodeling in asthma[

106] [107]

( Figure 18-2 ).

Neutrophils can also play a role in fatal asthma. A study comparing the cellular infiltrate in sudden-onset versus slow-onset cases of fatal asthma demonstrated significantly fewer eosinophils but more neutrophils in sudden-onset than in slow-onset cases.[

87] [108]

Such a relationship was also shown between the number of

109 episode.[ ]

eosinophils and the duration of terminal These results therefore suggest a rapid, possibly transient, involvement of neutrophils in the earlier phases of the asthma attack that is followed by the subsequent

Figure 18-2 Mechanisms of neutrophil activation during airway inflammation in asthma. Neutrophils express membrane receptors for immunoglobulin E (IgE) and T helper type 2 cytokines, such as interleukin-4 (IL-4), interleukin-9 (IL-9), interleukin-17 (IL-17), and grnaulocyte-macrophage colony-stimulating factor (GM-CSF). The functional consequence of this phenomenon is the release of a wide range of mediators, including chemokines, lipid mediators, proteases, oxygen radicals, and growth factors, which can contribute to the development of both acute and chronic airway inflammation and remodeling.

Figure 18-3 Hypothesis for a possible role of neutrophils in causing asthma. Neutrophil recruitment and activation are likely mediated by allergen exposure and release of T helper cell type 2 cytokines through activated T lymphocytes. In the airways, activated neutrophils release inflammatory mediators and proteases, which are able to increase neutrophil activation through autocrine or paracrine mechanisms or modulate other airway cells (e.g., macrophages). In turn, these latter cells can also contribute to neutrophil activation. Release of proteases and growth factors (e.g., TGF-β) causes damage and remodeling of the airways. These events may contribute to alterations of the immune system, leading to a greater susceptibility to both viral and bacterial infections that specifically target and activate the bronchial epithelium. These redundant and self-perpetuating mechanisms may play an important role in the development of an ongoing inflammatory process associated with airway remodeling and reduced responsiveness to corticosteroids (CS).

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145. Vignola AM, Riccobono L, Mirabella A, et al: Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis, Am J Respir Crit Care Med 158:1945, 1998. 146. Wenzel SE, Schwartz LB, Langmack EL, et al: Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics, Am J Respir Crit Care Med 160:1001, 1999. 147. Bousquet J, Jeffery PK, Busse WW, et al: Asthma: from bronchoconstriction to airways inflammation and remodeling, J Allergy Clin Immunol 105:1720, 2000. 148. Payne DN, Balfour LI: Children with difficult asthma: a practical approach, J Asthma 38:189, 2001. 149. Balfour LI: Difficult asthma: beyond the guidelines, Arch Dis Child 80:201, 1999. 150. Weissler JC: Syndromes of severe asthma, Am J Med Sci 319:166, 2000. 151. Chung KF: Difficult, therapy-resistant asthma: definition and clinical features, Monaldi Arch Chest Dis 55:459, 1999. 152. Vachier I, Roux S, Chanez P, et al: Glucocorticoids induced down-regulation of glucocorticoid receptor mRNA expression in asthma, Clin Exp Immunol 103:311, 1996. 153. Gibson PG, Simpson JL, Saltos N: Heterogeneity of airway inflammation in persistent asthma: evidence of neutrophilic inflammation and increased sputum interleukin-8, Chest 119:1329, 2001. 154. Oakley RH, Sar M, Cidlowski JA: The human glucocorticoid receptor beta isoform: expression, biochemical properties, and putative function, J Biol Chem 271:9550, 1996. 155. Hamid QA, Wenzel SE, Hauk PJ, et al: Increased glucocorticoid receptor beta in airway cells of glucocorticoid-insensitive asthma, Am J Respir Crit Care Med 159:1600, 1999. 156. Leung D, Hamid Q, Vottero A, et al: Association of glucocorticoid insensitivity with increased expression of glucocorticoid receptor beta, J Exp Med 186: 1567, 1997. 157. Strickland I, Kisich K, Hauk PJ, et al: High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids, J Exp Med 193:585, 2001. 158. Vignola AM, Chanez P, Chiappara G, et al: Transforming growth factor-β expression in mucosal biopsies in asthma and chronic bronchitis, Am J Respir Crit Care Med 156:591, 1997. 159. Fava RA, Olsen NJ, Postlethwaite AE, et al: Transforming growth factor beta 1 (TGF-β1 ) induced neutrophil recruitment to synovial tissues: implications for TGF-β-driven synovial inflammation and hyperplasia, J Exp Med 173:1121, 1991. 160. Cassatella MA: The production of cytokines by polymorphonuclear neutrophils, Immunol Today 16:21, 1995.

161. Fava RA, Casey TT, Wilcox J, et al: Synthesis of transforming growth factor-β1 by megakaryocytes and its localization to megakaryocyte and platelet αgranules, Blood 76:1946, 1990. 162. Chu HW, Trudeau JB, Balzar S, et al: Peripheral blood and airway tissue expression of transforming growth factor beta by neutrophils in asthmatic subjects and normal control subjects, J Allergy Clin Immunol 106:1115, 2000. 163. Rajah R, Nachajon RV, Collins MH, et al: Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle, Am J Respir Cell Mol Biol 20:199, 1999. 164. Stetler-Stevenson WG: Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention, J Clin Invest 103:1237, 1999. 165. Shipley JM, Wesselschmidt RL, Kobayashi DK, et al: Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice, Proc Natl Acad Sci U S A 93:3942, 1996. 166. Shipley JM, Doyle GA, Fliszar CJ, et al: The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases: role of the fibronectin type II-like repeats, J Biol Chem 271:4335, 1996. 167. Legrand C, Gilles C, Zahm JM, et al: Airway epithelial cell migration dynamics: MMP-9 role in cell-extracellular matrix remodeling, J Cell Biol 146:517, 1999. 168. Mautino G, Henriquet C, Oliver N, et al: Elevated levels of tissue inhibitor of metalloproteinase-1 in bronchoalveolar lavage of asthmatic patients, Lab Invest 79:39, 1999. 169. Hoshino M, Nakamura Y, Sim J, et al: Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation, J Allergy Clin Immunol 102:783, 1998. 170. Lemjabbar H, Gosset P, Lamblin C, et al: Contribution of 92•kDa gelatinase/type IV collagenase in bronchial inflammation during status asthmaticus, Am J Respir Crit Care Med 159:1298, 1999. 171. Bousquet J, Lacoste J, Chanez P, et al: Bronchial elastic fibers in normal subjects and asthmatic patients, Am J Respir Crit Care Med 153:1648, 1996. 172. Wright R: Elastic tissue of normal and emphysematous lungs: a tridimensional histologic study, Am J Pathol 34:355, 1961. 173. McCarthy DS, Sigurdson M: Lung elastic recoil and reduced airflow in clinically stable asthma, Thorax 35:298, 1980. 174. Brackel H, Bogaard J, Kerrebijn F: The compressibility of central airways in healthy subjects and patients with severe asthma, Am Rev Respir Dis 141:A848, 1990.

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Chapter 19 - Biology of Eosinophils

Hirohito Kita Cheryl R. Adolphson Gerald J. Gleich

*

The eosinophil granulocyte, although likely first observed by Wharton Jones in 1846 in unstained preparations of peripheral blood, was so named by Paul Ehrlich in 1 2

1879 because of the intense staining of its granules with the acidic dye eosin.[ ] [ ] Since then the eosinophil has been the subject of extensive investigation. Its occurrence in such disparate conditions as parasitic infections, presumably for the benefit of the host, and hypersensitivity diseases, to the detriment of the patient, 1

although paradoxical, has become better understood as a consequence of newer information. [ ] This chapter reviews the biologic and biochemical aspects of eosinophils and the mechanisms of their recruitment and activation, then discusses the potential roles of eosinophils in human diseases and host defense.

MORPHOLOGY, PRODUCTION, AND DISTRIBUTION OF EOSINOPHILS Morphology Although eosinophils share some structural similarities with other granulocytes, the mature human eosinophil is slightly larger than the neutrophil, having a diameter 3

of 12 to 17••m on blood films,[ ] and the nucleus is usually bilobed. In addition, eosinophils are characterized by their distinctive granules, as shown by their staining properties with acid dyes such as eosin and by their unique electron microscopic appearance ( Figure 19-1 ). The distinctive specific or secondary granules are 4

composed of an electron-dense core and an electron-lucent matrix. High-power views of the core show a crystalline lattice[ ] ; therefore the core is likely a single substance.

Figure 19-1 Electron photomicrograph of human eosinophil leukocyte with crystalloid-containing granules showing dense cores (C) of various shapes embedded in less dense matrix (M) (×10,000). (From Gleich GJ, Adolphson CR: Adv Immunol 39:177, 1986; photomicrograph courtesy M.S. Peters, MD.)

(From Gleich GJ, Adolphson CR: Adv Immunol 39:177, 1986; photomicrograph courtesy M.S. Peters, MD.) 5

The number of specific granules is around 30 per cross section.[ ] Three other types of eosinophil granules have been described, including primary granules, small granules, and secretory vesicles. Primary granules are round, uniformly electron dense, present at 1 to 3 granules per cross section, and characteristically seen in 4

immature eosinophilic promyelocytes. Small granules are present at 2 to 8 granules per cross section and contain acid phosphatase and arylsulfatase.[ ] Secretory vesicles, also referred to as microgranules or tubulovesicular structures, are the most abundant in number, about 160 per cross section, and are characterized by their small, dumbbell-shaped structures and their albumin content.[

5]

* Portions of this chapter were adapted from and used with permission from the following sources: Gleich GJ, Adolphson CR, Leiferman KM: Eosinophils. In Gallin JI, Goldstein IM, Snyderman R, editors: Inflammation: basic principles and clinical correlates, ed 2, New York, 1992, Raven Press, Chapter 32, pp 663–700; Gleich GJ, Kita H, Adolphson CR: Eosinophilis. In Frank MM, Austen KF, Claman HN, et al, editors: Samter's immunologic diseases, vol 1, Boston, 1995, Little, Brown, Chapter 14, pp 205–245; Gleich GJ: Eosinophil granule proteins and bronchial asthma, Allergol Int 45:35, 1996 (review); Kita H, Gleich GJ: The eosinophil: structure and functions. In Kaplan AP, editor: Allergy, ed 2, Philadelphia, 1997, WB Saunders, Chapter 11, pp 148–178; Gleich GJ, Adolphson CR, Kita H: The

eosinophil and asthma. In Busse WW, Holgate ST, editors: Asthma and rhinitis, ed 2, Malden, Mass, 2000, Blackwell Science, Chapter 30, pp 429–479.

306

Eosinophils also contain varying numbers of lipid bodies, which are non-membrane-bound, lipid-rich inclusions. The numbers of lipid bodies are increased in activated eosinophils and may play roles as extranuclear sites for formation of eicosanoid mediators.[

6]

Growth and Differentiation 3

Eosinophils are produced in bone marrow from pluripotential stem cells.[ ] The latter cells differentiate into a progenitor different from those for neutrophils or 7

3

monocytes. [ ] This progenitor is capable of giving rise to mixed colonies of basophils and eosinophils, pure basophil colonies, or pure eosinophil colonies,[ ] suggesting a common progenitor, but the degree of differentiation toward one or the other lineage is stochastic. A common eosinophil-basophil progenitor is further supported by several observations. Charcot-Leyden crystal protein and major basic protein, two proteins previously considered unique to eosinophils, are also found 4

in mature basophils.[ ] Normal cord blood precursors cultured with interleukin-3 (IL-3) and interleukin-5 (IL-5) in a plastic vessel coated with Matrigel developed into hybrid cells with both eosinophil and basophil characteristics. [

8]

Production of eosinophils in the human bone marrow involves a cascade of interdependent regulatory events driven by cytokines. Among various hematopoietic 4 7

factors, those important for eosinophil proliferation and differentiation are IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5.[ ] [ ] These cytokines are encoded by closely linked genes on chromosome 5q31 and bind to receptors that have a common beta (β) chain and different alpha (α) chains. IL-3 has the broadest specificity and can act directly on the CD34+ stem cell. IL-3 leads to the expansion of the megakaryocytes, erythrocytes, monocytes, basophils, and neutrophils, with eosinophils representing 10% of total colonies.[

3] [8]

When infused in the cynomolgus monkey, IL-3 alone leads to a modest leukocytosis, with 3

more than a tenfold increase in levels of circulating basophils and eosinophils. [ ] GM-CSF likely acts on an overlapping but more mature precursor population. In vitro stimulation of human bone marrow precursors with GM-CSF induces the formation of eosinophil, neutrophil, and macrophage colonies, with eosinophils representing up to 25% of all colonies. [

4] [7]

In acquired immunodeficiency syndrome (AIDS) patients, GM-CSF administration induces a major increase in

circulating and bone marrow eosinophils and neutrophils, with a modest increase in circulating monocytes.[

3]

Thus, IL-3 and GM-CSF are relatively nonspecific and stimulate proliferation of neutrophils, basophils, and eosinophils. In contrast, IL-5 potently and specifically 3

stimulates eosinophil production by the bone marrow.[ ] For example, IL-5 forces differentiation of human basophil or eosinophil progenitors into the eosinophil 7

9

lineage in vitro.[ ] High plasma levels of IL-5 occur in humans with eosinophilia, including patients with episodic angioedema with eosinophilia,[ ] recombinant IL-2 10]

therapy,[

and Onchocerca volvulus infection.[

11]

Furthermore, overproduction of IL-5 in transgenic mice results in profound eosinophilia, [

12]

and deletion of the

13 challenge.[ ]

IL-5 gene causes a reduction of eosinophils in the blood and lungs, even after an allergen In humans a study in allergic asthmatic patients treated with monoclonal antibody (mAb) directed against IL-5 has confirmed that blocking IL-5 greatly reduces blood and airway eosinophils and that this effect is long-lasting. [14]

The malignant expansion of T cell clones that produce IL-5 in some patients with lymphoma induces marked eosinophilia.[

15]

Some evidence indicates that

certain cytokines can inhibit eosinophil progenitor growth and differentiation. Transforming growth factor beta (TGF-β) suppresses the eosinophil pathway and aids 16]

in the switch from eosinophil to basophil commitment.[

Interferon alpha (IFN-α) inhibits colony formation by human bone marrow multipotential, erythroid, 7

granulocyte-macrophage, and eosinophil progenitor cells in vitro[ ] and has been used to treat certain patients with eosinophilia.[

17]

Life Cycle and Distribution The life cycle of the eosinophil may be divided into bone marrow, blood, and tissue phases. Although the eosinophil is a formed element of the peripheral circulation, 3

it is primarily a tissue-dwelling cell. In humans the tissue eosinophil/blood ratio is about 100:1.[ ] Furthermore, eosinophils tend to reside in those tissues where the epithelial surfaces are exposed to the external environment (gut); mast cells primarily reside in these tissues as well. Thus, eosinophils are considered merely to “pass 4

through” the circulation en route to the tissues.[ ] The specific localization of eosinophils and mast cells in mucosal tissues points to their roles in the mucosal immune response. 4

Eosinophil kinetics appear to be very complex, and methodologic differences may account for some of the differences in reported results.[ ] For example, a study of three hematologically normal patients (all with surgically treated cancer) in which dividing cells were labeled by a single injection of [3 H]-thymidine suggested two populations of eosinophils with differing proliferative activities: one with a rapid bone marrow emergence time of about 10 hours and a second with a slow emergence time of about 80 hours. Another study found a mean bone marrow maturation and storage time of about 4.3 days. Once the eosinophil has entered the blood, it has a short half-life, ranging from 8 to 18 hours. 4

After circulating in the blood, eosinophils migrate into the tissues, probably by diapedesis at endothelial intercellular junctions[ ] by a mechanism involving cytokines and adhesion molecules (see later discussion). Under normal conditions, once eosinophils enter the tissues, most do not recirculate. The tissue life span of eosinophils ranges from 2 to 5 days, depending partly on the tissue studied. However, cytokines increase eosinophil survival in vitro up to 14 days; thus, they might also prolong eosinophil survival in vivo. Finally, several possible mechanisms exist for clearing the tissues of eosinophils. These include shedding of cells across mucosal surfaces into the lumen of the gut or the respiratory tract, engulfment of apoptotic eosinophils by macrophages,[

18]

4

and lysis or degranulation with accompanying cell degeneration.[ ] The striking

extracellular deposition of eosinophil granule proteins, often in the virtual absence of intact eosinophils, shows that eosinophil degranulation occurs in tissues.[

19]

Under normal circumstances, a balance exists between bone marrow production and release of eosinophils, their circulation, and their entrance into the tissues. Changes in one or in several of these functional compartments may cause an increase or decrease in the number of circulating eosinophils. Eosinophilia is associated with hypersensitivity and helminth

307

infections. In rats given intravenous injections of Trichinella spiralis larvae, there was a delay of about 2 days before significant peripheral eosinophilia was detected; 4

maximum eosinophilia occurred at 6 to 7 days. [ ] In the bone marrow the percentage of morphologically recognizable eosinophils increased by 5 days, preceding the maximum peripheral eosinophilia by 1 to 2 days. In fact, patients exposed to a large quantity of antigens have a temporal eosinopenia, suggesting rapid recruitment 20

and sequestration of eosinophils from blood into tissues.[ ] Furthermore, eosinopenia from glucocorticoids, catecholamines, and a variety of acute bacterial or viral infections may result from eosinophil margination or failure of release from the bone marrow. Alternatively, very rapid increases in circulating eosinophils, as seen in certain helminth infections or acute hypersensitivity diseases, might result from redistribution of marginated eosinophils into the circulation or shortening of the bone 21]

marrow reserve emergence time rather than from accelerated proliferation of bone marrow precursors.[

EOSINOPHIL MEDIATORS Eosinophils are unique among circulating leukocytes in their prodigious capacity to wage chemical warfare. These cells are endowed with numerous highly basic and cytotoxic granule proteins that are released on eosinophil activation or during cell necrosis ( Box 19-1 ). Eosinophils also possess an arsenal of enzymes designed to inflict oxidative damage on biologic targets. Granule Proteins Mature eosinophils contain three major types of granules: the specific granules, small granules, and secretory vesicles. The specific granules contain major basic protein (MBP, localized in the crystalloid core), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), βglucuronidase, and secretory phospholipase A2 (PLA2 ); the latter five are localized in the matrix.

Box 19-1. Secretory Products of Eosinophils

Granule Proteins Major basic protein (MBP) MBP homologue (MBP-2) Eosinophil peroxidase (EPO) Eosinophil cationic protein (ECP) Eosinophil-derived neurotoxin (EDN) Charcot-Leyden crystal (CLC) protein Phospholipase A2 (PLA2 , secretory)

Acid phosphatase Arylsulfatase B β-Glucuronidase Bactericidal/permeability-increasing protein (BPI) Lipid Mediators Leukotriene B4 (LTB4 , minimal) Leukotriene C4 (LTC4 ) Leukotriene C5 (LTC5 ) 5-HETE 5,15- and 8,15-diHETE 5-Oxo-15-hydroxy-6,8,11,13-ETE Prostaglandins E1 and E2 (PGE1 , PGE2 ) 6-Keto-prostaglandin F1 (PGF1 ) Thromboxane B2 (TXB2 ) Platelet-activating factor (PAF) Reactive Oxygen Intermediates Superoxide radical anion Hydrogen peroxide (H2 O2 ) Hydroxy radicals Cytokines

*

Interleukin-1 alpha (IL-1α) Interleukin-2 (IL-2) Interleukin-3 (IL-3)

Interleukin-4 (IL-4) Interleukin-5 (IL-5) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interleukin-10 (IL-10) Interleukin-12 (IL-12) Interleukin-6 (IL-16) Granulocyte-macrophage colony-stimulating factor (GM-CSF) Tumor necrosis factor alpha (TNF-α) Transforming growth factor alpha (TGF-α) Transforming growth factor beta-1 (TGF-β1 ) Platelet-derived growth factor-b (PDGF-b) Vascular endothelial growth factor (VEGF) Nerve growth factor (NGF) RANTES Macrophage inflammatory protein-1 alpha (MIP-1α) Monocyte chemoattractant protein-1 (MCP-1) Eotaxin Leukemia inhibitory factor (LIF) Enzymes Collagenase 92-kD gelatinase Modified from Kita H, Gleich GJ: The eosinophil: structure and functions. In Kaplan AP, editor: Allergy, ed 2, Philadelphia, 1997, WB Saunders, p 153. HETE, Hydroxyeicosatetraenoic acid; ETE, eicosatetraenoic acid; diHETE, dihydroxyeicosatetraenoic acid; RANTES, regulated on activation, normal T cells expressed and secreted. * Physiologic significance of these cytokines needs to be confirmed.

308

The specific granules of human eosinophils also have lysosome-associated membrane protein (LAMP)-1 and LAMP-2, which are acidified upon cell activation, and the granule is a secretory lysosome.[

22]

The small granules contain enzymes, including acid phosphatase and arylsulfatase B. Secretory vesicles contain membrane23]

bound cytochrome b558 (phox-22), CD11b, the alpha chain of a β2 integrin, and albumin.[

The secretory vesicles of eosinophils may represent a compartment that

holds membrane-bound receptors and other proteins that can be rapidly mobilized after cellular activation. Table 19-1 summarizes the properties of the proteins purified from the specific crystalloid-containing granules. Major Basic Protein

Originally, major basic protein was so named because (1) it accounts for about 55% of the granule protein, (2) its isoelectric point (pI) is greater than pH 10, and (3) it is proteinaceous in nature. More recently a homologue for MBP, major basic protein-2 (MBP-2), has also been characterized.[

24] [25]

TABLE 19-1 -- Properties of Human Eosinophil Granule Proteins and Encoding Complementary DNA and Genes Site

Mr (×

Major Basic Protein (MBP)

10−3

)

Isoelectric Point

*

Cell Content

†

Molecular Biology Activities

cDNA

Gene

Core

13.8

11.4

7–20

Potent helminthotoxin and cytotoxin; unique, bactericidal, strong platelet agonist; causes histamine release from basophils and rat mast cells; neutralizes heparin; increases bronchial reactivity to methacholine in primates; provokes bronchospasm in primates; activates neutrophils

∼900•nt (preproMBP)

3.3•kb 5 introns 6 exons Centromere 11q12

8.7

4–10

Potent cytotoxin; causes histamine release release from basophils; causes and LTC4

∼900•nt

4.6•kb Centromere 11q12

Major Basic Protein-2 (MBP-2) ND

13.4

superoxide and IL-8 release from neutrophils Eosinophil Cationic Protein (ECP)

Matrix

18–21

10.8

5

Potent ∼725•nt (pre-EDN) helminthotoxin, in UTR potent neurotoxin, weak RNase activity, bactericidal, virucidal; inhibits cultures of peripheral blood lymphocytes; causes histamine release from rat mast cells; neutralizes heparin and alters fibrinolysis

8.9

3

Potent neurotoxin, potent RNase activity, virucidal, weak helminthotoxin; inhibits cultures of peripheral blood lymphocytes; identical to eosinophil protein X

∼1.2•kb 1 intron Chromosone 14 q24q31

Eosinophil-Derived Neurotoxin (EDN) Matrix

18–19

Eosinophil Peroxidase (EPO)

∼725•nt (pre-ECP) in ∼1.2•kb 1 intron UTR Chromosome 14 q24q31

Matrix

66

10.8

12

In presence of H2 O2 plus halide: kills microorganisms and tumor cells; causes histamine release and degranulation from rat mast cells; inactivates leukotrienes In absence of H2 O2

∼2500•nt (2106•nt ORF)

12•kb 11 introns 12 exons Chromosome 17

plus halide: kills Brugia microfilariae; damages respiratory epithelium; provokes bronchospasm in primates. Generates reactive oxidants and radical species Modified from Gleich GJ, Adolphson CR, Leiferman KM: Eosinophils. In Gallin JI, Goldstein IM, Snyderman R, editors: Inflammation: basic principles and clinical correlates, New York, 1992, Raven Press. Mr, Molecular weight; nt, nucleotides; kb, kilobases; ND, not determined; LTC4 , leukotriene C4 ; IL-8, interleukin-8; RNase, ribonuclease; H2 O2 , hydrogen peroxide; UTR, untranslated region; ORF, open reading frame. * Calculated from amino acid sequences deduced from the cDNAs. † •g/106 eosinophils.

309

26]

Human MBP is a 13.8-kD single polypeptide rich in arginine and has five unpaired cysteines, with a calculated pI of 11.4.[ does not show high sequence similarity to any known

27 proteins.[ ]

MBP does have weak sequence identity (23% to 28%) with C-type lectin domains of mannose-

binding protein, human lithostathine, and the lectin domain of the low-affinity IgE receptor, FcepsilonRII.[ different

29 promoters[ ]

With the exception of MBP-2, MBP

27] [28]

Finally, in humans the gene encoding MBP has two

generating two different transcripts of 1.0 and 1.6 kilobases (kb), respectively. The rationale for two promoter regions and the generation of

two transcripts is not known. The nucleotide sequence of the MBP complementary deoxyribonucleic acid (cDNA) indicates that MBP is translated as a 23-kD to 25.2-kD preproprotein with a 3

calculated pI of 6.0 to 6.2. The 9.9-kD pro section is rich in glutamic and aspartic acids and has a calculated pI of 3.9.[ ] The combination of the pro section and MBP yields a molecule of 207 amino acids, pro-MBP, with approximately equal numbers of strongly basic and strongly acidic amino acids and a resulting pI of 6.2. ProMBP lacks the cytostimulatory and cytotoxic properties of MBP. One possible function of the pro section of pro-MBP is that the pro section protects the cell during transport from the Golgi apparatus to the eosinophil granule.[ pro sections are highly acidic, and the MBP molecules are

30]

Interestingly, genes encoding mouse, guinea pig, and rat MBP and pro-MBP have been reported; the

28 basic.[ ]

Analyses of the maturation of eosinophil granules in cultures of IL-5–stimulated umbilical cord stem cells have indicated that pro-MBP localizes to large eosinophil 31

granules in developing cells.[ ] In these differentiating eosinophils, pro-MBP appeared between days 6 and 18, whereas MBP appeared between days 12 and 24. Western blots showed sequential processing of the 33-kD pro-MBP to an 18-kD intermediate form and finally to 13.8-kD MBP. By dual-label immunoelectron microscopy, pro-MBP was localized primarily to large uncondensed eosinophil granules, whereas MBP was localized to granules containing a condensed central area. Pro-MBP is expressed in differentiating eosinophils and processed as the granule condenses in a multistep process to 13.8-kD MBP. Overall, the pro section likely neutralizes the toxic properties of MBP during the transport of pro-MBP to the eosinophil granule. During eosinophil differentiation, posttranslational processing of the pro piece of pro-MBP occurs in the granule, followed by MBP condensation, ultimately forming the characteristic crystalline core of the mature eosinophil. 4 7

Studies of human eosinophils by immunoelectron microscopy have also shown that MBP is present in granule cores.[ ] [ ] MBP is not in other eosinophil organelles or in plasma cells, lymphocytes, or neutrophils. Recent immunoelectron microscopy studies located MBP uniformly distributed throughout the coreless granules of eosinophils; in the crystalloid granules, MBP was located only in the core.[ MBP, indicating a differentiation process that occurs in the

31 granule.[ ]

32]

These findings support those described earlier for the localization of pro-MBP and

When basophils are isolated by flow cytometry using fluorescein-labeled anti-IgE, the 4

isolated cells show immunofluorescence staining for MBP in a granular pattern. [ ] Furthermore, MBP is measurable in extracts of basophils by radioimmunoassay; the MBP content of basophils from normal individuals averages 0.14••g MBP/106 cells, whereas eosinophils from normal individuals contain 4.98••g MBP/106 cells. [4]

Thus, MBP and Charcot-Leyden crystal protein (see later discussion), as specific markers for eosinophils, should be used in conjunction with other criteria. MBP is also localized by immunofluorescence in mast cells close to eosinophils, for example, in nasal polyps, mastocytosis specimens, and ileal tissues, but not in normal skin mast cells. When injected into normal skin, MBP rapidly localizes to granules.[

33]

Recent studies found that human cord blood–derived mast cells express

abundant MBP messenger ribonucleic acid (mRNA), suggesting mast cells are also capable of producing MBP.[

34]

Pro-MBP is the predominant or only molecular form of MBP in the blood of pregnant women; pro-MBP circulates as a complex with pregnancy-associated plasma protein A (PAPP-A), with angiotensinogen, and with complement C3dg.[ cell in the

4 placenta,[ ]

and these cells intensely express MBP

trimester and usually show a marked rise just before

37 mRNA.[ ]

38 labor.[ ]

35] [36]

This pregnancy-associated MBP has been localized by immunofluorescence to the X

Serum levels of this pregnancy-associated pro-MBP become elevated during the first

However, the function of pro-MBP during pregnancy is obscure.

A breakthrough occurred with the discovery that PAPP-A is in the metzincin superfamily of metalloproteases and is able to cleave insulin-like growth factor (IGF)– 39]

binding protein 4.[

Cleavage of IGF-binding protein 4 by PAPP-A releases IGF activity; IGF-I and IGF-II have many biologic activities, including effects on cell

metabolism, proliferation, differentiation, and migration. Recombinant PAPP-A also cleaves IGF-binding protein 4.[ disulfide bonds to neutralize the protease activity of

40 PAPP-A.[ ]

40]

Pro-MBP appears to irreversibly bind through

Thus, pro-MBP functions as a novel enzyme inhibitor that is able to inhibit PAPP-A and to modify

its ability to release active IGF activity during primate pregnancy. Interestingly, pro-MBP serum levels predict Down's syndrome.[

41]

4

MBP and certain other basic proteins, including protamine, directly damage schistosomula of Schistosoma mansoni.[ ] When schistosomula are incubated with purified MBP, MBP binds to the membrane of the schistosomula and causes its disruption. Additionally, when eosinophils, in the presence of antibody from S. mansoni–infected patients, attack the schistosomula, MBP is released from eosinophils (as determined by the presence of MBP in the culture supernatants), and MBP 3

3

is deposited on the surface of the parasites.[ ] MBP has toxicity against other helminths, including T. spiralis newborn larvae,[ ] Brugia pahangi, Brugia malayi microfilariae, [

42]

4

and Trypanosoma cruzi.[ ] MBP and ECP killed stationary phase Staphylococcus aureus in a simple nutrient-free buffer. In addition, MBP killed 4

Escherichia coli. MBP, but not EPO, EDN, or ECP, stimulates histamine release from human basophils.[ ] This noncytolytic MBP-induced basophil histamine release is inhibited by several agents, such as calmodulin antagonist, theophylline, and inhibitors of PLA2 . Basophil pretreatment with pertussis toxin results in a concentration-dependent inhibition of the release, suggesting that a guanosine triphosphate (GTP) regulatory protein may be involved in the release mechanism.[ MBP, but not EDN or ECP, induces a concentration-dependent chemiluminescence in neutrophils. MBP also induces superoxide and lysozyme

3]

310

release from neutrophils.[

43]

44]

MBP and EPO, but not ECP or EDN, stimulate the release of platelet 5-hydroxytryptamine in a noncytolytic manner.[

IL-8 production and release from human intestinal

MBP induces

45 myofibroblasts.[ ]

4

Several other properties of MBP are known.[ ] MBP neutralized the effect of heparin on blood clotting (as did protamine), and MBP increased the clotting time from threefold to fivefold. The target of MBP action on coagulation is not known. The crystal structure of MBP indicates that it is a member of the C-type lectin family. [27]

However, MBP does not coordinate intercellular calcium (Ca++ ) and lacks the invariant residues involved in carbohydrate binding by other lectins. MBP does possess the capacity to bind sulfated sugars, and in vitro assays show that MBP binds specifically to heparin. MBP regulated the generation of both classic and alternative amplification pathway C3 convertases. However, the effect of MBP is more pronounced on the classic complement pathway than on the alternative pathway. 4

MBP is toxic to murine tumor cells and to other mammalian cells in a dose-related manner.[ ] MBP likely exerts its toxic effect by disrupting the integrity of lipid bilayers. [ ] Furthermore, MBP concentrations in the toxic range (1.5 × 10−6 to 10 × 10−6 M) are measurable in body fluids of patients with diseases associated with eosinophilia. Thus, eosinophils likely release granule proteins in vivo and, through release of MBP, probably damage tissues in hypersensitivity diseases. For 46

28]

example, MBP causes ciliostasis and exfoliation of respiratory epithelial cells, an effect that mimics the pathology of asthma.[ localized to sites of bronchial epithelium damage in patients with

47 asthma.[ ]

Furthermore, MBP has been

A recent analysis of peptides from MBP indicated that two peptides (18–45 and 89–

117) stimulated histamine release from basophils and caused cytotoxicity in K562 cells; this 89–117 peptide contains a unique carbohydrate-binding region.[

48]

The numbers of eosinophils in the peripheral blood of patients with asthma, as well as the numbers of eosinophils and the concentrations of MBP in bronchoalveolar lavage (BAL) fluid of patients with asthma and of monkeys, are correlated with the severity of bronchial hyperreactivity.[

28] [49]

Therefore, to test whether MBP

50 monkeys.[ ]

mediates bronchial hyperreactivity, MBP, EPO, EDN, and ECP were directly instilled into the trachea of cynomolgus MBP and EPO provoked a transient bronchoconstriction immediately after instillation that resolved by 1 hour; EDN and ECP were inactive. Furthermore, MBP instillation resulted in a tenfold dose-related increase in airway responsiveness to inhaled methacholine. None of the other eosinophil proteins had an effect on airway responsiveness. Thus, MBP likely has a direct role in the pathophysiology of airway hyperresponsiveness by interacting with respiratory epithelial cells. Interestingly, polyglutamic acid is a polyanion that neutralizes the toxic effects of MBP in vitro[ cynomolgus

50 monkeys,[ ]

51]

and antagonizes its in vivo ability to increase respiratory resistance and bronchial hyperreactivity in

suggesting that the cationic nature of MBP contributes significantly to its capacity to cause injury and physiologic changes. However,

recent studies using a novel MBP homologue indicate that the high net-positive charge of MBP is important but not essential for certain of its biologic activities.[ MBP is also an antagonist for M2 muscarinic receptors. [

28]

24]

In lungs the release of acetylcholine from the vagus nerve contracts smooth muscle by stimulating

muscarinic M3 receptors. At the same time the acetylcholine feeds back onto inhibitory muscarinic M2 receptors on the nerve endings, thus limiting further release of acetylcholine and preventing bronchoconstriction. In some patients with asthma, these M2 receptors are dysfunctional. A recent report studied airways from three patients with fatal asthma; eosinophils were abundant (196 eosinophils) and close to (less than 4••m) nerves, with extracellular MBP often seen adhering to the nerves; specimens from nonasthmatic patients showed no extracellular MBP and only one eosinophil in proximity (p < .001).[ cholinergic nerves through specific adhesion molecules, leading to eosinophil activation and degranulation.

[53]

52]

In vitro, eosinophils adhere to

Thus, MBP may alter M2 function in asthma. 24

MBP-2, the recently discovered homologue, has a calculated pI of 8.7 and thus is about 100 times less basic than MBP itself.[ ] Both MBP and MBP-2 have identity of 42 amino acids of the approximately 117 total amino acids. The MBP and MBP-2 genes have recently been localized to chromosome 11 in the centromere to 11q12 region.[

54]

These genes for MBP and MBP-2 have diverged considerably, likely after a gene duplication event. In terms of protein expression, pro-MBP is

expressed in bone marrow and placenta, whereas the proMBP-2 is expressed only in bone marrow.[

24]

Purified eosinophil granules contain both MBP and MBP-2, 25

and sufficient MBP-2 has been isolated from granule lysates to characterize this protein. MBP-2 has not been detected in basophils.[ ] Analyses comparing the biologic activities of MBP with MBP-2 showed that MBP-2 had effects similar to MBP in cell killing and neutrophil (superoxide anion production and IL-8 release) and basophil (histamine and leukotrieneC4 release) stimulation assays, but usually with reduced potency. Overall, MBP-2 has distinctive physical properties compared with MBP, but the lack of a high net positive charge for MBP-2 does not impair certain of its biologic activities. Eosinophil Cationic Protein

3 4 55

Eosinophil cationic protein is a remarkably basic protein, with pI of 10.8, and consists of a single polypeptide chain of 15.5•kD.[ ] [ ] [ ] The ECP amino acid sequence has 67% identity to EDN and 32% to human pancreatic ribonuclease (RNase) A. The native protein has three glycosylated forms, ranging from 18 to 21• kD. ECP possesses RNase activity and is also known as RNase-3, although its activity is about 100 times less than that of EDN. The 725–base pair (bp) cDNA for ECP shows 89% sequence identity with that reported for EDN. The cDNA sequence codes for a preprotein of 160 amino acids and a protein of 133 amino acids. Both EDN and ECP genes have been localized to the q24 to q31 region of human chromosome 14. It is likely that these two genes arose as a consequence of a gene 56

57

duplication event about 30 million years ago.[ ] The crystal structure of ECP has been recently reported and is similar to that of RNase A and EDN.[ ] Although certain residues are conserved as in all other RNase A homologues, considerable divergence is also evident. The distribution of cationic residues on the surface of the molecule may be important for understanding the cytotoxicity of ECP. In general, the RNase activity of ECP is required for its neurotoxic and antiviral properties, but not for its antibacterial and antihelminthic activities[

55]

(see Table 19-1 ).

During secretion, ECP has been thought to undergo a conformational change. This difference has been used to

311

differentiate resting eosinophils from activated eosinophils, in which the mAb EG1 recognizes the stored form from resting eosinophils, and the mAb EG2 recognizes 4

the activated state.[ ] However, more recent studies suggest that these two antibodies probably cannot differentiate between these forms.[ matrix of secondary eosinophil granules, but a recent report documented the presence of ECP in

58] [59]

ECP is found in the

60 neutrophils.[ ] 1 4

ECP is a potent toxin for parasites including schistosomula of S. mansoni, and in a comparative test, ECP was eightfold to tenfold more active than MBP.[ ] [ ] The ECP killing of schistosomula is qualitatively different from that of MBP. ECP produces complete fragmentation and disruption of schistosomula, whereas MBP produces a distinctive ballooning and detachment of the tegumental membrane. The MBP-induced phenomena mimics the changes observed with eosinophils. On a 4]

molar basis, ECP is a more potent helminthotoxin for schistosomula than MBP, but MBP is more abundant in the eosinophil granule.[ 3

ECP is also toxic for newborn larvae of T. spiralis[ ] and microfilariae of B. pahangi and B. malayi in vitro. MBP, ECP, and EDN kill B. pahangi and B. malayi in a dose-dependent manner.[

42]

3]

In the case of newborn larvae of T. spiralis, ECP is more toxic on a molar basis than MBP.[

Secretions from lower airways of patients infected with respiratory syncytial virus (RSV) contain ECP and EDN.[ suspensions in vitro, the cells produced a concentration-dependent reduction in virus infectivity.[ dependent antiviral

63 64 activity.[ ] [ ]

Eosinophil-Derived Neurotoxin

62]

61]

When eosinophils were added to RSV

Furthermore, both EDN and ECP showed ribonuclease-

Eosinophils contain a powerful neurotoxin that can severely damage myelinated neurons in experimental animals. M.H. Gordon first described this neurotoxic reaction in 1932, and it is still referred to as the Gordon phenomenon. Extracts of highly purified eosinophil granules produce the Gordon phenomenon. Patients with the idiopathic hypereosinophilic syndrome and patients with cerebrospinal fluid (CSF) eosinophilia due to a variety of causes exhibit varied neurologic abnormalities. [1] [4]

Neurotoxic eosinophil granule proteins may be important in central nervous system (CNS) disease in humans. Eosinophil-derived neurotoxin was initially 4

purified by centrifugation of sonicated human eosinophils and by chromatography of the supernatant. [ ] Subsequently, EDN has been isolated from human eosinophil granules and has a molecular mass of about 18.6•kD. [

65]

Immunoelectron microscopy localized EDN to the granule matrix.[

1] [4]

Recombinant EDN has also been prepared from bacteria, baculoviris, and eukaryotic cell cultures. With ECP, EDN belongs to the pancreatic RNase A superfamily and is also known as RNase-2 or RNase Us ; it is also found in human liver, lung, urine, and spleen. [

66]

EDN has also been detected in neutrophils.[

60]

EDN shows

3

36% amino acid sequence homology with RNase A and marked amino acid sequence homology (67%) with ECP.[ ] Significant differences still exist in the 4

sequences, however, which likely explains the differences in reactivity with polyclonal[ ] and monoclonal[ homologous to pancreatic RNase A, EDN has about 100 times more RNase activity than ECP.

[3]

65]

antibodies. Although both EDN and ECP are

Interestingly, pancreatic RNase by itself does not produce the

65 rabbits,[ ]

3

Gordon phenomenon when injected intrathecally into and it is not toxic to newborn larvae of T. spiralis. [ ] It seems likely that these targets do not possess a receptor for the RNase molecule but do so for EDN. The relationship of the RNase activity of EDN to its neurotoxic and helminthotoxic properties remains to be defined. A 725-bp full-length cDNA clone for EDN has been isolated. There is a 27–amino acid leader sequence preceding a 134-residue mature EDN with a predicted molecular mass of 15.5•kD. The encoded amino acid sequence is identical to that reported for urinary RNase and human liver RNase. The mRNA for EDN was 67

68

detected in both eosinophils and mature neutrophils.[ ] High-resolution crystal structures for native EDN and complexed EDN have been reported. [ ] The structures show differences between EDN and RNase A at the ribonucleolytic active site. In vitro recombinant EDN (50•nM) decreased by fortyfold the infectivity in 62 64

63

RSV group B suspensions. [ ] [ ] ECP also shows antiviral activity, but it is less potent on a molar basis.[ ] Semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) showed loss of viral genomic RNA after exposure to EDN; thus EDN could promote the direct ribonucleolytic destruction of extracellular 4

virions. Tests of purified EDN for toxicity to schistosomula of S. mansoni showed little reactivity. [ ] When EDN was tested against newborn larvae of T. spiralis, 3

significant toxicity was observed at 12 hours. However, in comparison with ECP, a tenfold greater concentration of EDN was required.[ ] Additionally, EDN is toxic 42]

to the microfilariae of B. pahangi and B. malayi, although less so than purified MBP, ECP, or EPO.[ Eosinophil Peroxidase

Eosinophil peroxidase is one of a family of mammalian peroxidases that includes myeloperoxidase, lactoperoxidase and thyroid peroxidase. EPO differs from neutrophil or monocyte myeloperoxidase by its distinctive differences in absorption spectra and heme prosthetic groups. Human EPO has been purified using either whole-blood leukocytes or eosinophil granules. Depending on the report, EPO has a molecular mass ranging from about 67•kD (by gel electrophoresis) to 77•kD (by gel filtration). EPO consists of two subunits, a heavy chain of 50 to 58•kD and a light chain of 10.5 to 15.5•kD in a 1:1 stoichiometry, and has pI of 10.8. By 4

radioimmunoassay the cell content of EPO is 15••g/106 eosinophils.[ ] The cDNA clone for EPO has been isolated and has an open reading frame, 2106•bp,

3

corresponding to a prosequence, L chain, and H chain, in this order.[ ] The human EPO gene maps to chromosome 17q23.1, which is 34•kb from the myeloperoxidase and lactoperoxidase genes.[

69]

Earlier studies have shown that myeloperoxidase (from neutrophils and monocytes) in the presence of hydrogen peroxide (H2 O2 ) and halide kills bacteria, viruses, Mycoplasma, and fungi. Because activated eosinophils produce a respiratory burst with generation of the superoxide anion and its dismutation product, H2 O2 , and 4

thus have an active halogenation capability, the killing of microorganisms by the EPO + H2 O2 + halide system has been tested.[ ] Purified EPO in the presence of H2 O2 is able to oxidize halides to form highly reactive hypohalous acids. Analyses of the preference of EPO for halides showed that eosinophils prefer bromide over 3

chloride.[ ] Serum contains concentrations of approximately 100•mM of chloride, 100 to 200••M bromide, and less than 1••M iodide. Therefore the physiologic concentration of bromide is high enough to generate hypobromous acid, a powerful

312

oxidant. However, serum also contains 20 to 120••M of thiocyanate, a pseudohalide whose peroxidative product, hypothiocyanous acid, is a weak, primarily sulfhydryl-reactive oxidant. Tests of EPO substrate preference showed that 1••M of thiocyanate is a potent inhibitor of the reactivity of EPO with bromide or iodide 70

(both present at 100••M).[ ] Furthermore, even subphysiologic concentrations of thiocyanate, 3.3 to 10••M, almost completely blocked the bromide-dependent toxicity of EPO for 51 Cr-labeled aortic endothelial cells. Thus, hypothiocyanous acid, best known as a bacteriostatic agent in saliva and milk, may be a major oxidant produced by EPO in physiologic fluids. At plasma concentrations of bromide (20 to 120••M) and thiocyanate (20 to 100••M), both hypobromous acid and 71]

oxidation products of thiocyanate are produced by EPO, but thiocyanate is preferred 2.8-fold over bromide as a substrate. [ 72]

EPO is a central participant in generating reactive oxidants and radical species by activated eosinophils.[

For example, eosinophil activation in vivo can produce

73 residues.[ ]

oxidative damage of proteins through bromination of tyrosine Biopsy specimens from asthma patients challenged with allergen showed increased numbers of eosinophils and levels of EPO; the BAL fluid from the same patients showed a greater than tenfold increase in 3-bromotyrosine content. Thus, eosinophil activation could result in oxidative damage of proteins through bromination of their tyrosine residues. Eosinophils are also a major source of nitric oxide (NO)– derived oxidants in specimens from patients with severe asthma.[

74]

Furthermore, EPO was identified as an enzymatic source of the nitrating intermediates leading to

protein nitration. A third role for EPO involves its oxidation of thiocyanate to hypothiocyanite (OSCN− ) and cyanate (OCN− ). [ covalently modify crucial cellular or extracellular SH-containing biologic targets. EPO in the presence of Br− can brominate bases in nucleotides and double-stranded DNA.[

76] [77]

Shen et al[

76]

75]

These two ions are thought to

exposed calf thymus DNA to low concentrations of

H2 O2 and plasma levels of halides in the presence and absence of EPO and detected brominated adenine only in the presence of EPO. Henderson et al[

77]

tested both

EPO and activated eosinophils in the presence of H2 O2 and bromide with the nucleobase uracil; both experiments showed the production of brominated uracil. Thus,

eosinophils could brominate nucleobases at sites of inflammation. Whether eosinophil-rich sites of inflammation (e.g., long-term parasitic infections) lead to EPOmediated generation of potentially mutagenic base analogues in vivo remains an open question. Both EPO and lactoperoxidase are enriched in airways of patients with asthma; in vitro these peroxidases act as catalytic sinks for NO, inhibiting its bronchodilator 78

function.[ ] Using preconstricted tracheal rings from rats, the NO-dependent bronchodilation could be reversibly inhibited by physiologic concentrations of EPO, lactoperoxidase, or myeloperoxidase. Thus a complicated bidirectional relationship may exist between these peroxidases and NO at sites of inflammation. EPO plus H2 O2 and halide (Cl− , Br− , or I− ) kills not only a variety of microorganisms, such as E. coli, schistosomula, microfilariae of B. pahangi and B. malayi, 4

trypanosoma, toxoplasma, and mycobacteria, but also mast cells and tumor cells (see below).[ ] A recent report investigated EPO and superoxide in the killing of E. 79

coli; when EPO was blocked by azide, the rate of killing decreased greatly.[ ] When both superoxide and EPO were inhibited, there was no further decrease in E. coli killing, suggesting that superoxide acts in conjunction with EPO. In the case of B. pahangi and B. malayi microfilariae, the EPO + H2 O2 + halide system is extremely toxic even at EPO concentrations as low as 5 × 10−9 M (in the presence of iodide).[

42]

42]

pahangi and B. malayi than were MBP, ECP, and EDN.[ −

over

On a molar basis, EPO + H2 O2 + halide was more toxic for B.

However, the biologic significance of these observations is tempered by the finding that EPO prefers SCN

70 Br− .[ ]

EPO binds to mast cell granules, and the EPO–mast cell granule complex catalyzes the iodination of proteins and the killing of microorganisms. EPO supplemented 4

by H2 O2 and halide induced a noncytotoxic rat mast cell degranulation and histamine release.[ ] The EPO–mast cell granule complex was more effective than free EPO in the stimulation of mast cell secretion. These findings led to the proposal that eosinophils either by secretion or by cell lysis release EPO, and that EPO in the presence of H2 O2 (generated by eosinophils or other phagocytes in the area) and halide (chloride, iodide, or bromide) initiates mast cell secretion. The cytokine tumor necrosis factor alpha (TNF-α) stimulates highly purified eosinophils to damage human umbilical vein endothelial cells (HUVECs) as a model of human endothelium. This effect is enhanced by adding physiologically relevant concentrations of Br− , which suggests the participation of EPO. Furthermore, superoxide generation by eosinophils is enhanced by TNF-α.[

80]

Thus, these results suggest a role for the EPO + H2 O2 + halide system in the inflammatory response. Whether

the proposed reactions occur in disease requires further study. One report has shown leakage of EPO from eosinophil granules into the cytoplasm and apparent 4]

extracellular release after allergen provocation of human nasal membranes.[

Binding of EPO to microbes such as Staphylococcus aureus, T. cruzi, and Toxoplasma gondii greatly potentiates their killing by mononuclear phagocytes. Interestingly, tumor cells adsorb EPO, and this binding potentiates their lysis by H2 O2 . The EPO-coated tumor cells are spontaneously lysed by activated macrophages, which are able to release H2 O2 , resulting in the possibility of a synergistic action between EPO and H2 O2 in the destruction of tumor cells. These findings plus the observations that MBP is toxic to tumor cells indicate that the eosinophil could function to limit the spread of tumors. The combination of EPO and 4

MBP has significant cytotoxicity on acute lymphocytic leukemia and IM-9 cells in vitro.[ ] Experiments demonstrating that EPO bound to pneumocytes resulted in cell lysis, whereas EPO in solution did not, also show the importance of EPO binding to targets. Thus the beneficial binding of EPO to tumor cells may be detrimental to the host when EPO binds to normal cells during the course of hypersensitivity reactions. As outlined in the MBP section, both EPO and MBP evoke a

44]

dose-dependent noncytolytic release of 5-hydroxytryptamine from platelets.[

Binding of EPO to neutrophils appears to inhibit reversibly EPO peroxidase activity and, on the other hand, to increase neutrophil aggregation and adhesion to endothelial cell monolayers.[

81]

Charcot-Leyden Crystal Protein Distinctive crystals were initially described in 1853 in a patient with leukemia and later in 1872 in the sputa of patients

313

with asthma. Since then the appearance in tissues and body fluids of these hexagonal bipyramidal crystals of Charcot-Leyden crystal (CLC) protein has been regarded as a hallmark of the eosinophil. CLC protein is a characteristic, although not unique, constituent of eosinophils, constituting 7% to 10% of total eosinophil 3

protein content.[ ] CLC protein is also a major basophil product. The quantities of CLC protein in basophils range from 3 to 32••g/106 cells, whereas eosinophils contain 9 to 14••g/106 cells. mRNA for CLC as well as that for EDN are among the most abundantly expressed mRNA by mature peripheral blood eosinophils, [ suggesting potential de novo synthesis of these proteins. 82]

According to its genomic organization, CLC belongs to a family of galactose-binding lectins (so-called galectins) and is also named galectin-10. [ however, the CLC protein showed no affinity for β-galactosides but bound mannose avidly.

[83]

CLC is localized to the primary

detect CLC protein in the nuclear matrix and the cytoplasm of eosinophils derived from IL-5–induced umbilical cord blood

3 granules.[ ]

84 cultures.[ ]

protein in eosinophil function is unknown; earlier reports found that CLC protein possesses weak lysophospholipase activity.

Interestingly,

It is also possible to

Furthermore, in transfected

COS cells expressing the recombinant form of the molecule, CLC protein was detectable in the nucleus, cytoplasm, and plasma membrane.[ [4]

34]

85]

The role of CLC

Any CLC-associated 85]

lysophospholipase activity could protect the cells from the lytic effects of lysophospholipids generated at the site of inflammation.[

Alternatively, it may degrade

pulmonary surfactant lysophospholipids and alter the surface tensile properties of surfactants and, in turn, contribute to the development of focal atelectasis.[

86]

A

recent report found that CLC protein binds to a lysophospholipase inhibitor and suggested that lysophospholipase activity can be dissociated from CLC protein.[ Furthermore, CLC may bind carbohydrates expressed on microorganisms or biologic molecules, such as immunoglobulin E (IgE) or laminin,[ like domains.

88]

87]

through its lectin-

Protease and Other Enzymes In addition to CLC protein, EPO, and the RNase activity associated with EDN and ECP, a variety of other enzymes have been associated with the eosinophil.

Arylsulfatase B is located predominantly in the small granules of the eosinophil and is present in eosinophils in greater amounts than in neutrophils. β-Glucuronidase activity in eosinophils is about twice that in neutrophils, and exposure of eosinophils to opsonized zymosan particles releases up to 24% of the total cellular β4

glucuronidase. [ ] Although the eosinophil was thought to be deficient in lysozyme and phagocytin, lysozyme can be localized to both normal and abnormal eosinophil granules and is present within the eosinophil crystalloids of normal eosinophils.[

89]

Because the eosinophil has been recognized as a proinflammatory cell in various human diseases, a number of studies have analyzed eosinophil proteases. Neutrophil elastase has been identified in human eosinophils and can be localized to eosinophil granules.[ detect elastase in eosinophils by

91 immunohistochemistry.[ ]

90]

However, this is a controversial issue because the authors could not

Eosinophil collagenase degrades type I and type III collagen, the two major connective tissue 4

components of human lung parenchyma. Eosinophils are present during wound healing, and their collagenase could be important in tissue remodeling.[ ] Eosinophils 92] [93]

are a major source of a 92-kD matrix metalloproteinase (MMP)-9 (gelatinase B) in basal cell carcinoma lesions.[

This gelatinase was also localized on

[94]

eosinophils infiltrating into the lesions of patients with bullous pemphigoid, and it cleaved the type XVII collagen, a transmembrane molecule of the epidermal hemidesmosome, suggesting that production and release of gelatinase by eosinophils contributes to tissue damage of bullous pemphigoid. Furthermore, in vitro 95

transmigration of eosinophils through the basement membrane compartment was blocked by a neutralizing anti-MMP-9 antibody,[ ] suggesting that this enzyme is required during the migration process of eosinophils. In a mouse model of asthma, when the tissue inhibitor of metalloproteinase-2 (TIMP-2), an inhibitor of MMP-2 and MMP-9, was administered to airways, it inhibited the antigen-induced infiltration of lymphocytes and eosinophils to airways and reduced airway hyperresponsiveness.[

96]

The phospholipase A2 superfamily of structurally discrete enzymes consists broadly of three subfamilies: 85-kD cytosolic PLA2 (cPLA2 , group IV); 14-kD secretory PLA2 (sPLA2 , groups I to III, V, and IX); and 40-kD and 80-kD Ca2+ -independent PLA2 (iPLA2 , group VI). The 85-kD cPLA2 appears to play an essential role in mediating hydrolytic cleavage of arachidonic acids (AAs) at the sn-2 position of membrane phospholipids and generating various lipid mediators (see below). 97] [98] [99]

Specific blockade of cPLA2 inhibited secretion of EPO, generation of superoxide anion, AA metabolism and adhesion in activated eosinophils,[

suggesting

critical roles of this enzyme in eosinophil effector functions. Eosinophils also express 14-kD sPLA2 at levels much higher than those found in other circulating 100]

leukocytes, such as neutrophils, basophils, monocytes, and lymphocytes [ 101]

fraction of human eosinophils.[ smooth muscle contraction.[ 105 (BPI),[ ]

104]

; sPLA2 is mainly located in specific granules and copurified with ECP in the granular

sPLA2 is known to degrade phospholipids of gram-negative bacteria[

102]

103]

and to cause airway inflammation[

and airway

Also, human eosinophil granules contain another bacteriostatic and bactericidal protein, bactericidal/permeability-increasing protein

suggesting that sPLA2 and BPI may act together for host defense against microorganisms. These findings allow speculation on the potential roles of

intracellular and extracellular PLA2 in eosinophil activation and mediator release as well as in inflammatory reactions in host defense and allergic diseases. Lipid Mediators Lipid bodies in eosinophils serve as sites for AA storage in the form of esterified arachidonate, which would be available for eicosanoid synthesis. [

106]

In general,

PLA2 hydrolyzes membrane phospholipids, phosphatidylcholine (PC), and phosphatidylethanolamine (PE) to produce AA and lyso-PC and lyso-PE. AA may be metabolized by cyclooxygenase (COX) or 5-lipoxygenase (5-LO)/5-LO–activating protein (FLAP) into prostaglandins or leukotrienes, as well as lipoxins and 5107]

hydroxyeicosatetraenoic acid (5-HETE). Lyso-PC is acetylated to form PAF.[ eosinophils.

Furthermore, 5-LO has been localized by immunocytochemical staining to

[108] [109]

In eosinophils the predominant metabolite through the 5-LO pathway is leukotriene C4 (LTC4 ), which in turn is metabolized to leukotriene D4 (LTD4 ) and the less 7

active LTE4 .[ ] When stimulated with calcium ionophore A23187,

314

eosinophils can generate relatively large amounts of LTC4 (70•ng/106 cells) but only negligible amounts of LTB4 (2•ng/106 cells). This finding is in contrast to 7

neutrophils, which can produce large amounts of LTB4 but little, if any, LTC4 . The other major 5-LO metabolite of eosinophils is 5-HETE.[ ] These mediators contract airway smooth muscle, promote secretion of mucus, alter vascular permeability, and elicit eosinophil and neutrophil infiltration. One of the eosinophil-active cytokines, IL-5, increases expression of FLAP and induces translocation of 5-LO into the nucleus, suggesting that IL-5 primes eosinophils for production of cysteinyl 110

leukotrienes.[ ] In eosinophils stimulated by the addition of exogenous AA, 15-LO metabolites are also detectable, including 5,15- and 8,15-dihydoxy-HETEs and a novel chemotactic lipid, 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid. There have been few studies of eosinophil COX metabolism. Eosinophils from patients with the hypereosinophilic syndrome synthesize prostaglandins E1 (PGE1 ) and E2 (PGE2 ), 6-keto-prostaglandin F1 (PGF1 ), and thromboxane B2 (TXB2 ).[

7]

Eosinophils contain high levels of ether phospholipids, the stored precursor to platelet-activating factor (PAF), about fourfold more than neutrophils, suggesting that 111]

the eosinophil is a good PAF producer. [

Much of this material is in the form of 1-alkyl-2-lyso-sn-glycero-3-phosphocholine. Eosinophils produce PAF by an 4

acetylation reaction using the enzyme 1-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl coenzyme A (CoA) acetyl transferase.[ ] Eosinophils produce at least three molecular species of PAF, the predominant species being 1-hexadecyl-2-acetyl-glycero-3-phosphocholine 16:0. Much of the PAF produced by eosinophils remains 112

cell-associated.[ ] PAF has a number of important pharmacologic activities, including the activation of platelets and neutrophils and induction of bronchoconstriction. However, the role of PAF produced by eosinophils in asthma requires further investigation. Cytokines Until fairly recently, the role of the eosinophil in immunoregulatory networks has been viewed as a target of cytokines. However, eosinophils are also sources of growth factors and regulatory or proinflammatory cytokines and chemokines (see Box 19-1 ). Eosinophils could be a source of various tissue growth factors. For example, human eosinophils infiltrating intestinal tissues and eosinophils from patients with eosinophilia express transforming growth factor alpha (TGF-α) and beta-

1 (TGF-β1 ).[

113] [114]

Eosinophils from nasal polyps also express TGF-β1 , suggesting that TGF-β1 synthesis by eosinophils may contribute to the structural 115]

abnormalities of nasal polyps, such as stromal fibrosis and basement thickening. [ synthesis of lung and dermal fibroblasts.

[116]

Indeed, eosinophil-derived TGF-β enhances proliferation and collagen 117]

TGF-α produced by cytokine-activated eosinophils increases mucin production by airway epithelial cells. [ 118

Furthermore, eosinophils produce and release nerve growth factor (NGF) and promote extension of neurites in nerve cells.[ ] Eosinophils also produce cytokines able to act on eosinophils themselves, the “autocrine” cytokines, including IL-3, IL-5, and GM-CSF. For example, human eosinophils stimulated in vitro with 119] [120]

interferon gamma (IFN-γ), the calcium ionophore A23187, or ionomycin produce GM-CSF and IL-3 protein.[ cytokines comparable with T cells.

[119]

Eosinophils are able to produce quantities of

Production of GM-CSF is also observed with eosinophils stimulated by lipopolysaccharide (LPS), and this production is

121 (IL-10).[ ]

inhibited by interleukin-10 Interestingly, fibronectin-coated surfaces enhance eosinophil survival by a mechanism that depends on the interaction between fibronectin with an integrin molecule, very late antigen-4 (VLA-4), on eosinophils. This phenomenon is inhibited by antibodies to GM-CSF and IL-3, suggesting an autocrine production of these cytokines by eosinophils.[ stabilization of mRNA for

122]

The increased GM-CSF production in response to fibronectin likely results from the

123 GM-CSF.[ ]

Another group of cytokines produced by eosinophils modulates the functions of other immune cells. Eosinophils produce interleukin-4 (IL-4), and by RT-PCR, IL-4 124 125

126

124

mRNA is detected in highly purified blood eosinophils. [ ] [ ] IL-4 protein is localized to tissue eosinophils in the airway[ ] and the skin[ ] from patients with atopy. Eosinophils also release stored IL-4 rapidly by vesicular transport to the local milieu when stimulated with eotaxin or RANTES (regulated on activation, 127

normal T cells expressed and secreted).[ ] Thus a subpopulation of eosinophils from allergic subjects likely produces IL-4, which may be important in local IgE production and other IL-4–dependent events associated with the allergic reaction. A number of other cytokines and chemokines are also transcribed and produced by eosinophils. By RT-PCR, constitutive expression of interleukin-6 (IL-6) and IL10 mRNA is detected in peripheral blood eosinophils.[ cytochalasin

130 B,[ ]

eosinophil

131 MBP,[ ]

125] [126] [128] [129]

Complement C5a and formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of

and GM-CSF plus RANTES or PAF,[

132]

can stimulate eosinophils to produce interleukin-8 (IL-8). Increased levels of 132

intracellular IL-8 are observed with eosinophils from patients with asthma or atopic dermatitis.[ ] Furthermore, eosinophils from the peripheral blood of normal individuals or patients with hypereosinophilic syndrome express a proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), and a chemokine, macrophage 133]

inflammatory protein-1 alpha (MIP-1α).[

Chemotactic cytokines active on eosinophils, including RANTES and interleukin-16 (IL-16), are also produced and 134

released by eosinophils themselves, suggesting an autocrine production of these chemotactic factors.[ ] Interestingly, RANTES is stored in at least two intracellular compartments in eosinophils; the matrix of specific granules and small secretory vesicles. On IFN-γ stimulation, RANTES may be mobilized and released by 135]

piecemeal degranulation (see below), which would involve transport through a putative pool of small secretory vesicles.[

Thus, eosinophils can provide a strikingly wide variety of cytokines and chemokines, suggesting that eosinophils are potentially involved in diverse biologic responses, from tissue remodeling to activation of resident and infiltrating cells. However, the relative contribution of eosinophil-derived cytokines and growth factors to the development and maintenance of inflammatory reactions associated with allergic reactions remains to be determined, because the amounts of cytokines generated by eosinophils appear to be less than those produced by lymphocytes or other cells. Nonetheless, the potential autocrine and paracrine use of eosinophilderived cytokines may have particular pathophysiologic relevance.

315

EOSINOPHIL RECEPTORS Eosinophils express various membrane receptors through which they communicate with the extracellular environment: adhesion receptors, receptors for the Fc portion of immunoglobulins (Fc receptors), and receptors for soluble mediators, such as cytokines and lipid mediators ( Table 19-2 ). Immunoglobulin Receptors IgG Receptors

Receptors recognizing the Fc portion of immunoglobulin G (IgG) (FcγR) on eosinophils have been extensively studied for TABLE 19-2 -- Eosinophil Surface Molecules and Ligands Eosinophil Structures

Surface Molecules (CD Designation)

Ligands

Immunoglobulin receptors

FcαR (CD89)

IgA

FcγRI (CD64)

*

IgG IgG

FcγRII (CD32) FcγRIII (CD16) FcepsilonRI

*

†

FcepsilonRII (CD23)

Complement receptors

IgG IgE †

IgE

epsilonBP (Mac-2)

IgE

C3a receptor

C3a

C5a receptor (CD88)

C5a

CR1 (CD35)

C3b

CR3 (Mac-1, CD11b/CD18)

C3bi, ICAM-1

p150, 95 (CD11c/CD18)

C3bi

CD46

Measles virus

DAF (CD55) MACIF (CD59) Cell adhesion molecules (CAMs) Integrins

Selectins and their ligands

αLβ2, LFA-1 (CD11a/CD18)

ICAM-1, ICAM-2, ICAM-3

αMβ2, Mac-1, CR3 (CD11b/CD18)

C3bi, ICAM-1, fibrinogen

αXβ2, p150, 95 (CD11c/CD18)

C3bi

αdβ2

VCAM-1

α4β1, VLA-4 (CD49d/CD29)

VCAM-1, fibronectin

α6β1, VLA-6 (CD49f/CD29)

Laminin

α4β7

MAdCAM-1, VCAM-1, fibronectin

L-selectin (CD62L)

GlyCAM-1, CD34

PSGL-1 (CD162)

P-selectin

LewisX (LeX , CD15)

P-selectin

Sialyl-LewisX (CD15s)

Immunoglobulin-like

E-selectin, P-selectin

Sialyl-dimeric LewisX

E-selectin

Leukosialin (CD43)

ICAM-1

PECAM-1 (CD31)

PECAM, heparan sulfate

ICAM-1 (CD54)

Other CAMs

‡

*

LFA-1, Mac-1, rhinovirus

ICAM-3 (CD50)

LFA-1, αdβ2

LFA-3 (CD58)

CD2

Pgp-1 (CD44)

Hyaluronic acid

Siglec-8

Sialic acid

Chemokine receptors

Cytokine receptors

§

CCR1

MIP-1α, RANTES

CCR3

Eotaxins, MCP-3 and -4, RANTES

CCR6

MIP-3α

CXCR1 or CXCR2

IL-8

CXCR3

IP-10

CXCR4

SDF-1

IL-1R (CDw121a)

IL-1

IL-2 receptor α chain (CD25)

IL-3

IL-4Rα (CD124)

IL-4

IL-5Rα (CD125)

IL-5

†

IL-9

IL-13Rα

IL-13

GM-CSF receptor (CDw116)

GM-CSF

Common β chain

IL-3, IL-5, GM-CSF

IFN-γ receptor (CDw119)

IFN-γ

TNF receptor (CD120)

TNF-α

CD4

IL-16

p24 kinase (CD9) Tyrosine phosphatase (CD45) Aminopeptidase N (CD13)

Miscellaneous

IL-2

IL-3Rα (CD123)

IL-9

Enzymes

*

CAMPATH-1 (CD52) gp53 (CD63) CD69

*

CD137 B7.2 (CD86)

*

Fas (CD95) HLA class I HLA-DR

*

CRTH2 Prostaglandin D2 + (PGD2 +) PAR-2

Serine proteases

PAF receptor Leukotriene B receptor Leukotriene D receptor fMLP receptor β-Adrenergic receptor Histamine receptor P2X and P2Y

Various nucleotides

Modified from Kita H, Gleich GJ: The eosinophil: structure and functions. In Kaplan AP, editor: Allergy, ed 2, Philadelphia, 1997, WB Saunders, p 151. FcαR, Fc alpha-chain receptor; IgA, immunoglobulin A; ICAM, intercellular adhesion molecule; DAF, decay-accelerating factor; MACIF, membrane attack complex inhibitory factor; LFA, Lymphocyte function–associated antigen; VLA, very late antigen; IL, interleukin; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; RANTES, regulated on activation, normal T cells expressed and secreted; IL-2Rα, interleukin-2 alpha-chain receptor; PSGL-1, Pselectin glycoprotein ligand-1; VCAM-1, vascular cell adhesion molecule-1; MAdCAM-1, mucosal adressin cell adhesion molecule-1; GlyCAM-1, glycosylated cell adhesion molecule-1; PECAM-1, platelet-endothelial cell adhesion; IFN-γ, interferon gamma; IP-10, IFN-γ–inducible protein-10; TNF-α, tumor necrosis factor alpha; GM-CSF, granulocyte-macrophage colony-stimulating factor; HLA, human leukocyte antigen; SDF-1, stromal cell–derived factor-1; PAR-2, proteaseactivated receptor-2; PAF, platelet-activating factor. * Expressed only on activated eosinophils. † Controversial findings. ‡ Levels of expression are very low. § Expression is partly based on biologic activities of the cytokines.

their abilities to mediate eosinophil functions, including phagocytosis, antigen-dependent cytotoxicity, oxygen metabolism, leukotriene C4 (LTC4 ) production, and 7

136

degranulation.[ ] Although most of these studies have been performed using IgG-coated targets, Graziano et al [ ] showed that FcγR directly mediated eosinophil cytotoxicity. In their system, eosinophils, preincubated with GM-CSF, killed both an anti-FcγRII-bearing hybridoma cell line and chicken erythrocytes coated with antibody covalently coupled to Fab fragments of anti-FcγRII. However, eosinophils could not kill hybridomas expressing anti-FcγRI or anti-FcγRIII. The interaction between IgG and FcγRII may also be important in eosinophil degranulation in allergic diseases. The sera from patients with hay

316

fever contained elevated levels of allergen-specific IgG1 and IgG3, and these antibodies caused eosinophil degranulation in vitro in an allergen-dependent manner. This reaction was abolished when eosinophil FcγRII was blocked by an mAb.[

137]

Flow cytometry analyses of human cells revealed that freshly isolated eosinophils possess only FcγRII (CD32),[ and neutrophils possess FcγRII and FcγRIII

139 (CD16).[ ]

rarely expressed on the surface of nonactivated cells.

138]

monocytes possess FcγRI (CD64) and FcγRII,

Eosinophils produce FcγRIII protein intracellularly; however, this protein is released extracellularly and

[140]

The expression of FcγR is regulated by cytokines. For example, treatment of eosinophils with IFN-γ for

more than 24 hours enhances their expression of FcγRII and thus augments FcγRII-mediated tumor cell killing by eosinophils.[ of FcγRIII (a phosphatidylinositol-linked form) as well as FcγRI on induce expression of FcγRI or

142 eosinophils.[ ]

141]

IFN-γ also induces the expression

In contrast, culture with IL-3 causes an up-regulation of FcγRII but does not

142 FcγRIII.[ ]

IgE Receptors

Several reports suggest that eosinophils have IgE receptors that stimulate the eosinophil's cytotoxic function and degranulation.[ of schistosomula depends on IgE antibody or a factor that resembles

4] [7]

For example, eosinophil killing

143 IgE.[ ]

317

Eosinophils can potentially express three types of IgE receptors. First, an mAb has been produced by immunization with eosinophils from patients with eosinophilia, and this antibody completely inhibits IgE-dependent cytotoxicity and the binding of IgE to eosinophils. This mAb, called BB10, has identified 105 binding sites per 3

eosinophil and has recognized different determinants from those recognized by antibodies to CD23, the low-affinity IgE receptor, on B cells and monocytes.[ ] Second, eosinophils from patients with eosinophilia express another lectin type of low-affinity IgE-binding molecule, Mac-2/epsilonBP, and the cytotoxic function of

144

eosinophils against parasites was abolished by the antibody against this molecule.[ ] Third, studies indicate that the high-affinity IgE receptor, FcepsilonRI, is present on eosinophils from patients with eosinophilia, and that various functions of eosinophils, including degranulation and parasite cytotoxicity, are mediated through this receptor.[

145]

On the other hand, the number of high-affinity receptors expressed on the surfaces of eosinophils from patients with allergic diseases or

airway eosinophilia was minimal or undetectable; [

146]

147]

ligation of FcepsilonRI did not result in detectable eosinophil degranulation.[

Furthermore, eosinophils 146

make protein for the α chain of FcepsilonRI, but this protein is released extracellularly instead of being expressed on the cellular surface. [ ] Thus the nature of eosinophil IgE binding and its significance in eosinophil function remain to be clarified. In addition, the possible heterogeneity of eosinophils among different classes of diseases (e.g., eosinophilia and allergic disease) and eosinophils from different sources (e.g., tissue, BAL, peripheral blood) needs to be considered. IgA Receptors 3

Evidence from flow cytometric analyses and degranulation studies suggests that eosinophils have IgA receptors.[ ] Comparison of neutrophils and eosinophils 148

suggests that neutrophils express much higher levels of FcαR (CD89), detected by the binding of IgA and by anti-FcαR antibody.[ ] The FcαR on eosinophils has a higher molecular mass (range 70 to 100•kD) than that on neutrophils (range 55 to 75•kD). The FcαR on eosinophils and neutrophils likely is highly glycosylated with a major protein core of 32•kD for both cell types. Interestingly, eosinophils from allergic individuals display enhanced FcαR expression, whereas neutrophils do not.[

148]

Sepharose 4B beads coated with IgA or secretory IgA (sIgA) induce degranulation of eosinophils. When compared with IgE, IgG, and IgA, sIgA is the most 3

potent immunoglobulin stimulant for eosinophil degranulation.[ ] Although the exact mechanism to explain why sIgA is a more potent stimulus than IgA is unknown, eosinophils have been shown to possess binding site(s) for secretory component.[ enhanced proinflammatory functions of eosinophils, but not of

150 neutrophils.[ ]

149] [150]

Furthermore, the interaction with secretory component greatly

The interaction of eosinophils with IgA was enhanced by T helper cell type 2 (Th2)

151

cytokines, such as IL-4 and IL-5.[ ] Collectively, these findings along with the localization of eosinophils at epithelial surfaces[ sIgA in mediating the eosinophil's effector function in vivo.

20]

suggest an important role for

Complement Receptors Eosinophils express the CR1 and CR3 (Mac-1, CD11b/18) complement receptors (see Table 19-2 ); their levels of expression can be increased by incubating 3

152]

eosinophils with LTB4 ,[ ] fMLP, PAF, or GM-CSF.[

Furthermore, compared with neutrophils, eosinophils have a larger total pool of CR3 (Mac-1) within the

cells, which is mobilized to the cell surface in response to IL-5.[ molecule (see below).

153]

CR3 plays a critical role in the eosinophil's effector functions, especially as a versatile adhesion

The complement anaphylatoxins, C3a and C5a, activate human eosinophils through a receptor-mediated process. Both C3a and C5a induce elevations in intracellular Ca++ , degranulation and production of oxygen radicals from eosinophils.[ receptor is constitutively expressed on human Platelet-Activating Factor Receptors

158 eosinophils.[ ]

154] [155] [156] [157]

C3a and C5a also induce chemotaxis of eosinophils.[

154]

The C3a 7]

The receptor for C5a on eosinophils appears to be distinct from the C5a receptor on neutrophils.[

PAF receptors on human leukocytes have been cloned and expressed, and the resulting transfectants were stimulated by PAF and bound radiolabeled PAF. [

159]

The

7 studies.[ ]

action of PAF on eosinophils has been investigated in a number of in vitro For example, PAF is one of the most potent chemoattractants for eosinophils and selectively induces the migration of eosinophils over neutrophils. PAF increases the binding of IgE to human eosinophils and enhances cytotoxicity towards schistosomula of Schistosoma mansoni. Furthermore, PAF evokes the release of granule proteins, reactive oxygen species, and LTC4 from eosinophils. Despite these in vitro activities, the physiologic roles of PAF still need to be elucidated. Leukotriene Receptors 7

LTB4 induces several functional responses of eosinophils. [ ] LTB4 potently stimulates chemotaxis and chemokinesis in guinea pig eosinophils and elicits AA metabolism. Although LTB4 stimulates a respiratory burst in human and guinea pig eosinophils, LTB4 does not seem to induce degranulation of eosinophils, as assessed by the release of β-glucuronidase.[ express this receptor.[

160]

Eosinophil adhesion induced by LTD4 is inhibited by a cysLT1 receptor antagonist, suggesting that eosinophils

161]

Chemokine Receptors The known CC chemokine receptors are members of the G protein–coupled receptor superfamily; two of these receptors, CCR3 (40,000∼400,000 receptors/cell) and 162 163

a much smaller number of CCR1, are found on eosinophils.[ ] [ ] CCR3 binds eotaxins, monocyte chemotactic protein (MCP)-3, MCP-4, and RANTES. CCR1 binds to MIP-1α, RANTES, and MCP-3. Importantly, CCR3 is expressed by eosinophils but not by neutrophils or monocytes, which could explain the recruitment of eosinophils to allergic inflammation sites.[

164]

stromal cell–derived factor-1 alpha (SDF-1α),

Human eosinophils also express CXCR3 and CXCR4 that bind to interferon-γ–inducible protein-10 (IP-10)[ [166]

165]

and

respectively. Interestingly, at allergic inflammation sites, the expression on eosinophils of CCR3 decreased and 167]

that of CXCR4 increased, suggesting these receptors may have pivotal roles in eosinophil accumulation.[

318

EOSINOPHIL RECRUITMENT Adhesion Receptors and Ligands Leukocyte migration through the endothelium is a process involving sequential steps. Initially the cells are lightly tethered to the endothelium and roll along its surface, followed by leukocyte activation, which allows a firmer bond to develop between the leukocyte and the endothelial cell, resulting in successful adhesion and

transmigration. The receptors and mediators involved in eosinophil migration have been characterized (see Table 19-2 ). A family of adhesion receptors termed selectins and their carbohydrate ligands mediate the initial attachment and rolling steps. Integrins expressed on eosinophils bind to adhesion receptors belonging to the immunoglobulin superfamily on endothelial cells and are implicated in the firmer adhesion and transendothelial transmigration.[ Eosinophils constitutively express L-selectin and shed it on cell activation.[

169]

168]

Eosinophils use a functional epitope of L-selectin for ligand binding different from

170 171 neutrophils.[ ] [ ]

Furthermore, neutrophils bind more avidly than eosinophils to purified E-selectin. Neutrophils express large amounts of sialyl-LewisX , a ligand for E-selectin, whereas the expression of this epitope on eosinophils is extremely low or undetectable; a major proportion of the E-selectin ligand on the surface of eosinophils appears to be sialyl-dimeric LewisX .[

172]

The counterligands for P-selectin on eosinophils and neutrophils are similarly sialylated, protease-

sensitive, endo- β-galactosidase-resistant structures, P-selectin glycoprotein ligand-1 (PSGL-1).[ selectin is reduced by cell activation as PSGL-1 is shed.[

172] [173]

Eosinophil and neutrophil binding to E-selectin and P-

174]

175

Integrins are composed of α and β transmembrane subunits selected from among 16 α and eight β subunits.[ ] Cells are able to regulate the function of integrins by signaling in the opposite direction (inside-out signaling), and the receptors are converted from a form with low affinity for their ligands to one with high affinity. This 176

so-called activation of the integrin is required for firm interaction between integrins and their ligands,[ ] which is also the case with eosinophils. The levels of integrin expression are also dependent on the cell's activation state. Eosinophils express several integrins that can bind to immunoglobulin family receptors expressed on the endothelium and to components of extracellular matrix proteins (see Table 19-2 ). The resultant bond is much firmer than the selectin-carbohydrate bond and results in the eosinophil's flattening and transmigration between endothelial cells.[

177]

7

Eosinophils express β1 (CD29), β2 (CD18), and β7 integrins. [ ] Among the β1

integrins, VLA-4 (α4 /β1 , CD49d/CD29), which binds vascular cell adhesion molecule-1 (VCAM-1), plays an important role for eosinophil migration from the blood stream into the tissues (see below). Among the β2 integrins, lymphocyte function–associated antigen-1 (LFA-1) (CD11a/CD18) and Mac-1 (αM /β2 , CD11b/ CD18) integrins both bind intercellular adhesion molecule-1 (ICAM-1) expressed on various types of cells and also play important roles for eosinophil migration, as well as effector functions of the cells. Another β2 integrin, αd β2 , binds VCAM-1.[

178]

The expression of these integrins, especially VLA-4, by eosinophils may

contribute to their preferential recruitment into the sites of allergic diseases. For example, in contrast to neutrophils, eosinophils, as with lymphocytes, can emigrate into inflammatory sites in patients with the leukocyte adhesion deficiency (LAD) syndrome, indicating that eosinophils can migrate into tissues by mechanisms not dependent on β2 integrins. Eosinophils also express α4 β7 ,[

179]

and the principal ligand for this receptor is the gut mucosal addressin cell adhesion molecule-1

180

(MAdCAM-1).[ ] The expression of this integrin is intriguing, given the potential role of this integrin in mucosal recruitment of eosinophils. Integrins also function as receptors for extracellular matrix proteins and plasma proteins, such as laminin, fibronectin, and fibrinogen, and many integrins recognize a common sequence in these proteins, as described later in detail. Another adhesion molecule expressed by eosinophils is CD44, a hyaluronic acid receptor. The presence of CD44 on eosinophil progenitors is well documented,[ 182

181]

but it appears to have little if any hyaluronic acid binding function on mature eosinophils, even though it is considered a marker of eosinophil activation.[ ] One additional adhesion structure recently found exclusively on mast cells, basophils, and eosinophils, but not other leukocytes, is SAF-2, also known as siglec-8, one of 183]

the sialic acid–binding immunoglobulin-like lectins (siglecs).[

Eosinophil Chemotactic Factors 4

An essential component of eosinophil migration into the tissues in allergic reactions is an effective and selective chemoattractant.[ ] Because of the association of immediate hypersensitivity reactions with tissue eosinophilia, considerable attention has been focused on mast cell–derived chemotactic factors, such as histamine 4

4

and LTB4 .[ ] Histamine has chemotactic and chemokinetic activities for eosinophils in vitro.[ ] However, in vivo experiments do not appear to support histamine as 4

a significant eosinophilotactic agent.[ ] Although LTB4 is chemotactic for both eosinophils and neutrophils, recent evidence suggests that LTD4 is chemotactic for eosinophils but not for neutrophils in the under-agarose leukocyte assay.[ 186 oxoETE)[ ]

184]

Among LO-derived products, the diHETEs[

185]

and 5-oxo-eicosatetraenoic acid (5-

also have chemotactic activity for eosinophils. Interestingly, inhaled LTE4 generates airway eosinophilia, although it is not clear whether this is a direct 187

or indirect effect.[ ] PAF, produced by various types of cells, including mast cells, neutrophils, macrophages, eosinophils, and endothelial cells, is more potent as an eosinophil chemotactic factor than histamine and LTB4 over a dose range of 10−8 to 10−6 M. PAF is one of the most potent and effective chemoattractants for eosinophils and selectively induces the migration of eosinophils over neutrophils. In vivo, PAF injected into the skin caused the accumulation of eosinophils in atopic individuals, but neutrophils were prominent in nonatopic individuals. T cell products also play 4

a role in eosinophil chemotaxis. IL-5, GM-CSF, and IL-3 possess eosinophil chemotactic activity, but at nanomolar concentrations.[ ] Although the chemotactic activities of these cytokines are relatively weak compared with well-established eosinophil chemoattractants such as PAF, it is important to note that they can effectively prime eosinophils for enhanced chemotactic responsiveness to suboptimal concentrations of PAF and LTB4 and enhance the generally negligible activity 188]

that fMLP and IL-8 have for unactivated eosinophils.[

For example, preincubation of eosinophils with GM-CSF caused an increased response to

319

LTB4 and induced a response toward IL-8 and fMLP; similarly, preincubation of eosinophils with IL-3, IL-5, or GM-CSF enhanced PAF-induced chemotaxis.[ [190]

189]

191

Similarly, IL-5 and chemokines synergistically induced eosinophil migration through an endothelial cell monolayer.[ ] These stimulatory effects of cytokines on eosinophil responses to chemotactic factors appear to be relevant in vivo because eosinophils from allergic subjects are more responsive to some chemotactic stimuli than eosinophils from normal subjects.[

190]

Indeed, despite the relatively weak activity of IL-5 in the in vitro system, inhalation of IL-5 in animals and

humans leads to eosinophil recruitment into the airways,[ guinea pigs.[

195] [196]

192] [193] [194]

and antibodies against IL-5 inhibit eosinophil migration into the lungs of allergen-challenged

Furthermore, eosinophil mobilization from the bone marrow into the blood, as investigated by in situ perfusion of the guinea pig hind limb, is 197

strongly stimulated by IL-5 and eotaxin.[ ] Therefore a selective priming by IL-5 combined with a highly effective chemoattractant may result in selective eosinophil migration in allergic inflammation.

As described earlier, chemokines are certain to play a major role in cell recruitment during inflammation. Chemokines are 8-kD to 10-kD proteins that have 20% to 198

55% similarities in their amino acid sequences. To date, approximately 40 chemokines and 16 chemokine receptors have been characterized.[ ] The family is broadly divided into two main subfamilies, including CXC and CC subfamilies, by the position of the first two of the conserved cysteines. The CXC subfamily, 199]

typified by IL-8, is particularly active on neutrophils, although IL-8 has chemotactic activity for primed eosinophils.[ and eotaxin, is potently chemotactic for eosinophils, but not for

200 neutrophils.[ ]

Comparison of the chemotactic activity of four members of the CC subfamily, MCP-

1, RANTES, MIP-1α, and MIP-1β, found that RANTES is the most potent chemokine for eosinophils.[ effects on eosinophils similar to RANTES, although MCP-2 was less potent than MCP-3 and allergen-challenged guinea [205] [206]

204 pigs.[ ]

The CC subfamily, typified by RANTES

201]

Studies of MCP-2 and MCP-3 also show that they have

202 203 RANTES.[ ] [ ]

Eotaxin was originally identified in BAL fluid from

A second and third eotaxin, eotaxin-2 and eotaxin-3, with similar biological characteristics as eotaxin, have also been identified.

Human eotaxin and eotaxin-3 were more effective at inducing eosinophil infiltration than RANTES when injected into the skin of a rhesus monkey.[

[208]

207]

209

Intranasal administration of eotaxin to patients with allergic rhinitis specifically induced influx of eosinophils.[ ] Besides chemotactic activities, another important function of chemokines may be to regulate cell adhesion. Eotaxin and other CCR3-active chemokines induced detachment of eosinophils from fibronectin 210] [211]

or VCAM-1, suggesting they facilitate the movement of eosinophils during their tissue migration process by changing their integrin usage. [ human CC chemokine, designated monocyte chemoattractant protein-4 (MCP-4), has been

212 identified.[ ]

Another novel

MCP-4 shares 60% amino acid sequence identity with

MCP-3 and eotaxin and is highly active on eosinophils, as well as lymphocytes and monocytes, as a chemoattractant in vitro.[

212]

The rank order of potency in

191 RANTES.[ ]

eosinophil migration across unstimulated human umbilical vein endothelial cells was eotaxin ∼ eotaxin-2 > MCP-4 ∼ Thus the CC chemokines, including eotaxins, MCP-3, and probably RANTES and MCP-4, all of which act through CCR3, may act as selective and effective eosinophil chemokines in vitro and in vivo. Interestingly, another CC chemokine, macrophage-derived chemokine (MDC), appears to induce chemotaxis of eosinophils as potently as eotaxin but independently of CCR3 or CCR4.[

213]

Although the physiologic significance remains to be elucidated, a number of other factors can induce chemotaxis of eosinophils. For example, activation of the 4

complement system generates proteolytic fragments, such as C5a and C3a, which are chemotactic for eosinophils and of significant biologic relevance.[ ] Although C5a is chemotactic for both eosinophils and neutrophils, C3a is chemotactic only for eosinophils.[

154]

Activated eosinophils can express CD4 and thus can interact

[7]

with lymphocyte chemoattractant factor (LCF), also called interleukin-16, a tetrameric glycoprotein produced by activated T lymphocytes. LCF elicits eosinophil migration at low LCF concentrations. Although IL-2 does not affect the viability or other functions of eosinophils, IL-2 is a potent attractant for eosinophils. It is also possible to detect p55/CD25 low-affinity IL-2 receptors on the eosinophil's surface. [ patients with atopic dermatitis; eosinophils from normal donors do not

214]

Finally, IL-4 is chemotactic for eosinophils, but not neutrophils, from

215 respond.[ ]

Eosinophil Trafficking from Bone Marrow to Tissues Eosinophils are normally found in low numbers in the circulation. Therefore, recruitment from bone marrow is a prerequisite for their accumulation in tissues during 197 216

inflammation. IL-5 injected intravenously into guinea pigs stimulates a rapid mobilization of eosinophils from the bone marrow into the blood.[ ] [ ] Furthermore, airway challenge of sensitized guinea pigs with allergen induces a rapid release of eosinophils from the bone marrow, which is abolished by pretreatment of animals with anti-IL-5 antibody,[

217]

again suggesting a critical role for IL-5 in eosinophil recruitment from bone marrow to peripheral blood.

As described earlier, the migration of circulating eosinophils into the tissues involves several steps, including rolling, firm adhesion, and transmigration. When viewing microvascular endothelial cells in the rabbit mesentery with intravital video microscopy, eosinophils rolled in venules, and this rolling was stimulated by activating endothelial cells with IL-1. Eosinophil rolling was mediated by L-selectin and VLA-4, whereas neutrophil rolling was predominantly mediated by L218]

selectin.[

Eosinophil adhesion to nasal polyp tissues placed on a rotating platform (Stamper-Woodruff assay) was almost totally inhibited by an antibody against

219 P-selectin.[ ]

Human eosinophils express about twice as much PSGL-1 than do neutrophils, and the interaction of eosinophils with P-selectin is generally of greater

magnitude than that for neutrophils,[

220] [221]

suggesting a preferential role for P-selectin over E-selectin in eosinophil recruitment. Mediators released at sites of

allergic inflammation, such as histamine, leukotrienes, IL-4, and IL-13, induce P-selectin expression.[

222] [223] [224]

In contrast, neutrophils favor interaction with E172]

selectin because they express approximately 10 times as much sialyl-dimeric LeX , a ligand for E-selectin, than do eosinophils.[ on HUVECs stimulated with TNF-α are mediated largely by VLA-4 and

225 E-selectin[ ]

Eosinophil attachment and rolling

; P-selectin plays a minor role in this

320

model. Furthermore, α4 integrins are involved in specific migration of eosinophils through HUVECs stimulated with IL-4,[

226]

suggesting that eosinophil use of

adhesion molecules may be dependent on how endothelial cells are activated. Overall, L-selectin, P-selectin, E-selectin and VLA-4 may be involved in the rolling interactions of eosinophils with endothelial cells under flow conditions; however, in allergic inflammation, P-selectin and VLA-4 may be the principal receptors involved in this stage. The β2 integrins LFA-1 and Mac-1 are particularly important during the process of firm adhesion and transendothelial migration. In this regard the activation states 176

of these integrins are more important than their relative levels of cell surface expression.[ ] For example, eosinophil adhesion to unstimulated HUVECs was enhanced by PAF and other eosinophil-active inflammatory mediators, including IL-3, IL-5, and TNF, and their enhancement was totally inhibited by mAb to the 7

leukocyte integrin Mac-1.[ ] Hypodense eosinophils exhibited significantly greater ability to adhere spontaneously to endothelial cells than did normodense eosinophils.[

227]

PAF stimulation increased adhesiveness of hypodense eosinophils; this could be virtually completely blocked by monoclonal antibody to CD18, 7

thus implicating β2 integrin in the adhesion process.[ ] In contrast, eosinophil adhesion to IL-1–stimulated or TNF-α–stimulated HUVECs was inhibited by mAbs against ICAM-1 and VCAM-1 on the endothelial cells and LFA-1, Mac-1, and VLA-4 on the eosinophils, suggesting that both β1 integrin and β2 integrin are involved in adhesion of eosinophils to the activated endothelial cells.[

7] [228]

Furthermore, the activation state of eosinophils is also likely critical for selection of

integrins because exposure to IL-5 shifts integrin usage away from β1 integrin-mediated pathways while up-regulating β2 integrin–mediated events.[

178]

Eosinophil 211]

exposure to chemokines has been reported to enhance or reduce eosinophil adhesion to VCAM-1, or both, depending on the methods and duration of exposure.[ [229]

Because cells must detach during directional migration,[

230]

other molecular events, such as regulation of function and redistribution of integrins on the cell

surface, may be involved during eosinophil adhesion to endothelial cells. [

231]

Eosinophil transmigration through endothelial cells has been investigated with artificial models of the blood vessel wall. In these studies, eosinophils from allergic 232

donors showed an increased migration capacity, which was mimicked by in vitro culture of eosinophils from normal donors with GM-CSF, IL-3, or IL-5.[ ] The receptors that mediate transmigration of eosinophils through IL-1–stimulated or TNF-α–stimulated HUVECs appear to be the LFA-1 and Mac-1 on eosinophils 233]

binding to ICAM-1 on the endothelium.[ antibodies against β2 integrins.

[234]

In contrast, eosinophil migration through IL-4– stimulated HUVECs was inhibited by anti-VLA-4 antibody, as well as

Further, the combinations of IL-1 and IL-4 or TNF and IL-4 (which are synergistic for VCAM-1 expression) were also

synergistic in their abilities to enhance endothelial transmigration.[ 234]

through IL-4–activated endothelial cells.[

235]

Thus, both the β2 and the β1 integrins appear to be involved in the eosinophil transmigration

Because neutrophils do not express VLA-4, and because IL-4 is presumably produced during the inflammatory reaction 236

associated with asthma, the ability of IL-4 to up-regulate VCAM-1[ ] is an attractive mechanism by which eosinophils could be selectively recruited into the airways. This hypothesis has been strengthened by the observation that eosinophil, but not neutrophil, adhesion and transmigration through HUVECs were enhanced 237]

by IL-4. [

Eosinophil Interaction with Extracellular Matrix Proteins After migration through the endothelium, eosinophils come into contact with the extracellular matrix (ECM) proteins. Fibronectin binds to eosinophils through VLA4 and Act-1 (α4 β7 ), [

122]

laminin through VLA-6,[

238]

and hyaluronate through CD44[

181]

(see Table 19-2 ). This adhesion of eosinophils to ECM proteins modifies

the functions of the cells. For example, as a result of autocrine stimulation of IL-3 and GM-CSF production, eosinophils survived for prolonged periods when cultured on fibronectin. [

122]

However, the effects of ECM proteins on eosinophil effector functions are contradictory. Dri et al[

239]

demonstrated that eosinophil

superoxide production induced by fMLP or substance P is decreased when tissue culture wells are coated with fibronectin or laminin. In contrast, Anwar et al[ demonstrated enhancement of calcium-ionophore–stimulated LTC4 generation by eosinophils adherent to fibronectin. Neeley et al[

241]

240]

have reported that VLA-4–

mediated interaction with fibronectin results in increased degranulation of eosinophils stimulated by fMLP in the presence of cytochalasin B. Furthermore, eosinophil interaction with hyaluronate through CD44 enhanced eosinophil survival and production of TGF-β.[ likely playing important roles in the modulation of eosinophil functions in the tissues.

242]

Thus, although more studies are needed, ECM proteins are

EOSINOPHIL ACTIVATION AND DEGRANULATION While infiltrating into the tissue lesions, eosinophils are exposed to various cytokines, adhere to the endothelium, migrate through the blood vessels, and move toward the targets. During these processes, eosinophils are primed (partially activated), and they exert their full function at the site of the inflammation. Effects of Cytokines Several cytokines have a marked effect on eosinophil function. Eosinophils possess receptors for IL-3, IL-5, and GM-CSF (approximately 1000 receptors per cell).[

4]

The receptors for these cytokines consist of two noncovalently associated chains, namely a ligand-specific alpha (α) chain that binds the cognate ligand with low 7

affinity (dissociation constant [Kd ] = 10•nM)[ ] and a common beta (β) chain that confers high-affinity binding to the ligand and signal transduction.[

243]

The

affinity of the complete GM-CSF receptor (dissociation constant [Kd ] = 44•pM) on human eosinophils is approximately 10 times higher than for IL-3 (Kd = 470• 7

pM) and IL-5 (Kd = 518•pM).[ ] The sharing of a common β chain among the receptors for GM-CSF, IL-3, and IL-5 may explain in part the cross-competition among these cytokines.[

244] [245]

Interestingly, the association constant for the 125 I-labeled IL-5 to human hypodense eosinophils is higher than the association constant for the binding of IL-5 to 4

normodense cells; the number of receptor sites per cell is similar. [ ] The alpha chain of the IL-5 receptor (IL-5Rα) is encoded on chromosome 3

321

and occurs as two soluble isoforms. The membrane-anchored form of the IL-5 receptor is produced by alternative splicing.[ IL-5Rα has been localized in a promyelocytic leukemia cell line committed to eosinophil

247 lineage.[ ]

246]

Among human peripheral blood leukocytes, eosinophils are

the only cells having detectable levels of IL-5 receptors in agreement with the specific action of IL-5 on human eosinophils. [ regulated by TGF-β1 .[

250]

The promoter regulating the genes for

248] [249]

mRNA for IL-5Rα is down-

The expression of IL-5Rα on CD34+ stem cells increases after allergen challenge, suggesting that this receptor may contribute to

development of blood and tissue eosinophilia during allergic inflammation.[

251]

IL-3, IL-5, and GM-CSF, besides being growth and maturation factors for eosinophils, also stimulate several mature human eosinophil functions. IL-5, IL-3, and GMCSF antagonize the programmed cell death of eosinophils in culture, thus prolonging survival in vitro.[ inhibited by glucocorticoids, including dexamethasone and of eosinophils toward chemokines or lipid mediators human microvascular endothelial

176 cells[ ]

254 255 methylprednisolone.[ ] [ ]

256 257 synergistically[ ] [ ]

7] [252] [253]

The effects of these cytokines on eosinophils are

IL-5 is chemokinetic for eosinophils; it enhances the chemotactic response

; it enhances the integrin-dependent adhesion of eosinophils to plasma-coated glass and

; and it activates LTC4 and superoxide generation, phagocytosis, and helminthotoxic activity, as well as Ig-induced

4 258

degranulation.[ ] [ ] IL-5 provokes biochemical changes in human eosinophils, including activation of various kinases (e.g., Jak-2, lyn, Ras, Raf-1) and mitogenactivated protein (MAP) kinase, activation of adapter proteins (e.g., Shc, Grab-2), and induction of a DNA-binding complex containing signal transducer and activator of transcription-1 (STAT-1) protein,[

259] [260] [261] [262] [263]

providing pharmacologic bases for the action of IL-5 on eosinophils. In fact, activation of lyn 264

tyrosine kinase is likely crucial for the maintenance of eosinophil viability in vitro.[ ] MAP kinases also potentially activate many cytosolic proteins, including PLA2 , PLC, and cytoskeletal proteins, and are involved in eosinophil effector functions. IL-5 is present in various biologic fluids, including BAL fluids from patients with allergic rhinitis after endobronchial challenge[

265]

and sera from patients with eosinophilia of various causes.[

predominant eosinophil active cytokine in the antigen-induced pulmonary late-phase

266 reaction.[ ]

4] [9]

Moreover, IL-5 appears to be the

A role for IL-5 in bronchial asthma has been studied using animal

models treated with neutralizing antibodies and soluble receptors. In these studies, administration of either anti-IL-5 antibodies or soluble IL-5 receptors has prevented airway eosinophilia and bronchial hyperreactivity in a variety of models involving antigen challenges or virus challenges.[ inhalation in mice caused airway eosinophilia followed by airway hyperreactivity and mucosal

194 exudation.[ ]

196] [267] [268]

Conversely, IL-5

Finally, IL-5 production from human T cells was

enhanced by LTB4 , but not by its stereoisomer, suggesting a further link between this cytokine and allergic inflammation.[

269]

Thus, IL-5 enhances various functions

of eosinophils as inflammatory cells, including adhesion, chemotaxis, survival, and mediator release. IL-5 is detected in the biologic fluids associated with eosinophilia, and antagonism of IL-5 prevents pathologic changes associated with eosinophilia in vivo; therefore, IL-5 may be the most critical cytokine in the pathophysiology of eosinophil-associated inflammation. GM-CSF also activates and enhances eosinophil function, such as cytotoxic killing, superoxide production, leukotriene production, phagocytosis of serum-opsonized zymosan, and Ig-induced degranulation. In a similar manner, IL-3 enhances eosinophil cytotoxicity, phagocytosis, superoxide production (in response to stimulation 4

with fMLP) and Ig-induced degranulation.[ ] When eosinophils are maintained in culture with murine fibroblasts and GM-CSF, they synthesize and express CD4[ and the class II major histocompatibility complex (MHC) protein human leukocyte antigen-DR (HLA-DR)[ CD4 ligand,

271 LCF,[ ]

and to present antigen to CD4+

272 273 274 lymphocytes.[ ] [ ] [ ]

270]

7]

; these eosinophils therefore are able to respond to the

CD69, which is expressed on activated T cells and natural killer cells, is also

[275]

141

expressed on eosinophils cultured with GM-CSF or IL-3. IFN-γ activates eosinophils but does so in a delayed manner.[ ] For example, in short-term cytotoxicity assays, eosinophils are maximally activated by GM-CSF, followed in order of potency by IL-3, IL-5, TNF-α, and IL-4, whereas after a 24-hour incubation, maximal eosinophil activation is caused by IFN-γ followed by GM-CSF, IL-3, and IL-5. IFN-α and IFN-β enhance cytotoxicity as well. Furthermore, 142]

culture of eosinophils with IFN-γ for 1 to 2 days causes expression of FcγRIII (CD16) (not expressed on freshly isolated peripheral blood eosinophils)[ significant increase in FcγRII (CD32)

141 expression[ ]

and a

(see earlier discussion). In contrast, treatment of eosinophils with IFN-γ for 1 hour inhibited release of H2 O2 7

(induced by opsonized zymosan) and decreased eosinophil binding to complement-sensitized erythrocytes. [ ] The degranulation response of eosinophils to sIgA4

coated Sepharose beads was also inhibited by preincubation with IFN-γ for 3 hours.[ ] Thus, IFN-γ appears to inhibit eosinophil activation over short incubation times and to stimulate function and receptor expression when incubation is prolonged for days. 141]

The proinflammatory cytokines TNF-α and IL-1 also modulate eosinophil functions. TNF-α prolongs eosinophil survival in vitro,[ of LTC4

276 ,[ ]

increases eosinophil toxicity toward S. mansoni larvae,

[7]

and increases eosinophil adhesion for endothelial

80 cells.[ ]

enhances eosinophil synthesis

Although it is clear that TNF-α

7

itself has only modest activity toward eosinophils compared with the eosinophil hemopoietins (e.g., IL-5),[ ] the activity of TNF-α synergizes with that of IL-5. For 273

example, TNF-α and IL-5 synergistically induce the expression of ICAM-1 on mature eosinophils.[ ] Therefore the mechanism of action of TNF-α on eosinophils seems to be different from that of the eosinophil hemopoietin. The effects of IL-1 on eosinophils are more complex. IL-1 increases or decreases eosinophil release of 7

superoxide or the granule arylsulfatase and β-glucuronidase, depending on the choice and concentration of PMA or calcium ionophore used as co-stimuli.[ ] Although enzyme release from IgG-stimulated eosinophils is inhibited by preincubation with IL-1β, the enzyme release from IgE-stimulated eosinophils is enhanced by IL-1β.[

277]

In addition to their chemotactic activities, the chemokines also stimulate eosinophil effector functions. RANTES, MCP-3, and MIP-1α induce

elevations of Ca++ and, at relatively high cytokine concentrations, cause ECP release from eosinophils. [

201]

7

Little information is available regarding cytokines that inhibit the functions of eosinophils. TGF-β, an antiinflammatory cytokine,[ ] decreases the number of 16]

eosinophils in human bone marrow suspension cultures.[

TGF-β also inhibits

322

eosinophil survival induced by the eosinophil hemopoietins in vitro.[

278]

Furthermore, IFN-α suppresses antigen-induced eosinophilia and CD4+ T cell recruitment

279 mice.[ ]

into airway tissue in IFN-α has also been successful in treatment of certain subgroups of patients with eosinophilia.[ important regulatory roles in eosinophil-associated inflammation.

280]

Thus, TGF-β and IFN-α may play

The effects of Th2 cytokines such as IL-4 and IL-13 on eosinophils could be bimodal. IL-4 reduces eosinophil expression of IgG, but not IgE, receptors and IgGdependent release of granule enzymes or cytotoxic functions.[

281]

synergistically with TNF-α or IL-5 for increased expression of dependent adhesion of eosinophils, but not neutrophils, to

In contrast, IL-4 up-regulates binding of eosinophils to IgA.[

282 CD69[ ]

224 HUVECs.[ ]

and with chemokines for

283 migration.[ ]

151]

IL-4 or IL-13 works

Furthermore, IL-13 induces PSGL-1/P-selectin–

Interestingly, IL-13, but not IL-4, enhances survival of eosinophils and induces eosinophil

284 chemotaxis.[ ]

Recent work has shown the potential importance of another Th2-type cytokine, IL-9, in allergic responses. The development of transgenic mice overexpressing IL-9 has suggested a key role for this cytokine in the development of the asthmatic phenotype, including eosinophilic inflammation, bronchial hyperresponsiveness, 285

elevated IgE levels, and increased mucus secretion.[ ] IL-9 may act synergistically with other eosinophil-active cytokines. The addition of IL-9 to CD34+ cells cultured in IL-3 and IL-5 enhances eosinophil development. Moreover, IL-9 alone up-regulates the expression of IL-5Rα on human CD34+ cord blood progenitor 286

cells.[ ] Thus, eosinophil functions may be up-regulated or down-regulated by cytokines and their combinations. In the future the physiologic effects of these cytokines must be investigated in systems that consider the mucosal microenvironment surrounding eosinophils at sites of inflammation. Effects of Other Biologic Molecules Recent studies also suggest eosinophil activation can be induced by various biologic molecules. For example, an important mediator in the nervous and cardiovascular system, adenosine triphosphate (ATP), and other nucleotides induced Ca++ mobilization, oxygen radical production, and CD11b up-regulation of human eosinophils through P2X and P2Y purinoreceptors.[

287] [288]

Prostaglandin D2 (PGD2 ) is the major prostanoid released by mast cells during an allergic

response. Exposure of eosinophils to PGD2 induces rapid morphologic changes, chemokinesis, and cellular degranulation of human eosinophils through a newly 289 290

discovered seven-transmembrane receptor, chemoattractant receptor–homologous molecule expressed on Th2 cells (CRTH2).[ ] [ ] Proteolytic enzymes may also induce activation and noncytotoxic degranulation of eosinophils. For example, neutrophil elastase and trypsin induce degranulation of human eosinophils likely 291] [292]

through protease-activated receptor-2 (PAR-2),[

suggesting protease(s) released at the sites of inflammation or those derived from allergens may induce

release of proinflammatory mediators from eosinophils. Galectins are Ca++ -dependent lectins and are defined by their affinity for β-galactosides and by their homologous carbohydrate recognition domain. [

293]

Currently, 12 members of the galectin family have been identified, and they might be involved in a variety of 294]

allergic or immune responses. Indeed, galectin-9, also called ecalectin, induces superoxide production, chemokinesis, and aggregation of human eosinophils.[ discussed earlier, galectin-10, found abundantly at allergic inflammation sites, is the CLC protein produced by eosinophils and basophils.

As

Phenotypic Changes of Activated Eosinophils 4

Eosinophil exposure to activating cytokines leads to the development of hypodense eosinophils, with a specific gravity less than 1.085•g/ml. [ ] Cytokine exposure also leads to new expression or up-regulation of cell surface receptors through de novo synthesis or recruitment from intracellular stores. For example, after culture with GM-CSF alone, IL-5 plus TNF-α, or IL-3 plus IFN-γ, eosinophils express HLA-DR antigen and increased amounts of ICAM-1, giving these eosinophils the capacity to process and present antigens to T cells.[ 295]

of an early activation antigen, CD69,[

273] [274]

In vitro stimulation of peripheral blood eosinophils with IL-3, IL-5, or GM-CSF induces the expression

and IL-2Rα (p55, CD25),[

214]

296]

it up-regulates the expression of the β2 integrin, CD11b,[

and it down-regulates L-

174

selectin, platelet–endothelial cell adhesion molecule-1 (PECAM)-1, and PSGL-1 expression.[ ] Eosinophils from patients with asthma or allergy show similar phenotypic changes to those activated in vitro. For example, peripheral blood eosinophils from patients with asthma or allergy overexpress an adhesion molecule, CD11b,[

138]

and the expression is reduced by treatment of patients with glucocorticoids.[

increased expression of CD66, CD69, and [299] [300]

298 CD81.[ ]

297]

Blood eosinophils from patients with helminth infection also showed 295]

Eosinophils in sputum or BAL fluid from patients with asthma express ICAM-1, CD69, and HLA-DR.[

After allergen challenge, eosinophils in BAL fluids from patients with allergic asthma show even more dramatic changes. These eosinophils, compared

with blood eosinophils, show up-regulation of CD11b, CD11c, CD67, and a granule-associated antigen, CD63. [ CD45 seem to show no change and remain similar between the blood and BAL

302 eosinophils.[ ]

169] [301]

In contrast, the levels of CD32, CD11a, and

Because eosinophil transendothelial migration induces expression of 303

various activation markers, such as CD69, HLA-DR, and CD54, the migration step from blood into tissue likely plays a role, at least in part, for activation.[ ] Indeed, CD44 expression in circulating eosinophils was higher in patients with uncontrolled asthma compared with patients with controlled asthma and even higher 304

in sputum eosinophils. [ ] Thus, eosinophils from patients with asthma, especially those derived from the lungs, show some activated phenotypes, and these phenotypic changes may be associated with functional activation. Priming and Partial Activation The eosinophil respiratory burst is induced by fMLP, using two different mechanisms. First, at high concentrations, fMLP by itself can cause a respiratory burst in 4

305]

eosinophils.[ ] Second, with suboptimal concentrations of fMLP, the respiratory burst is greatly enhanced after pretreatment of eosinophils with PAF,[ that eosinophils are readily responsive to a secretagogue once they have been exposed to an activator. This phenomenon is called priming. several cytokines appear to prime eosinophils.

[306]

suggesting

In addition to PAF,

323

For example, after preincubation with eosinophil growth factors, such as IL-3 and IL-5, eosinophils produced LTC4 on stimulation by fMLP, C5a, and PAF. [

307]

In

contrast, normal eosinophils produce LTC4 after stimulation with fMLP but not after stimulation with C5a or PAF. Eosinophils can also be primed in vivo. One strategy to obtain in vivo primed eosinophils is segmental allergen challenge followed by BAL in patients with upper airway allergy. Eosinophils in the BAL after 300

allergen challenge show a greater respiratory burst and adherence response to fMLP.[ ] Stimulation of these BAL eosinophils with fMLP causes a sustained elevation in intracellular Ca++ ; stimulation of blood eosinophils with fMLP causes only transient elevations in cellular Ca++ . Eosinophils also can be primed within the peripheral blood in patients with disease. For example, eosinophils isolated from blood of patients with asthma also demonstrate many enhanced proinflammatory 308]

properties compared with those from normal individuals, including enhanced adhesion, chemotaxis, transendothelial migration, and LTC4 production.[

Furthermore, the treatment of asthma patients with a combination of inhaled glucocorticoids and β-adrenergic agonists not only improves lung function but also inhibits markers of eosinophil priming, such as the respiratory burst and PAF releasability. [

309]

Because eosinophils are exposed to the local cellular milieu consisting of cytokines, chemotactic factors, and ECM proteins, priming of eosinophils is important to 265

understand the pathophysiology and activity of disease.[ ] Furthermore, priming is likely a preparation step while eosinophils are recruited from bone marrow into the tissues so that they can be promptly and fully activated in response to secretagogues at the inflammatory sites. Degranulation Eosinophil degranulation in tissues is thought to make important contributions to the pathogenesis of disease. One of the striking features of the eosinophil-rich inflammatory reaction is the marked deposition of granule proteins, often in the virtual absence of intact eosinophils. However, the mechanism of eosinophil secretion in vivo is still poorly understood. Electron microscopic studies suggest that the release of granule proteins from eosinophils involves three different mechanisms: cytolytic degranulation or necrosis, regulated secretion or compound exocytosis, and piecemeal degranulation. Cytolytic degranulation is often seen in biopsies of patients with hypereosinophilic syndrome, episodic angioedema with eosinophilia, bullous pemphigoid, and IgE-mediated cutaneous late-phase reactions. [4] [7]

These eosinophils display centralization of granules, plasma and organelle membrane breakage, and chromatolysis of their nuclei. Occasionally, “cytolytic 310]

degranulation” is applied to the free granules seen in association with necrotic eosinophils in the tissues.[ or a result of eosinophils succumbing from environmental factors is not known.

Whether cytolytic degranulation is a regulated process

Regulated secretion, or compound exocytosis, resembles secretion of granulated secretory cells, especially of the anaphylactic degranulation of mast cells and 311

basophils.[ ] The individual granule membranes and portions of the plasma membrane fuse around the periphery of cells, and specific granule matrix and core contents are extruded through these degranulation pores ( Figure 19-2 ). Occasionally, granule

Figure 19-2 Electron photomicrograph showing a human eosinophil after 4 hours of incubation with 1.0-•M platelet-activating factor at 37°C. The lucency of the granule cores (in contrast to Figure 19-1 ) and the granule fusion among certain granules (white arrows) are evident.

(Photomicrograph courtesy J. Bankers-Fulbright, PhD.)

(Photomicrograph courtesy J. Bankers-Fulbright, PhD.) membranes may fuse with each other within the cytoplasm to form degranulation chambers. These chambers also open to the cell exterior through degranulation pores. Regulated secretion may take place into a phagosome/phagolysosome, containing an internalized microbe, within the cytoplasm of the cell.[ Piecemeal degranulation, which may occur in the peripheral blood of patients with allergy,[ partially empty granule chambers in the

311 cytoplasm.[ ]

313]

312]

is characterized by vesicular transport of granule contents and

Vesicles containing granule proteins seem to bud off from the specific granules. Although the mechanism of 314]

piecemeal degranulation is not fully elucidated, the role of a fusion protein, vesicle-associated membrane protein-2 (VAMP-2), is suggested.[

Thus, eosinophils can use three different mechanisms to release granule proteins; however, it is unknown whether any of these degranulation mechanisms are regulated by different stimuli and signal transduction mechanisms. Most observations suggest that cell injury (cytotoxic degranulation) occurs frequently in vivo. Also, degranulation, especially regulated secretion, can be a highly focused event in which an adherence step precedes granule release onto an appropriately opsonized target, such as complement-coated or Ig-coated larvae of the parasitic helminth S. mansoni. The adhesive interaction with the larval tegument is thought to be crucial for effective cytotoxicity against the parasite. Initial studies of eosinophil degranulation in vitro were performed using a parasite model. Eosinophils incubated with antiserum-coated schistosomula of S. mansoni

degranulate and release MBP.[

1] [4]

However, investigation of eosinophil degranulation in this system was complicated by the presence of the viable worm.

Sepharose beads coated with IgG, IgA, and sIgA stimulate eosinophil degranulation[ eosinophil killing of S. mansoni.[

316]

3] [315]

; sIgA is the most effective. IgA2 is a highly potent stimulus for

IgE may be also important for

324

TABLE 19-3 -- Activators and Regulators of Eosinophil Function

Degranulation

Lipid Mediator Production

Superoxide Production

IgG

↑

↑

↑

IgE

↑

↑

IgA

↑

↑

↑

Secretory IgA

↑

↑

↑

C3a

↑

↑

C5a

↑

Zymosan

↑

↑

↑

↑

↑

↑

Activators and Regulators

Priming

*

Chemotaxis, Chemokinesis

Adhesion

Survival

Cytokine Production

Immunoglobulins ↑

↑ ↑

Complement ↑ ↑

↑

†

↑

‡

Lipid Mediators PAF

↑

↑

LTB4

↑

↑

↑

Cytokines IL-1

↑↓

↑↓

IL-3

↑

↑

↑

↑

§

↑

↑

IL-4

↓

IL-5

↑

↑ ↑

↑

↑

↑

‡

IL-13 GM-CSF

↑

↑

↑

↑

‡

↑

TNF-α IFN-γ

↑

↑

↑

↑

↑

↑

↑

↑

↑

↑

↑

↑↓

↑

†

↑

↓

TGF-β Chemokines IL-8

↑

†

RANTES

↑

*

↑

MCP-3

↑

*

↑

*

↑

MCP-4 Eotaxin

↑

↑

↑

*

Cell Adhesion Molecule Ligands β2 integrin

↑

↑

↑

↑

ligands Fibronectin

↑

↑

↑

VCAM-1

↑

↑

↑

Other Activators and Regulators fMLP

↑

↑

↑

↑

↑ ↑

Ecalectin Trypsin/elastase

↑ ↑

ATP PGD2

↑

↑

↑

↑

↑

*

↑

MBP

↑

Modified from Kita H, Gleich GJ: The eosinophil: structure and functions. In Kaplan AP, editor: Allergy, ed 2, Philadelphia, 1997, WB Saunders, p 151. ↑, Induction/up-regulation of eosinophil function; ↓, inhibition of function; fMLP, formyl-methionyl-leucyl-phenylalanine; ATP, adenosine triphosphate; see Tables 19-1 and 19-2 for other abbreviations. * Enhancement of eosinophil function (e.g., superoxide production, chemotaxis) induced by other agonists. † In the presence of cytochalasin B. ‡ In the presence of priming factors. § At high concentrations.

325

1 4

eosinophil activation. Eosinophils isolated from patients with eosinophilia degranulated in response to anti-IgE antibody or IgE-coated parasites.[ ] [ ] However, using sera from patients with ragweed-sensitive hay fever, the authors found that ragweed-specific IgG, but not IgE, stimulates allergen-dependent eosinophil 137]

degranulation in vitro.[

The ability of eosinophils to secrete granule proteins is greatly enhanced by priming with relatively low concentrations of cytokines such 7

as IL-5, GM-CSF, and IL-3.[ ] Eosinophil degranulation can also be induced by soluble stimuli themselves. For example, eosinophils incubated for 4 days with IL-5 258

release 30% to 60% of their granule proteins.[ ] The release of granule proteins during culture with IL-5 may be an important mechanism for deposition of these cationic toxins in various diseases where IL-5 plays a role. Eosinophil-chemotactic cytokines, such as RANTES and MIP-lα, also induce eosinophil degranulation, although the effects are less pronounced than those of IL-5 or GM-CSF. [

201] [317]

Interestingly, eosinophil granule proteins themselves, including MBP and EPO, 131

stimulate eosinophils and cause degranulation in a noncytotoxic manner, suggesting an autocrine mechanism of eosinophil degranulation.[ ] The other stimuli for eosinophil degranulation include serum-opsonized zymosan, fMLP, the lipid mediator PAF (see Figure 19-2 ), the complement fragments C5a and C3a, naturally occurring peptides (e.g., substance P, mellitin), calcium ionophore A23187, and PMA.[

4] [154] [156] [157]

4

Although LTB4 stimulates a respiratory burst in human and

guinea pig eosinophils, [ ] LTB4 does not seem to induce degranulation of eosinophils, as assessed by the release of β-glucuronidase [

160]

( Table 19-3 ).

Eosinophils are primarily found in the tissues rather than in circulating blood. Therefore, not surprisingly, eosinophils' adhesive responses can modulate their proinflammatory functions. An adhesion molecule, Mac-1 (CD11b/CD18), likely plays critical roles for eosinophil degranulation (see Table 19-2 ). Historically, receptor ligands immobilized to relatively large surfaces, such as IgG-coated Sepharose beads and parasites, but not particulate ligands, such as aggregated IgG and 1] [4]

bacteria, are effective stimuli for eosinophil degranulation. [

By using IgG immobilized to tissue culture plates or Sepharose beads, the authors found that β2

integrins, especially Mac-1, play a crucial role in the activation of eosinophils stimulated by IgG.[

318]

Similarly, the eosinophil functional response to PAF or GM-

CSF is greatly influenced by the availability of cellular adhesion. The authors found that PAF or GM-CSF induced eosinophils to release EDN when the eosinophils were incubated in stationary wells of tissue culture plates, and the degranulation was blocked by antibody against Mac-1 on eosinophils.[ these stimuli to induce eosinophil degranulation was minimal when cells were kept in suspension by continuous can be induced by direct ligation of integrins by antibodies or ligands to mediated by Mac-1, plays a critical role in eosinophil degranulation.

320 Mac-1[ ]

or

321 VLA-4.[ ]

319 mixing.[ ]

319]

In contrast, the ability of

Furthermore, eosinophil granule release

These findings suggest that eosinophil adhesion, especially that

A number of pharmacologic approaches have been taken to regulate the secretory process of eosinophils. Pretreatment of eosinophils with pertussis toxin (PTX) 322

abrogates sIgA-induced degranulation and increases in PLC activity,[ ] suggesting that eosinophils contain PTX-sensitive G proteins implicated in activation of the cells. PTX-sensitive G protein is also involved in the interaction of eosinophils with chemokines, lipid mediators, and complement fragments. Glucocorticoids do not suppress eosinophil degranulation. However, cyclic adenosine monophosphate (cAMP) analogues, phosphodiesterase inhibitors, and β-adrenergic agonists inhibit degranulation processes.[

323]

Among them, type IV phosphodiesterase inhibitors (rolipram, denbufylline) potently inhibit eosinophil activation, suggesting a potential

use of these drugs as antiinflammatory agents. [

324]

ECP release by eosinophils stimulated with eotaxin involves the activation of cell signaling pathways associated 325]

with extracellular signal–regulating kinase-2 (ERK-2) and p38 mitogen-activated protein kinases.[ Production of Lipid Mediators

Eosinophils produce the sulfidopeptides LTC4 and LTD4 in response to calcium ionophore A23187, fMLP, PAF, and IgG-dependent stimuli. Furthermore, eosinophils release LTC4 on exposure to S. mansoni coated with parasite-specific IgE or IgG antibodies. In addition, eosinophils from patients with asthma produce 4

substantially larger quantities of LTC4 than do eosinophils from normal subjects.[ ] Furthermore, stimulated eosinophils release substantial quantities of PAF.[

4] [111]

[112]

Eosinophils contain high levels of ether phospholipids (the stored precursor of PAF), about fourfold more than neutrophils. The secretion of PAF from eosinophils is caused by stimulation with chemotactic stimuli, such as C5a, as well as zymosan and A23187. PAF biosynthesis by eosinophils is increased threefold to fourfold by preincubation of eosinophils with GM-CSF for 72 hours.

EOSINOPHILS IN DISEASE MECHANISMS Host Defense Although capable of phagocytosing and killing bacteria and other small microbes in vitro,[

79]

eosinophils cannot effectively defend the host against bacterial

326 deficient.[ ]

infections when neutrophil function is Rather, eosinophils appear to defend against large, nonphagocytosable organisms, most notably the multicellular helminthic parasites. Based on a number of in vitro studies demonstrating human eosinophils functioning as helminthotoxic effector cells and the cytotoxicity of eosinophil granule proteins against several nematodes, it has been hypothesized that a major beneficial function of eosinophils is to participate in host defense against helminthic parasites. Furthermore, in addition to host-derived immunoglobulins and complement components on the surface of their targets, eosinophils may bind and respond to carbohydrate ligands expressed on the parasites, such as the LewisX -related molecules, and cell adhesion molecules (CAMs) similar to selectins.[

4]

Such a putatively beneficial role is contrasted with some of the deleterious effects eosinophils exert in allergic diseases. However, studies with anti-IL-5-treated, helminth-infected mice have questioned this role, because neutralizing anti-IL-5 antibody has abrogated infection-induced blood, marrow, and tissue eosinophilia, but not the intensities of primary or secondary infection.[ differences between mouse and human

329 eosinophils.[ ]

327] [328]

However, these results need to be interpreted carefully in the light of potential functional

Furthermore, mice are not the natural hosts of many of the parasites tested experimentally.

326

Another potentially protective role for eosinophils may be against certain viral infections. It has been recognized for several years that the numbers of eosinophils and 330

concentrations of eosinophil granule proteins are increased in the respiratory tracts of patients with respiratory syncytial virus (RSV), an RNA virus.[ ] As described previously, EDN and ECP are ribonucleases, and purified eosinophils, EDN and ECP added to RSV viral suspensions reduced the viral titer, dependent on 62 63

their ribonuclease activities. [ ] [ ] Interestingly, ribonuclease A lacked this antiviral activity, suggesting that RNase activity is necessary but not sufficient for the antiviral effects of EDN and ECP. Furthermore, in guinea pigs infected with parainfluenza, pretreatment with anti-IL-5 and reduction of eosinophils strikingly 331

increases viral content in the airways,[ ] suggesting a potential role for eosinophils in viral immunity. Thus a beneficial function for eosinophils in host defense is possible; this concept remains to be fully validated in humans. Elicitation of Host Dysfunction and Damage The effector response of eosinophils may also contribute to the physiologic and pathologic reactions associated with eosinophilia, including immediate hypersensitivity and other allergic diseases (see Chapter 63 ). Eosinophil products that are most damaging to the host are likely the specific granule's cationic proteins. Elevated concentrations of these proteins can be detected in the sputum of patients with asthma, in nasal and BAL fluids after the experimental inhalation of allergens, and within involved tissues. As discussed earlier, in allergic diseases and asthma, eosinophil granule proteins may cause damage and desquamation of airway epithelial cells, alter airway hyperreactivity (AHR) and cilial function, and elicit local edema. Moreover, eosinophil-derived eicosanoids, PAF, and LTC4 may contribute to airway bronchial constriction and inflammation. Additional evidence from a guinea pig model suggests that neutralization of endogenously secreted 332]

MBP with antibodies to MBP can prevent antigen-induced AHR.[

Therefore, some of the mechanisms used by eosinophils in host defense may also have detrimental effects to the host; the dysfunction and damage caused by eosinophil granule proteins may contribute to the pathogenesis of diseases in which large numbers of eosinophils are found in the involved tissues. Indeed, the association of airway eosinophilic inflammation and asthma has been so strong that researchers have suggested that asthma should be renamed “eosinophilic desquamative bronchiolitis.” However, this concept has been queried recently by a study that examined the effects of treatment with a humanized anti-IL-5 mAb (SB14

240563) on allergen-induced airway response and inflammation in atopic subjects. [ ] The study demonstrated that treatment with anti-IL-5 mAb significantly reduced blood eosinophils and sputum eosinophils for at least 4 weeks, but it did not have any significant effect on allergen-induced late response or histamine airway responsiveness. These results then questioned the role of eosinophils in mediating the late asthmatic response and causing AHR. Subsequently, concerns have also been raised regarding several methodologic limitations of the study.[

333]

A recent study found that repeated anti-IL-5 treatment of patients with asthma only

334

reduced bronchial mucosal eosinophils by 50%.[ ] Furthermore, a complex interplay of various inflammatory mediators and inflammatory cells may operate in the pathologic processes of asthma, and the removal of one cell type may be insufficient to reverse the airway's physiology. Further studies are required to dissect the potential detrimental roles of eosinophils in human diseases. Other Functions In addition to host defense and dysfunction, eosinophils likely have other roles in the immune response. For example, the eosinophils that usually reside in normal mucosal tissues probably participate in mucosal innate immunity, but their specific functions in individual responses at mucosal surfaces have not yet been delineated. Eosinophils probably have roles in wound healing and repair and maintenance of tissue homeostasis and are associated with fibrotic disorders. Finally, eosinophils have the capacity to influence other immune and inflammatory responses, including T cell–dependent responses. As noted previously, eosinophils can be 335

a source of cytokines, such as IL-4 and IL-16, and can function as antigen-presenting cells (APCs).[ ] These potential roles for eosinophils may be particularly important in mucosal tissues exposed to the external environment, sites where eosinophils are abundant.

CONCLUSIONS The realization that eosinophils can release proinflammatory mediators, such as platelet-activating factor and eicosanoids, and the observation that eosinophil granule proteins are toxic for airway epithelium and other cells have led to a consensus that eosinophils are major effector cells for tissue inflammation in various allergic diseases. Recent advances in molecular biology and immunology are helping to elucidate the mechanisms of eosinophil proliferation, recruitment, activation, and effector functions. Future studies will shed additional light on the pathophysiologic roles of eosinophils in bronchial asthma, on the mechanisms of inflammation in diseases associated with eosinophilia, and on the means to disrupt the inflammatory processes caused by eosinophils.

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Chapter 20 - Biology of Mast Cells

John J. Ryan Thomas F. Huff

Mast cells are a central component of allergic disease and innate immunity to parasite and bacterial infections. This chapter reviews their biology and pathobiology, focusing primarily on human mast cells because of their relevance to human disease. Select aspects of rodent mast cells are considered, since a controlled genetic background and easy access to high-purity cells allow experimental manipulations not readily performed in humans. Not covered in this chapter but found elsewhere in this text are the essential aspects of signal transduction ( Chapter 8 ), mast cell–derived mediators ( Chapter 13 ), lipid mediators involved in hypersensitivity ( Chapter 14 ), and mast cell activation ( Chapter 16 ).

HISTORICAL PERSPECTIVE The first definitive description of mast cells is attributed to Paul Ehrlich, who observed the metachromatic staining of mast cell granules while he was still a medical student at Freiburg University, Germany, in 1877. Ehrlich applied aniline dyes to connective tissues and noticed that certain cells had prominent granules that stained 1

not only in the basic tone (blue) but also metachromatically (reddish purple), and that the stain of the granule was insoluble in alcohol.[ ] Even today, metachromasia remains an important criterion for a cell to be called a mast cell or a basophil. Ehrlich chose the name mastzellen, the German word mast referring to feeding, because he believed that the mast cell inclusion bodies were the result of overfeeding. Ehrlich described the association of mast cells with blood vessels, inflamed tissues, nerves, and neoplastic foci. He also provided the first description of mast cell degranulation. Ehrlich described the basophil separately as a metachromatic cell that circulated in the blood. Because mast cells and basophils share the attributes of metachromasia, histamine content, and expression of the high-affinity receptor for immunoglobulin E (IgE), past textbooks have reflected a long-standing assumption that the two cells are intimately related in lineage. However, this assumption appears incorrect, since other features identify mast cells and basophils as distinctly different ( Table 20-1 ). These distinguishing features include nuclear morphology, life span, proliferative ability, location in vivo, differentiation factors, cell surface adhesion molecules, mediator content, and response to chemical activating agents. Basophils are terminally differentiated granulocytes that complete their development in the bone marrow before being released as mature cells to circulate in the blood. They can be recruited into sites of inflammation. In contrast, mast cells leave the bone marrow and circulate in the blood as progenitors, completing their differentiation in mucosal or connective tissues. The differences between mast cells and basophils can have important implications for diagnosis and treatment and should be appreciated by the clinician (see Chapter 21 ).

MAST CELL MORPHOLOGY Mature human mast cells are usually 9 to 12••m in diameter and appear as round, spindly, or spiderlike cells in tissues. They have numerous cytoplasmic granules and thin elongated folds of their plasma membrane. Mast cell nuclei are unsegmented ovals, elliptically placed, and rarely show mitotic figures in tissue. The morphologic feature most definitive for both mast cells and basophils is a striking array of metachromatic granules. In mature mast cells these granules may 2

constitute much of the dry weight of the cell and half its volume.[ ] At the ultrastructural level, differences in granule morphology appear to exist among different species; human mast cell granules have intricate and varied patterns, whereas rodent mast cell granules have an amorphous appearance. Some differences in granule morphology correspond to different protease compositions in the two human mast cell subpopulations, called MCT and MCTC ( Figure 20-1 ). Granules with chymase exhibit a grating/lattice substructure, whereas regions lacking this protease exhibit complete scroll patterns. Neither pattern appears in basophils, which exhibit

granules more heterogeneous in size with amorphous and particulate features. Mast cells reside in tissues at potential portals of entry of noxious substances, such as skin, conjunctiva, gut, and respiratory mucosal surfaces. Moreover, mast cells are not randomly dispersed in these tissues. In the skin, for example, mast cells are found at the epidermal/dermal junction and are concentrated around blood vessels, 3

nerves, and appendages.[ ] Consequently, allergic symptoms are best appreciated at such sites. In most species, including humans, bone and cartilage have few mast cells. Parenchymatous organs such as liver, brain, kidney, and adrenals generally have modest numbers of mast cells. Mast Cell Types Two types of mast cells, differing in their granule contents, have been consistently observed in humans and rodents (see Table 20-1 ). Contradictory data describing mast cell

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TABLE 20-1 -- Preformed Mediators of Mast Cells and Basophils Cell Type

Biogenic Amine

Protein

Proteoglycan

MCT

Histamine

α- and β-Tryptase

Heparin, CS A and E

MCTC

Histamine

α- and β-Tryptase Chymase Carboxypeptidase Cathepsin G

Heparin CS A and E

Basophil

Histamine

2D7 antigen

CS A

Mucosal (RBL-1)

Histamine

Chymase II

CS A and di-B

Connective tissue

Histamine Serotonin

Chymase II TryptaseCarboxypeptidase A

Heparin CS

Human

Rat

Mouse

Mucosal

Histamine

Chymases MMCP-1,2,4,9

CS A and E

Connective tissue

Histamine Serotonin

Chymases MMCP-3,4,5,8

Heparin CS

Dog

Histamine

Chymase Tryptase

Heparin CS

CS, Chondroitin sulfate(s); MMCP, murine mast cell protease. heterogeneity, worsened by nomenclature that differs in human and rodent systems, has yet to offer a consensus on how these two lineages develop and why this matters to biology. The reader's patience, however, is advised; it is unlikely that the two forms of mast cells evolved without purpose, and their function most likely is not yet fully understood. The great strides made in this relatively new science offer hope that a full developmental and clinically significant picture will evolve from this work.[

4]

In rodent systems the terms mucosal mast cells and connective tissue mast cells refer to certain phenotypic characteristics. Although describing the site where these cells are most frequently found, the terms should be used to describe mast cell phenotype, not location. Each type may in fact be found in both mucosal and connective tissues. Rodent mucosal mast cells (MMCs, atypical) and connective tissue mast cells (CTMCs, typical) are defined by differences in fixation and histochemical staining that relate to the selective production of heparin proteoglycan in CTMC, whereas chondroitin sulfate proteoglycans reside in both MMC and 5

CTMC. A single gene encodes the core peptide of both types of proteoglycan,[ ] suggesting that the class of glycosaminoglycan added onto the core may be regulated by signals dependent on mast cell type. After staining with Alcian blue (blue) and safronin O (red), mature CTMCs fixed in either formalin or Carnoy's fluid are red, whereas mature MMCs stain blue and only after fixation in Carnoy's fluid. [

6]

Besides staining criteria, a more precise and complex definition of rodent MMC and CTMC is based on expression of mast cell–specific proteases (see Table 20-1 ). 7

Neutral protease expression qualitatively distinguishes rat CTMCs, which contain chymase I and carboxypeptidase A, from rat MMCs, which contain chymase II.[ ] More complex discriminations based on expression of mouse mast cell proteases (MMCPs) have been reported in the mouse, which express at least seven different chymases (MMCP-1 through MMCP-5; MMCP-8 and -9) and two tryptases (MMCP-6 and -7). One essential difference is the selective expression of MMCP-5 in CTMC and MMCP-1 expression in MMC.[ is outside the scope of this chapter.

8] [9]

Much detail is now known about control of MMCP expression under physiologic and pathologic conditions, but this

Human mast cell nomenclature is based on neutral protease composition. Whereas MCT cells express tryptase alone, MCTC cells produce tryptase, chymase, mast 10

cell carboxypeptidase, and cathepsin G.[ ] Although a particular mast cell type often predominates in a particular tissue, smaller amounts of the other mast cell type are usually present, and the relative abundance of each may change with tissue inflammation or fibrosis. Therefore, it is impossible to base a subtype designation on location alone. With this caveat, it can be said that MCT cells predominate in normal lung, particularly alveoli, and in the small intestine mucosa. MCTC cells are the predominant type found in normal skin, blood vessels, gastrointestinal submucosa, and synovium. Also, two reports have described a chymase-positive, tryptase11] [12]

negative (MCC ) human mast cell that can be derived in vitro and is found as a minority population (7%–17%) in the bowel mucosa and submucosa.[

Little

more is currently known about the role of this cell or its development. Human MCTC and MCT bear similarities to mouse CTMC and MMC, respectively, both in terms of tissue distribution and the selective expression of human chymase in MCTC and MMCP-5 chymase in CTMC. However, the mouse tryptases MMCP-6 and MMCP-7 are generally found only in CTMC,

335

Figure 20-1 Ultrastructural features of MCTC and MCT types of mast cells in human tissues. A, MCT cell from human lung showing scroll-rich granules. B, High magnification of scroll-rich granule. C, Scroll-rich granule from MCT cell stained with antitryptase associated with 5-nm gold particles and antichymase associated with 20-nm gold particles. Labeling only with the 5-nm gold particles is detected ultrastructural features of MCTC and MCT types of mast cells in human tissues. D, MCTC cell from human skin showing electron-dense granules with regions of gratings and lattices. E, High magnification of grating/lattice structure in granules of MCTC cell. F, Granule from MCTC cell stained as in C showing 5-nm and 20-nm gold particles labeling the granules. (Courtesy Shirley Craig, PhD, Medical College of Virginia.)

(Courtesy Shirley Craig, PhD, Medical College of Virginia.) 8 9 10

unlike the shared expression of tryptase in almost all human mast cells.[ ] [ ] [ ] All human mast cells appear to express heparin proteoglycan, unlike its selective expression in rodent CTMC. Also, histamine levels in human mast cell types appear to be similar, whereas rodent CTMC can possess tenfold more histamine than 13]

MMC.[

Consequently, the rodent and human nomenclature systems are similar but distinct. Table 20-2 summarizes the distribution of MCT and MCTC cells in

tissue sections obtained from apparently normal human tissues.

Function and Pharmacologic Differences

Although much remains to be learned about the selective distribution of mast cell types, several studies imply possible clinical importance to this distribution. For example: T cell deficiency results in selective loss of MCT cells [ are associated with areas of dense fibrosis.[ to their type.

18]

14]

; asthma and allergic rhinitis can selectively increase MCT numbers;[

15] [16] [17]

and MCTC cells

Such studies make relevant a brief discussion of the different aspects of mast cell activation and regulation according

Studies of mast cells derived from mature human foreskin are of interest because the mast cells here, although somewhat immature, are almost exclusively of the MCTC type. Such mast cells release histamine in response to immunologic stimulation (antigen and anti-IgE) and a wide variety of nonimmunologic agents, such as calcium ionophore A23187, compound 48/80, basic polypeptides (polylysine, polyarginine), morphine sulfate, formyl-methionyl-leucyl-phenylalanine (fMLP) peptides, substance P, and the anaphylatoxin C5a. In contrast,

336

TABLE 20-2 -- Distribution and Morphology of Human Mast Cells MCTC

MCT

Skin

++

−

Intestinal submucosa

++

+

Small intestine mucosa

+

++

Alveolar wall

−

++

Bronchi/bronchioles

+

++

Nasal mucosa

++

++

Conjunctiva

++

+

Synovium

++

−

Vascular wall

++

−

Distribution/Morphology Normal Tissue Distribution

Heart

++

+

Granule Morphology

Grating/lattice structures

Complete scroll rich

Complete scroll poor

Lamellar/particulate structures

Lamellar/particulate structures First/Second-Degree SCIDs

Normal numbers/distribution in intestine

Deficient in intestine

++, Wide distribution; +, some distribution; -, no distribution; SCIDs, severe combined immunodeficiency diseases. 19]

mast cells isolated from lung (approximately 90% MCT ) are activated by anti-IgE and calcium ionophore A23187 but not by any of the other agents listed. [

Compound 48/80 and the anaphylatoxins C3a and C5a also are ineffective in activating dispersed human adenoidal mast cells, where MCT cells predominate.[

20]

Furthermore, activation of skin MCTC cells corresponded to C5a receptor (CD88) expression, whereas lung mast cells were not activated by C5a and did not express 21

CD88.[ ] Such studies are based on the caveats that they frequently employ samples with low mast cell purity and can be biased from microenvironmental effects. However, the sum of this work offers a picture of MCTC cells as generally more responsive to non-IgE-mediated stimuli than MCT cells. This may mean that MCTC cells have a broader role in immunity. Disodium cromoglycate is frequently used for the treatment of allergic asthma, rhinitis, and conjunctivitis. Its local effects are believed to be caused by inhibition of 22 23

24 25

mast cell and mediator release and possibly cytokine production, [ ] [ ] although cromoglycate's clinical effectiveness has been debated.[ ] [ ] Several studies have demonstrated modest (10%–20%) cromoglycate-mediated reductions in mast cell histamine release. These studies used tissues where MCT cells predominate, including bronchoalveolar lavage (BAL),[

26]

20]

adenoids,[

27] [28]

and dispersed mast cell preparations from lung or intestinal tissue.[

In contrast, histamine release

[29]

from human skin after in vitro antigen challenge was not inhibited by disodium cromoglycate. The addition of 2% cromolyn to ragweed antigen preparations did not inhibit the skin reaction to local injection of the antigen in ragweed-sensitive individuals, as determined by histamine release, ultrastructural alterations of mast cells, and ingress of eosinophils.[

30]

Such studies indicate a differential, although weak, inhibitory effect of disodium cromoglycate on MCT but not MCTC cells.

Interestingly, this indication contrasts with the situation in rodents, where CTMCs but not MMCs are affected.

MAST CELL GROWTH AND DEVELOPMENT The cellular origin and lineage of mature mast cell subpopulations have been studied in both human and rodent species. Although clinical interest focuses on human mast cell development, rodent systems offer a wealth of information through transplantation and gene-knockout approaches. Therefore the rodent system is considered first and contrasted with human mast cell development. Rodent Studies

31

The bone marrow origin of mast cells was first demonstrated through a series of in vivo reconstitution studies.[ ] Injection of bone marrow cells from beige mice, whose mast cells are marked by giant granules, into the dermis of normal mice resulted in tissue mast cells arranged in clonal patterns of either donor cells (giant granules) or host cells (normal granules), but cell types were never mixed within the same cluster.[

32]

33

Injection of normal bone marrow cells into mast cell– deficient mice bearing mutations at the white spotting locus (W) reconstitutes mast cells in many tissues.[ ] Mice with homozygous mutations at the steel locus (SI) are also profoundly mast cell deficient but cannot be cured by injection of normal bone marrow cells. These and many other results suggested that SI mutants possessed microenvironmental defects, whereas W mutants were defective at the level of the progenitor cell.[ molecular explanation for these mast cell deficiencies was provided when the W and the SI genes were protooncogene, which

34 35 36 37 38 39 40 cloned.[ ] [ ] [ ] [ ] [ ] [ ] [ ]

31]

The

W contains the c-kit

337

encodes Kit, a receptor tyrosine kinase highly expressed on mast cells. SI encodes a hematopoietic growth factor called stem cell factor (SCF; also called steel factor, mast cell growth factor, or Kit-ligand). SCF is highly expressed on the surface of fibroblasts, stromal cells, and endothelial cells. It is also produced as a soluble 41

protein.[ ] Interestingly, SId mice produce soluble SCF but not membrane SCF. Therefore it appears that membrane SCF is essential for normal mast cell development. Mast cell differentiation, unlike basophil differentiation, can be separated into a bone marrow phase and a peripheral phase, which may depend on the presence of fibroblasts or stromal cells. Both phases are mediated by growth factors acting together or in sequence. Within the bone marrow the precise lineage of mast cells is still unknown, unlike the lineage of basophils. Expression of number of cell surface markers indicates that mast cells are related to the myeloid lineage. In rodent bone marrow, CD34+ , interleukin-3 (IL-3R)+ , Kitlow , FcepsilonRI− stem cells become CD34+ , IL-3R+ , Kithigh , FcepsilonRI− and then CD34− , IL-3R+ , Kithigh , 42

FcepsilonRI+ under the influence of IL-3 and SCF.[ ] SCF and IL-3 were both originally defined as mast cell growth factors but were later defined as growth factors capable of promoting self-renewal of stem cells. In fact, if naive bone marrow from rodents is cultured continuously with either SCF or IL-3 without other lineagespecific growth factors, the cultures yield greater than 90% mast cells after a few weeks. Interestingly, although IL-3 is a potent mast cell growth factor, IL-3– deficient mice have normal mast cell development. It appears that the nonredundant in vivo functions of IL-3 are related to mast cell expansion during some parasitic infections in rodents.[

43]

To begin the peripheral phase of the mast cell lineage, multipotential bone marrow progenitors progress to the level of unipotential mast cell–committed progenitors. [44] [45]

These committed progenitors leave the marrow and migrate to and settle in fibroblast-rich connective tissues or mucosal sites by a process that may be 46]

mediated by the binding of their laminin receptors[

47]

with laminin in tissues or of c-kit with SCF on fibroblasts. [

Studies using fetal blood indicate that the 42

circulating mast cell–committed progenitor is faintly granulated and expresses mRNAs encoding mast cell proteases, but lacks FcepsilonRI expression. [ ] These committed progenitors undergo limited clonal expansion and final differentiation into fully granulated, phenotypically distinct mast cells. Once differentiated, mature mast cells can be stimulated to undergo limited proliferation by many of the same factors that act on the progenitors. For example, mature peritoneal mast cells can

respond to SCF to form small colonies in semisolid medium.[

48]

Rodent studies have demonstrated that in addition to Kit and SCF, two transcription factors, the microphthalmia locus-encoded transcription factor (MITF) and GATA-2, appear to be particularly important in mast cell development and gene expression. MITF is a basic helix-loop-helix leucine zipper type of deoxyribonucleic acid (DNA)–binding protein that is important early in mast cell development.[

49]

Mice with the microphthalmia mutation are mast cell deficient, although not as 50

severely as mice with the W or SI mutation. MITF controls c-kit transcription, but apparently only in mast cells, not in melanocytes, neurons, or stem cells.[ ] MITF activity may explain why Kit expression increases during mast cell development while other hematopoietic lineages lose Kit expression. MITF also regulates expression of the tryptase, MMCP-6, the earliest protease expressed during mouse mast cell development. [

51]

The other transcription factor, GATA-2, is a DNA-binding protein that uses two novel zinc finger domains to activate transcription of messenger ribonucleic acid (mRNA) for various gene products associated with mature mast cells. The (W)GATA(R) consensus-binding motif is found in the mouse mast cell carboxypeptidase 52

promoter; the mast cell–specific IL-4 enhancer; the promoters for MMCP-1, -2, -4, -5, and -6; and IgE and IgE receptor β-chain promoter.[ ] Loss of GATA-2 expression prevents mast cell development from embryonic stem cells in culture. In vivo studies of mice have not been possible because GATA-2's genetic deletion 53]

results in severe anemia and death on embryonic days 10 or 11.[

How mast cells develop into two distinct phenotypes is not entirely clear, but several studies indicate the importance of cytokines and the microenvironment in this process. For example, culturing bone marrow-derived mast cells in SCF induced a CTMC phenotype that could be prevented by including IL-3 in the culture 54]

medium. [ [55]

Peritoneal mast cells showed bidirectional changes in phenotype when cultured ex vivo in IL-3 plus IL-4 and reinjected into mouse peritoneal cavities. 56]

Peritoneal mast cells injected into the skin or glandular stomach of the same mouse developed CTMC or MMC phenotypes, respectively.[

Also, a recent study

57 spiralis.[ ]

demonstrated that mast cell phenotype can be reversibly altered during infection with Trichinella These studies indicate that CTMC and MMC are likely derived from a common progenitor and that changes in phenotype largely result from the tissue microenvironment. Human Studies Studies of human mast cell development are complicated by the variety of tissues employed, which include fetal liver, umbilical cord blood, adult bone marrow, and peripheral blood. Unfortunately, similar studies using different starting populations occasionally have disparate results, making consensus difficult. Again, the reader's patience is urged; the details of mast cell development have important therapeutic implications for the tens of millions with allergic disease, but such studies in the human system are fraught with technical and ethical issues, making contradictory results part of the process. With these caveats in mind, some central themes appear to distinguish human and rodent mast cell development. First, SCF appears to be the central mast cell growth factor in both rodents and humans.[ [61]

findings in vitro.

58] [59] [60]

SCF injection into human subjects with metastatic breast cancer results in mast cell hyperplasia, consistent with the

Second, although IL-3 is a potent growth factor for both mast cell progenitors and mature mast cells in the rodent, its role in human mast cell

development has been questioned. Human mast cell precursors apparently use IL-3, but IL-3 receptor expression is lost on mature human mast cells.[ Instead, IL-3 is a potent stimulus of basophil growth in humans,[

65] [66]

a cell that is exceedingly rare in mice.

62] [63] [64]

As in rodents, the human mast cell progenitor exhibits bone marrow and peripheral phases in its development. The human mast cell progenitor in the circulation appears to be

338

CD34+ /Kit+ and clearly distinct from cells in the basophil or monocyte lineages ( Figure 20-2 ).[ and mucosal tissues, giving rise to the two mature mast cell types.

67]

This progenitor completes its differentiation in the connective

Human MCT and MCTC development is incompletely understood. Although it is a reasonable assumption that the two types are derived from a common uncommitted precursor, distinct committed progenitors may exist for each “lineage” of mast cells. Two pieces of data support this possibility. First, patients with inherited severe combined immunodeficiency disease (SCID) or acquired immunodeficiency syndrome (AIDS) have a marked decrease in MCT cell numbers in the 14]

bowel, whereas MCTC numbers and distribution are unaffected. [

This suggests that the appearance in tissues of MCT cells depends on functional T lymphocytes

and that MCTC cell development and recruitment proceed independently. Second, ultrastructural studies of immature human mast cells demonstrated different granule structures in cells that progressed to become mature MCT versus MCTC .[

68]

These structures correlated with expression of the expected proteases, implying

that distinct developmental pathways

Figure 20-2 Developmental pathways for human mast cells and basophils. Basophils (Baso) also develop from an early myeloid progenitor and complete their development in the bone marrow, largely in response to interleukin-3 (IL-3R). HSC, Hematopoietic stem cell. Mast cell development in its earliest stages is still poorly understood. This figure depicts mast cell progenitors deriving from a myeloid progenitor, although the exact lineage is unknown. Early mast cell differentiation appears to involve both IL-3 and stem cell factor (SCF). Progenitor mast cells differentiate to mast cell–committed progenitors (MCCP), which migrate through the blood to tissues, where they complete their development. Full differentiation depends on SCF stimulation of Kit receptors. The signals leading to differential protease and resulting MCT versus MCTC phenotype are still under study. This schema depicts these two mast cell types as developing from a common committed progenitor, with ultimate phenotype determined by local tissue influences (e.g., cytokine stimulation).

TABLE 20-3 -- Cytokines in Mouse Mast Cell (MC) Growth and Survival Cytokine

Receptor

Effect on Mast Cells

Stem cell factor (SCF; Kit ligand, steel factor, mast cell growth factor)

Kit 145-kD single chain RTK Ig superfamily

Primary growth factor for MC lineage in mouse/ human; prevents apoptosis; chemotactic for MCs; adhesion molecule for MCs; primes IgE-dependent mediator release; directs IgEindependent mediator release

Interleukin-3 (IL-3)

IL-3R α/βc ; HR superfamily

Major proliferation factor for mouse MCs and progenitors; may act on primitive human progenitors

Interleukin-4 (IL-4)

IL-4R α/γ c ; HR superfamily

Down-regulates Kit expression; induces FcepsilonRI expression on human MCs; inhibits mouse MC progenitors; induces/ stabilizes cyclooxygenase-2 and phospholipase A2

Interleukin-9 (IL-9)

IL-9R α/γc ; HR superfamily

Induces expression of NK cell granzyme B; costimulates mouse MC proliferation with multiple cytokines

Interleukin-10 (IL-10, CSIF)

IL-10R 90–110•kD IFN-γ receptor family

Co-stimulates mouse MC colony formation with IL3, SCF, or IL-4; reversibly induces MMCP-2; inhibits cytokine production in mouse MCs. Induces mouse MC death with IL-4

Nerve growth factor (NGF)

NGF-R TNF receptor family

Promotes survival (suppresses apoptosis); increases cytokine/c-fos/Bcl-2 mRNA in mouse MCs; promotes MC colony formation in mice

IgE immune complexes

FcepsilonRI α/β/γ2

Promotes growth of mouse connective tissue MCs; IgE strongly up-regulates expression of FcepsilonRI on mouse/human MCs; activates MCs

IgG immune complexes

FcγRIII

Mediator/cytokine release from mouse MCs

α/β/γ2 FcγRII

*

Co-ligation with FcepsilonRI inhibits mediator release from mouse MCs

†

Transforming growth factor beta (TGF-β)

TGF-βR

Interferon gamma (IFN-γ)

IFN-γ 477 aa α chain 332 aa β chain IFN receptor family

For mouse MCs, inhibits IL-3–dependent proliferation and serotonin release; enhances IgEmediated adherence to laminin, chemotaxis, expression of β7 integrins, and FcepsilonRIdependent lymphotactin mRNA expression Inhibits proliferation of mouse/human MCs; depresses steady-state levels of mRNA for TNF-α; inhibits antigen-induced histamine secretion; upregulates MHC II

RTK, Receptor tyrosine kinase; HR, hemopoietin receptor; NK, natural killer; TNF, tumor necrosis factor; MHC, major histocompatibility complex. * Single chain; multiple isoforms. † Single-chain serine/threonine kinase; cysteine rich.

be clinically important, although further study is needed to clarify their roles. IL-6 and prostaglandin E2 (PGE2 ) have been shown to enhance human mast cell development.[

74] [75] [76]

IL-15 was recently reported to block mouse mast cell apoptosis through regulation of Bcl-xL .[ 76

77]

78

In contrast, IL-4 inhibits human mast cell development[ ] and induces apoptosis of mouse mast cells when combined with IL-10.[ ] Interestingly, IL-4 and IL-10 are produced during Th2 responses and thus could play a role in dampening mast cell function during allergic disease. Although data to support this theory are needed, Daley and colleagues recently showed that expression of a gain-of-function mutation in the IL-4 receptor correlates with a better prognosis in human mastocytoma patients, possibly because prolonged IL-4 signaling is inhibiting mast cell survival.[

79]

Current understanding of mast cell homeostasis and its regulation is rudimentary. Further studies should clarify how mast cell survival is controlled and the impact on allergic disease. These findings could have clinical value because many molecules involved are soluble proteins with therapeutic potential.

340

MAST CELL PATHOBIOLOGY Since mast cells were described in 1877, scientists have struggled to determine their teleologic justification, including storage, ion exchange, and detoxification. After the discovery of IgE in the mid-1960s, most considerations of mast cell function centered on their pathologic role in allergic disease. This dogma is still strong

at present, but a new image of the mast cell as an essential effector cell in protective immunity is emerging from animal studies. Although these studies await clinical comparisons, the import of the data is clear: in particular infections, mast cells make a life-and-death difference to the host. These new data offer a view of mast cell functions as beneficial, implying that the pathologic role of the mast cell may be a matter of protective responses gone awry, a common feature in autoimmune disease. This discussion first reviews current knowledge of protective mast cell functions, then relates this to the role of the mast cell in disease. Responses to Parasites Intestinal parasites were once common in humans and currently infect a considerable portion of the third world population. Infectious agents such as Trichinella spiralis, Strongyloides species, and Nippostrongylus brasiliensis have been widely used in animal studies to assess the immune response to intestinal parasites. Studies with agents such as N. brasiliensis showed a profound mast cell hyperplasia correlating with parasite expulsion. Knowledge of mast cell mediators (e.g., histamine, neutral proteases, cytokines) made these mediators logical tools for attacking such pathogens. These data gave credence to the theory that mast cells are an essential aspect of immunity to parasites, a response that also involves eosinophils, Th2 cells, and numerous cytokines. Recent findings, however, have challenged the importance of mastocytosis in worm expulsion. Perhaps not surprisingly, technical advances have led to a more complicated view of immunity to parasites, a view in which layers of redundancy appear to exist. Studies using mast cell–deficient mice have revealed that mast cells 80 81

82 83

are not essential for expulsion of N. brasiliensis and Trichuris muris but are essential for immunity to T. spiralis.[ ] [ ] The roles of eosinophils[ ] [ ] and intestinal goblet cells are similarly debated. It is now clear that Th-helper lymphocyte type 2 (Th2) cytokine signaling is important to parasite immunity and that the 84

85

shared receptor for IL-4 and IL-13 (IL-4 receptor α) is critical (reviewed in references[ ] and[ ] ). Interpreting these findings is complicated by the ability of Th2 cytokines to impact on many facets of the inflammatory response, including IgE synthesis, Th cell proliferation and development, mastocytosis, eosinophilia, and intestinal mucus production and contractility. One view of the current knowledge is that mast cells are an important but somewhat redundant mediator of parasite immunity. Responses to Bacteria The newest and most exciting role for mast cells is in innate immunity to some bacterial pathogens. Two separate studies showed that mast cell–deficient W/WV mice 86 87

died from experimental bacterial peritonitis or pneumonia, whereas mast cell transfer into these animals allowed survival. [ ] [ ] Mast cell function in skin and bladder infections, analogous to the lung and peritoneal studies, is being investigated. These findings point to a strikingly important role for mast cells in innate immunity. Moreover, they support the location of mast cells at points of bacterial invasion, as well as their production of inflammatory mediators. Mast cell–mediated resistance to bacterial infection has been linked to their production of tumor necrosis factor alpha (TNF-α). This proinflammatory cytokine is a potent inducer of neutrophil chemotaxis and activation. Neutrophil influx correlated with survival and was dependent on mast cell–derived TNF-α in these studies. [87]

A role for TNF-α is particularly intriguing because mast cells produce this cytokine as both a preformed granule-associated protein and a newly synthesized

mediator. [

88]

Thus, mast cells could secrete TNF-α rapidly on activation and continue production for some time.

Enterobacteriaceae were shown to directly induce mast cell TNF-α production in these studies. Mast cell–bacteria interaction has since been demonstrated to involve bacterial expression of the adhesion protein FimH, binding to mast cell–expressed CD48.[

89]

These data have led to an entire area of research on CD48-type

90

molecules and their role in immunity, with new findings that these molecules may be involved in many host-pathogen interactions (reviewed in reference[ ] ). This story is far from complete. For example, the most recent data indicate that mast cells can phagocytize bacteria through CD48 interactions, but that this phagocytosis is not followed by bacterial killing and may even protect the pathogens. Further, mast cells are now argued to activate T cells by antigen presentation, using major histocompatibility complex (MHC) antigens I and II (reviewed in references[

90]

and[

91]

).

These new data are an exciting but, again, incomplete picture. Mast cell responses to bacterial infection appear to be an essential form of rapid and innate immunity protecting the host until the delayed, acquired response is engaged. The sum offers a view of mast cells that is more analogous to phagocytic, antigen-presenting cells than has been appreciated. Such a theory argues that mast cell responses in allergic disease may represent little more than improper cell activation, subverting a protective response to produce a pathologic outcome. Responses in Allergic Disease: Mast Cell Mediators as Clinical Assessment Tool Because purified mast cells and basophils are difficult to obtain in large numbers, it has been difficult to assess their involvement in clinical events. This fact has led to a search for clinically useful indicators of mast cell activation. Lipid mediators, such as leukotriene C4 (LTC4 ) and prostaglandin D2 (PGD2 ), or their associated 92]

metabolites lack sufficient cell specificity and are rapidly metabolized. Histamine is also not suitable as a specific marker; it is present in basophils,[ [93]

94 types.[ ]

is rapidly

metabolized in the circulation, may be derived from certain ingested foods, and reportedly is produced and secreted by other cell Nevertheless, elevated levels of histamine along with either tryptase or PGD2 suggest mast cell activation, and elevated levels of histamine in the absence of tryptase and PGD2 suggest basophil activation. 95]

The most useful clinical markers of mast cell involvement may be α-tryptase and β-tryptase.[

Tryptase immunoassays

341

TABLE 20-4 -- Immunoassays for Human Tryptase G5 Capture Mab

Parameter

B12 Capture Mab

Tryptase gene product recognized

β•α

β=α

Normal serum/plasma level (ng/ml)

50

46

Unknown

Psi c 2

>50

16

Peptidyl-prolyl isomerase

Coprinus comatus

Psilocybe cubensis

See Table 36-1 footnote.

* Frequency determined in ABPA patients.

is a low-molecular-weight, alkaline protein that has eukaryote-2 initiation factor (EIF) α-kinase activity and thus may play a role in protein synthesis. Asp f 1 is homologous to the fungal cytotoxin mitogillin (from A. restrictus) and α-sarcin (from A. gigantus) and is found in both spores and mycelium. These low-molecularweight proteins are nonglycosylated, cytotoxic enzymes with purine-specific ribonuclease activity. The Asp f 2 allergen is a protein showing homology with the C. albicans 54-kD mannoprotein, which has been shown to bind to human fibrinogen. Asp f 4 is a binding protein associated with

593

peroxisomes, which are self-replicating organelles similar in size to lysosomes. Peroxisomes play an important metabolic role in removing toxic substances from cells due to their specific collection of enzymes. The major allergens from Penicillium, Candida, and Trichophyton species are serine peptidases, dipeptidyl peptidases, aspartate peptidases, enolases, and peroxisomal membrane proteins or allergens of unknown function. The serine proteases are similar in structure to the bacterial subtilisins, and two types have been identified, the secreted 33-kD alkaline peptidases (e.g., Asp f 13, Asp fl 13, Pen c 13, Pen ch 13, Tri r/t 2) and the 39-kD vacuolar peptidases (e.g., Pen ch 18) involved in protein processing within vacuoles. The dipeptidyl peptidase from Trichphyton species (Tri r 4) is also a secretory protein but shares sequence similarity with enzymes from Aspergillus species implicated in aspergilloma. The function of the 30-kD Trichophyton allergen, Tri t 1, is unknown, although it may be an exo1,3-β-glucanase, given the limited sequence homology data. The peroxisomal membrane protein allergens (e.g., Asp f 3, Pen c 3, Mala f 2, 3, 5) possess thioldependent peroxidase activity. The enolases are glycolytic enzymes involved in the dehydration of glycerate-2-phosphate to produce phosphoenolpyruvate and represent a major group of cross-reacting allergens from a variety of fungal species (e.g., Cla h 6, Asp f 22w, Alt a 11).[

17]

In addition to these allergens, several minor allergens have been described that are associated with specific cellular functions and therefore share significant sequence identity with proteins in many diverse species, including humans. For example, several belong to the group of proteins termed molecular chaperones, which play a role in protein assembly and include protein disulphide isomerases (e.g., Alt a 4), peptidyl-prolyl isomerases (e.g., Asp f 11, Psi c 2, Mala f 6), and heat shock proteins (HSPs; e.g., Alt a 3, Asp f 12, Pen c 19). Some of these proteins work in concert to facilitate the folding of proteins after they are synthesized, for example, 18

HSPs and peptidyl-prolyl isomerases.[ ] Such pairs of proteins are found in spores from Aspergillus and Alternaria species, and one or the other of these has been shown to be allergenic in other species (e.g., C. herbarium), suggesting that the corresponding member yet to be isolated will also be allergenic. Other common groups of fungal allergens include the 60S P1 and P2 ribosomal proteins involved in protein chain elongation (e.g., Cla h 3/4, Alt a 6, Asp f 8, Alt a 12, Cla h 12), superoxide dismutase (e.g., Asp f 6), dehydrogenases (e.g., Alt a 10, Cla h 3, Cand a 1), aldehyde-forming enzyme (e.g., Asp f 7), 1,4-benzoquinone reductase (e.g., Alt a 7, Cla h 5), aldolase (37-kD allergen from Candida), thioredoxin (Cop c 2), proteins showing homology with the 88-kD mannoprotein from Filobasidiella (Cryptococcus) neoformans (e.g., Mala f 8), and a possible serine protease (e.g., Asp f 15, referred to as Asp f 13 in database) showing homology with Coccidioides immitis 19-kD antigen. The functions of some of the other minor fungal allergens that have been described remain to be determined (e.g., Asp f 16; Cla h 2; Mala f 7, 9; Cop c 1, 3, 5, 7).

Animal-Derived Aeroallergens The clinically important animal allergens in both domestic and occupational settings are derived from cats, dogs, cows, rats, mice, horses, rabbits, mice, gerbils, and guinea pigs and accumulate in dusts in both settings ( Table 36-7 ). The allergens are derived from dander, epithelium, fur, urine, or saliva, although the allergens may originate from the same source (e.g., saliva or urine), and are then distributed to other sites by grooming. The major mammalian dander allergens, except for 19

those derived from the cat, belong to the lipocalin (also referred to as calycin) superfamily.[ ] Lipocalins play a role in the binding and transport of small hydrophobic molecules and comprise proteins such as β-lactoglobulin, bilin-binding proteins, α1 -microglobin, and odorant-binding proteins. Allergens in this group include those from horse (e.g., Equ c 1, 2), cow (e.g., Bos d 2, Bos d 5− β-lactoglobulin), dog (e.g., Can f 1 and 2, which may also have cysteine protease inhibitory activity), rabbit (e.g., Ory c 1), mouse (e.g., Mus m 1), and rat (e.g., Rat n 1) dander or urine. These proteins may exist as monomers (Bos d 2 and Mus m 1) or homodimers (Ecu c 1 and Bos d 5). The nature of the ligands bound by the mammalian lipocalin allergens is unclear, although the mouse allergen is thought to bind pheromones, and the horse allergen may bind histamine. Recently, however, it has been shown that the Bos d 5 lipocalin allergen, along with other nonallergenic lipocalins, possesses nonspecific endonuclease activity. The catalytically important glutamic acid at position 134 in Bos d 5 necessary for phosphodiester bond cleavage is also conserved in Bos d 2, Equ c 1, Can f 1 (but not Can f 2), and Mus m 1, as well as in the cockroach Bla g 4 lipocalin allergen, suggesting that these allergens may also possess such activity. [

20]

In addition to the major allergens already described, several minor mammalian allergens have been demonstrated and include the serum albumins (e.g., Ecu c 3; Can f 3; rat, mouse, and rabbit albumin), immunoglobulin G, an oligomycin sensitivity-conferring protein (e.g., AS1) of the mitochondrial adenosine triphosphate (ATP) synthase complex, a calcium-binding psoriasin-like allergen (e.g., BDA 11), a horse allergen showing homology to rat mandibular gland protein A (e.g., Ecu c 4), and a protein of unknown function (e.g., Ecu c 5). The sequence identities observed between similar proteins from different species (e.g., the albumins) account for the marked allergenic cross-reactivity observed in clinical studies. In contrast to the animal-derived allergens, the major cat allergens appear to be nonlipocalin proteins. Fel d 1 is a heterodimeric protein comprising two distinct peptides, designated chains 1 and 2, in which chain 1 shows sequence homology with proteins belonging to the uteroglobin family, thought to play a role in inflammation. Allergens corresponding to Fel d 1 appear to be restricted to other cat species and to the lagomorphs such as rabbit. The function of this group of 21

allergens is unclear, but recent data suggest that they may possess protease activity.[ ] If this activity is confirmed, such allergens may represent a novel group of proteases. The other major cat allergen is the cystatin allergen Fel d 3, a cysteine protease inhibitor. Cat dander also has been shown to contain a minor serum albumin allergen (Fel d 2). Arthropod-Derived Aeroallergens 22

The main arthropod allergen sources are to be found in Insecta (e.g., midges, cockroaches, moths, butterflies, flies, silverfish, locusts) and Arachnida (mites).[ ] Allergy may arise in the home or in scientific institutions where many arthropods are reared or studied. Of the arthropods, house-dust mites and cockroaches are the most clinically important sources, and

594

TABLE 36-7 -- Physicochemical and Biochemical Characterisation of Animal and Dander Allergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Cat (Felix domesticus) Fel d 1

>80

33–39

Heterodimer; chain 1 shows homology with 10-kD secretory protein from human Clara cells, mouse salivary androgen-binding protein subunit; rabbit uteroglobin, Syrian hamster protein

Fel d 2

15–22

69

Serum albumin

Fel d 3

60–90

11

Cystatin

Can f 1

>70

19–25

Lipocalin; shows homology with von Ebner's gland protein, which has cysteine protease inhibitory activity

Can f 2

70

27

Lipocalin; shows homology with other lipocalin allergens

Can f 3

35–77

69

Serum albumin

88

150

Immunoglobulin G

Equ c 1

100

22

Lipocalin; shows homology with rodent urinary proteins

Equ c 2

100

16

Lipocalin; shows homology with rodent urinary proteins

Equ c 3

?

67

Serum albumin

Equ c 4

?

19

Shows homology with rat mandibular gland protein A

Dog (Canis familiaris)

IgG Horse (Equus caballus)

Equ c 5

?

17

Unknown

Bos d 2

97

18

Shows homology to the rodent lipocalin allergens

AS1

31

21

Oligomycin sensitivity-conferring protein of mitochondrial adenosine triphosphate synthase complex

BDA 11

?

12

Shows homology with human calciumbinding psoriasin protein

>80

19

Major urinary protein; shows homology with lipocalins: βlactoglobulin, odorant-binding proteins, Rat n 2 (Rattus novegicus)

Rat n 1

>80

21

Lipocalin; shows homology with lipocalins: β-lactoglobulin, odorantbinding proteins, Mus m 1

Albumin

24

69

Serum albumin

Ory c 1

?

18

Odorant-binding protein, lipocalin; shows homology with Ory c 2

Ory c 2

?

21

Odorant-binding protein

8-kD allergen

?

8

Shows homology with rabbit uteroglobin

90

25

Cysteine protease

Group 2 (Der p 2)

>90

14

Shows homology with putative human epididymal protein; possible cholesterol-binding protein

Group 3 (Der p 3)

90

25

Trypsin

Group 4 (Der p 4)

25–46

56

Amylase

Group 5 (Der p 5)

9–70

13

Unknown

Group 6 (Der p 6)

39

25

Chymotrypsin

Group 7 (Der p 7)

53–62

11–29

Group 8 (Der p 8)

40

26

Glutathione transferase

Group 9 (Der p 9)

>90

28

Collagenase-like serine protease

Group 10 (Der p 10)

81

33

Tropomyosin

Group 11 (Der f 11)

82

98

Paramyosin

Unknown

Group 12 (Blo t 12)

50

16

May be chitinase; shows homology with Der f 15

Group 13 (Lep d 13)

11–23

15

Fatty acid–binding protein

Group 14 (Der f 14)

84

190

Vitellogenin or lipophorin

Group 15 (Der f 15)

95

63/98

Group 16w (Der f 16)

35

53

Gelsolin

Group 17w (Der f 17)

35

30

Calcium-binding protein

Group 18w (Der f 18)

?

60

Chitinase

Mag29

?

67

Heat shock protein

*

Chitinase; shows homology with Blot 12 allergen

See Table 36-1 footnote. * Glycosylated and nonglycosylated forms; frequency determined in dogs with atopic dermatitis.

amylase (e.g., Der p 4), as well as a chitinase that has been shown to be an allergen for humans (Der f 18w) and dogs (Der p 15) with atopic dermatitis. In this regard the region comprising residues 505 to 543 shows limited homology with the C-terminal third of Blo t 12 allergen, suggesting that the latter is enzymatically similar although of lower molecular weight. The actin-binding allergens include tropomyosin (e.g., Der p 10) and paramyosin (e.g., Der f 11), both of which form part of the cytoskeleton of most cells, suggesting that cellular debris is a significant source of allergen and is derived either from the whole body or from fecal pellets. The mite tropomyosin allergens show marked sequence identity with similarly allergenic proteins from other invertebrates (see Table 36-12 ). In addition to these allergens, the group 2 allergens (e.g., Der p 2, Lep d 2, Gly d 2) are also classified as major allergens, although their function in mites is still to be determined. The group 2 allergens may play a role in reproduction because they show significant sequence similarity with a group of phylogenetically conserved mammalian epididymal proteins. However, this role has been difficult to reconcile on the basis of data showing that the allergen is restricted to the mite gut and fecal pellets rather than reproductive organs. Despite this, recent studies have shown that the mammalian epidymal equivalent proteins bind cholesterol, raising the possibility that the group 2 allergens are involved in the transfer of this or similar ligands in either the mite gut or reproductive system. More recently, mites have been shown to produce the high-molecular-weight group 14 allergen, which encompasses two previously described allergens (Mag 1 and 3) that show sequence identity with a number of lipid-binding proteins, including lipophorins and vitellogenin. Finally, mites have been shown to possess two major groups of allergens of unknown function (e.g., Der p 5 and 7). Mites also produce a number of minor allergens (see Table 36-8 ), including gelsolin (e.g., Der f 16), fatty acid–binding protein (e.g., Lep d 13, Aca s 13, Blo t 13), glutathione Stransferase, HSP, and an EF-hand calcium-binding protein (e.g., Der f 17w).[ Insect Aeroallergens

3]

22

A number of insect-derived proteins have been shown to be clinically significant aeroallergens.[ ] Allergens from bloodworms (Chironomus thummi thummi), midges (Cladotanytarsus lewisi, Polypedium nubifer, Chironomus kiiensis), cockroaches (Blattella germanica, Periplaneta americana), and moths (Plodia interpunctella) have been described ( Table 36-9 ). These allergens may be derived from whole insect bodies or from feces, and evidence indicates that the spectrum and concentrations of specific allergens in these different sources may vary. In midges the major allergens are hemoglobins (e.g., Chi t 1, Cla l 1, Pol n 1), whereas in cockroach, the major allergens are enzymes, such as glutathione S-transferase (e.g., Bla g 5) and an inactive aspartate protease (e.g., Bla g 2), lipocalin (e.g., Bla g 4), troponin C, a calcium-binding protein (e.g., Bla g 6), tropomyosin (e.g., Per a 7), hexamerin (an insect storage protein belonging to the hemocyanin superfamily; e.g., Per a 3), or proteins of unknown function (e.g., Bla g 1, Per a 1; structurally related to a group of mosquito proteins induced in the gut after a blood meal[

23]

).

The Bla g 2 allergen shows marked sequence similarity to porcine pepsin, although it is thought to be proteolytically inactive due to amino acid substitutions in the catalytic region. However, the allergen is related to a large group of pregnancy-associated glycoproteins of unknown function

596

TABLE 36-9 -- Physicochemical and Biochemical Characteristics of Insect Aeroallergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Chironomidae Bloodworm (Chironomus thummi thummi) Chi t l–9

>50

15

Hemoglobin

>50

17

Hemoglobin

>50

17

Hemoglobin

?

33

Tropomyosin

Midges Cladotanytarsus lewisi Cla l 1 Polypedium nubifer Pol n 1 Chironomus kiiensis Chi k 10 Blattidae

German cockroach (Blattella germanica) Bla g 1

50

25

Shows homology with Per a 1 allergen, Cr PII, and ANG12 secretory mosquito protein

Bla g 2

58

36

Aspartate protease (inactive)

Bla g 4

40–60

21

Lipocalin

Bla g 5

70

23

Glutathione transferase

Bla g 6

50

27

Troponin C

Per a 1

50

24

Shows homology with Bla g 1 allergen, Cr-PII, and ANG12 secretory mosquito protein

Per a 3

83

78

Hexamerin; subunit protein showing homology with larval insect storage proteins

Per a 7

50

72

Tropomyosin

American cockroach (Periplaneta americana)

Indianmeal moth (Plodia interpunctella) Plo i 1

25

40

Arginine kinase

See Table 36-1 footnote. found in the mononuclear and invasive binucleate cells of the epithelial layer of the trophectoderm in artiodactyl species such as cow and sheep. These aspartate protease–related proteins possess the characteristic bilobed aspartate protease structure with a cleft capable of binding peptide ligands. In the cockroach, however, as with the mite group 2 allergens, which show homology with proteins present in the mammalian reproductive system, the allergen is expressed solely in the gut. In addition to cockroach and midges, an arginine kinase allergen from the Indianmeal moth has been described (Plo i 1) that possesses an actin-binding site and is involved in energy storage.[

24]

shrimp Parapenaeus fissures.

This allergen shows marked homology with similar enzymes from various invertebrates, including an ingested allergen from the

[25]

Occupational Aeroallergens The major occupational allergens include fungal, bacterial, and mammalian hydrolytic enzymes, egg powder, latex products, and flours derived from rice, wheat, barley and rye seeds, castor bean and mustard seeds, green coffee beans, Ispaghula, and soybeans ( Table 36-10 ). Allergy results from exposure to such materials in

industries where they are produced, used, or added to other products such as washing detergents and pancreatic supplements as well as doughs, which are associated with the clinical condition known as baker's asthma.[

26]

Although the respiratory tract is the major route, exposure may also occur through the skin, as with latex

allergens present in latex gloves and other materials used in spina bifida and other patients undergoing multiple surgical procedures[

27]

(see Table 36-11 ).

Fungal-, Bacterial-, Mammalian-, and Plant-Derived Hydrolytic Aeroallergens

The major enzymatic aeroallergens include the bacterial subtilisins and amylases used in the detergent industry, mammalian serine proteases (e.g., trypsin, chymotrypsin, pepsin) used in pancreatic supplements in the treatment of cystic fibrosis patients, the plant cysteine proteases (e.g., Car p 1, Ana c 2) used in the pharmaceutical industry, fungal amylases (e.g., Asp o 21), and various other carbohydrases, such as β-xylosidase (Asp f 14), cellulase, and glucoamylase, used in the baking industry ( Table 36-10 ). Egg Aeroallergens 28

The major egg allergens are derived from either the white or yolk. [ ] Those contained within egg white include ovomucoid (Gal d 1), ovalbumin (Gal d 2), conalbumin (Gal d 3), and lysozyme (Gal d 4), whereas that in yolk is α-livitin (chicken albumin, Gal d 5). However, the most frequently recognized egg allergens are Gal d 3 and Gal d 5. The biochemical activities of each of these proteins, with the

597

TABLE 36-10 -- Physicochemical and Biochemical Characteristics of Occupational Aeroallergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Fungal Allergens Aspergillus niger ••Pectinase

?

35

Poly(1,4)-α-d-galacturonidase

••Cellulase

8

26

1,4-(1,3;1,4)-β-d-Glucan glucanhydrolase

••Glucoamylase

5

66

1,4-α-d-Glucan glucanhydrolase

>50

50

Histidine acid phosphatase

••Phytase

?

105

β-Xylosidase

••Asp o 13

?

34

Alkaline serine protease; belongs to subtilase family

••Asp o 21

56

52

α-Amylase

••Lactase

?

?

1,4-β-d-Galactoside galactohydrolase

?

34

Aspartate protease; shows homology with mammalian and cockroach pepsins

>50

28

Subtilisin serine protease

>50

28

Subtilisin serine protease

>50

68–125

?

23

Papain, cysteine protease

100

30

Actinidin, cysteine protease

?

23

Bromelain cysteine protease

••Asp n 14 Aspergillus oryzae

Cryphonectira parasitica ••Renin

Bacterial Proteases Bacillus subtilis ••Alcalase Bacillus licheniformis ••Esperase Clostridium histolyticum Collagenase

*

Metalloprotease

Plant Proteases Caricaceae ••Pawpaw (Carica papaya) ••••• Car p 1 ••Kiwifruit (Actinidia chinensis) ••••• Act c 1 Bromelaceae ••Pineapple (Ananas comosus) ••••• Ana c 1

Mammalian Proteases Trypsin (porcine)

?

24

Serine protease; shows homology with mite groups 3, 6, and 9 allergens

Chymotrypsin (bovine)

?

25

Serine protease; shows homology with mite groups 3, 6, and 9 allergens

Pepsin (porcine)

?

35

Aspartate protease; shows homology with Bla g 2 and renin

Gal d 1

34–38

20

Ovomucoid, protease inhibitor

Gal d 2

32

43

Ovalbumin; function unknown but protein shows homology with serine protease inhibitors

Gal d 3

47–53

76

Conalbumin (ovotransferrin), iron transport protein

Gal d 4

15

14

1,4-β-N-acetylmuramidase (lysozyme)

>50

65–70

Chicken Egg Allergens Chicken (Gallus domesticus) Egg white

Egg yolk Gal d 5

Serum albumin (α-livetin)

Brassicaceae Yellow mustard seed (Sinapis alba L.) Sin a 1

?

2S albumin

• Short chain

4

• Long chain

10

Oriental mustard seed (Brassica juncea) Bra j 1 • Short chain

?

2S albumin 4

• Long chain

10

Oilseed rape (Brassica napus) BnIII (Bra r 1)

?

2S albumin

• Short chain

4

• Long chain

10

Euphorbiaceae Castor bean (Ricinus communis) Ric c 1

96

2S albumin

• Short chain

4

• Long chain

7

Ric c 2

?

47

11 S crystalloid protein

Ric c 3

?

47–51

95

8

Cysteine-rich hydrophobic seed protein, member of lipid transfer protein family

95

8

Unknown

Trypsin inhibitor (B)

86

20

Kunitz protease inhibitor

Lipoxygenase

?

94

Lipoxygenase

Unknown

Leguminosae Seed husk allergens Soybean (Glycine max) Gly m 1

†

Gly m 2 Flour Soybean (Glycine max)

‡

Poaceae Barley (Hordeum vulgare)

Hor v 15

?

14

α-Amylase/trypsin inhibitor; shows homology with wheat allergens and 2S albumin allergens

Hor v 16

>96

64

α-Amylase (1,4-α-d-Glucan glucanohydrolase)

Hor v 17

>96

60

β-Amylase (1,4-α-d-Glucan maltohydrolase)

Hor v 21

91

§

34

Hordein; shows homology with rye secalins and wheat gliadins

>90

15

α-Amylase inhibitor; shows homology with wheat and barley α-amylase/ trypsin inhibitor allergens

?

33

Glyoxylase I

>50

13

Wheat α-amylase/trypsin inhibitor; shows homology with barley allergens and 2S albumin allergens

WMAI-1

?

13

Wheat α-amylase/trypsin inhibitor; shows homology with barley allergens and 2S albumin allergens

Agglutinin

?

17

Lectin

27-kD allergen

?

27

Shows homology with acyl-CoA oxidase from barley and rice

35-kD allergen

60

35

Peroxidase

Tri a 3

?

13

Unknown; found in wheat ovaries; shows homology with pollen allergens

Tri a 19

100

65

ω-Gliadin; shows homology with rye secalins and barley hordeins

Gliadin

72

40

α-Gliadin

Rice (Oryza sativa) Ory s 1

33-kD protein Wheat (Triticum spp.) CM16

37-kD allergen

?

37

Fructose biphosphate-aldolase

Sec c 1

>50

14

α-Amylase/trypsin inhibitor; shows homology with wheat allergens and 2S albumin allergens

34-kD protein

83

§

34

Rye γ-35 secalin; shows homology with wheat gliadins and barley hordeins

70-kD protein

91

§

70

Rye γ-70 secalin; shows homology with wheat gliadins and barley hordeins

Rye (Secale cereale)

See Table 36-1 footnote. * Represents a mixture of proteases. 3]

† Note that this designation has also been used for the ingested cysteine protease allergen from soybean.[ ‡ See Table 36-13 for ingested allergens from this source. § Frequency based on patients with wheat-dependent, exercise-induced anaphylaxis.

exception of ovalbumin, are known: ovomucoid is a protease inhibitor, conalbumin (also known as ovotransferrin) an iron transport protein, lysozyme a bacteriostatic agent, and α-livitin a carbohydrase with bacteriolytic properties and a ligand transport protein. Many of the egg- (and flour)-derived allergens are also important as ingested allergens and are described later. Latex Aeroallergens

Latex allergens are absorbed into the starch powder used to facilitate the donning of gloves or leach from latex-derived materials used in surgical procedures; 11 29

allergens have been described ( Table 36-11 ).[ ] The major allergens include the rubber-associated proteins prohevein (Hev b 1, 3), prohevein (Hev b 6), and a protein of unknown function (Hev b 5). The minor allergens include a patatin-like protein (Hev b 7), enolase (Hev b 9), manganese superoxide dismutase (Hev b 10), profilin (Hev b 8), and carbohydrases (Hev b 2, 11w). Seed-Derived Aeroallergens

The major seed-derived aeroallergens are proteins that play important roles in defense, storage, or metabolism. The major defense-related proteins include 12-kD to 15-kD amylase/trypsin inhibitory albumin proteins (e.g., Hor v 15, Ory s 1, Sec c 1), the 2S seed storage albumins (napins; e.g., Sin a 1, Ric c 1), vicilin-related proteins (which may possess chitin-binding activity with consequent antifungal properties), and the glycinins, or these proteins are enzymes involved in essential

30 31

plant biochemical processes, such as amylases (e.g., Hor v 16, 17), peroxidase, fructose biphosphate-aldolase, glyoxylase, and lipoxygenase ( Table 36-10 ).[ ] [ ] The types of seed proteins involved in allergic disease vary according to the source. For example, the amylase/trypsin inhibitors are characteristic of the Poaceae; the 2S storage albumins are prominent in the Brassicaceae and Euphoriaceae; and trysin inhibitor, lipoxygenase, and LTP are characteristic of the Leguminosae. The LTPs are associated with soybean husks

599

TABLE 36-11 -- Physicochemical and Biochemical Characteristics of Latex Allergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Hev b 1

22–82

15

Rubber elongation factor; exists as homotetramer of 58•kD and pI of 8.5

Hev b 2

20–61

30

Endo-1,3-β-glucosidase

Hev b 3

79

24

Shows some homology with rubber elongation factor

Hev b 4

65–77

50–57

Hev b 5

56–92

16

Hev b 6

83

20

Hev b 7

23–49

43

Euphorbiaceae Rubber tree (latex; Hevea brasiliensis)

*

Microhelix component Shows homology with acidic protein from kiwifruit and potato Prohevein; chitin-binding lectin, causes latex agglutination Patatin-like protein with lipid acylhydrolase and phospholipase A2 activity; shows cross-reactivity with Sol t 1

Hev b 8

24

14

Profilin; plays role in actin polymerization

Hev b 9

15

51

Enolase

Hev b 10

4

26

Manganese superoxide dismutase

Hev b 11w

3

30

Class I chitinase

See Table 36-1 footnote. * Hevein is a 5•kD chitin-binding domain from this precursor.

600

(Gly m 1; see also Table 36-13 ), and these abundant, hydrophobic, and cysteine-rich seed surface proteins reduce the wetability of surfaces and are therefore thought to play a role in defense. These proteins and the 2S storage albumins described earlier are similar to plant LTPs. The gliadins (e.g., Tri a 19, rye gliadins) and the related secalins (rye) and hordeins (e.g., Hor v 21) are characteristic of the Poaceae, as are glyoxylase, acyl-CoA oxidase, peroxidase (a PR protein), and fructose biphosphate-aldolase.

INGESTED ALLERGENS A variety of food sources contain allergens provoking IgE-mediated symptoms, but seven appear to be the most clinically important sources, accounting for more 32]

than 90% of food-induced allergies.[

In decreasing order of frequency, these sources are eggs, peanut, milk, nuts, soy, fish, and wheat. More recently, however, 33

allergens from animal meats have been shown to be clinically important as well.[ ] Allergy to ingested food proteins may result in a range of symptoms, such as dermatitis, asthma, anaphylaxis, angioedema, and abdominal symptoms, and certain sources are often associated with a particular allergic manifestation. For example, peanuts, fish, and crustaceans are associated with anaphylaxis; egg and milk are associated with atopic dermatitis; and wheat allergens may be associated with exercise-induced anaphylaxis. Animal-Derived and Fish-Derived Allergens The ingested mammalian meat allergens include the serum albumins (e.g., Bos d 6), immunoglobulins (e.g., Bos d 7), and transferrin in raw meat. In cow's milk the allergens are principally α-lactalbumin (Bos d 4), β-lactoglobulin (Bos d 5, a lipocalin), and casein (Bos d 8), with casein particularly important ( Table 36-12 ). In fish the major allergens are the calcium-binding parvalbumins (e.g., Gal c 1), whereas in shellfish and mollusks such as shrimp, crab, and snails the major allergens are tropomyosins (e.g., Pen a 1, Met e 1, Cha f 1) or actin-associated arginine kinases ( Table 36-12 ). Similar allergens from different species show high sequence similarity and are therefore immunologically cross-reactive. With the tropomyosins this cross-reactivity extends to invertebrates such as cockroaches and dust mites 34]

and the nematode parasite Anisakis simplex.[

Seed-Derived Allergens The major ingested seed-derived allergens are similar to those described previously as occupational aeroallergens (see Table 36-10 ). As ingested allergens, however, the most clinically important are those derived from peanuts and include the major vicilin storage protein (Ara h 1), conglutin (Ara h 2), and peanut agglutinin allergens (see Table 36-13 ). The minor peanut allergens include profilin (Ara h 5), the glycinin allergens (Ara h 4, 5), and proteins similar to conglutins (Ara h 6, 7). The most important ingested allergens from soybean include Gly m 1, Gly m Bd 28K, and Gly m 3. Gly m 1 is a cysteine protease that shows homology with members of the papain family (although it lacks the essential catalytic cysteine residue at position 25), including the house-dust mite allergen Der p 1. This enzyme may play a role in mobilizing seed storage proteins during germination. In contrast, Gly m 3 is a profilin, and Gly m Bd 28K shows homology with a range of vicilinrelated storage proteins, including Ara h 1, and may possess chitin-binding properties. Allergens belonging to the TABLE 36-12 -- Physicochemical and Biochemical Characteristics of Ingested Mammal-Derived and Fish-Derived Food Allergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Mammal-Derived Cow (Bos domesticus) Bos d 4

6

14

α-Lactalbumin, lactose synthase

Bos d 5

13

18

β-Lactoglobulin, lipocalin

Bos d 6

29

67

Serum albumin

Bos d 7

83

160

Immunoglobulin

Bos d 8

100

20–30

75-kD allergen

16

75

Transferrin

100

12

Parvalbumin, calcium-binding protein

Group 1 (e.g., Met p 1)

>50

34–36

39-kD allergen

70

39

Arginine kinase

>50

34

Tropomyosin

Casein

Fish-Derived Atlantic salmon (Salmo salar), cod (Gadus callarias) Group 1 Shrimp (Metapenaeus species, Penaeus species) Tropomyosin

Crab (Charybdis feriatus) Group 1

Squid (Todarodes pacificus) Group 1

>50

38

Tropomyosin

See Table 36-1 footnote.

601

vicilin family are also present in walnut (Jug r 2) and coconuts. Interestingly, profilins are usually regarded as minor allergens, although in both soybean (Gly m 3) 35

and Mercurialis annua pollen (Mer a 1), they are recognized by more than 50% of sensitized individuals.[ ] The soybean minor allergens include members of the glycinin family (G1 and G2 glycinins) that share homology with Ara h 3 and the alpha subunits of β-conglycinin. Seed proteins belonging to a group of methioninerich 2S albumins have been described in walnut (Jug r 1) and brazil nuts (Ber e 1) as well as sunflower and sesame seeds. Fruit-Derived and Vegetable-Derived Allergens A range of allergens have been described from fruits and vegetables such as plum, banana, pear, cherry, kiwifruit, apple, peach, plum, apricot, mango, avocado, tomato, potato, celery, zucchini, and carrot ( Table 36-13 ). However, sensitivity to these allergens is thought to arise from prior sensitization to pollen allergens. Oral allergy syndrome (OAS) arises from cross-reactivity between proteins in the respiratory allergen and those in food.[ uncooked food rather than cooked foods because processing results in protein denaturation, and symptoms

36]

OAS is associated predominantly with

TABLE 36-13 -- Physicochemical and Biochemical Characteristics of Ingested Seed and Fruit Allergens Allergen

Frequency of Reactivity (%)

Molecular Weight (kD)

Function

Leguminosae Peanut (Arachis hypogaea) Ara h 1

>90

70

Vicilin seed storage protein

Ara h 2

>90

18

Conglutin seed storage protein

Ara h 3

?

60

Glycinin seed storage protein

Ara h 4

43

36

Glycinin seed storage protein

Ara h 5

16

14

Profilin

Ara h 6

38

16

Similar to conglutin

Ara h 7

43

15

Peanut agglutinin

50

27

Gly m 1 (Gly m Bd 30•K)

90

34

Seed vacuolar protein; cysteine protease; shows homology with mite group 1 allergen, papain, and bromelain (EC 3.4.22.–)

Gly m Bd 28•K

>50

22

Vicilin-like glycoprotein; shows homology with Ara h 1

Gly m 3

69

14

Profilin

21-kD allergen

?

22

Member of G2 glycinin family

G1 glycinin

?

?

Member of G1 glycinin family; shows homology with Ara h 3

Gly m Bd 60•K

25

60

β-conglycinin seed storage protein

100

9

2S albumin

Soybean (Glycine max)

*

Similar to conglutin Lectin

†

Juglandaceae Brazil nut (Bertholletia excelsa) Ber e 1 Lecythidaceae English walnut (Juglans regia) Jug r 1

?

?

2S albumin

Jug r 2

60

47

Vicilin-like glycoprotein

Polygonaceae Buckwheat (Fagopyrum esculentum Moench) Fag e 1

>50

26

β chain of 11S globulin

19-kD protein

78

19

2S albumin

16-kD protein

90

15

Phospholipase A2

Api m 2

95

41

Hyaluronidase

Api m 3

>50

49

Acid phosphatase

Api m 4

>50

3

Melittin

Api m 6

>42

8

Unknown

Bom p 1

?

?

Phospholipase A2

Bom p 4

?

?

Protease

Bom t 1

?

49

Acid phosphatase

Venom Allergens Aidae Honeybee (Apis mellifera)

Bumblebee (Bombus pennsylvanicus/terrestris)

Vespidae White-faced and yellow hornets (Dolichovespula spp.), paper wasps (Polistes spp.), yellow jackets (Vespula spp.)

Group 1

46

34

Phospholipase A1

Group 2

26

39

Hyaluronidase

Group 3

?

49

Acid phosphatase

Group 5

8

26

Shows homology with cysteine-rich secretory protein in testis, epididymis, and salivary gland

Sol i 1

26

37

Phospholipase A1

Sol i 2

87

13

Unknown

Sol i 3

17

24

Shows homology with vespid group 5 allergens

Sol i 4

26

13

Shows homology with Sol i 2

Myr p 1

>50

9

Unknown; pilosulin 1

Myr p 2

35

5

Unknown

Aed a 1

29–43

68

Apyrase

Aed a 2

11

37

Unknown

Aed a 3

32

30

Unknown; shows homology with odorant-binding proteins

80

20

Unknown

Formicidae Fire ant (Solenopsis invicta)

Australian jumper ant (Myrmecia pilosula)

Salivary Allergens Culicidae Mosquito (Aedes aegypti)

Pulicidae Flea (Ctenocephalides felis) Cte f 1

Cte f 2

?

30

Salivary protein; shows homology with ant Sol i 3 allergen and vespid group 5 allergens

89

19

Procalin; member of lipocalin family; shows homology with triabin (thrombin inhibitor)

Reduviidae Kissing bug (Triatoma protracta) 19-kD allergen

See Table 36-1 footnote. but more recently, route of exposure and inflammatory events occurring in the vicinity of antigen deposition have been recognized as playing similarly important roles. The recognition of inflammation has given rise to the concept of the “danger signal,” which hypothesizes that immune responses do not occur unless 45

inflammatory signals are provided, particularly from the host tissue.[ ] A number of cell types are likely to provide danger signals and are thus essential in the generation of both innate and adaptive immunity. The most important cell types likely to interact with aeroallergens in the respiratory tract and generate the appropriate signals include epithelial cells, mast cells, macrophages, and dendritic cells. These interactions may occur directly through specific receptors or indirectly through humoral factors such as surfactant and protease inhibitors. Furthermore, functional interactions between different allergens may of critical significance to the outcome of allergen exposure, as might interactions with the many poorly characterized components that occur together with

605

TABLE 36-16 -- Clinically Important Allergens of Known Three-Dimensional Structure Allergen

*

PDB Number

Function

Technique

Bet v 1

Ribonuclease

X-ray diffraction

1BV1

Bet v 1-Fab complex

—

X-ray diffraction

1FSK

Bet v 2

Profilin

X-ray diffraction

IG5U

Pollens Birch

Timothy grass

Phl p 2

Unknown

X-ray diffraction

1WHO

Unknown

NMR

2BBG

PR-5 protein

Modeling

Based on 1AVN, structure of mouse urinary protein, Mus m 1

Profilin

X-ray diffraction

1BV1

Gly m 1

Lipid transfer protein

X-ray diffraction

1HYP

Wheat CM16

2S trypsin/amylase inhibitor

X-ray diffraction

1HSS

Ribonuclease

NMR

1EO9

Manganese superoxide dismutase

Modeling

Based on 1ABM, structure of human MnSOD

Hyaluronidase

X-ray diffraction

1FCQ

Unknown

X-ray diffraction

IQNX

Lipocalin

X-ray diffraction

1EW3

Ragweed Amb t 5 Mountain cedar Jun a 3 Latex Heb v 8 Seed

Fruit Cherry Pru av 1 Fungi Asp f 6 Stinging Insect Venom Bee Api m 2 Wasp Ves v 5 Mammalian Dander Horse Equ c 1 Cow

Bos d 2

Lipocalin

X-ray diffraction

1BJ7

Lipocalin

X-ray diffraction

1MUP

Cystatin, cysteine protease inhibitor

Modeling

Based on human cystatin A (1CYU), stefin A (1DVC), stefin B (1STF)

Der p 1

Cysteine protease

Modeling

Based on structure of papain (9PAP) and actinidin (2ACT)

Der p 2

Unknown

NMR

1A9V

Unknown

NMR

1AHK

α-Lactabumin

Lactose synthase

X-ray diffraction

1F6S

β-Lactoglobulin

Lipocalin

X-ray diffraction

1J8W

Ovalbumin

Unknown

X-ray diffraction

1OVA

Lysozyme

Carbohydrase

X-ray diffraction

1DPX

Ovotransferrin

Iron transporter

X-ray diffraction

1 AIV

Mouse Mus m 1 Cat Fel d 3 Dust Mites Dermatophagoides pteronyssinus

Dermatophagoides farinae Der f 2 Milk

Egg

PDB, Protein Data Base; NMR, nuclear magnetic resonance; PR, pathogenesis related. * Technique used to determine three-dimensional structure.

606

TABLE 36-17 -- Summary: Potential Consequences of Biochemical Properties of Allergens Property of Allergen

Potential Consequence

Protease activity

Epithelial permeability caused by: • Mast cell degranulation • Tight junction degradation • Kinin generation • Apoptosis

Macrophage/monocyte receptor interaction Cytokine release Adhesion molecule up-regulation Complement activation Receptor interaction T helper cell polarization Protease inhibitor

Macrophage/monocyte receptor interaction

Lipocalin

Epithelium receptor interactions

Glycosylation

Macrophage/monocyte receptor interaction

High focal concentration

Epithelial permeability caused by osmotic effects

established allergens. Detailed exploration of these possibilities is still in its infancy but seems to be a fertile area for investigation. Some of these studies have shed new light on understanding the earliest events of allergic sensitization, and to date the majority of this work has been performed with house-dust mite allergens, particularly those with peptidase activity. Biologic Actions of Mite Peptidases: Transepithelial Allergen Delivery

Dendritic cells (DCs) are the major antigen-presenting cells (APCs) of the respiratory tract and thus are a vital component in the development of allergy. In the process of sensitization, inhalant allergens must penetrate the barrier formed by the epithelial cells lining the airways to contact DCs that lie beneath it. Surprisingly, little information exists about this event, and a ma jor challenge in understanding the development of sensitization is to establish how this fundamental event occurs. There are two potential routes to deliver substances across the epithelial barrier: transcellularly through cells and paracellularly through gaps between them. Cell membranes have low intrinsic permeability to mite allergens, and transcellular transporters have not been identified thus far. Paracellular routes offer a logical route for the delivery of inhaled allergens to DCs, particularly because DCs are thought to insert their dendrites into paracellular spaces. In epithelia, however, access to paracellular spaces is restricted by tight junctions (TJs) that seal the paracellular channels apically and therefore regulate the paracellular permeability. Studies of mite peptidase allergens in whole airways and in cultured cells from airway epithelium indicate that TJs represent the key target in the transepithelial delivery of these allergens. Central to this discovery was the theory that allergens are bioactive moieties whose IgE-independent effects are significant factors the development 43 46 47

of allergy.[ ] [ ] [ ] These IgE-independent effects include those that control delivery of allergen to DCs. This hypothesis emphasizes the functional properties of molecules as being an important key to allergy and thus avoids the proposition that allergenicity is related simply to macromolecular structure, charge, or some other physical property. A significant breakthrough was made by studying the effects of allergens in a carefully controlled cell culture model where functional events and cellular imaging were performed in the same cells. Application of small numbers of mite fecal pellets to epithelial cells resulted in the cleavage of TJs. This effect could be replicated by the allergenic mite group 1 cysteine peptidases,[

48] [49]

the group 3 tryptic serine peptidases, the group 6 chymotryptic serine peptidase, and the group 9

50 peptidases.[ ]

collagenolytic serine In all cases, cleavage occurred by proteolysis of the extracellular domains of the TJ adhesion proteins, and transepithelial delivery of mite allergen occurred in proportion to the extent of TJ cleavage, both of which were reduced when the peptidase activity was inhibited. In the case of fully inactive peptidase allergen, transepithelial delivery could not be detected. Tight Junctions as Targets

TJs are supramolecular assemblies situated at the most apical point of the lateral membrane of epithelial cells, where they occlude the paracellular space between adjacent cells. TJs are composed of proteins that span the cell membrane and others that are localized at the cytoplasmic face of the junction, where they interact with the cytoskeleton ( Color Plate 2 ). The transmembrane (TM) proteins appear to have roles in intercellular adhesion and the regulation of paracellular permeability, 51

whereas the cytoplasmic face proteins may be involved in transducing “inside-out” and “outside-in” intercellular signaling.[ ] The TM proteins of TJs are occludin, claudins, and junctional adhesion molecules (JAMs). Occludin and claudins share similar membrane topography in having four TM α-helices that create two 51

extracellular domains. [ ] The extracellular domains of claudins contain notable sequence similarity, perhaps indicative of conserved functional significance, whereas the regions of difference presumably establish the basis for functional diversity. In occludin the first extracellular domain is rich in Tyr and Gly residues, but the second domain is compositionally more diverse. The amino-terminal and carboxy-terminal domains of occludin and claudins are intracellular and are longer in occludin than in most claudins. The carboxy-termini of occludin and claudins are established as a site of interaction with zona occludens-1 (ZO-1), ZO-2, and ZO-3. These are related membrane-associated, guanylate kinaselike proteins (MAGUKs) and are cytoplasmic components of the TJ plaque. Occludin interacts with the GUK domain of these proteins, whereas claudins and JAMs interact at their PSD95/DL9/ZO-1 (PDZ) domains.[

51]

51

Claudins are adhesive and create the distinctive anastomosing strands that characterize the appearance of TJs when viewed as freeze-fracture replicas.[ ] Occludin is adhesive, associates with claudins, and regulates paracellular permeability and cell migration. JAM differs from the other TM proteins by being a 40-kD member of

the immunoglobulin superfamily with a single membrane-spanning domain and two Ig-like extracellular domains. Several different JAMs may exist, but little is known of their role in TJs. Although it seems unlikely that JAMs contribute greatly to the appearance of TJ strands, they may have a significant role in TJ (re) assembly, because direct and indirect interactions can occur with the cytoplasmic TJ proteins cingulin and ZO-1, respectively.

607

At concentrations that mimic daily exposures to these allergens, mite peptidases attack peptide bonds in the extracellular domains of occludin and claudins ( Color 48 49 50

48 49

Plate 3 ).[ ] [ ] [ ] This causes cleavage of TJ and initiates the proteolytic processing of the TJ plaque protein ZO-1.[ ] [ ] The consequence of TJ cleavage is an increased epithelial permeability that enables allergens to cross the epithelial barrier. When TJ cleavage is blocked, transepithelial delivery of allergen does not occur. [48]

The increase in epithelial permeability is nonspecific, and transepithelial delivery of any allergen will be increased, provided the allergen is smaller than the diameter of the paracellular channels at the cleaved junctions. This finding would explain why nonpeptidase mite allergens evoke allergic sensitization and why allergic reactivity to multiple allergens from diverse sources is common. In addition to facilitating transepithelial allergen delivery, the cleavage of TJs might provide signals that are required to drive DCs into orchestrating adaptive immune responses.[ prophylaxis might be achieved. The effects of mite peptidase allergens on TJs are potentially reversible.[

48]

45] [52]

If correct, this idea suggests a number of novel ways in which allergy

Pulse-chase labeling of occludin established that de novo synthesis occurs rapidly after

TJ cleavage is initiated and that the acute repair process is largely independent of changes in occludin gene expression.[

49]

The process of TJ reassembly under these

conditions occurs in an ordered manner, with a reticular scaffold of ZO-1 being established before rings of occludin are reinstated.[

48]

Other Interepithelial Junction Targets 49]

TJs are not the only lateral intercellular adhesions of epithelia that are affected by mite peptidases.[

Adherens junctions (zonulae adherentes, ZAs) encircle cells in

[53]

a continuous manner, forming homotypic interactions with neighboring cells. These junctions are closely associated with TJs. Although they have a contiguous ar rangement at the cell surface, however, adherens junctions do not form a seal that occludes the paracellular channel. In the airways, ZAs consist of E-cadherin, a Ca2 + -binding protein with a single transmembrane domain (see Color Plate 2 ). E-cadherin is a classic cadherin in which the extracellular region contains five repeating domains with internal homology.[

53]

The cytoplasmic domain of E-cadherin forms complexes with α, β, and γ catenins, linking ZAs to the actin cytoskeleton and 54

thus, indirectly, to other membrane-associated proteins such as those of TJs.[ ] E-cadherin is vulnerable to cleavage by mite peptidase allergens, but despite an abundance of theoretic cleavage sites, proteolysis of E-cadherin is much less extensive than that of occludin, suggesting that peptidase allergen access to E-cadherin is sterically hindered until TJs are cleaved.[

49]

Desmosomes constitute a third type of intercellular adhesion studied in relation to the effects of mite peptidases. Desmosomes anchor intermediate filaments to sites of intercellular adhesion (see Color Plate 2 ), conferring resilience to mechanical stress and thus giving desmosomes an important role in processes such as

55 56

morphoregulation.[ ] [ ] Desmosomes are adhesive because they contain desmogleins and desmocollins, which are single-pass TM glycoproteins belonging to the family of desmosomal cadherins. These interact with the phosphoproteins plakoglobin and desmoplakin in the desmosomal plaque to form a bridge to the keratin 56

intermediate filaments.[ ] The punctate nature of desmosomal adhesion does not create a significant barrier to paracellular solute permeability, and the response of desmosomes to peptidase allergens is less striking than effects at TJs. However, using 2-photon molecular excitation microscopy and digital analysis has shown that 50

the intensity of desmosomal puncta increases as other cell adhesions are destroyed.[ ] Neither the molecular basis nor the functional significance of this phenomenon is known, a situation that reflects the relatively undefined nature of the signal transduction pathways that regulate desmosomal assembly and clustering under any conditions. Whatever the molecular basis of these events, it might represent an acute adaptation to the injury produced by the allergen. Other potentially adaptive responses are also apparent, including changes in the actin cytoskeleton. Mite Peptidase Allergens Apoptosis

Mite peptidase allergens exert a gradual and reversible effect on the intercellular adhesions of epithelia, and when only TJs are cleaved, resynthesis of junctional proteins and TJ reassembly are rapid and complete. When cleavage is more extensive, other adaptational changes occur, but these may be insufficient to maintain 52

epithelial integrity and lead to apoptosis.[ ] Adherent cells can become apoptotic by disruption of homotypic and heterotypic adhesions, but it is currently uncertain if this is the mechanism by which mite peptidase allergens induce apoptosis. During the exfoliation of apoptotic cells, the permeability of the epithelial barrier may be very high in the immediate vicinity of these events, further facilitating allergen delivery. Proinflammatory Cytokine and Mediator Production

Peptidase allergens are potentially capable of producing other effects in or around the airway mucosa that could be relevant to allergic sensitization and the perpetuation of allergy. In addition, evidence suggests these allergens could have other actions that involve cellular components of the adaptive immune response. For inhalant allergens, one difficulty in establishing the relevance of these effects is determining the concentrations of allergens that can be achieved in the body after typical environmental exposures. In the case of mite allergens in the airway mucosa, it is possible to estimate the amounts of allergen delivered to defined regions of the airways. Indeed, such modeling was used as a basis to determine the amounts of allergen used in the studies of their effects on TJs. However, it is presently unclear whether sufficient amounts of allergen are inhaled for nonmucosal effects to occur. Mite peptidase allergens (usually Der p 1) have been implicated in a variety of events at the airway interface, ranging from the production of inflammatory mediators by epithelial cells to actions on molecules present in epithelial lining fluid. For example, α1 -antitrypsin, a serpin that protects the airways from injury by serine peptidases (see Chapter 42 ), is a target of Der p 1, which attacks the serpin at its reactive center loop and proximal to its NH2 terminus, causing it to become inactive. [

57]

This interaction is of potential significance because α1 -antitrypsin would be predicted to inhibit the effects of the serine peptidase allergens in vivo.

When α1 -antitrypsin is inactivated by Der p 1, the mite serine peptidases may then have unopposed action on epithelial TJs.

608

Because serine peptidase activity may account for up to 70% of the proteolytic activity in mite faecal pellets, loss of an endogenous inhibitor may swing the balance toward conditions that favor transepithelial allergen delivery. Mite peptidases have also been shown to stimulate the release of various cytokines and prostaglandin E2 (PGE2 ), as well as initiating the IgE-independent degranulation of mast cells.[

58] [59]

Both Der p 1 and Der p 3 elicit a net release of histamine and interleukin-4 (IL-4) from human lung mast cells.[

Der p 1 and Der p 9 up-regulate cytokine gene transcription and cytokine production (IL-6, IL-8, and GM-CSF) in bronchial epithelial effects have been reported for peptidase-containing extracts of Aspergillus

61 fumigatus.[ ]

60 cells.[ ]

59]

Similarly, both

Interestingly, similar

Unlike the actions on TJs, this effect of mite peptidase allergens is caused 62

by the activation of signal transduction pathways coupled to protease-activated receptors (PARs).[ ] Der p 1, Der p 3, and Der p 9 have been shown to cleave the NH2 terminus of the PAR-2 receptor, leading to an increase in phoshoinositide turnover, increase in intracellular Ca2+ , and release of eotaxin and granulocytemacrophage colony-stimulating factor (GM-CSF) by cultured epithelial cells.[

63] [64]

More chronically, peptidase allergen induced cytokine release might involve the

phosphorylation of I-κBα, a cytoplasmic inhibitor of the transcription factor NF-κB. [

65]

Mite Peptidases and Immune Responses

The proteolytic action of Der p 1 can skew immune responses toward an allergic phenotype, as demonstrated in mice sensitized to Schistosoma mansoni.[ 67]

Additionally, catalytically active peptidase allergens produce heightened allergic responses compared with their enzymatically inactive forms,[

66]

and responses to a

68 1.[ ]

nonpeptidase bystander allergen are also increased by the peptidase activity of Der p Several investigations have attempted to determine whether peptidase allergens might skew adaptive responses. Initially it was suggested that IgE production might be enhanced by Der p 1 cleaving the low-affinity IgE receptor, CD23 69 70

71

on B lymphocytes.[ ] [ ] However, there is no convincing evidence that immune responses are modified as predicted. [ ] Skewing might also occur by cleavage of the α subunit of the IL-2 receptor (CD25) by Der p 1, suppressing IL-2dependent proliferation of T cells into the T helper type 1 (Th1) phenotype, and possibly 72

favoring the development or persistence of T helper cell type 2 (Th2) responses.[ ] This is a potentially interesting mechanism, particularly in view of current thinking regarding neonatal programming of adaptive responses. However, the differences between commitment to Th1 and Th2 responses are more clearly distinguishable in mice than in humans, and it remains to be established whether such peptidase allergendependent skewing occurs in people.

CONCLUSIONS Clinically important information is now known about the allergens associated with allergic disease, and these data continue to complement information emerging from many other scientific disciplines. It can be seen that the protein allergens fall, not unexpectedly, into previously delineated protein groups. Because of the efforts directed toward the identification, purification, and characterization of clinically important allergens, it is now possible to appreciate some of the reasons why these substances may lead to disease. It is clear that allergens are functionally and structurally diverse, but the application of molecular techniques has also revealed important clues and relationships between certain major allergens. These approaches have led to the first mechanism-based description of how one important group

of allergens may modulate immune function, such as facilitating contact with dendritic cells, a step essential to allergic sensitization. Similarly, studies have revealed how their IgE-independent effects are likely to create conditions that favor the promotion of an allergic phenotype and a host inflammatory response. Besides relating specifically to mite allergy and asthma, in which peptidases appear to dictate key steps in transepithelial allergen delivery and inflammation, these findings also serve as an important reminder that interactions are likely to be important in defining the outcome of sensitization. These interactions may occur between allergens from diverse sources (e.g., mite allergens and cockroach, fungal, pollen, or pet allergens) or between one set of allergens and associated materials from the same matrix that potentially act as adjuvants. We are now entering a new phase where biotechnological advances combined with an increased understanding of the immune system, the pharmacology of allergic diseases, and the genetics underlying mediator production promise much in the new millennium. Acknowledgment Our work described in this chapter was supported by the Australian NHMRC, the Asthma Foundation of Western Australia, the National Asthma Campaign (UK), the Wellcome Trust, and the Medical Research Council (UK).

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15. Makimura K, Hanazawa R, Takatori K, et al: Fungal flora on board the Mir-Space Station, identification by morphological features and ribosomal DNA sequences, Microbiol Immunol 45:357, 2001. 16. Mitakakis TZ, Barnes C, Tovey ER: Spore germination increases allergen release from Alternaria, J Allergy Clin Immunol 107:388, 2001. 17. Breitenbach M, Simon B, Probst G, et al: Enolases are highly conserved fungal allergens, Int Arch Allergy Immunol 113:114, 1997. 18. Nadeau K, Das A, Walsh CT: Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases, J Biol Chem 268:1479, 1993. 19. Mantyjarvi R, Rautiainen J, Virtanen T: Lipocalins as allergens, Biochem Biophys Acta 1482:308, 2000. 20. Stewart GA, McWilliam AS: Endogenous function and biological significance of aeroallergens: an update, Curr Opin Allergy 1:1, 2001. 21. Ring PC, Wan H, Schou C, et al: The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity, Clin Exp Allergy 30:1085, 2000. 22. Arlian LG: Arthropod allergens and human health, Annu Rev Entomol 47:395, 2002. 23. Arruda LK, Vailes LD, Ferriani VP, et al: Cockroach allergens and asthma, J Allergy Clin Immunol 107:419, 2001. 24. Binder M, Mahler V, Hayek B, et al: Molecular and immunological characterization of arginine kinase from the Indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen, J Immunol 167:5470, 2001. 25. Lin RY, Shen HD, Han SH: Identification and characterization of a 30thinsp;kd major allergen from Parapenaeus fissurus, J Allergy Clin Immunol 92:837, 1993.

26. Baur X, Degens PO, Sander I: Baker's asthma: still among the most frequent occupational respiratory disorders, J Allergy Clin Immunol 102:984, 1998. 27. Posch A, Chen Z, Dunn MJ, et al: Latex allergen database, Electrophoresis 18:2803, 1997. 28. Bernstein DI, Smith AB, Moller DR, et al: Clinical and immunologic studies among egg-processing workers with occupational asthma, J Allergy Clin Immunol 80:791, 1987. 29. Kurup VP, Fink JN: The spectrum of immunologic sensitization in latex allergy, Allergy 56:2, 2001. 30. Sanchez-Monge R, Garcia-Casado G, Lopez-Otin C, et al: Wheat flour peroxidase is a prominent allergen associated with baker's asthma, Clin Exp Allergy 27: 1130, 1997. 31. Menendez-Arias L, Moneo I, Dominguez J, et al: Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I, Eur J Biochem 177:159, 1988. Ingested Allergens 32. Ring J, Brockow K, Behrendt H: Adverse reactions to foods, J Chromatogr B Biomed Sci Appl 756:3, 2001. 33. Han GD, Matsuno M, Ito G, et al: Meat allergy: investigation of potential allergenic proteins in beef, Biosci Biotechnol Biochem 64:1887, 2000. 34. Pascual CY, Crespo JF, San Martin S, et al: Cross-reactivity between IgE-binding proteins from Anisakis, German cockroach, and chironomids, Allergy 52:514, 1997. 35. Vallverdu A, Asturias JA, Arilla MC, et al: Characterization of recombinant Mercurialis annua major allergen Mer a 1 (profilin), J Allergy Clin Immunol 101:363, 1998. 36. Ortolani C, Ispano M, Pastorello E, et al: The oral allergy syndrome, Ann Allergy 61:47, 1988. 37. Aalberse RC, Akkerdaas J, van Ree R: Cross-reactivity of IgE antibodies to allergens, Allergy 56:478, 2001. Injected Insect Allergens 38. Hoffman DR: Allergens in Hymenoptera venom XIII: Isolation and purification of protein components from three species of vespid venoms, J Allergy Clin Immunol 75:599, 1985. 39. King TP, Spangfort MD: Structure and biology of stinging insect venom allergens, Int Arch Allergy Immunol 123:99, 2000. 40. Wu CH, Lan JL: Immunoblot analysis of allergens in crude mosquito extracts, Int Arch Allergy Appl Immunol 90:271, 1989. 41. Lee SE, Johnstone IP, Lee RP, et al: Putative salivary allergens of the cat flea, Ctenocephalides felis felis, Vet Immunol Immunopathol 69:229, 1999.

42. Paddock CD, McKerrow JH, Hansell E, et al: Identification, cloning, and recombinant expression of procalin, a major triatomine allergen, J Immunol 167:2694, 2001. Allergen Biochemistry, Immunogenicity, and Inflammation 43. Robinson C, Kalsheker NA, Srinivasan N, et al: On the potential significance of the enzymatic activity of mite allergens to immunogenicity: clues to structure and function revealed by molecular characterization, Clin Exp Allergy 27:10, 1997. 44. Stewart GA, Thompson PJ, McWilliam AS: Biochemical properties of aeroallergens: contributory factors in allergic sensitization? Pediatr Allergy Immunol 4:163, 1993. 45. Gallucci S, Matzinger P: Danger signals: SOS to the immune system, Curr Opin Immunol 13:114, 2001. 46. Herbert CA, Holgate ST, Robinson C, et al: Effect of mite allergen on permeability of bronchial mucosa, Lancet 336:1132, 1990. 47. Herbert CA, King CM, Ring PC, et al: Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1, Am J Respir Cell Mol Biol 12:369, 1995. 48. Wan H, Winton HL, Soeller C, et al: Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions, J Clin Invest 104:123, 1999. 49. Wan H, Winton HL, Soeller C, et al: Quantitative structural and biochemical analyses of tight junction dynamics following exposure of epithelial cells to house dust mite allergen Der p 1, Clin Exp Allergy 30:685, 2000. 50. Wan H, Winton HL, Soeller C, et al: The transmembrane protein occludin of epithelial tight junctions is a functional target for serine peptidases from faecal pellets of Dermatophagoides pteronyssinus, Clin Exp Allergy 31:279, 2001. 51. Tsukita S, Furuse M, Itoh M: Multifunctional strands in tight junctions, Nat Rev Mol Cell Biol 2:285, 2001. 52. Winton HL, Wan H, Cannell MB, et al: Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium, Br J Pharmacol 124:1048, 1998. 53. Takeichi M: Cadherin cell adhesion receptors as a morphogenetic regulator, Science 251:1451, 1991. 54. Rajasekaran AK, Hojo M, Huima T, et al: Catenins and zonula occludens-1 form a complex during early stages in the assembly of tight junctions, J Cell Biol 132:451, 1996. 55. Garrod DR: Desmosomes and hemidesmosomes, Curr Opin Cell Biol 5:30, 1993. 56. Green KJ, Jones JC: Desmosomes and hemidesmosomes: structure and function of molecular components, FASEB J 10:871, 1996. 57. Kalsheker NA, Deam S, Chambers L, et al: The house dust mite allergen Der p 1 catalytically inactivates alpha 1-antitrypsin by specific reactive centre loop cleavage: a mechanism that promotes airway inflammation and asthma, Biochem Biophys Res Commun 221:59, 1996.

58. Stewart GA, Boyd SM, Bird CH, et al: Immunobiology of the serine protease allergens from house dust mites, Am J Ind Med 25:105, 1994. 59. Machado DC, Horton D, Harrop R, et al: Potential allergens stimulate the release of mediators of the allergic response from cells of mast cell lineage in the absence of sensitization with antigen-specific IgE, Eur J Immunol 26:2972, 1996. 60. King C, Brennan S, Thompson PJ, et al: Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium, J Immunol 161:3645, 1998. 61. Kauffman HF, Tomee JF, van de Riet MA, et al: Protease-dependent activation of epithelial cells by fungal allergens leads to morphologic changes and cytokine production, J Allergy Clin Immunol 105:1185, 2000. 62. Asokananthan N, Graham PT, Fink J, et al: Activation of protease-activated receptor (PAR)-1, PAR-2, and PAR-4 stimulates IL-6, IL-8, and prostaglandin E2 release from human respiratory epithelial cells, J Immunol 168:3577, 2002. 63. Sun G, Stacey MA, Schmidt M, et al: Interaction of mite allergens Der p 3 and Der p 9 with protease-activated receptor-2 expressed by lung epithelial cells, J Immunol 167:1014, 2001. 64. Asokananthan N, Graham PT, Stewart DJ, et al: House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1, J Immunol 169:4572, 2002. 65. Stacey MA, Sun G, Vassalli G, et al: The allergen Der p 1 induces NF-κB activation through interference with IκB alpha function in asthmatic bronchial epithelial cells, Biochem Biophys Res Commun 236:522, 1997. 66. Comoy EE, Pestel J, Duez C, et al: The house dust mite allergen, Dermatophagoides pteronyssinus, promotes type 2 responses by modulating the balance between IL-4 and IFN-γ, J Immunol 160:2456, 1998. 67. Gough L, Schulz O, Sewell HF, et al: The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response, J Exp Med 190:1897, 1999. 68. Gough L, Sewell HF, Shakib F: The proteolytic activity of the major dust mite allergen Der p 1 enhances the IgE antibody response to a bystander antigen, Clin Exp Allergy 31:1594, 2001. 69. Hewitt CR, Brown AP, Hart BJ, et al: A major house dust mite allergen disrupts the immunoglobulin E network by selectively cleaving CD23: innate protection by antiproteases, J Exp Med 182:1537, 1995. 70. Schulz O, Laing P, Sewell HF, et al: Der p I, a major allergen of the house dust mite, proteolytically cleaves the low-affinity receptor for human IgE (CD23), Eur J Immunol 25:3191, 1995. 71. Shakib F, Schulz O, Sewell H: A mite subversive: cleavage of CD23 and CD25 by Der p 1 enhances allergenicity, Immunol Today 19:313, 1998. 72. Schulz O, Sewell HF, Shakib F: Proteolytic cleavage of CD25, the alpha subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity, J Exp Med 187:271, 1998.

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Chapter 37 - Laboratory Tests for Allergic and Immunodeficiency Diseases

Robert G. Hamilton

Personnel in the diagnostic immunology laboratory perform an array of analytic measurements that facilitate the diagnosis and management of human allergic and immunodeficiency diseases. These assays can be subdivided into (1) immunologic tests that measure humoral and cellular components of the human immune system and (2) tests that measure antigenic proteins (allergens) that drive the allergic disease process. This chapter focuses on methods used in the diagnostic immunology laboratory that have particular relevance for measuring immunoglobulin E (IgE) antibodies, cell-derived mediators of inflammation, markers of anaphylaxis, and lymphocyte subsets and function.

TERMINOLOGY The terms antibody and antigen are generically used throughout this chapter as reagents in clinical assays that are used to quantify blood-borne and environmental analytes. Antibody is a protein that is formed as part of an immunologic response to a foreign substance and that specifically binds with that foreign substance. As such, the assay principles discussed for antibody are analogous to the properties of assays that use a receptor to bind antigen, including cell surface and intracellular receptors and plasma transport proteins. Antigen is a substance (usually a protein) that is capable of inducing antibody formation and reacting specifically with this antibody. The more generic descriptor for antigen is ligand, which is a substance bound by receptor, such as an antibody or cell surface receptor.

HUMORAL IMMUNE RESPONSES TO ALLERGIC AND IMMUNODEFICIENCY DISEASES Of the five human immunoglobulin isotypes, human IgE, IgG, and IgA antibodies have been those principally measured to assess humoral immune responses to allergens in individuals who manifest immediate-type hypersensitivity reactions and to protein and carbohydrate antigens administered as vaccines to individuals suspected of immunodeficiency disease. There has been minimal interest in IgM and IgD immune responses in individuals suspected of either allergic or immunodeficiency disease.

Immunoglobulin E IgE antibody is the humoral “gatekeeper” of the human immediate-type hypersensitivity response. It possesses the power of an estimated 106 to 108 antigen-binding 1

specificities, [ ] which are defined by its variable region, and biologic effector functions (e.g., mast cell/basophil Fcepsilon cell receptor binding), which are determined by its epsilon (epsilon) heavy-chain constant-region domains. IgE (190•kD) circulates in the blood as a four-chain monomeric protein. Quantitation of the total IgE level in serum is usually reported in international units per milliliter (IU/ml) or converted to mass units using a conversion factor of 1 IU = 2.4 nanograms (ng) of protein. More recently, using the International System of Units (Système International d'Unités, SI) has been proposed, in which 1 SI unit = 1 microgram per liter (•g/L); however, these units are slow to appear in the peer-reviewed literature. Serum IgE concentrations are highly age dependent. Cord serum IgE levels are low, usually less than 2•kU/L, because IgE does not pass the placental barrier in significant amounts.[

2] [3]

Progressive increases in mean serum IgE levels occur up to age 10 to 15 years, rising at a rate slower than IgG but comparable to IgA.

From the second to eighth decades of life, humans experience an age-dependent decline in total serum IgE.[

4] [5]

Interestingly, IgE does not appear to be necessary 6

for maintaining health in parasite-nonendemic countries because adults may remain healthy for years with no detectable serum IgE. [ ] Atopic infants have an 4

apparent earlier and steeper rise in serum IgE levels during their early years of life compared with nonatopic controls,[ ] but high infant IgE levels are not always predictive of atopy in adult life. Clinically, adult serum IgE levels are reported in comparison with those from age-adjusted healthy, nonatopic individuals. After age 14, serum IgE levels greater than 333•kU/L (800••g/L) are considered abnormally elevated and strongly associated with atopic disorders such as allergic rhinitis, 2 3 4 5

extrinsic asthma, and atopic dermatitis. [ ] [ ] [ ] [ ] In a population of adults with allergic asthma, mean serum IgE levels of 1589••g/L (range 5 to 12,750••g/L) have been reported, with only about half these asthmatic patients having IgE concentrations above 800••g/L (age-adjusted upper limit for nonatopic adults). In a population of individuals with atopic dermatitis, high serum IgE levels have been observed (mean 978•kU/L; range 1.3 to 65,208•kU/L). Although high levels of serum IgE are common in allergic diseases, total serum IgE has become less frequently used as an indicator for atopy because of the wide overlap in the IgE distributions of atopic and nonatopic populations. In contrast, the presence of allergen-specific IgE antibodies to environmental allergens has been more strongly associated with

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allergic symptoms and the risk of systemic allergic reactions in individuals with a positive history of adverse reactions. The clinical utility of IgE antibody measurements in the diagnosis of human allergic disease is limited to diseases in which the role of IgE has been documented, as in allergic rhinitis, extrinsic (allergic) bronchial asthma, urticaria/anaphylaxis to insect venoms, drugs, and foods (see Chapter 62 ). The measurement of IgE has little value in the diagnosis of immunodeficiency. The significance of total serum IgE levels after allogeneic bone marrow transplantation (BMT) has been studied in patients with aplastic anemia, leukemia, Wiskott-Aldrich syndrome, and infantile agranulocytosis. In one study, total

7

serum IgE levels increased from 7- to 2000-fold in 10 of 12 individuals as early as 14 days after BMT.[ ] The IgE levels, however, returned to baseline in each subject by 60 days. In more than half these subjects the increase in IgE was associated with clinical and biochemical evidence of graft-versus-host disease (GVHD). In another study of 135 BMT recipients, statistically significant increases in IgE levels coincided in time with engraftment and acute GVHD, but not with herpes 8

simplex virus (HSV) or cytomegalovirus (CMV) infections or chronic GVHD.[ ] After BMT, some nonatopic recipients developed persistently raised total serum IgE 9

levels with antigen-specific IgE to the same allergen specificities as their atopic donors.[ ] Mechanisms inducing transient changes in plasma IgE were investigated in 100 patients admitted to the intensive care unit (ICU) for trauma. Substantial but transient increases in serum IgE concentrations were observed in association with the degree of sepsis after trauma. The temporal IgE increase in these individuals was associated with the magnitude of infection and level of plasma interleukin-4 (IL4) rather than the actual traumatic event.[

10] [11]

Immunoglobulin G IgG antibody responses are considered by some to be a useful marker of antigen exposure, especially in allergic individuals receiving allergen immunotherapy or individuals receiving protein and polysacharide vaccines to diagnose immunodeficiency. In healthy adults the four-polypeptide-chain IgG monomer (150•kD) 12

constitutes approximately 75% of the total serum immunoglobulins.[ ] IgG is equally distributed between intravascular and extravascular serum pools, and it thus affords protection to the fetus and newborn because of its ability to pass across the placental barrier. Human IgG can be divided into four subclasses (IgG1 to IgG4) on the basis of unique antigenic determinants on their heavy-chain constant-region domains. Any two IgG subclasses show greater than 95% amino acid sequence homology. The greatest differences are found in the number of amino acids in the hinge region (IgG1, 15 amino acids; IgG2, 12; IgG3, 62; IgG4, 21) and the number of cysteine residues probably involved in inter-heavy-chain disulfide bridges (IgG1, two cysteine residues; IgG2, four; IgG3, 11; IgG4, two). The IgG subclasses circulate in the blood in the following relative percentages: 60% to 70% IgG1; 14% to 20% IgG2; 4% to 8% IgG3; and 2% to 6% IgG4.[

13] [14]

The structural differences among the four IgG subclasses translate into differences in their biologic effector functions involving complement activation and Fc receptor binding. For example, IgG3 and IgG1 activate complement efficiently, whereas IgG2 is less efficient and IgG4 does not appear to bind C1 or activate complement. Of the four IgG subclasses, IgG4 antibodies have been of particular interest to investigators of allergic disease disorders because of their observed 15

potential for blocking Ag-induced basophil histamine release in vitro,[ ] especially after immunotherapy. In fact, some IgG4 antibodies have been shown to function as monovalent antibodies as a result of asymmetry caused by unstable inter-heavy-chain disulfide bonds of IgG4 that are in equilibrium with intrachain disulfide 16 17

bonds. As a result, some IgG4 molecules possess two different antigen-binding sites and functional bispecificity. [ ] [ ] The clinical importance of these bispecific IgG4 antibodies has yet to be shown, but IgG4's apparent functional monovalency is an attractive feature that may produce antigen-blocking activity in vivo. The diagnosis of primary or secondary immunodeficiency can be made in an individual 2 years or older with recurrent infections if the total serum IgG concentration is less than 30% of the individual's age-adjusted normal range. In these individuals, confirmation of immunodeficiency by further testing of IgG antibody responses 18

after intentional vaccination is generally considered unnecessary. [ ] Lesser reductions in circulating IgG levels, however, should be confirmed with an impaired ability to produce Ag-specific IgG antibody responses to both protein (e.g., tetanus toxoid) and polysaccharide (e.g., pneumococcal) antigens after active immunization. For children under 2 years of age it is not always possible to distinguish between a permanent antibody deficiency and a developmental delay in IgG production, especially because IgG antibody responses to carbohydrate antigens are often deficient in children of this age. Although no absolute standards exist for

determining what constitutes an “adequate” IgG antibody response after vaccination, some reference laboratories provide useful “protective” IgG anti-pneumococcal serotype ranges for children and adults. Some clinicians use the criteria of a fourfold or greater rise in IgG antibodies specific for tetanus toxoid and in two or more pneumococcal polysaccharide serotypes as a mark of a successful IgG antibody response.[

18]

Immunoglobulin A IgA is the predominant immunoglobulin detected in human colostrum, saliva, tears, bronchial secretions, nasal mucosa, vaginal secretions, prostatic fluid, and mucous secretions of the small intestine.[

19]

In serum, IgA circulates primarily in a monomeric form (160•kD, 90%), although 10% is polymeric with 2 four-chain

basic units (400•kD) combined by a J chain. IgA1 and IgA2 have been identified with two allotypic forms of IgA2, m(1) and m(2).[

20]

Total IgA and Ag-specific IgA immune responses have been measured in fluids lavaged from nasal passages, bronchi, and alveoli of allergic individuals with rhinitis after antigen challenge.[

21]

In one study, ragweed-specific IgA in bronchoalveolar lavage (BAL) fluid that was collected after a segmental lung challenge correlated 22

significantly with the release of eosinophil cationic protein (ECP), a marker of eosinophil degranulation.[ ] Ragweed-specific IgE in serum from the same subjects also correlated with ECP release, indicating a role for IgE antibodies in allergic pulmonary inflammation and a potential role for antigen-specific IgA in eosinophil degranulation in the lung after antigen challenge. More recently, evidence indicates that ragweed-specific IgE and

613

IgA antibodies are independently involved in the pathogenesis of the late-phase reaction (LPR) induced in the lung by an antigen insult.[

23]

ANTIBODY-ANTIGEN–BINDING REACTIONS The interaction of bivalent or multivalent antibody with multivalent antigen in gels and solutions is the basis of quantitative immunochemical assays used in the diagnostic immunology laboratory. This binding reaction is governed by the law of mass action, which in this case states that free antigen [Ag] and free antibody [Ab] reversibly interact to form antigen-antibody complexes [Ag-Ab]. The relationship between free and antibody-bound antigen is determined by the equilibrium, or affinity, constant Ka , which equals the ratio of the association (k1 ) and dissociation (k2 ) constants, as follows:

The addition of increasing amounts of antigen to a fixed quantity of antibody leads to the formation of increasing amounts of [Ag-Ab] complexes. At a certain

concentration of antigen, half the antibody-binding sites become saturated, and [Ag-Ab] = [Ab]. This results in an affinity constant equal to the reciprocal of the free antigen concentration at half-maximum saturation binding, or Ka = 1/[Ag]. As the concentration of antigen increases further, molar excess quantities of antigen drive the binding reaction toward complete saturation of the antibody-binding sites. At that point the molar ratio of bound antigen to total antibody approximates 2, which is the valence of monomeric antibody ( Figure 37-1 ). 24

Scatchard analysis is performed by plotting the bound/free molar ratio of antigen that is unitless versus the molar concentration of bound antigen.[ ] The affinity constant, or strength of binding of the antibody, is defined numerically as the negative inverse of the slope of the curve. Figure 37-1 displays a representative Scatchard analysis plot for a human insulin-specific antibody. Because most antigens are complex mixtures of proteins with multiple antigen-binding epitopes, Scatchard plots of polyclonal antisera and complex antigens are usually not linear. Each antigen possesses multiple binding sites and thus binds to a spectrum of antibodies of different isotypes, each with a slightly different affinity. With polyclonal antisera, high-affinity antibodies define the slope of the Scatchard plot at low antigen concentrations. Low-affinity antibodies contribute more to the slope of the Scatchard plot when the antigen concentrations are high. The interaction of multivalent antigens with polyclonal antisera is not as easily described mathematically with Scatchard analysis. The term avidity is used to describe the strength of binding of complex multivalent interactions. Binding at one site or epitope on an antigen may affect the rate of binding to other antigenbinding epitopes on the same antigen. When the binding of antibody to one antigenic

Figure 37-1 Calculation of the affinity constant (Ka ) of the antigen (human insulin in microunits per ml or nanograms per ml) and antibody (polyclonal anti–human insulin) binding reaction (see law of mass action in text) by means of Scatchard analysis (left) and the concentration of unbound ligand at half-saturation of antibody (right). In Scatchard plot, Ka is derived from the slope of the line (y intercept/x intercept) in which the concentration of bound insulin (abscissa) is plotted against the molar ratio of bound insulin to free insulin ([AgB ]/[Ab][AgF ]). In the saturation plot (right), Ka is calculated at the inverse of the free hormone concentration at halfsaturation of the antibody. (From Thorell JI, Larson SM: Radioimmunoassay and related techniques: methodology and clinical applications, St Louis, 1978, Mosby, p 29.)

TABLE 37-1 -- Performance Documentation for Allergosorbent Reagents Using Natural Rubber Latex Allergen Parameter

Method/Source Information

Allergen extract name: genus, species, common name, code

Hevea brasiliensis, natural rubber latex, K82 (occupational allergen)

Allergen extract source

••••Clone 600, H. brasiliensis, rubber tree sap, Malaysia (nonammoniated); identity test confirmed by supplier

*

Binding capacity

10 ng IgE antibody per tube, based on plateau of dilution recovery analysis

Parallelism

17.5

>35.1

>22,501

Class

†

Qualitative Interpretation

kIUa/L

0

Absent/undetectable

100.00

Class

Qualitative Interpretation

% Reference

CAP System Scoring Scheme (Pharmacia)

Alternate Scoring System: Pharmacia CAP System

‡

0

Absent/undetectable

6000

* Normalized counts (“modified RAST”) uses 25•IU/ml total IgE standard (PRIST) as positive “time control” to define gamma-counting time appropriate tonormalize assay by accumulating 25,000 counts with PRIST standard (see Figure 37-5 ). Normalized counts associated with each PRU/ml calibrator are reportedbased on data from 17 assays in which birch reference serum and 25-IU/ml PRIST positive control were run in tandem in the same assay. † Calibrator standardized against WHO IgE standard in kIU/L. This is the preferred calibration method for clinical allergen-specific IgE antibody assays. ‡ Designed to report results in classes equivalent to those obtained in modified RAST above. Enzyme activity generated from patient specimens is comparedwith total IgE standard of 0.35•kU/L. Fluorescence unit (FU) ratio (% reference)for each patient test is obtained by dividing the result expressed as FU by meanFU for IgE 0.35-kU/L standard.

limit for their peer group. IgE antibody results are reported as qualitative grade or class results that reflect the presence and relative levels of IgE antibodies in the test sera. IgE antibody results in kIU/L are also produced by the quantitative IgE antibody immunoassays. Gross outlier laboratories are identified when a laboratory detects allergen-specific IgE antibody in a serum from a nonatopic person (clinical history negative, puncture skin test negative) by reporting a class result other than zero. Moreover, a reported class “0” for an IgE antibody–containing serum from an atopic individual (positive clinical history, positive puncture skin test) is considered an “outlier.” The problem arises with a class 1 IgE antibody result that is considered equivocal or positive by some laboratories and negative by others. The CAP surveys operate on the basis of peer group consensus. Thus, if more than 90% of laboratories performing an allergen-specific IgE antibody assay method do not agree with the result, that measurement is not graded in that cycle of the survey for that assay. Each diagnostic allergy survey participant receives a summary of the SE survey data for each cycle with a commentary that critiques general performance trends and problem areas.

IMMUNOCHEMICAL METHODS FOR ALLERGEN-SPECIFIC IgG ANTIBODY MEASUREMENT Allergen-specific IgG antibodies were identified during the 1930s in the sera of immunized allergic persons. Subsequently, these antibodies were shown to block the release of histamine from IgE-sensitized basophils in the presence of allergen. Unimmunized individuals who are minimally exposed to clinically important environmental aeroallergens (e.g., from weed, grass, and tree pollens and mold/animal

623

TABLE 37-4 -- Classification of IgE Antibody Assays Classification

Results

Method of Standardization

Calibrators/Controls

One reference sample. Negative and positive control

Qualitative

Single or dual calibrators used to Reactive or nonreactive normalize assay Positive or negative Indeterminant (equivocal) zone may be No calibration curve present. Based on preassigned positive threshold

Semiquantitative

Arbitrary units or classes Single-point calibration curve: modified or alternative scoring system. Multipoint calibration curve: classes 0, I, II, III, IV, V, VI; PRU/ml

Single or dual calibrators to normalize assay

Negative and bilevel positive control sera References unique for test system; made from pooled patient sera

Quantitative

Units (kIU/L or ng/ml) related to recognized reference preparation (e.g., WHO IgE standard 75/702)

Multipoint homologous or heterologous standard curve used for interpolation of response data into dose results

Trilevel positive controls. Reference calibrated by comparison with widely available, quality reference preparation

Modified from Matsson P, Hamilton RG: Evaluation methods and analytical performance characteristics of immunological assays for human immunoglobulin.E (IgE) antibodies of defined allergen specificities. National Committee on Clinical Laboratory Standards (NCCLS) Task Force., 1/LA20-A, Wayne, Pa, 1997, NCCLS. epidermal antigens) produce serum levels of specific IgG antibody that are low (ng/ml) or undetectable. Increases in serum IgG antibody to •g/ml levels are observed after injection of antigen either by intentional or accidental exposure (e.g., drug injection, insect sting) or by active allergen immunotherapy. Quantitative immunoassays for allergen-specific IgG antibody have been developed to evaluate an allergic patient's relative allergen exposure by measuring the magnitude of their humoral immune response to parenterally administered allergen injections given during immunotherapy. Clinically successful immunotherapy is accompanied by 47]

high serum levels of allergen-specific IgG antibody,[ remains unclear at present.

even though the precise role of IgG-“blocking” antibodies in conferring protection against allergic reactions

Knowledge of the presence or absolute levels of IgG antibodies in human serum has not been always clinically useful. Serum levels of IgG antibodies specific for food antigens, for instance, have shown no correlation with the frequency of reactions observed during double-blind placebo-controlled food challenges. The clinical care role for IgG antibody measurements in the field of allergy has been their use in the evaluation of Hymenoptera venom–allergic patients who have been receiving 48

maintenance immunotherapy injections.[ ] In these cases, Hymenoptera venom–specific IgG antibody levels are reportedly useful in individualizing venom immunotherapy doses. Although the clinical utility of IgG antibody measurements appears to be restricted to less than 4 years of venom therapy, the IgG antivenom level can be valuable for individualizing the venom dose and frequency of venom injections to maximize the protective effects of venom immunotherapy (see Chapter 81 ). Analytically, the presence of •g/ml levels of IgG antibody permit the dilution of a serum before immunoassay analysis. This dilution procedure has tended to minimize nonspecific binding and enhance the assay's analytic sensitivity. The optimization of venom-specific IgG assays therefore has focused less on the issue of sensitivity of the assay and more on ensuring parallelism, precision, and accuracy and the development of human IgG antibody standards. The first quantitative

immunoassay used to measure venom-specific IgG antibody was the radioimmunoprecipitation assay, or double-antibody assay. The first incubation of this assay involved a noncompetitive liquid-phase reaction between radioiodinated purified allergen (e.g., phospholipase A2 from honeybee venom) and antibody-containing 49

serum. After an incubation period, IgG was selectively precipitated from other human antibody isotypes with polyclonal anti–human IgG.[ ] Both the isotopic and the nonisotopic versions of this assay have remained useful analytic tools for quantitation of IgG antibody specific for purified protein antigens. Unfortunately, most allergen preparations are biologic extracts that contain complex mixtures of proteins that are not purified and do not radiodinate or enzyme-label uniformly. Because the radioimmunoprecipitation assay performs best under labeled antigen–limiting conditions, this tends to accentuate nonparallelism and differential plateauing when polyclonal sera with heterogenous IgG antibodies populations are being evaluated. To address these concerns, a noncompetitive solid-phase immunoassay was developed in which complex antigen mixtures were covalently insolubilized onto a solid phase (e.g., carbohydrate particles, paper disk). The solid-phase radioimmunoassay (SPRIA) binds antigen-specific antibodies of all isotypes from the reference or 50

test sera onto an antigen-sorbent. After a buffer wash, 125 I-labeled protein A from Staphylococcus aureus detects bound human IgG.[ ] In direct comparison to the radioimmunoprecipitation assay, SPRIA has displayed superior performance for the detection of IgG antibodies, especially those specific for multiple epitopes on 51]

different proteins in complex allergen extracts.[

More recently, IgG antibodies against purified allergens have been

624

measured using a microtiter plate–based noncompetitive enzyme immunoassay in which allergenic protein is insolubilized on the plastic microtiter wells. Antibodies are bound to insolubilized protein on the microtiter plate. After buffer wash, bound IgG is detected with an enzyme-labeled mAb anti–human IgG Fc reagent.[ Because of the limited binding capacity of plastic microtiter plate wells, this assay format has been best suited for the detection of IgG antibodies specific for individual purified allergens.

15]

IgG SUBCLASS PROTEIN AND ANTIBODIES IgG has achieved special importance because it is the sole immunoglobulin that is transported across the placenta in any significant quantities. As such, IgG confers humoral immunity on the neonate. The levels of IgG1, IgG2, IgG3, and IgG4 protein in serum have been quantified by a number of immunologic methods, including RID, nephelometry, and immunometric assays.[

52]

The concentrations of IgG subclass proteins are known to increase throughout childhood, reaching adult levels by

53 54

12 years of age.[ ] [ ] Interest in selective IgG subclass deficiencies has stemmed from their functional differences associated with the binding to antigen (valency), activation of complement via the classic pathway, and binding to cell surface receptors. Decreased levels of all four IgG subclasses have been observed in common variable hypogammaglobulinemia, severe combined immunodeficiency, and WiskottAldrich syndrome. More selective deficiencies of IgG2 and IgG4 proteins are seen in some individuals with IgA deficiency. In the assessment of allergic patients, IgG4 antibodies in particular have received special attention. IgG subclass protein measurements were extensively performed in the latter decades of the twentieth 55]

century,[

leading to extensive focus on IgG-subclass deficiencies.[

56]

However, most clinicians and researchers no longer view IgG-subclass protein or antibody

measurements as useful in the clinical work-up of their patients for immunodeficiency. Total IgG, IgA, and IgM levels in serum are most useful in evaluating a patient for immunodeficiency. IgG4 constitutes approximately 4% of the total IgG in serum of adults, preferentially produced as a result of chronic antigenic challenge, such as occurs during immunotherapy. Research interest therefore has focused on IgG4 antibodies in patients with type 1 hypersensitivity who receive immunotherapy or chronic natural allergen exposures. Allergen-specific IgG4 antibody has been measured by several immunoassay configurations. The SPRIA type assay uses allergen that is insolubilized on an allergosorbent (microtiter plate or particle) to bind specific antibodies from serum. Bound human IgG4 antibodies are then detected with a purified, radiolabeled or enzyme-labeled, anti–human IgG4 reagent. In an alternative assay, IgG4 is selectively bound from serum with a solid phase containing IgG4specific capture antibody. Allergen-specific IgG4 antibodies are then detected with a radiolabeled allergen. The latter assay avoids interference by IgG antibodies of other isotypes and subclasses saturating limited antigen-binding sites on the allergosorbent. However, interference in the labeled-antigen assay can still occur if the allergen-specific IgG4 is a small percentage of total IgG4 antibody population. 57

Allergen-specific IgG4 measurements continue to be of interest in research studies because of their in vivo blocking potential.[ ] However, their application to the clinical management of patients with allergic disease remains limited because comparable frequencies of IgG4 antibodies specific for food and inhalant allergens are observed in both healthy adults and adults with asthma. [

58]

SERUM TRYPTASE Mast cells in connective tissue throughout the human skin and the respiratory and digestive tracts store 10 to 35 pg of tryptase per cell. Tryptase is a 134-kD serine 59

esterase with four subunits, each with its own enzymatically active site.[ ] When dissociated from heparin, tryptase spontaneously degrades into enzymatically inactive monomeric subunits. In contrast to skin and lung mast cells, human basophils contain 300- to 700-fold less tryptase. Thus the activation of mast cells through an IgE-mediated hypersensitivity reaction is believed to be the primary source of tryptase detected in human serum. Tryptase is released from activated mast cells in parallel with prestored histamine and other newly generated vasoactive mediators. Two tryptase genes code for the alpha (α) and beta (β) forms of tryptase. Of the two forms, α-tryptase in blood is constitutively secreted by all mast cells and is therefore considered a reflection of total mast cell number; it is estimated by subtracting β-tryptase from total tryptase in serum assays. The β-tryptase levels in blood are a measure of mast cell activation. Tryptase is intentionally converted to its enzymatically inactive form. The total serum level of mast cell tryptase is then 60

measured in a noncompetitive fluorescent enzyme immunoassay (CAP System, Pharmacia) that uses a capture mAb that binds both α-protryptase and β-tryptase.[ ] Serum levels of total tryptase in healthy (nondiseased) individuals range from 1 to 10•ng/ml (average 5•ng/ml). Systemic mastocytosis should be suspected if baseline serum levels exceed 20•ng/ml. Quantitation on β-tryptase is accomplished with a solid-phase noncompetitive immunoassay that uses a β-tryptase-specific capture mAb. However, because the β-tryptase-specific antibody reagent weakly cross-reacts with α-protryptase, high levels of α-protryptase in serum may result in falsely lower levels of β-tryptase as a result of competitive inhibition. Normal levels of β-tryptase are less than 1•ng/ml. β-Tryptase levels greater than 1•ng/ml indicate mast cell activation. After systemic anaphylaxis induced by an insect sting, β-tryptase levels tend to peak above 5•ng/ml within 30 to 60 minutes after the sting, then decline, with a biologic half-life of about 2 hours.[ suspected mast cell–mediated systemic be listed as a probable cause of death.

62 reaction.[ ]

61]

For optimal test results, blood samples should be collected from 0.5 to 4 hours after initiation of a

A peak β-tryptase level greater than 10•ng/ml in a postmortem serum indicates that systemic anaphylaxis should

OTHER ANALYTES IN ALLERGIC DISORDERS AND ASTHMA A number of other analytes are infrequently measured in human serum to support the differential diagnosis of allergy, asthma, or hypersensitivity pneumonitis and assist in the management of asthmatic patients. These include cotinine, ECP, and precipitating human IgG antibody for antigens in organic dusts.

625

Cotinine Nicotine in the blood of smokers or individuals passively exposed to smoke is directly excreted in the urine or converted by cytochrome P-450 enzymes to 5hydroxynicotine, which is then converted by aldehyde oxidase to cotinine. As the major metabolite of nicotine, cotinine has been shown to have a longer biologic half-life than nicotine in the blood. The rate of clearance of cotinine from the urine of smokers (15.4 ± 1.2 hours) is significantly shorter than that of nonsmokers 63

(27.3 ± 1.9 hours). Cotinine has therefore become the serum marker of choice for monitoring asthmatic patients for passive smoke exposure.[ ] In one study the use of preset positive threshold levels of 13.7, 14.2, and 49.7•ng/ml for cotinine levels in plasma, saliva, and urine specimens, respectively, produced a diagnostic 64

sensitivity of 97% to 99% and diagnostic specificity of 81% to 83% for passive smoke exposure.[ ] Plasma, saliva, and urine have all been successfully used in clinical studies to evaluate subjects for passive smoke exposure. Although each specimen type has its advantages and limitations, serum is often considered the specimen of choice. When venipuncture is not possible (e.g., in children), saliva and urine appear to be satisfactory alternative specimens. Cotinine is currently measured by enzyme immunoassay. One set of commercial reagents (STC Technologies) for cotinine detection in urine exhibits 10% cross-reactivity with 3hydroycotinine and no detectable cross-reactivity to nicotine, nicotinic acid, penicillin-G, ascorbic acid, acetylsalicylic acid, or caffeine. Eosinophil Cationic Protein Eosinophilia is associated with a variety of inflammatory disorders, including asthma, in which primed and activated eosinophils are detectable in high numbers. Tissue destruction, such as the shedding of epithelial cells in airways, may be caused by products released from eosinophil granules. Solid-phase noncompetitive 65

immunoassay methods (Pharmacia) have been developed to quantify ECP, a 18.5- to 22-kD basic protein that is present in eosinophil granules.[ ] Sera from 100 healthy subjects contained ECP levels of 2.3 to 16•ng/ml (95% range), with a geometric mean of 6•ng/ml. When released, ECP acts as a potent neurotoxin and cytotoxic agent that can kill parasites (e.g., Schistosoma mansoni), possibly through membrane damage. Elevated levels of ECP have been detected in serum, sputum, and nasal secretions of individuals undergoing late phase reactions (LPRs), usually 6 to 24 hours after allergen exposure, when eosinophil influx predominates at the reactive site.[

66]

Results correlate strongly with total blood eosinophil counts.

At present, ECP measurements have demonstrated limited clinical utility in the monitoring of patients with extrinsic asthma and other allergic diseases in which eosinophils are thought to induce tissue damage.

Precipitating IgG Antibodies (Precipitins) Diagnostic immunology laboratories continue to perform the precipitin test to evaluate individuals suspected of having extrinsic allergic alveolitis or hypersensitivity 67

pneumonitis (see Chapter 75 ). This hypersensitivity reaction involves the lung interstitum and terminal bronchioles.[ ] Within hours after a heavy exposure to antigenic organic dusts (e.g., molds, bird droppings), a sensitized individual experiences chills, fever, malaise, cough, and shortness of breath. Although cellmediated pathology is suggested from the histology of the lung lesions, most individuals with hypersensitivity pneumonitis have high levels of IgG antibody in their serum to the offending antigen. Serum is evaluated by the double-diffusion method (Ouchterlony) for precipitating antibodies. More specifically, a crude antigen extract and antibody (control or patients serum) are pipetted into closely spaced wells in a porous agarose gel. Precipitating antibodies in visible white precipitin lines are confirmed by lines of identity with known human antibody controls. In one study, precipitating antibodies or precipitins were detected in the serum of almost all 68

ill patients, but also in the serum of as many as 50% of asymptomatic individuals exposed to the relevant organic dusts.[ ] Immunoassays for IgG antibody to the organic dust antigens are available but appear to be too analytically sensitive and diagnostically nonspecific. Precipitin assays are clinically available with specificities for pigeon serum, Aureobasidium pullulans, fecal particles from parakeets and a variety of exotic household birds (Amazon, cockatiel, blue-front parrot), thermophillic Actinomyces, and Aspergillus fumigatus. A positive precipitin to one or several Aspergillus antigens is also an important diagnostic criterion for allergic bronchopulmonary aspergillosis (ABPA). Together with an elevated total serum IgE, typically greater than 1000•ng/ml, and a positive skin or blood test for Aspergillus-specific IgE antibody, a positive Aspergillus precipitin supports the diagnosis of ABPA (see Chapter 74 ).

ASSESSMENT OF ENVIRONMENTAL ALLERGENS The treatment of allergic diseases involves the combined use of allergen avoidance, pharmacotherapy for management of allergic symptoms, and allergen-specific immunotherapy. Of these, separation of the patient from the allergen through avoidance strategies is preferred and possibly the least expensive and most effective approach for reducing allergy symptoms and minimizing further sensitization. The immunology laboratory is often asked to quantify the level of aeroallergens in both outdoor and indoor environments. Outdoor Aeroallergens Daily pollen and mold spore levels in the outdoor air are evaluated in most major cities across the United States by aerobiology stations (see Chapter 33 ). The Aerobiology Committee of the American Academy of Allergy, Asthma, and Immunology has established an aerobiology network that certifies participating laboratories, monitors their performance, and collates longitudinal pollen and spore data. Fungal spores are relatively small compared with pollen grains. The mold spore size varies from 1 to more than 100•• (•m), with most spores in the 7-•m to 12-•m range. In contrast, pollen grains are larger, with diameters from 20 to 70••m. Until recently, the rotorod has been the primary collection device used by most aerobiology stations. To address the poor efficiency of the rotorod in collecting mold 69

spores,[ ] aerobiology stations have been replacing their rotorod samplers with a suction impactor that pull particles from the air and embeds them on a moving paper tape. These devices have the advantage of being able to collect longitudinal samples over 24 hours or 5 days.

626

The clinical value of the pollen/mold count resides in its diagnostic value, because it defines the type of pollen and mold that can be found in the air in different U.S. regions at times when patients experience allergic symptoms. This information may aid the allergist in determining which allergen specificities are inducing allergy symptoms and then may facilitate selection of the allergen specificities for investigation with skin and blood testing. Indoor Aeroallergens Children with asthma, in particular, have a high prevalence of IgE antibodies specific for environmental allergens found indoors. Dust mite (Dermatophagoides pteronyssinus, D. farinae), cat (Felix domesticus), dog (Canis familiaris), cockroach (Blatella germanica), mouse (Mus musculus), and molds (e.g., Alternaria, Aspergillus, Cladosporium, Penicillium) are the primary sources of indoor aeroallergens [

70]

(see Chapter 34 ). For dust mite allergens a dose-response relationship 71

has been shown between increasing exposure and the likelihood of a positive skin test response to mite allergens.[ ] Even exposure to low levels of mite allergen (0.02 to 2••g/g of dust) was shown to be a significant risk factor for sensitization. Moreover, the level of cockroach allergen exposure was also an important determinant for sensitization to cockroach allergens. The indoor environment has therefore become an important target for control of these allergens to facilitate avoidance therapy. A number of clinical immunology laboratories perform a house-dust analysis to provide an assessment of the level of dust mite, cat, dog, cockroach, mouse, and mold allergen exposure to inhabitants. A “gross” dust specimen is collected from carpeted areas of homes, workplaces, and schools using one of several inexpensive dust collectors attached to a standard household vacuum cleaner. Once sent to the clinical laboratory, the crude dust is processed (e.g., sieved, agitated with physiologic buffer, 1:20 wt/vol) to prepare an extract of soluble proteins. The allergen content of the extracted dust is determined by analysis in noncompetitive (twosite) mAb-based IEMAs. The following indoor aeroallergens can be quantitatively measured in environmental dust with available commercial reagents: Der f 1, Der p 1, Fel d 1, Can f 1, Bla g 1/g 2, and Mus m 1. Mold/Fungus Evaluation Accurate assessment of the mold content in an indoor environment is problematic because mold spores are always present in outdoor air. Fungal spores can be viable and nonviable, and laboratory methods for evaluating each form of mold spores are different. Viable mold spores have been considered more clinically important by some allergists because they can colonize indoor environments and mass-produce spores for release into the air. A qualitative viable mold spore analysis can be performed on 5•mg of fine dust in a microbiologic culture plate containing Sabouraud's dextrose agar. The total number of mold colonies growing at 24 and 48 hours are quantified by visual inspection, and these results are reported as a colony count. The species of the predominant molds growing can then be determined by repetitive subculture and morphologic identification. Immunoassays for indicator mold allergens (e.g., Alt a 1) are not always useful clinically because molds that inhabit indoor environments do not always produce the same repertoire of proteins; growth depends on the specific environmental conditions present on any given day (e.g., nutrients, temperature, humidity). Interpretation of Indoor Aeroallergen Measurements Indoor aeroallergen levels are used to identify allergen sources in homes of individuals with asthma, workplaces, schools, and especially “environmentally tight” buildings. Levels of Der p 1 or Der f 1 allergen greater than 2000•ng/g of fine dust are associated with increased risk for allergic symptoms in sensitized individuals,

whereas levels greater than 10,000•ng/g of fine dust are associated with increased risk for sensitization. For cat, greater than 8000•ng/g of Fel d 1 in fine dust is suggested as the threshold for sensitization. Because cat allergen can be transported on clothing, the Fel d 1 content in the localized air space of an individual may be clinically important and not effectively assessed by environmental testing. Comparable risk targets have also been used for dog (Can f 1) allergen levels in indoor environments. Any detectable cockroach allergen in an indoor environment (>0.5 units/g fine dust) places a cockroach allergic individual at risk for symptoms and further sensitization. For fungal contamination, no established mold spore contamination levels are known to identify an environment as a risk to a mold-allergic person because of the multiple variables associated with heterogeneity of mold spores, differential growth of mold, aerosolization of spores, and variable specificity of IgE antibody. Variations in these parameters minimize the use of mold spore levels in predicting a clinical outcome from any tested environmental exposure. Mold spore levels above 25,000 colonies/g of fine dust place a home environment in the 75th percentile for homes monitored across the United States. When any of these proposed threshold levels are exceeded, the allergic individual is encouraged to remediate their environment. [

70] [72]

(see Chapter 34 ).

LABORATORY METHODS IN CELLULAR IMMUNOLOGY Lymphocytes serve to eliminate foreign antigens from the host and modulate cellular responses through the release of cytokines. The peripheral blood contains several subpopulations of mature lymphocytes: thymus-derived (T) cells, B cells, and natural killer (NK) cells. Each of these subpopulations can be broadly defined by the presence of one or more glycoproteins on their cell membranes. Human leukocyte subsets are enumerated and evaluated for function to identify abnormal cellular immune responsiveness. Enumeration of Lymphocyte Subpopulations Immunophenotyping is a method that is used to detect peripheral blood cells that possess specific cell surface glycoprotein antigens. In its most basic form the technique involves incubating cells with a fluorochrome-labeled antibody and detecting fluorescence-positive cells. Initially a fluorescence microscope was used to detect fluorescent cells, but the flow cytometer quickly assumed this primary role in the clinical laboratory because of its capacity to analyze thousands of cells in less than a minute and its greater sensitivity than the human eye. By the mid-1970s, mAbs had permitted the development of highly

627

specific reagents to identify cell surface antigens. This led in the early 1980s to the first important lymphocyte immunophenotyping clinical test, used to identify human immunodeficiency virus (HIV)–infected patients deficient in peripheral blood CD4+ T cells. 73

The flow cytometer has three components: an optical, a fluid, and an electronic system ( Figure 37-6 ).[ ] The basic optical system consists of an argon laser that emits a single wavelength of light at 488•nm (blue region). Cells are labeled with one of several fluorochromes, including fluorescein (FITC), phycoerythrin (PE), and peridin chlorophyll protein (PerCP), as a result of mAb-fluorochrome conjugate binding to specific cell surface epitopes. The fluorochromes are excited by the

laser and emit green (FITC), orange (PE), and red (PerCP) light that is measured through optical filters designed to capture their specific wavelength. Most clinical flow cytometers measure five parameters on each cell: two nonfluorescence measures (magnitude of forward and side scatter) and three fluorescence measures (green, orange, and red light intensity). The fluid system introduces cells in suspension into a pressurized sheath of fluid that travels through a clear cuvette. The laser light intersects a stream of cells that pass single file through the cuvette. The electronic system measures electronic signals from the detectors that provide measures of the magnitude of fluorescence intensity and the

Figure 37-6 Schematic of flow cytometer. Optical, fluid, and electronic components work together to determine whether any given cell in a stream of cells has fluorophor-labeled antibody attached to its surface. Cells are sorted based on degree of their forward scatter and magnitude of their fluorescence. (Modified from Homburger HA, Katzmann JA: In Middleton E Jr, Reed CE, Ellis EF et al, editors: Allergy: principles and practice, ed 3, St Louis, 1988, Mosby.)

TABLE 37-5 -- Antibody Specificities Used in Flow Cytometry for Immunodeficiency Assessment Antibody Designation

Detection on Normal Cells

CD2

Pan T cell, natural killer (NK) cells (80%–95% of T lymphocytes)

CD3

Mature pan T cell (95% of T lymphocytes)

CD4

Helper/inducer T cells (65% of T lymphocytes), monocytes

CD8

Cytoxic/suppressor T cells (35% of T lymphocytes)

CD14

Maturing monocytes

CD16

Granulocytes, NK cells

CD19

Pan B cell lymphocyte

CD20

Maturing B cells (surface immunoglobulin-positive B lymphocytes)

CD34

Immature hematopoietic cells, stem cells

CD41

Platelets, megakaryocytes

CD45

Leukocyte common antigen

CD56

NK cells, some stem cell disorders

HLA-DR

Myeloid blasts, B cells, activated T cells (90% of B lymphocytes and monocytes)

Common Monoclonal Antibody Panel Specificities To evaluate lymphocyte purity and recovery

CD45, CD14 (two color)

To evaluate helper/inducer T cell population

CD3, CD4 (two color) CD3, CD4, CD45 (three color) CD3, CD4, CD45, CD8 (four color)

To evaluate cytoxic/suppressor T cell population

CD3, CD8 (two color) CD3, CD8, CD45 (three color)

To evaluate B cell lymphocyte population

CD3, CD19 (two color) CD3, CD19, CD45 (three color) CD3, CD19, CD45, CD16 (four color)

To evaluate NK cell population

CD3, CD16, and/or CD56 (two color)

standard. The mean channel numbers for maximal scatter and fluorescence peaks are monitored on a quality control chart. Moreover, the analysis of a mixture of microbeads with different fluorescence intensities from zero to very bright allows linearity to be assessed. A regression line is drawn between the fluorescence channels in which each of the beads is detected versus the fluorescence intensity of the beads. If the investigator analyzes two or more fluorochromes at one time, spectral compensation is performed by the instrumentation with beads containing multiple fluorochromes (e.g., unlabeled, PE-/FITC-labeled microbeads) to ensure that the photomultiplier tube gains are adjusted to minimize overlapping detection of the different fluorochrome spectra. For the mAb-labeling step an isotypematched antibody, with no specificity for lymphocyte surface antigens and matched for protein concentration and specific activity of the fluorescence, is run as a negative control. A fluorescent mAb specific for CD34, an antigen found on the surface of all immature hematopoietic cells and stem cells, is analyzed as a positive control. A processing control is run each time a patient's test specimen is prepared to ensure that immunoreactive fluorescent mAb reagent and an effective lysing procedure are employed.

Functional Evaluation of Lymphocytes Human lymphocyte function is evaluated by in vitro stimulation with antigen or polyclonal activators. Antigen challenge of lymphocytes has been used to identify cell populations that proliferate in individuals previously exposed to a particular antigen. In contrast, polyclonal activators or plant mitogens induce proliferation of cells by binding to carbohydrate groups in glycoproteins on their membranes. The process of activating lymphocytes can induce biochemical changes, including calcium influx, activation of phospholipase C, inositol triphosphate–induced calcium release, and gene activation. These events can ultimately lead to membrane receptor redistribution, secretion of lymphokines, cell mobility, and initiation of cell division. The activation stimulus determines whether T cells, B cells, or both cell 76

types proliferate.[ ] Clinically, in vitro proliferation of lymphocytes is viewed as a laboratory correlate of skin testing for delayed-type hypersensitivity to recall antigens. Abnormal lymphocyte proliferation has been associated with congenital immunodeficiencies, but also with infectious diseases, malnutrition, cancer, surgery, shock, and autoimmune disease. In cases of severe combined immunodeficiencies, both B cell and T cell functions are impaired. One clinical assay provides an estimate of the amount of new deoxyribonucleic acid (DNA) produced after a stimulus to assess the amount of lymphocyte proliferation. Mononuclear cells are separated from the whole blood by a centrifugation on a Ficoll-hypaque density gradient. Cells are then resuspended in growth medium containing serum. Cells containing about 0.5 × 106 mononuclear cells/ml are pipetted into multiple wells in the microtiter plate. Either culture medium alone (negative control) and appropriate dilutions of plant mitogens (positive control) or recall antigens (Candida albicans, tetanus toxoid) are pipetted into triplicate wells containing cells, and the plate is incubated in a humidified

629

atmosphere of 5% CO2 at 37° C. Phytohemagglutinin-, pokeweed mitogen–, or concanavalin A (mitogen)−containing wells are incubated for up to 72 hours, whereas wells receiving recall antigens are incubated longer (e.g., 7 to 8 days). Six hours before harvesting, all cultures are pulsed with 25••l of tritiated thymidine (1 microcurie per well). After the incubation period, cultures are cooled to 4° C, harvested onto glass filter paper disks, placed into vials containing scintillation fluid, and counted on a beta scintillation counter. Mean net counts per minute are computed and the quantity of 3 H-thymidine incorporated into the cell's DNA after 77

antigen or mitogen stimulation is compared to the level observed with the control cells in medium alone.[ ] The precise definition of a “positive proliferation test” (e. g., threefold to fivefold count increase incorporated in the cell over the buffer condition) varies among different reported studies.

CONCLUSIONS Immunologic methods used to measure analytes in human blood allow more effective characterization of the immunologic status of patients suspected of allergic disease or immunodeficiency. Of the analytes measured for an allergy work-up, the presence and level of IgE antibody specific for a defined allergen specificity have the highest predictive value in supporting the clinical history of allergic disease. Total immunoglobulins and lymphocyte enumeration and functional assays are the most useful laboratory assays available for the diagnosis of an immunodeficiency disease.

An important but often-overlooked issue is that the assays for these analytes are considered complex tests. Ultimately, the physician ordering the test has the responsibility to the patient to ensure that the blood sample is sent to a clinical laboratory that performs accurate measurements with the least amount of variability 78

and that reports immunologic test results in the most quantitative manner.[ ] Selection of the clinical laboratory should be based on the technical ability and diligence of the laboratory personnel performing the assays and on the quality of the assay reagent source used. The laboratory should be certified under CLIA-88 statutes and should successfully participate in external proficiency surveys, such as CAP-conducted diagnostic allergy, immunology, and flow cytometry surveys.

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27. Ouchterlony O: In vitro method for testing the toxin-producing capacity of diphtheria bacteria, Acta Pathol Microbiol Scand 25:186, 1948. 28. Mancini G, Carbonara AO, Heremans JF: Immunochemical quantitation of antigens by single radial immunodiffusion, Immunochemistry 2:235, 1965. 29. Yalow RS, Berson SA: Immunoassay of endogenous plasma insulin in man, J Clin Invest 39:1157, 1960. 30. Ekins RP: The estimation of thyroxine in human plasma by an electrophoretic technique, Clin Chim Acta 5:453, 1960. 31. Sobotka AK, Valentine MD, Ishizaka K, Lichtenstein LM: Measurement of IgG blocking antibodies: development and application of a radioimmunoassay, J Immunol 117:84, 1976. 32. Greenwood FC, Hunter WM, Glover JS: The preparation of 131 I-labelled human growth hormone of high specific activity, Biochem J 89:114, 1963. 33. Marchalonis JJ: An enzymatic method for the trace iodination of immunoglobulins and other proteins, Biochem J 113:299,1969. 34. Catt K, Tregear GW: Solid phase radioimmunoassay in antibody coated tubes, Science 158:1570, 1967. 35. Axen R, Porath J, Ernback S: Chemical coupling of peptides and proteins to polysaccharides by cyanogen halides, Nature 214:1302, 1967. Immunochemical Methods for IgE Protein Measurement 36. Zetterstrom O, Johannsson SG: IgE concentrations measured by PRIST in serum of healthy adults and in patients with respiratory allergy, Allergy 35:51, 1976. 37. Seagroatt V, Anderson SG: The second international reference preparation for human serum immunoglobulin E and the first British standard for human serum immunoglobulin E, J Biol Stand 9:431, 1981. 38. Evans R: A U.S. reference for human immunoglobulin E, J Allergy Clin Immunol 68:79, 1981. 39. Hamilton RG, Adkinson NF Jr: Immunological tests for the diagnosis and management of human allergic disease: total and allergen-specific IgE and allergen specific IgG. In Rose HR, de Macario DC, Folds JD, et al, editors: Manual of clinical laboratory immunology, ed 5, Washington, DC, 1997, American Society for Microbiology, p 881.

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40. Diagnostic Allergy (SE-C) Survey, 60093-2750, Northfield, Ill, 2001, College of American Pathologists. Immunochemical Methods for IgE Antibodies of Defined Allergen Specificity

41. Ishazaka K, Ishizaka T: Physiochemial properties of reaginic antibody. I. Association of reaginic activity with an immunoglobulin other than gamma A or gamma G globulin, J Allergy 37:169, 1967. 42. Johansson SG: Raised levels of a new immunoglobulin class (IgND) in asthma, Lancet 2:951. 1967. 43. Wide L, Bennich H, Johansson SG: Diagnosis of allergy by an in vitro test for allergen antibodies, Lancet 2:1105, 1967. 44. Hamilton RG, Adkinson NF Jr: Measurement of total serum immunoglobulin E and allergen specific immunoglobulin E antibody. In Rose HR, de Macario EC, Fahey JL, et al, editors: Manual of clinical laboratory immunology, ed 4, Washington, DC, 1992, American Society for Microbiology, p 689. 45. Engvall E, Perlmann P: Enzyme-linked immunosorbent assay (ELISA): quantitation of specific antibodies by enzyme-linked anti-immunoglobulin in antigen coated tubes, J Immunol 109:129, 1972. 46. Hamilton RG, Adkinson NF Jr: Natural rubber latex diagnostic skin testing reagents: comparative performance of non-ammoniated latex, ammoniated latex and latex rubber glove extracts, J Allergy Clin Immunol 98:872, 1996. Immunochemical Methods for Allergen-Specific IgG Antibody Measurement 47. Lichtenstein LM, Norman PS, Winkenwerder WL: A single year of immunotherapy of ragweed hay fever: immunologic and clinical studies, Ann Intern Med 75:663, 1971. 48. Golden DBK, Lawrence ID, Hamilton RG, et al: Clinical correlation of the venom specific IgG antibody level during maintenance venom immunotherapy, J Allergy Clin Immunol 90:386, 1989. 49. Kagey-Sobotka A, Valentine MD, Ishizaka K, Lichtenstein LM: Measurement of IgG blocking antibodies: development and application of a radioimmunoassay, J Immunol 117:84, 1976. 50. Hamilton RG, Adkinson NF Jr: Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human serum with I-125 protein A from Staphylococcus aureus, J Immunol 122:1073, 1979. 51. Hamilton RG, Adkinson NF Jr: Quantitation of antigen-specific IgG in human serum. II. Comparison of radioimmunoprecipitation and solid phase radioimmunoassay techniques for the measurement of IgG specific for a complex allergen mixture (yellow jacket venom), J Allergy Clin Immunol 67:14, 1981. IgG Subclass Protein and Antibodies 52. Hamilton RG: Human immunoglobulins. In Leffell MS, Donnenberg, AD, Rose NR, editors: Handbook of human immunology, Boca Raton, Fla, 1997, CRC Press, p 65. 53. Morell A, Skvaril F, Hitzig WH, Barandum S: IgG subclasses: development of the serum concentrations in normal adults and children, J Pediatr 80:960, 1972. 54. Oxelius V: IgG subclass levels in infancy and childhood, Acta Pediatr Scand 68:23, 1979. 55. Shaikib F, editor: Basic and clinical aspects of IgG subclasses, Monogr Allergy 19, 1986.

56. Hanson LA, Soderstrom T, Oxelius VA, editors: Immunoglobulin subclass deficiencies, Monogr Allergy 20, 1985. 57. Aalberse RC, Schuurman J: IgG4: breaking the rules, Immunology 105:9, 2002. 58. Homburger HA, Mauer K, Sachs MI, et al: Serum IgG4 concentration and allergen specific IgG4 antibodies compared in adults and children with asthma and non-allergic subjects, J Allergy Clin Immunol 77:427, 1986. Serum Tryptase 59. Schwartz LB, Bradford TR: Regulation of tryptase from human lung mast cells by heparin: stabilization of the active tetramer, J Biol Chem 261:7372, 1986. 60. Enander I, Matsson P, Andersson AS, et al: A radioimmunoassay for human serum tryptase released during mast cell activation, J Allergy Clin Immunol 85:154, 1990. 61. Van der Linden PW, Hack CE, Poortman J, et al: Insect sting challenge in 138 patients: relation between clinical severity of anaphylaxis and mast cell activation, J Allergy Clin Immunol 90:110, 1992. 62. Schwartz LB, Yunginger JW, Miller J, et al: Time course of the appearance and disappearance of human mast cell tryptase in the circulation after anaphylaxis, J Clin Invest 83:1551, 1989. Other Analytes in Allergic Disorders and Asthma 63. Haley NJ, Sepkovic DW, Hoffmann D: Elimination of cotenine from body fluids: disposition of smokers and non-smokers, Am J Public Health 79:1046, 1989. 64. Jarvis MJ, Tunstall-Pedoe H, Feyerabend C, et al: Comparisons of tests used to distinguish smokers from non-smokers, Am J Public Health 77:1435, 1987. 65. Gleich GJ, Loegering DA: Immunobiology of eosinophils, Annu Rev Immunol 2:429, 1984. 66. Hallgren R, Bigelle A, Venge P: Eosinophil cationic protein in inflammatory effusions as evidence of eosinophil involvement, Ann Rheum Dis 43:556, 1984. 67. Pepys J: Hypersensitivity diseases of the lungs due to fungi and organic dusts, Monogr Allergy 4:69, 1972. 68. Fink JN, Zacharisen MC: Hypersensitivity pneumonitis. In Middleton E Jr, Reed CE, Ellis EF, et al, editors: Allergy: principles and practice, ed 5, St Louis, 1998, Mosby, p 994. Assessment of Environmental Allergens 69. Edmonds RL: Collection efficiency of rotorod samplers for sampling fungus spores in the atmosphere, Plant Dis Reporter 56:704, 1972. 70. Hamilton RG, Chapman MD, Platts-Mills TAE, Adkinson NF Jr: House dust aeroallergen measurements in clinical practice: a guide to allergen-free home and work environments, Immunol Allergy Pract 14:9, 1992.

71. Huss K, Adkinson NF, Eggleston P, et al: House dust mite and cockroach exposure are strong risk factors for positive allergy skin tests in the Childhood Asthma Management Program, J Allergy Clin Immunol 107:48, 2001. 72. Wood RA, Eggleston PA, Lind P: Antigenic analysis of household dust samples, Am Rev Respir Dis 137:358, 1988. Laboratory Methods in Cellular Immunology 73. Homburger HA, Katzmann JA: Methods in laboratory immunology: principles and interpretation of laboratory tests for allergy. In Middleton E Jr, Reed CE, Ellis EF, et al, editors: Allergy: principles and practice, ed 3, St Louis, 1988, Mosby. 74. National Committee for Clinical Laboratory Standards: Clinical applications of flow cytometry: quality assurance and immunophenotyping of peripheral blood lymphocytes, Wayne, Pa, 1998, NCCLS, H42-A. 75. Nicholson JKA, Green TA: Selection of anticoagulants for lymphocyte immunophenotyping, J Immunol Methods 165:31, 1993. 76. Fletcher MA, Klimas N, Morgan R, Gjerset G: Lymphocyte proliferation. In Rose HR, de Macario EC, Fahey JL, et al, editors: Manual of clinical laboratory immunology, ed 4, Washington, DC, 1992, American Society for Microbiology, p 213. 77. Waldmann TA, Broder S: Polyclonal B-cell activators in the study of the regulation of immunoglobulin synthesis in the human system, Adv Immunol 32:1, 1982. Conclusions 78. Hamilton RG: Responsibility for quality IgE antibody results rests ultimately with the referring physician, Ann Allergy Asthma Immunol 86:353, 2001 (invited editorial).

631

Chapter 38 - In Vivo Methods for Study of Allergy Skin Tests, Techniques, and Interpretation

Pascal Demoly Vincent Piette Jean Bousquet

Since the recognition that immunoglobulin E (IgE)–mediated allergic diseases are caused by exposure to allergens, it has been a common practice to establish a diagnosis by reexposure of the individual to the allergen. Skin tests have represented the primary diagnostic tool in allergy since their introduction in 1865 by 1

2

3

Blackley.[ ] The intracutaneous test proposed by Mantoux[ ] in 1908 was rapidly applied to immediate allergy. Lewis and Grant[ ] described the prick test in 1924. These methods have been used for decades without major modifications, but in recent years they have been refined, and a number of standardized devices have been proposed for prick and puncture testing. Skin tests can provide useful confirmatory evidence for a diagnosis of specific allergy that has been made on clinical grounds. Their characteristics—simplicity, rapidity of performance, low cost, and high sensitivity—define their key position in allergy diagnosis. When improperly performed, however, cutaneous testing can lead to false-positive or false-negative results. The main limitation of the skin test is that a positive reaction does not necessarily mean that the symptoms are caused by an IgE-mediated allergic reaction, because symptom-free subjects may have allergen-specific IgE. Skin tests have many uses other than the strict diagnosis of IgE-mediated allergy, including epidemiologic studies. Changes in skin sensitivity are used for the standardization of allergen extracts and for pharmacologic studies. Skin tests and methods derived from them can also be used to understand better the pathophysiology of the allergic reaction and assist in evaluating the mechanisms of action of antiallergy treatments.

PATHOPHYSIOLOGY OF SKIN RESPONSE The IgE-mediated allergic reaction of the skin results in a wheal-and-flare reaction dependent on both proinflammatory and neurogenic mediators (immediate reaction). It is inconstantly followed by a late-phase reaction (LPR) starting 1 to 2 hours later, peaking at 6 to 12 hours, and resolving in approximately 24 to 48 hours. The LPR is represented by an erythematous inflammatory reaction. Immediate Reaction 4

The immediate reaction is essentially induced by mast cell degranulation after allergen challenge.[ ] Release of histamine and tryptase begins about 5 minutes after 5

allergen injection and peaks at 30 minutes.[ ] The injection of histamine into the skin by prick or intradermal technique mimics the allergen-induced wheal-and-flare reaction. Histamine is the major mediator of the wheal-and-flare reaction but not the exclusive mediator; the size of the wheal usually is not correlated to the 6]

concentration of histamine release, and some subjects show no significant histamine release during that phase, as assessed by the microdialysis technique.[ 6

Neurogenic and cellular inflammatory components interact in the generation of the immediate reaction. Substance P[ ] and, to a lesser extent, neurokinin A or 5

calcitonin gene-related peptide (CGRP) produce dose-related wheal-and-flare reactions in humans.[ ] In addition, CGRP induces a slow-onset, intense vasodilatation 7

in human skin that persists for several hours and is associated with leukocyte infiltration.[ ] More than any other mediator, histamine can trigger by axon reflex the release of substance P, and this neuromediator enhances the immediate reaction by causing the release of histamine by skin mast cells (positive feedback loop). 8

9

Nitric oxide[ ] and corticotropin-releasing hormone[ ] may also be involved. Late-Phase Reaction

The LPR after allergen challenge is caused by IgE antibodies, [ accumulation in developing LPR

[5]

10]

11]

and type III mechanisms (immune complex) make no significant contribution.[

is accompanied by altered expression of adhesion molecules on local vascular endothelium.

[12] [13]

Granulocyte

The cell inflammatory

[14]

infiltrate has been extensively characterized in the skin LPR ; however, the same cellular pattern may be found after an immediate wheal-and-flare reaction that does not lead to a macroscopic LPR. The exact mechanisms of the erythematous inflammatory LPR are not completely characterized. Mast cells appear to be the trigger cell, releasing chemotactic mediators and cytokines and attracting inflammatory cells to the site of the allergic reaction. The release of mast cell vasoactive mediators increases vascular permeability, allowing increased progression of inflammatory cells and exposure of those cells to chemoattractant factors. Histamine accounts for only a limited portion of the LPR.[

15]

16

Lymphocytes, predominantly CD4+ T cells,[ ] play a key role in the generation and regulation of the LPR by the generation and release of cytokines. [ infiltration is a more diffuse process in delayed-type hypersensitivity than in

17]

T cell

632

the LPR, with the appearance of CD8+ T cells.[

18]

Eosinophils are activated during skin LPR[

19]

inflammation because they release RANTES (regulated on activation, normal T expressed and

and produce cytotoxic proteins.[

21 secreted)[ ]

20]

These cells can perpetuate

and interleukin-4 (IL-4).[

22]

Several mediators have been recovered from skin blister fluids after allergen challenge during the LPR, primarily histamine, kallikrein, thromboxane B2 , prostaglandin D2 (PGD2 ), leukotriene C4 (LTC4 ), and small amounts of platelet-activating factor (PAF)–acether. [

23]

The individual injection of these mediators

into the skin indicates that most of them are likely to contribute to LPR IgE-dependent skin reactions. Mediators capable of activating the coagulation, fibrinolytic, and bradykinin pathways may also be involved.[

24]

The use of some inhibitors has also given insights in the pathophysiology of skin tests. Prednisone inhibits the appearance of inflammatory mediators and the influx of eosinophils and basophils associated with the cutaneous LPR to allergen.[ 26]

inhibitors, [

On the other hand, variable results have been obtained using cyclooxygenase

although misoprolol, a prostaglandin E2 (PGE2 ) analog, reduces the cutaneous LPR to allergen.[

TECHNIQUES FOR SKIN TESTS Precautions

25]

27]

Before initiating any skin test, some precautions should be taken ( Box 38-1 ). Two major methods are routinely used. In the first method the antigen is placed onto the skin and introduced in the epidermis (prick test), and in the other method the antigen is injected into the dermis (intradermal reaction) ( Figure 38-1 ). Scratch testing should no longer be performed. Prick-Puncture Tests 3

28

Prick testing was first described by Lewis and Grant[ ] in 1924 and became widespread in the 1970s after its modification by Pepys.[ ] The modified prick test is performed by placing a small drop of each test extract and control solution on the volar surface of the forearm (or occasionally on the back). The drops are placed 2• cm or more apart to avoid false-positive reactions.[

Figure 38-1 Common methods of skin testing.

29]

A disposable hypodermic needle (25 or

Box 38-1. Skin Testing Precautions 1. Never perform skin tests unless a physician is immediately available to treat systemic reactions. 2. Have emergency equipment readily available;1:1000 epinephrine should be drawn up in syringe. 3. Be careful treating patients with current allergic symptoms. 4. Determine the value of allergenic extracts used, and assess their stability. 5. Be certain that the test concentrations are appropriate. 6. Include a positive and a negative control solution. 7. Perform tests in normal skin. 8. Evaluate the patient for dermographism. 9. Determine and record medications taken by the patient and time of last dose. 10. Record the reactions at the proper time: 10 minutes for histamine control, 15 minutes for allergens.

26 gauge) is passed through the drop and inserted into the epidermal surface at a low angle with the bevel facing up. The needle tip is then gently lifted upward to elevate a small portion of the epidermis without inducing bleeding. The needle is then withdrawn and the solution gently wiped away with a paper tissue approximately 1 minute later. A separate needle must be used for each test to avoid mixing of solutions. Using the same needle or lancet wiped with dry cotton wool [30]

31

or a paper tissue moistened with 75% ethanol[ ] between tests for several prick tests to save time and money provokes an unacceptable number of false-positive results. Smearing of test solutions to adjacent test sites must be avoided. Box 38-2 lists common errors in prick testing. Other prick-puncture test methods in which the test instrument is inserted perpendicular to the skin have been introduced by different investigators to decrease the variability of the prick.[

32]

The most popular instruments are the Morrow Brown standardized needle,[

633

33]

the Allergy Pricker,[

34]

Box 38-2. Common Errors in Prick-Puncture Testing 1. Placing tests too close together (0.05•ml) High concentration leading to false-positive results “Splash” reaction caused by air injection Subcutaneous injection leading to false-negative test (no bleb formed) Intracutaneous bleeding site read as an adequate test Too many tests performed at same time, inducing systemic reactions

Modified from Mansmann HC Jr, Bierman CW, Pearlmman D, editors: Allergic diseases in infancy, childhood, and adolescence, Philadelphia, 1980, WB Saunders, p 289.

49 52 53

50 54

under 0.5% of the tested patients.[ ] [ ] [ ] Some fatalities have been reported during skin testing.[ ] [ ] Thus, although tests may be done by a nurse or a technician, a physician should always be available. A waiting period of 20 minutes in the office of the physician before the patient is released has been 55

recommended, and this time should be extended for high-risk patients.[ ] Particular care should be taken in patients being treated with β-blocking agents, angiotensin-converting enzyme (ACE) inhibitors, or monoamine oxidase inhibitors (MAOIs), which may increase the risk of systemic reactions and make them more difficult to treat. Performing prick-puncture tests before intradermal tests and using serial tenfold dilutions of the usual test concentration, especially in patients with history of anaphylaxis, are useful ways to avoid untoward adverse local and systemic reactions in the routine skin testing. In the patient with generalized anaphylactic reaction, a rubber tourniquet should be placed above the test site on the arm and 1:1000 aqueous epinephrine administered intramuscularly in the opposite arm. Because of the 56] [57]

risk of infectious bacterial or viral diseases, allergy testing of multiple patients should not be performed with a common intradermal skin test syringe.[

As a general rule, the starting dose of intracutaneous extract solutions in patients with a preceding negative prick test should range between 100-fold and 1000-fold dilutions of the concentrated extract used for prick-puncture test. [

58]

With potent standardized allergen extracts of 100,000 allergy units per milliliter (AU/ml), the

range of starting intradermal skin tests in patients with a negative prick-puncture test is 10 to 100 AU. For less potent allergens of 10,000-AU/ml concentration, range of starting intradermal skin tests in patients with a negative prick-puncture test is 100 to 1000 AU. Prick-Puncture versus Intradermal Tests The value of prick-puncture tests is limited by low-potency extracts inducing false-negative results. The concentration of allergen extract required to elicit a positive reaction with intradermal testing is 1000 to 30,000 times smaller than that

634

TABLE 38-1 -- Comparison of Prick-Puncture and Intradermal Testing Factor

Prick-Puncture

Simplicity

+++

++

Speed

++++

++

Interpretation of +/− reactions

++++

++

Pain/discomfort

Minimal

False-positive results

Rare

False-negative results

Possible

Rare

Reproducibility

+++

++++

Sensitivity

+++

++++

Specificity

++++

+++

Detection of IgE antibodies

Yes

Yes

Safety

++++

++

Testing of infants

Yes

++, Least; +++, intermediate; ++++, most.

Intradermal

Moderate to severe Possible

Difficult

necessary for a positive prick-puncture test. With standardized and potent extracts, however, the prick-puncture test appears to have several advantages over the intradermal test ( Table 38-1 ). It is generally accepted that prick-puncture tests are less sensitive and less reproducible but more specific than intradermal tests. Prickpuncture tests rarely induce irritant reactions, unlike intradermal skin tests. Prick-puncture test results correlate better with symptoms,[ low sensitivity, intradermal skin tests may produce the only positive results.

59]

although in patients with

Skin prick-puncture tests are recommended as the primary test for the diagnosis of IgE-mediated allergic diseases and for research purposes by the European 60]

Academy of Allergology and Clinical Immunology[

and the U.S. Joint Council of Allergy Asthma and Immunology.[

58]

Negative and Positive Control Solutions Because of interpatient variability in cutaneous reactivity, it is necessary to include negative and positive controls in every set of skin tests. The negative control solutions are the diluents used to preserve the allergen extracts. The rare dermographic patient will have wheal-and-erythema reactions to the negative control. The negative control also detects traumatic reactivity induced by the skin test device (wheal size may approach a diameter of 3•mm with some devices) and the technique 58]

of the tester. Any reaction at the negative control test sites makes interpretation of the allergen sites much more difficult. [

Positive control solutions are used to (1) detect suppression by medications or disease, (2) detect the exceptional patients who are poorly reactive to histamine, and (3) determine variations in technician performance. In the United States the usual positive control for prick-puncture testing is histamine phosphate, used at a concentration of 5.43•mmol/L (or 2.7•mg/ml, equivalent to 1•mg/ml of histamine base). Wheal diameters with this preparation range from 2 to 7•mm. However, a tenfold greater concentration may be more appropriate, with a mean wheal size ranging between 5 and 8•mm. For the intradermal test the concentration routinely used is 0.0543•mmol/L. The mean wheal size elicited ranges from 10 to 12•mm. Mast cell secretagogues such as codeine phosphate (9% solution) may also be used. Grading of Skin Tests Measurement

Skin tests should be read at their peak. Whatever the method, the immediate skin test induces a response that reaches a peak in 8 to 10 minutes for histamine, 10 to 15 58] [61]

minutes for mast cell secretagogues, and 15 to 20 minutes for allergens. LPRs are not recorded often because their exact significance is unknown. [

Skin tests should be read in a standard manner. When the reactions are mature, the size of each reaction is measured with a millimeter rule. The largest and smallest diameter of the wheal and erythema are measured. Because the reactions are often oval or irregular in shape, the diameters measured are necessarily at right angles to each other. Both diameters are recorded, summed, and divided by 2. To obtain a permanent record, the size of the reaction may be outlined with a pen, blotted onto a cellophane tape, and stored on paper. The area of the cutaneous response may be estimated by planimetry,[ 63 software.[ ]

62]

by weighing the cut-out tape with a precision balance, 64]

or may be scanned by a computer and the surface areas measured using commercial Ultrasonic measurements permit assessment of other parameters,[ such as the wheal thickness and wheal volume, which might improve the distinction between different tests. The quantification also may be done with laser Doppler

flowmetry, which determines the blood flow in the wheal and erythema.[

65]

66]

The erythematous reaction of the LPR may be evaluated by thermography.[

Criteria of Positivity

The wheal or the erythema are both used to assess the positivity of skin tests. Using the prick-puncture test, when control sites are completely negative, small wheals 28

of 1 to 2•mm with flare and itching may represent a positive immunologic response and the presence of specific IgE antibodies.[ ] Although significant in immunologic terms, small positive reactions do not necessarily indicate the presence of a clinically relevant allergy. Using prick-puncture test, reactions generally regarded as indicative of clinical allergy are usually greater than 3•mm in wheal diameter (corresponding to a wheal area of 7•mm2 ) and more than 10•mm in flare diameter.[

67]

Another criterion is a ratio of the size of the test induced by the allergen to the size of that elicited by the positive control solution.

Grading Systems

Several grading systems have been proposed. For prick-puncture tests, the sizes of the wheal and erythema often are not greatly considered, although a grading 59]

system based on the relation between the reactions induced by allergen and the histamine reference (5.43•mmol/L) has been used in Scandinavia for many years.[ Because of the log/log

635

relationship between changes in skin prick-puncture test response and allergen concentrations, only large differences in skin sensitivity will be detected using a fixed 68

concentration of allergen. More information is obtained if a threshold dilution titration skin test is performed employing serial dilutions.[ ] End-point titrations, estimated as the concentration of allergen resulting in a wheal size comparable to that of a positive control solution, have been performed but should be replaced by methods based on parallel-line bioassay or on median slope.[

59]

For intradermal tests, one of the most widely used grading system derives from Nacleiro.[

69]

Although a single concentration of a specific extract may be selected for 70

testing and the reaction graded for positivity, more information is obtained if a threshold dilution titration skin test is performed employing threefold[ ] or tenfold dilution series. The grading of the reaction may be studied using the wheal and/or the erythema. The size of a wheal for a single allergen dose is not accurate, because identical reactions may be observed for tests performed with allergenic extracts whose potency differs by 100-fold. Several methods of evaluation have therefore been proposed. The least dilution required for 1+ or 2+ reaction is considered the end point. Other investigators have 71

considered the end-point titer to be the dilution of extract that results in a wheal identical to the histamine-positive control. Norman et al. [ ] introduced the midpoint method by establishing a dose-response curve and determining the dose of allergen extract producing a wheal 7•mm in diameter. They found that the midpoint skin test correlated with serum IgE levels, leukocyte histamine release, provocative challenges, and symptoms.[

72]

Van Metre et al[

73]

examined the effect of effective

immunotherapy with ragweed extracts on skin sensitivity and found no effect using the end-point titration, whereas the midpoint skin tests were significantly reduced. Turkeltaub et al[

70]

used the erythema diameter, not the wheal diameter, because the slope of the regression line of the former is steeper.

For routine diagnosis of allergy, a single allergen concentration is usually sufficient, but for research purposes, more sophisticated techniques are required. Studies also indicate that the LPR is also a measure of IgE sensitivity, [

74] [75]

but its grading is not yet standardized.

Number of Tests and Frequency of Testing The number of skin tests varies according to (1) age of the patient (fewer prick-puncture tests are necessary in infants, concentrating on common food allergens, house dust mites, indoor molds, indoor insects, and animal danders rather than pollen),[ allergic disease (perennial symptoms, clear causative factors).

58]

(2) geographic location and mobility of the patient, and (3) history of the

58

Skin tests may be repeated for a variety of reasons,[ ] including (1) age of the patient (allergic children acquire new sensitivities, initially foods and indoor allergens and later pollens and outdoor molds), (2) patient exposure to new allergens (acquisition of new pet, geographic relocation) and particularly if symptomatology has changed, and (3) immunotherapy. However, with the exception of venom immunotherapy, routine periodic skin test titration is not recommended. Other Methods Passive Transfer Test

The passive transfer test is a technique of the past. Before the discovery of IgE, it was used to demonstrate the presence of reagins. Now the passive transfer 23]

technique is unacceptable because of the risks of transferring serum.[ Skin Window Techniques and Derived Methods

The skin window technique proposed by Rebuck and Crowley[

76]

in 1955 was modified by Dunsky and Zweiman,[

77]

who developed the skin chamber technique.

FACTORS AFFECTING SKIN TESTS Allergenic Extracts The skin reaction depends on a number of variables, most importantly the quality of the allergen extract. Some false-negative reactions are caused by the lack of allergens in some nonstandardized extracts. Although many years ago the skin test materials were often made directly in hospital laboratories or in physicians' offices

by extracting allergen raw materials, this practice cannot be recommended and should be abandoned. When possible, it is advised to use allergen extracts 58

standardized using biologic methods and labeled in biologic units.[ ] Allergen extracts should be marketed only if their potency, composition, and stability have been documented as extracts from a single source material, mixtures of related, cross-reacting allergens such as grass pollen, deciduous tree pollen, related ragweed pollen and related mite allergen extracts. Using mixes of unrelated allergens is not recommended; this practice may result in false-negative responses due to overdiluted allergenic epitopes in some mixes or enzyme degradation. With marketed mixtures, the relative amounts of each component of the mixture and stability data should be indicated on the label. 60 78 79 80

Recombinant allergens used for in vivo diagnosis should have the same IgE binding activity as their natural counterparts. [ ] [ ] [ ] [ ] For safety reasons, the starting dose of recombinant allergens should be low and carefully determined, and prick tests should be used first. Such an approach may be of great importance for the diagnosis of allergy to unstable allergen extracts such as fruits and to cross-reacting allergens.[

79] [80]

Some extracts, such as Hymenoptera venoms, can induce false-positive reactions by nonimmunologic mechanisms. Preservatives used in allergenic extracts also may be irritative; for example, it was shown that sodium merthiolate can elicit a wheal-and-flare reaction in nonsensitized subjects.[

81]

Area of Body The site of skin testing may affect the results. The midback and upper back are more reactive than the lower back. The back as a whole is more reactive than the forearm.[

29]

The antecubital fossa is the most reactive portion of the arm, whereas the wrist is the least reactive. The ulnar side of the arm is more reactive than the

radial side. It is recommended that tests should not be placed in areas 5•cm from the wrist or 3•cm from the antecubital fossae.[

58]

636

Age Skin reactions vary with age. Infants react predominantly with a large erythematous flare and a small wheal. Using prick-puncture testing, a significant wheal is 82

detectable after 3 months of age in most infants tested with histamine, codeine phosphate, or allergen extracts.[ ] Performing skin tests to diagnose allergic disorders in infancy is therefore possible, but the size of the wheal is often reduced, and criteria of positivity should always compare the size of the wheal induced by allergen extracts with that elicited by positive control solutions. 83]

Skin test wheals increase in size from infancy to adulthood and then often decline after age 50.[ Gender

There is no gender difference in skin test reactivity. Women exhibit the weakest histamine whealing capacity during the first day of the menstrual cycle and a second weak response about the twentieth day. However, these differences have no clinical significance.[

84]

Race The whealing capacity to histamine is significantly greater in healthy nonatopic blacks with darkly pigmented skin than it is in whites with light skin pigmentation. [85]

The flare may be difficult to measure in patients with pigmented skin.

Circadian Rhythms 86] [87]

The circadian variation of skin reactivity is minimal and does not affect the clinical interpretation of skin tests.[ Seasonal Variations Seasonal variations related to specific IgE antibody synthesis have been demonstrated in pollen[ especially for tree pollen allergy,[

91]

88] [89]

and house dust mite allergy.[

90]

The skin sensitivity,

increases after the pollen season and then declines until the next season. This effect has some importance in patients with a low

sensitivity and for allergen extracts of weak potency. Ultraviolet B (UVB) radiation significantly reduces wheal intensities. [

92]

Pathologic Conditions 93

94

Atopic dermatitis is known to diminish the skin reactivity to histamine,[ ] but this finding is not consistently observed.[ ] It seems reasonable not to perform skin tests in areas where there is any skin lesion that might interfere with skin reactivity. Patients with chronic renal failure and those receiving chronic hemodialysis usually have decreased skin reactivity, and the texture of their skin makes testing difficult.[ 97

95] [96]

Some patients with cancer have a decreased skin reactivity more

pronounced on the flare than on the wheal.[ ] Patients with spinal cord injuries or peripheral nerve abnormalities such as a diabetic neuropathy[ in reactivity to skin tests. In persons who have experienced a recent anaphylactic reaction, skin tests should be deferred for 1 week.

98]

show a decrease

Drugs Some drugs can interfere with the performance of skin tests and can modulate either the wheal or the flare, making the interpretation of skin tests difficult. Conversely, other drugs used in allergic and asthmatic patients do not modify the cutaneous responsiveness and can be continued ( Table 38-2 ). Antihistamines

23]

The H1 -antihistamines inhibit the wheal-and-flare reaction to histamine, allergen, and mast cell secretagogues.[

The duration of the inhibitory effect appears to be

linked to the pharmacokinetics of the drug and its metabolites. First-generation H1 -antihistamines reduce skin reactivity for up to 24 hours.[ generation agents, such as cetirizine, ebastine,[

103]

loratadine, mizolastine, and terfenadine,[

Astemizole (no longer available in most countries) has an effect for up to 60

105 106 days.[ ] [ ]

104]

these drugs during seasonal allergic

101] [110]

Second-

are potent drugs in blocking the skin reaction for 3 to 10 days.

Tachyphylaxis, as defined by a reduction of the inhibitory effects on skin

tests of these drugs prescribed for long-term therapy, has been reported with chlorpheniramine [ Although some antihistamines inhibit skin tests more than others,[

99] [100] [101] [102]

99]

but not with second-generation H1 -antihistamines.[

107] [108] [109]

skin test reactivity to allergen or histamine is not predictive of the clinical efficacy of

111 112 rhinitis.[ ] [ ]

Topical H1 -antihistamines such as levocabastine may reduce skin tests because plasma concentrations of the drug can be measured within 1 to 2 hours of administration of single doses of nasal spray and eye drops.[

113]

H2 -antihistamines used alone have a limited inhibitory activity on skin tests.[

114] [115]

conflicting results, with some reporting an enhancement of the inhibitory effect.[

Studies on the co-administration of H1 - and H2 -antihistamines have produced

115] [116]

Discontinuation of H2 -antihistamines on the day of testing is probably

sufficient to prevent significant suppression of skin tests. Ketotifen

Ketotifen suppresses skin test responses for more than 5 days. [

99] [117]

Imipramines, Phenothiazines, and Tranquilizers

Tricyclic antidepressants exert a potent and sustained decrease of skin reactions to histamine.[ agents of the phenothiazine class have H1 -antihistaminic activity and can block skin days of therapy and up to 11 days after the last dose of doxepin.[

118]

119 tests.[ ]

This effect may last for a few weeks. Tranquilizers and antiemetic

Topical doxepin has been shown to abolish skin reactivity after 1 to 3

120]

Corticosteroids

Short-term (less than 1 week) oral administration of corticosteroids at the therapeutic doses used in asthmatic patients does not modify the cutaneous reactivity to histamine, compound 48/80, or allergen.[ 122 responses[ ]

121]

Long-term corticosteroid therapy does not alter histamine-induced vascular reactivity in skin but affects skin mast cell

and modifies the skin texture, therefore making the interpretation of immediate skin tests difficult in some cases. However, allergen-induced skin tests

can be accurately performed in asthmatic patients receiving long-term oral corticosteroid treatment. [

123]

637

TABLE 38-2 -- Inhibitory Effect of Various Treatments on IgE-mediated Skin Tests Suppression Drug

Degree

Duration (days)

Significant

H1 Antihistamines 102]

++++

30–60

Yes

++++

3–10

Yes

++++

3–10

Yes

++

1–3

Yes

Clemastine[

+++

1–10

Yes

Cyproheptadine

0 to +

1–8

Yes

0 to +

1–3

Yes

Astemizole[ [103]

102]

Azelastine[ Cetirizine[

102]

[108]

Chlorpheniramine [99] [105] 99]

[102] [105]

Diphenhydramine [105]

Doxepin[

121]

++

3–11

Yes

Ebastine[

103]

++++

3–10

Yes

+++

1–10

Yes

Hydroxyzine

†

*

Levocabastine

Possible

Yes

[113] 102]

++++

3–10

Yes

++++

3–10

Yes

++++

3–10

Yes

++

1–3

Yes

Terfenadine[

102]

++++

3–10

Yes

Tripelennamine

0 to +

1–3

Yes

Loratadine[

Mequitazine 109]

Mizolastine[

Promethazine[

105]

[105]

H2 Antihistamines Cimetidine[ Ranitidine[

114]

116]

0 to +

No

+

No

Corticosteroids Systemic, short

0

122 term[ ]

Systemic, long

Possible

Yes

123 124 term[ ] [ ]

Inhaled

0

Topical[

125] [126]

0 to +

Yes

β2 -Adrenergic Agonists Inhaled[

128] [129]

0 to +

No

0 to ++

No

[130] [132]

Oral, injection [128] [129] [131]

†

132]

Formoterol[

Unknown

Salmeterol

Unknown

Others Ketotifen[

99] [118]

Imipramines[

119]

Phenothiazines

++++

>5

Yes

++++

>10

Yes

++

Yes

0 to +

No

[120]

Theophylline[

127]

[128] 134]

0

135]

+

136]

++

Cromolyn[

Dopamine[ Clonidine[

Montelukast[

138]

0

Specific Immuno- 0 to ++ therapy

No

[74] [140]

[141] [143] [144]

0, None; +, low; ++, moderate; +++, high; ++++, very high. * Clinical significance for skin testing. † Regulatory approval for these H1 antihistamines has been withdrawn in most countries.

Inhaled corticosteroids have not been tested for suppressive effects on skin tests, but because therapeutic doses produce fewer systemic effects than oral steroids, it may be predicted that they should not modify skin tests. Application of topical dermal corticosteroids for 1 week reduces both the immediate reaction and LPR 124] [125]

induced by allergen.[ Theophylline

Theophylline slightly reduces skin test reactions[

126] [127]

23]

but does not need to be stopped before skin testing.[

Beta-Adrenergic Agents

Short-acting inhaled β2 -adrenergic agonists in the usual doses used for the treatment of asthma do not usually inhibit allergen-induced skin tests.[ terbutaline was shown to decrease the allergen-induced wheal,[ agonists such as formoterol[

131]

130]

128] [129]

Oral

but this inhibitory effect has little significance in clinical practice. Newer long-acting inhaled β2 -

and salmeterol may significantly decrease skin test reactivity, but definitive results are lacking. Conversely, β-blocking agents such

as propranolol can significantly increase skin reactivity.[

132]

Cromones

Inhaled cromolyn and nedocromil do not alter the whealing response to skin tests with allergens or degranulating agents.[

133]

Other Drugs

Other drugs, such as dopamine[

134]

and clonidine,[

135]

136]

have been shown to decrease skin test reactivity. Nifedipine[

reactivity. ACE inhibitors increase skin reactivity to allergen, histamine, codeine, and

137]

and montelukast[

have no effect on skin

138 bradykinin.[ ]

Specific Immunotherapy A decreased wheal-and-flare reaction has been noticed in patients undergoing specific immunotherapy with inhalant allergens[ and in professional beekeepers who are spontaneously desensitized.[

142]

139]

140] [141]

or Hymenoptera venoms[

However, these effects were seen mostly when skin tests were carried out using several

dilutions. Specific immunotherapy with pollen extracts induces a decreased LPR.[

74] [143] [144]

INTERPRETATION OF SKIN TESTS Positive Results in Population without Clinical Allergy The occurrence of positive skin tests does not necessarily imply that the patient is allergic, because many studies have shown that skin tests can be positive in patients with asymptomatic sensitization to common allergens.[

23]

In some patients the presence of irritants or nonspecific mast cell secretagogues may explain positive

responses with concentrated extracts, especially when the intradermal route is used.[

145]

Using prick-puncture tests, positive skin tests probably detect the presence of

specific IgE antibodies to environmental allergens, although their presence may not always coincide with clinically significant allergic disease. The presence of 146] [147]

positive skin tests in asymptomatic subjects may predict the onset of allergic symptoms.[ without sensitivity on the basis

To discriminate patients with symptomatic sensitivity from those

638

148

of both their skin prick tests and their specific serum IgE to five common aeroallergens, Pastorello et al[ ] have shown that the optimal cutoff values for skin test results are a wheal area of 32.4•mm2 for seasonal allergens and 31.2•mm2 for Dermatophagoides pteronyssinus. False-Positive and False-Negative Results Both false-positive and false-negative skin tests may occur because of improper technique or material. False-positive skin tests may result from dermographism or from “irritant” reactions or a nonspecific enhancement from a nearby strong reaction.[ common aeroallergens, such as house dust mites, may also need to be ruled out.

149] [150]

The possibility of a contamination of an allergen extract by other

59

False-negative skin tests can be caused by (1) extracts of poor initial potency or subsequent loss of potency, [ ] (2) drugs modulating the allergic reaction, (3) diseases attenuating the skin response, (4) decreased reactivity of the skin in infants and elderly patients, and (5) improper technique (no or weak puncture). The use of positive control solutions may overcome some of the false-negative results, because reactions will be either decreased or abolished in patients with weakly reactive skin. A positive intracutaneous test preceded by a negative prick-puncture test could denote clinical allergy in a less sensitive patient or the presence of an 58]

immunoglobulin G (IgG) reagin that cannot be detected by prick-puncture tests. [ Correlation with Other Tests in Allergy Diagnosis

Many tests have been proposed for the diagnosis of IgE-mediated allergic diseases, but they do not have the same significance as skin tests ( Figure 38-2 ). In Vitro Tests

Test comparisons using the titration of specific IgE depend on the quality and standardization of allergens used in both tests and to a lesser extent on the method of skin testing.[ allergens.

23] [59]

Using standardized extracts, the percentage agreement between IgE tests and skin prick-puncture tests ranges from 85% to 95% depending on

[151] [152] [153] [154]

In Vivo Tests

However, skin tests appear to be more sensitive but less specific than IgE tests.[

155]

60

When both tests are compared with bronchial, nasal, or oral challenges, prick-puncture tests are more specific but less sensitive than intradermal tests. [ ] With a suggestive history and strongly positive skin tests, the correlation between skin tests and bronchial or nasal challenges is highly significant. Poor correlations often are observed with unstandardized allergenic extracts or when a discrepancy exists between history and skin tests. Diagnostic Value of Skin Tests 60]

The position papers on Skin Tests from the European Academy of Allergy and Clinical Immunology[ 58 Immunology[ ]

and the U.S. Joint Council of Allergy Asthma and

agree that, when properly performed, prick-puncture tests are generally considered to be the most convenient and least

Figure 38-2 Differences between in vivo and in vitro tests used in the diagnosis of IgE-mediated allergic diseases.

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Immunol 103:591, 1999. 113. Heykants J, Van Peer A, Van deVelde V, et al: The pharmacokinetic properties of topical levocabastine: a review, Clin Pharmacokinet 29:221, 1995. 114. Harvey RP, Schocket AL: The effect of H1 and H2 blockade on cutaneous histamine response in man, J Allergy Clin Immunol 65:136, 1980. 115. Meyrick-Thomas RH, Browne PD, Kirby JD: The effect of ranitidine, alone and in combination with clemastine, on allergen-induced cutaneous wheal-and-flare reactions in human skin, J Allergy Clin Immunol 76:864, 1985. 116. Hutchcroft BJ, Moore EG, Orange RP: The effects of H1 and H2 receptor antagonism on the response of monkey skin to intradermal histamine, reverse-type anaphylaxis, and passive cutaneous anaphylaxis, J Allergy Clin Immunol 63:376, 1979. 117. Esau S, del Carpio J, Martin JG: A comparison of the effects of ketotifen and clemastine on cutaneous and airway reactivity to histamine and allergen in atopic asthmatic subjects, J Allergy Clin Immunol 74:270, 1984. 118. Sullivan TJ: Pharmacologic modulation of the whealing response to histamine in human skin: identification of doxepin as a potent in vivo inhibitor, J Allergy Clin Immunol 69:260, 1982. 119. Wolfe HI, Fontana VJ: The effect of tranquilizers on the immediate skin wheal reaction, J Allergy 35:271, 1964. 120. Karaz SS, Moeckli JK, Davis W, Craig TJ: Effect of topical doxepin cream on skin testing, J Allergy Clin Immunol 96:997, 1995. 121. Slott R, Zweiman B: A controlled study of the effect of corticosteroids on immediate skin test reactivity, J Allergy Clin Immunol 54:229, 1974. 122. Olson R, Karpink MH, Shelanski S, et al: Skin reactivity to codeine and histamine during prolonged corticosteroid therapy, J Allergy Clin Immunol 86:153, 1990. 123. Des Roches A, Paradis L, Bougeard Y, et al: Long-term oral corticotherapy does not alter the results of immediate type allergy skin prick tests, J Allergy Clin Immunol 98:522, 1996. 124. Andersson M, Pipkorn U: Inhibition of the dermal immediate allergic reaction through prolonged treatment with topical glucocorticosteroids, J Allergy Clin Immunol 79:345, 1987. 125. Pipkorn U, Hammarlund A, Enerback L: Prolonged treatment with topical glucocorticoids results in an inhibition of the allergen-induced wheal-and-flare response and a reduction in skin mast cell numbers and histamine content, Clin Exp Allergy 19:19, 1989. 126. Fine SR, Fogarty M, Goel Z, Grieco MH: Correlation of serum theophylline levels with inhibition of allergen and histamine-induced skin tests, Int Arch Allergy Appl Immunol 61:241, 1980. 127. Chipps BE, Sobotka AK, Sanders JP, et al: Effect of theophylline and terbutaline on immediate skin tests, J Allergy Clin Immunol 65:61, 1980. 128. Imbeau SA, Harruff R, Hirscher M, Reed CE: Terbutaline's effects on the allergy skin test, J Allergy Clin Immunol 62:193, 1978.

129. Spector SL: Effect of beta-adrenergic agents on skin test responses and bronchial challenge responses, Chest 73:976, 1978. 130. Gronneberg R, Hagermark O, Strandberg K: Effect in man of oral terbutaline on cutaneous reactions induced by allergen and cold stimulation, Allergy 35:143, 1980. 131. Gronneberg R, Zetterstrom O: Inhibitory effects of formoterol and terbutaline on the development of late phase skin reactions, Clin Exp Allergy 22:257, 1992. 132. Lamkin N Jr, Lieberman P, Shereff R, et al: Effects of beta-adrenergic stimulation and blockade on cutaneous reactivity to histamine, J Allergy Clin Immunol 57:449, 1976. 133. Ting S, Zweiman B, Lavker RM: Cromolyn does not modulate human allergic skin reactions in vivo, J Allergy Clin Immunol 71:12, 1983. 134. Casale TB, Shelhamer JH, Parrillo JE, Kaliner MA: Dopamine inhibition of histamine-mediated cutaneous responses, J Allergy Clin Immunol 73:837, 1984. 135. Miadonna A, Tedeschi A, Leggieri E, et al: Clonidine inhibits IgE-mediated and IgE-independent in vitro histamine release from human basophil leukocytes, Int J Immunopharmacol 11:473, 1989. 136. Fernandez-Rivas M, Puyana J, Quirce S, et al: Effect of nifedipine on skin prick tests, Allergol Immunopathol Madr 18:79, 1990. 137. Anderson MW, deShazo RD: Studies of the mechanism of angiotensin-converting enzyme (ACE) inhibitor-associated angioedema: the effect of an ACE inhibitor on cutaneous responses to bradykinin, codeine, and histamine, J Allergy Clin Immunol 85:856, 1990. 138. Simons FER, Johnston L, Gu X, Simons KJ: Suppression of the early and late cutaneous allergic responses using fexofenadine and montelukast, Ann Allergy Asthma Immunol 86:44, 2001. 139. Bousquet J, Michel F: Specific immunotherapy in allergic rhinitis and asthma. In Busse W, Holgate S, editors: Asthma and rhinitis, Oxford, UK, 1995, Blackwell, p 1309. 140. Graft DF, Schuberth KC, Kagey-Sobotka A, et al: The development of negative skin tests in children treated with venom immunotherapy, J Allergy Clin Immunol 73:61, 1984. 141. Bousquet J, Knani J, Velasquez G, et al: Evolution of sensitivity to Hymenoptera venom in 200 allergic patients followed for up to 3 years, J Allergy Clin Immunol 84:944, 1989. 142. Bousquet J, Menardo JL, Aznar R, et al: Clinical and immunologic survey in beekeepers in relation to their sensitization, J Allergy Clin Immunol 73:332, 1984. 143. Parker W Jr, Whisman BA, Apaliski SJ, Reid MJ: The relationships between late cutaneous responses and specific antibody responses with outcome of immunotherapy for seasonal allergic rhinitis, J Allergy Clin Immunol 84:667, 1989. 144. Varney VA, Hamid QA, Gaga M, et al: Influence of grass pollen immunotherapy on cellular infiltration and cytokine mRNA expression during allergeninduced late-phase cutaneous responses, J Clin Invest 92:644, 1993.

Interpretation of Skin Tests 145. Roane J, Crawford L, Triplett F, Brasher G: Intradermal tests in nonatopic children, Ann Allergy 1968;26:443. 146. Hagy G, Settipane G: Prognosis of positive allergy skin tests in an asymptomatic population: a three-year follow-up of college students, J Allergy 48:200, 1971. 147. Horak F: Manifestation of allergic rhinitis in latent-sensitized patients: a prospective study, Arch Otorhinolaryngol 242:239, 1985. 148. Pastorello EA, Incorvaia C, Ortolani C, et al: Studies on the relationship between the level of specific IgE antibodies and the clinical expression of allergy. I. Definition of levels distinguishing patients with symptomatic from patients with asymptomatic allergy to common aeroallergens, J Allergy Clin Immunol 96:580, 1995. 149. Voorhorst R: Perfection of skin testing technique: a review, Allergy 35:247, 1980. 150. Terho EO, Husman K, Vohlonen I, Heinonen OP: Atopy, smoking, and chronic bronchitis, J Epidemiol Community Health 41:300, 1987. 151. Bousquet J, Chanez P, Chanal I, Michel FB: Comparison between RAST and Pharmacia CAP system: a new automated specific IgE assay, J Allergy Clin Immunol 85:1039, 1990. 152. Ewan PW, Coote D: Evaluation of a capsulated hydrophilic carrier polymer (the ImmunoCAP) for measurement of specific IgE antibodies, Allergy 45:22, 1990. 153. Crobach MJJS, Hermans JO, Kaptein AA, et al: The diagnosis of allergic rhinitis: how to combine the medical history with the results of radioallergosorbent tests and skin prick tests, Scand J Prim Health Care 16:30, 1998. 154. Wood RA, Phipatanakul W, Hamilton RG, Eggleston PA: A comparison of skin prick tests, intradermal skin tests, and RASTs in the diagnosis of cat allergy, J Allergy Clin Immunol 103:773, 1999. 155. Van der Zee JS, de Groot H, van Swieten P, et al: Discrepancies between the skin test and IgE antibody assays: study of histamine release, complement activation in vitro, and occurrence of allergen-specific IgG, J Allergy Clin Immunol 82:270, 1988. 156. Onorato J, Merland N, Terral C, et al: Placebo-controlled double-blind food challenge in asthma, J Allergy Clin Immunol 78:1139, 1986. 157. Workshop on experimental methodology for clinical studies of adverse reactions to foods and food additives, J Allergy Clin Immunol 86:421, 1990. 158. Anhoej C, Backer V, Nolte H: Diagnostic evaluation of grass- and birch-allergic patients with oral allergy syndrome, Allergy 56:548, 2001. 159. Rance F, Juchet A, Bremont F, Dutau G: Correlations between skin prick tests using commercial extracts and fresh foods, specific IgE, and food challenges, Allergy 52:1031, 1997. 160. Bock SA: The natural history of adverse reactions to foods, N Engl Reg Allergy Proc 7:504, 1986. 161. Sampson HA: Comparative study of commercial food antigen extracts for the diagnosis of food hypersensitivity, J Allergy Clin Immunol 82:718, 1988.

162. Burks A, Cockrell G, Stanley J, et al: Recombinant peanut allergen Ara h 1 expression and IgE binding in patients with peanut hypersensitivity, J Clin Invest 96:1715, 1995. 163. Breiteneder H, Hoffmann-Sommergruber K, O'Riordain G, et al: Molecular characterization of Api g 1, the major allergen of celery (Apium graveolens), and its immunological and structural relationships to a group of 17-kDa tree pollen allergens, Eur J Biochem 233:484, 1995. 164. Bousquet J, Muller UR, Dreborg S, et al: Position paper, Working Group on Immunotherapy, European Academy of Allergy and Clinical Immunology: Immunotherapy with Hymenoptera venoms, Allergy 42:401, 1987. 165. Georgitis JW, Reisman RE: Venom skin tests in insect-allergic and insect-nonallergic populations, J Allergy Clin Immunol 76:803, 1985. 166. Harries MG, Kemeny DM, Youlten LJ, et al: Skin and radioallergosorbent tests in patients with sensitivity to bee and wasp venom, Clin Allergy 14:407, 1984. 167. Sicard H, Turjanmaa K, Palosuo T, et al: Latex allergy diagnosis: standardization of a natural rubber latex extract, J Allergy Clin Immunol 97:323, 1996. 168. Blanco C, Carrillo T, Ortega N, et al: Comparison of skin-prick test and specific IgE determination for the diagnosis of latex allergy, Clin Exp Allergy 29:133, 1999. 169. Vervloet D, Arnaud A, Vellieux P, et al: Anaphylactic reactions to muscle relaxants under general anesthesia, J Allergy Clin Immunol 63:348, 1979.

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170. Grammer LC, Schafer M, Bernstein D, et al: Prevention of chymopapain anaphylaxis by screening chemonucleolysis candidates with cutaneous chymopapain testing, Clin Orthop 234:12, 1988. 171. Fisher MM, Bowey C: Intradermal compared with prick testing in the diagnosis of anaesthetic allergy, Br J Anaesth 79:59, 1997. 172. Gadde J, Spence M, Wheeler B, Adkinson N Jr: Clinical experience with penicillin skin testing in a large inner-city STD clinic, JAMA 270:2456, 1993. 173. Blanca M, Vega M, Garcia J, et al: Allergy to penicillin with good tolerance to other penicillins: study of the incidence in subjects allergic to beta-lactams, Clin Exp Allergy 20:475, 1990. 174. Romano A, Quaratino D, Di Fonso M, et al: A diagnostic protocol for evaluating nonimmediate reactions to aminopenicillins, J Allergy Clin Immunol 103:1186, 1999. 175. Sarlo K, Clark ED, Ryan CA, Bernstein DI: ELISA for human IgE antibody to subtilisin A (Alcalase): correlation with RAST and skin test results with occupationally exposed individuals, J Allergy Clin Immunol 86:393, 1990. 176. Moser M, Crameri R, Brust E, et al: Diagnostic value of recombinant Aspergillus fumigatus allergen I/a for skin testing and serology, J Allergy Clin Immunol

93:1, 1994. Skin Tests for Nondiagnostic Purposes 177. Bousquet J, Guerin B, Michel FB: Units of allergen extracts, Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf AM 85:105, 1992. 178. Nordic Council on Medicines: Registration of allergen preparations, ed 2, NLN Pub No 23, Nordiska Läkemedelsnämnden, 1989, Uppsala, Sweden. 179. Shall L, Marks R: Non-invasive instrumental techniques to detect terfenadine and temelastine induced suppression of histamine weals in man, Br J Clin Pharmacol 24:409, 1987. 180. Muller P, Keller R, Imhof P: Laser Doppler flowmetry, a reliable technique for measuring pharmacologically induced changes in cutaneous blood flow? Methods Find Exp Clin Pharmacol 9:409, 1987. 181. Michel L, De Vos C, Rihoux JP, et al: Inhibitory effect of oral cetirizine on in vivo antigen-induced histamine and PAF-acether release and eosinophil recruitment in human skin, J Allergy Clin Immunol 82:101, 1988. 182. Charlesworth EN, Kagey-Sobotka A, Norman PS, Lichtenstein LM: Effect of cetirizine on mast cell-mediator release and cellular traffic during the cutaneous late-phase reaction, J Allergy Clin Immunol 83:905, 1989. 183. Burney PG, Luczynska C, Chinn S, Jarvis D: The European Community Respiratory Health Survey, Eur Respir J 7:954, 1994. 184. Tschopp JM, Sistek D, Schindler C, et al: Current allergic asthma and rhinitis: diagnostic efficiency of three commonly used atopic markers (IgE, skin prick tests, and Phadiatop). Results from 8329 randomized adults from the SAPALDIA Study. Swiss Study on Air Pollution and Lung Diseases in Adults, Allergy 53:608, 1998. 185. International Study of Asthma and Allergies in Childhood Steering Committee: Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema, Lancet 351:1225, 1998. 186. Peat JK, Tovey E, Toelle BG, et al: House dust mite allergens: a major risk factor for childhood asthma in Australia, Am J Respir Crit Care Med 153:141, 1996. 187. Postma DS, Bleecker ER, Amelung PJ, et al: Genetic susceptibility to asthma-bronchial hyperresponsiveness coinherited with a major gene for atopy, N Engl J Med 333:894, 1995. 188. Oryszczyn MP, Annesi I, Neukirch F, et al: Longitudinal observations of serum IgE and skin prick test response, Am J Respir Crit Care Med 151:663, 1995. 189. Gottlieb DJ, Sparrow D, O'Connor GT, Weiss ST: Skin test reactivity to common aeroallergens and decline of lung function: the Normative Aging Study, Am J Respir Crit Care 153:561, 1996.

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Chapter 39 - Nasal Provocation Testing

Karalasingam Rajakulasingam

Respiratory allergic diseases such as rhinitis and asthma are unique because their symptoms and signs can be reproduced in a laboratory set up under controlled conditions. This approach has become feasible because the agents suspected of causing or contributing to the pathogenesis of allergic disease can be used as provocative agents. In patients with atopic rhinitis, the accessibility of the nose simplifies such a task. The nasal challenge test or nasal provocation test (NPT) involves the administration of suspected allergens or mediators directly into the nose and has long been used in the study of both allergic and nonallergic rhinitis. NPT has been crucial for the scientific investigation of the pathophysiology, immunology, and pharmacotherapy of rhinitis. The NPT model mimics naturally occurring disease in a controlled laboratory setting and has become the best experimental way to assess the role of allergens in rhinitis. It is also useful to investigate the pathophysiology of bronchial hyperreactivity because of the similarities of the response to allergen challenge in the upper and lower airways.

LIMITATIONS AND BENEFITS Several techniques of NPT have been used depending on the purpose of the investigation. Each method has its limitations and advantages. At present, no standardized method exists for administering the allergens or mediators into the nose. Other limitations include the variety of test techniques and the lack of validated direct comparisons between methods. The method of administering allergens and mediators varies from one center to another, with nasal drops, nebulized droplets, powders, and paper disks all used. Also, the mode of exposure is not as natural as that occurring in the course of the disease. Finally, dose and concentration of agents used in most NPTs are well above the environmental or physiologic levels within the short time span of the experimental setting. To select the most appropriate method for nasal provocation and to make a proper evaluation of the results, the investigator should be familiar with the characteristics of the method employed and its reproducibility. Most NPT models monitor several parameters after provocation, including physiologic changes, symptoms, generated mediators, cytokines, and cells. These models have helped in understanding the events that occur after nasal provocation with a variety of stimulants. Furthermore, study of different treatment regimens has also been possible, and several medications currently used for rhinitis have been tested in these models to elucidate efficacy and mechanisms before marketing. NPT also has allowed comparisons of treatment strategies. Nasal provocation testing has provided significant insight into the pathophysiology and treatment of the several forms of rhinitis. However, several factors need to be considered before undertaking NPT.

SUBJECT SELECTION The selection of subjects for NPT is a crucial step. If the aim of the study is to investigate the events that follow nasally administered allergen, it is ideal to use subjects with seasonal allergies (“hay fever”) and to undertake the studies outside pollen season. Although many patients have sensitization to perennial allergens such as dust mite, many seasonal allergen studies have included patients with positive skin tests to perennial allergens, as long their nasal symptoms are not perennial. Although asymptomatic, such patients may have subclinical inflammation that may have compromised the conclusions reached. On the other hand, some studies do call for subjects with established nasal mucosal inflammation to undergo NPT in order to study outcomes such as nasal priming effect. Criteria for subject selection should be strictly followed to minimize problems.

INDICATIONS NPT is mainly used for clinical and scientific indications ( Boxes 39-1 and 39-2 ). In some countries, NPT is used for clinical evaluation as well. The indications for nasal provocation range from diagnosis of specific/nonspecific hyperreactivity and the study of nasal pathophysiology to therapeutic purposes. NPT is not used widely in the day-to-day management of rhinitis because the diagnosis of specific hypersensitivity to common allergens (e.g., pollens) usually can be accurately made clinically with an adequate medical history, skin tests, and radioallergosorbent test (RAST). However, the performance of NPT may be valuable in diagnosing specific hyperreactivity when skin tests and RAST are unhelpful.[ skin reactivity to multiple allergens, a role for a specific allergen may be determined only by undertaking NPT using the allergen in

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1] [2]

In the subject who shows

Box 39-1. Clinical Indications for NPT 1. To assess the role of allergens implicated by the patient's history when allergy skin testing and radioallergosorbent test (RAST) are negative.[

9]

[10]

2. To confirm the clinical relevance of a specific allergen in causing rhinitis when the patient demonstrates multiple positive skin test results. [6]

3. To confirm the role of a specific occupational agent in causing rhinitis, [7] [8]

11

including food-induced nasal symptoms.[ ] 4. To identify a role for a nonstandardized or novel allergen in the nasal target organ, possibly by undertaking NPT using a preparation of the 6

allergen.[ ] 5. To determine if nasal application of allergen can induce symptoms in 12]

other organs (e.g., conjunctiva, middle ear, sinus, lower airways).[ [13] [14]

6. To confirm the role of allergen in asthmatic patients for whom 15]

bronchial challenge may not be safe.[

15

7. To determine nasal hyperreactivity using bradykinin.[ ] 8. To determine nasal reactivity before starting immunotherapy against the specific allergen causing rhinitis.[

16]

3

question.[ ] Because most atopic asthmatic patients are sensitive to multiple allergens, it may be difficult to ascribe one particular allergen as the causal/contributory agent of asthma symptoms. NPT may therefore be used as a tool in predicting bronchial responses if both upper and lower airways are sensitized and responsive to the same allergen.[

4]

3] [4]

NPT may also be used as a safer alternative to bronchial challenge in evaluating the role of specific allergens in asthmatic patients.[ 5 6 7 8 required.[ ] [ ] [ ] [ ]

It is also a useful tool in the

diagnosis of occupational rhinitis, especially when positive confirmation of the agent in question is In the lower airways, methacholine and histamine challenges are usually carried out to distinguish patients with nonspecific bronchial hyper-reactivity (or hyperresponsiveness, BHR) from normal subjects. [9]

Although similar studies have been carried in the

Box 39-2. Scientific Indications for NPT 1. To investigate the physiologic, morphologic, and cellular spectra of allergen-induced immediate and late-phase responses and the dose17 18 19 20 21 22 23]

dependent nature of these reactions. [ ] [ ] [ ] [ ] [ ] [ ] [ 2. To assess the response of nasal airways to allergens and other provocative agents and study resulting changes in bronchial 24 25 26

responsiveness.[ ] [ ] [ ] 3. To examine the therapeutic effects of drugs on early, late-phase, nonspecific, and other aspects of airway disease.[

27]

upper airways to differentiate patients with nonspecific nasal hyperreactivity (or hyperresponsiveness, NHR) from normal subjects, there is a considerable overlap 10] [11] [12] [13] [14]

between both groups, and therefore NPT with histamine and methacholine cannot be recommended in the diagnosis of rhinitis.[

On the other hand, nasal challenge with incremental doses of bradykinin results in exaggerated increase in nasal airway resistance and rhinorrhea, indicating selective 15

NHR to bradykinin in subjects with perennial allergic rhinitis.[ ] Bradykinin may therefore be of use as an agent to identify subjects who may have underlying NHR. However, large-scale studies are required before recommending bradykinin as an agent for the diagnosis of NHR. Allergen-specific immunotherapy is a widely used form of treatment for allergen-induced rhinitis. NPT with specific allergen is undertaken in clinical studies before and after immunotherapy to assess the effectiveness of allergen immunotherapy.[ symptoms in other organs, such as the conjunctiva, middle ear, and

16]

NPT has also been used to determine if nasal application of allergen can induce

17 18 sinus.[ ] [ ]

Unlike bronchial challenge studies, nasal challenge can be carried out as either unilateral or bilateral challenges. The ability to study both unilateral and contralateral nasal responses allows evaluation of both direct effects and neural reflexes. For example, unilateral nasal challenge with allergen or other materials lead to local vascular and neural responses as well as contralateral neural responses. This model also permits study of certain aspect of nasal responses depending on the nasal 19 20

stimulant used.[ ] [ ] Substance P, for example, causes mainly glandular secretion, whereas calcitonin gene-related peptide (CGRP) causes vasodilation, resulting in increased nasal resistance to airflow. Studies using NPT models with allergen have detailed both early and late responses.[

21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]

The spontaneous recurrence of a late 32

nasal response after initial allergen challenge without further provocation, termed late-phase response (LPR) and first reported by Blackley[ ] in 1873, has gained increased attention in recent years. The prevalence of nasal LPR differs 5% to 50% among studies. LPRs in the nose are more difficult to quantify and usually 21] [22]

manifest as nasal obstruction and a late increase in some proinflammatory mediators in nasal lavage.[

Using NPT models, investigators can also study changes in cells and cytokines and novel mechanisms.[ help find new therapeutic interventions in the treatment of allergic

37 rhinitis.[ ]

Nasal provocation using cytokines may

NPT models also allow assessment of the response of nasal airways to allergens and

38 39 responsiveness.[ ] [ ]

other provocative agents and resulting changes in bronchial mechanisms underlying the pathogenesis of both asthma and rhinitis.

23] [33] [34] [35] [36]

NPT therefore provides a suitable in vivo human model for exploring the

40]

Both NPT and allergen-induced skin test models have also been extensively used for comparison of antihistamine effects.[ study the therapeutic effects of all other drugs and other novel therapies for

Moreover, NPT model is widely used to

41 rhinitis.[ ]

CONTRAINDICATIONS It is imperative that an investigator understands the factors known to influence the outcome of NPT. There are specific

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Box 39-3. Contraindications for NPT 1. 2. 3. 4. 5. 6. 7.

Episode of rhinitis in last 2 to 4 weeks Exacerbation of allergic disease Use of allergen known to have caused anaphylactic reaction Pregnancy Nasal surgery in last 6 to 8 weeks Coexisting severe general disease, especially cardiopulmonary diseases Treatment with certain medications

and relative contraindications for NPT ( Box 39-3 ). Because symptoms and pathophysiologic changes from a recent episode of acute rhinitis tend to persist, NPT soon after a rhinitis episode may result in nasal priming to allergen and may alter NPT outcome measures. NPT may also result in exacerbation of both nasal and bronchial pathologies. It is therefore recommended not to undertake NPT for 2 to 4 weeks after an episode of acute rhinitis.

Patients with a history of severe reactions or anaphylaxis to the suspected allergen should not undergo NPT with the same allergen. Some laboratories, however, perform NPT under extreme monitoring conditions using the same allergen when other test results are not helpful to establish the cause of anaphylaxis. NPT is not advised during pregnancy because of the danger of anaphylaxis resulting in miscarriage. A number of medications can interfere with the outcome measures of NPT, including false-negative results. It is therefore recommended to withdraw these 42 43 44

medications before NPT. The period of withdrawal varies depending on the medication used ( Box 39-4 ).[ ] [ ] [ ] However, it is routine practice to pretreat the nose with a topical decongestant before NPT to assist in the recovery of nasal lavage fluid. Nasal patency or symptom recording therefore should not be undertaken in NPT models using pretreatment with topical nasal decongestants. Although medications such as oral Box 39-4. Recommended Withdrawal Period for Medications Known to Interfere with NPT 1. 2. 3. 4. 5. 6. 7. 8.

Antihistamines (general): 48 hours to 1 week Ketotifen: 2 weeks Nasal steroids: 48 hours or 3 to 6 weeks Topical α-adrenergic agonists: 1 day Oral steroids (>10•mg): 2 to 4 weeks Nonsteroidal antiinflammatory drugs (NSAIDs): 1 week Antihypertensives (e.g., reserpine, clonidine): 3 weeks Antidepressants (e.g., imipramines, tricyclics): 2 to 3 weeks

contraceptives, certain eye drops, and solutions containing preservatives (e.g., sulfites) may also interfere with NPT by altering nasal resistance and cellular function, [45]

the need for withdrawal of these medications before NPT is not clearly defined. Nasal pathologies such as polyps, atropic rhinitis, and deviated septum may also compromise outcome measures. Patients who have undergone nasal surgery in the last 6 to 8 weeks should not have NPT.

NASAL PROVOCATION MODELS Various investigators have used different NPT laboratory methods, and therefore comparisons may be difficult. Unlike bronchial asthma, at present there is no widely accepted, standardized procedure for performing NPTs. The important elements are delivery method of test material and monitoring of the subject's response. Although no nasal challenge system seems to address all questions regarding the pathophysiology of rhinitis, the availability of multiple techniques increases the ability of gain further understanding of this prevalent condition. However, some methods have been cited more often in the literature. The few published provocation models have been used mainly used for research purposes. Regardless of the model employed, the main objective should be to measure the subject's response objectively after nasal provocation. Ideally, measurements should include (1) physiologic changes in nasal airway resistance, nasal

airway caliber, air space volume, and nasal mucosal blood flow; (2) mediators and cytokines in nasal lavage; and (3) cellular sampling techniques. Certain provocation models allow all these responses and measures. Nasal Lavage Method 46

The nasal lavage approach involves spraying the nasal cavity with stimulants and collecting the resultant secretions by lavage of both nostrils.[ ] This method provides more readily accessible material for studying pathologic responses of the nose. To improve the recovery of generated nasal fluid, the nose is pretreated with topical α-adrenergic agonists. Subjects are then instructed to tilt their neck backward about 30 degrees from the vertical while in the sitting position, hold their breath, and refrain from swallowing. From 2.5 to 5•ml of prewarmed saline is then instilled into each nostril, and after 10 seconds the subjects flex their neck and expel the mixture of mucus and saline into a collection vessel. The procedure can then be repeated in the other nostril. The returned volume is recorded and the sample immediately sieved to remove mucus processed, according to study protocol. This method usually samples both nasal and nasopharyngeal airways. The advantages of this model include ease of challenge and collection of secretions and obtaining large amounts of recovered lavages, which allows the simultaneous measurement of several mediators and the evaluation of cells. Disadvantages include that premedication with a decongestant may be required, which precludes using nasal airway caliber/resistance or blood flow measurements. The nasal lavage also dilutes generated secretions and therefore makes it difficult to appreciate the exact amount of nasal secretions produced by the actual nasal challenge. Because this model samples lavage from both nasal cavities and nasopharynx simultaneously, it does not allow the investigation of nasonasal reflex.

647

Aspiration/Nasal Lavage This model involves an aspiration and nasal lavage technique during which a soft, rubber catheter is placed along the floor of the nasal cavity and used to aspirate 47

46

lavage fluid, which is administered by aerosol spray.[ ] The advantages of this method are the same as those of nasal lavage method,[ ] with the additional benefit of sampling each nostril separately. The main limitation is the inability to measure changes in nasal airway resistance and caliber because of repeated nasal lavages and the presence of a catheter in the nostril. Nasal Pool Device 48

This method further refines nasal challenge and lavage by using a compressible plastic container that contains the solution of provocation material. [ ] The tip of the container has a nasal adapter that is tightly placed inside the nostril. While the head is held bent forward, the challenge solution is squeezed inside the nostril and left inside for a desired period. It is then pulled back into the container. The nasal pool approach has the advantage of delivering a known concentration of allergen to one nasal cavity.

Filter Paper Disk Method 49

This third common method uses the technique of filter paper disk placement on the nasal mucosa to deliver stimulants and collect generated secretions. [ ] This method allows study of the nasonasal reflex. Smaller doses of allergen can be used to provoke a response, and volume of nasal secretions after provocation and nasal airway resistance/caliber can be measured. The main limitation of the filter disk method is the inability to sample cells from the nasal secretions after challenge. However, nasal biopsies after lavage allow simultaneous study of both cellular changes and mediators.

NASAL CHALLENGE TECHNIQUES Before nasal challenge with an agent, the relevant patient history should be obtained, skin test/RAST performed, and the indication for nasal challenge clearly stated. A routine rhinoscopic examination is necessary to study the baseline condition of the nose and evaluate any local structural pathology (e.g., nasal polyp, septal deviation). Several protocols have been used in various laboratories, and this section describes a safe, simple, and reproducible method ( Figure 39-1 ). 50 51 52 53 54

Only well-trained personnel should undertake nasal challenge under standard conditions.[ ] [ ] [ ] [ ] [ ] The investigator should be able to identify any nonspecific effects, should be trained in dealing with potential side effects after nasal challenge, and should be familiar with any methodologic limitations. The most suitable time for nasal challenge is usually in the morning, to minimize effects on the nasal responses caused by other factors, such as diet, fumes, cold air, and exercise. The subjects should adapt to the laboratory temperature for about 20 minutes, and baseline nasal functions should be evaluated. Because nasal lavage is known to interfere with nasal physiology, it is advisable either to monitor physiologic measurements/symptoms or to perform nasal lavage after nasal challenge in 1 day. Therefore, at least two visits are necessary if both physiologic measures and lavage are required. As mentioned, nasal challenge may be performed either unilaterally or bilaterally. The nasal provocation usually begins with challenge by a known volume of diluent solution. As with bronchial challenge, this is undertaken to detect any nonspecific nasal responses. The subject is usually monitored for the next 15 minutes, with sneezes counted, nasal discharges collected, and nasal symptoms (e.g., itch, blockage, rhinorrhea, eye symptoms) scored either on a 10-cm visual analog scale or 4point severity scale. The patient should have no new clinical symptoms and less than 15% to 20% variability in nasal patency measurements in order to proceed to nasal challenge with the agent. The initial dose of allergen is estimated, and incremental doses of allergen are given in about threefold increases at 15-minute intervals. Doses are increased until a positive response is obtained or maximum concentration is reached. Unlike bronchial provocation studies with histamine, methacholine, or allergen, a positive nasal provocation response may be difficult to define, and criteria vary among laboratories. Before allergen challenge, skin testing should be performed to determine whether the subject is atopic to the allergen in question. For subjects with a negative skin 52

test, the initial dose of allergen may be in the range of 1:10,000 to 1:5000 weight/volume (w/v).[ ] This is equivalent to an overall protein concentration of 50 to 100 protein nitrogen units per milliliter (PNU/ml). As mentioned, further allergen challenges can be carried out in threefold dilutions every 15 minutes. For subjects with 55

a positive skin test, the concentration of allergen causing a 3-mm wheal size can be used as a dose for NPT,[ ] and the extracts are made in threefold dilutions. If intradermal allergen testing is done, it is necessary to find out the lowest concentration generating a wheal, and 10 times this concentration's dilute usually is used for 55

NPT.[ ] The amount of allergen solution required to deliver a certain dose may vary depending on the delivery device used. In general, concentrations higher than or equal to 1:500•w/v may usually result in nonspecific irritant effects. Therefore, it may be difficult to differentiate between nonspecific and specific allergen nasal effects. The investigator must be careful to differentiate a true positive reaction from a nonspecific irritant reaction.

However, within the confinement of their laboratory setup, investigators have used NPT to measure nasal reactivity in childhood asthma[

56]

and determine the

2

allergen dose for nasal immunotherapy and assess the effects before and after immunotherapy.[ ] Only a limited number of studies have investigated the comparative effects of nasal challenge with house dust and dust mite allergen in atopic subjects. A recent study using both dust mite and dust mite allergen showed varied nasal sensitivity in atopic subjects, but all subjects demonstrated a reaction to the final dose of allergen. [

57]

The allergen dose depends on the technique of delivery, allergen concentration, and aim of the study. There is no single standardized measure for allergen dose. Allergen doses are generally expressed in allergy units (AUs), biologic units (BUs), and PNUs. Studies have therefore become difficult to compare because varying allergen standardization methods have been used in different laboratories.

NASAL CHALLENGE ALLERGENS Several different substances have been applied in the nose as part of challenge procedures, including allergens, putative

648

Figure 39-1 Standard nasal allergen challenge protocol. w/v, Weight/volume.

Box 39-5. Nasal Provocation Agents

Allergens Pollen House-dust mite Anti-immunoglobulin E (IgE) Pet allergens (cat) Occupational allergens

Mediators Histamine Leukotrienes Prostaglandins Platelet-activating factor (PAF) Serotonin Kinins

Neuromediators Substance P Methacholine

Cytokines

Interleukin-6 (IL-6), IL-8, others

Irritants Cigarette smoke Ammonia (NH3 ) Glutaraldehyde Diesel particles

Physical stimuli Osmolarity Cold/warm air Water content

In contrast to nebulizers used for bronchial challenge, nasal challenge units deliver particles of larger size and tend to deposit far anteriorly in the nose, from where 67

much is carried by mucociliary action back to the nasopharynx over the ensuring 10 to 20 minutes.[ ] Handheld spraying might give significantly less uniform aerosol delivery. The aerosol is usually delivered during gentle inspiration. However, when primed properly and used with clear instructions, handheld nasal sprays 68 69

provide reproducible amounts of drug delivery.[ ] [ ] Furthermore, this device also improves the contact and distribution of challenge solution over the anterior nasal cavity surface. The devices also deliver small volumes of solution in the order of about 100••l and therefore do not lead to bronchospasm. The allergen meter50

70

dose pump is often used in many European centers.[ ] Larger particle– generating handheld automizers are also used.[ ] In general, devices delivering larger particles help to avoid the delivery of challenge material to the lower airways. Furthermore, this device generates mist, which may be more readily absorbed by the nasal mucosa than with mass application methods such as syringes. The use of syringes and droppers do not allow even distribution of challenge solutions, and therefore the area of distribution cannot be predicted. These methods of application are likely to result in rapid drainage of the stimulants into the nasopharynx. Furthermore, challenge solution may be aspirated, causing side effects such as cough and bronchospasm. To avoid or minimize this problem, patients should be advised not to inhale vigorously or bend their head backward after challenge solution is placed inside the nose. Nasopharyngeal and middle ear symptoms occur if challenge solution is carried posteriorly by mucociliary clearance.[

65]

Pipettes

allow deposition of microliter volumes onto the nasal mucosa, as achieved with a rhinoscope. 23 24 49 71

Another alternative is to place small cotton pledgets or paper disks with applied test agents directly on the nasal mucosa.[ ] [ ] [ ] [ ] Filter paper disks about 3• mm in size impregnated with allergen can be placed unilaterally or bilaterally by using forceps on the anterior part of the inferior turbinate or septum under direct 23 24 71

vision. To collect nasal secretions, larger dry-filter disks 8•mm in size are used.[ ] [ ] [ ] Although it may trigger some nasal reflex mechanisms due to local effect, the main advantages of this method are that (1) undiluted nasal secretion can be collected, (2) increasing doses of allergen can be placed stepwise, (3) smaller doses of allergen can be used to provoke a response, and (4) the nasonasal reflex can be studied by sampling nasal secretions from each nasal cavity. However, the mechanical application of materials onto the nasal mucosa may perturb its physiology and induce local nasal reflexes. The other disadvantage is the requirement for direct vision using head mirror and forceps. 48

The nasal pool device, a compressible plastic container with nasal adapter, holds a soluble agent of known volume and concentration.[ ] The adapter is pressed to the nostril and the fluid pushed into the nostril by compressing the container for a defined time and in the particular space of the anterior nasal cavity. A main benefit of this system is that the lavage fluid is collected when the container is reexpanded. Also, any soluble agents can be used, and the device is somewhat easy to use in children. The main limitation is the unknown dilutional effect of collected nasal lavage. Pollen grain nasal insufflation is undertaken to imitate natural exposure. In view of the hygroscopic properties of pollen, inhalation of intact pollen grains from stirred, dry suspensions[

72]

is usually the preferred method of nasal delivery, and the dose of pollen grains can also be well controlled. A later study used pollen 46

grains mixed with lactose from a gelatin capsule placed in a Spinhaler.[ ] The lactose used may cause nonspecific nasal reactions. The amount of pollen required to elicit nasal response varies individually, and a large amount of pollens is required to elicit a significant nasal response outside the pollen season.

OUTCOME MEASUREMENTS Objective physiologic and cellular changes and subjective symptomatic changes occur after nasal challenge ( Table 39-1 and Figure 39-2 ). The measurements of these changes involve various techniques and depend on the type of nasal challenge model used and the study's goal. Expertise is required to ensure that the measurements are made with an understanding of the inherent problems in the methodology used.

650

TABLE 39-1 -- Physiologic Responses Observed after Nasal Provocation Objective

Subjective

Increased nasal/airflow resistance

Nasal stuffiness

Increased nasal secretions

Runny nose

Decreased mucociliary transport

Postnasal drip

Impaired eustachian tube function

Ear fullness

Changes in lavage mediators and cytokines Changes in cellular pattern Nasal Patency Nasal resistance to airflow is primarily controlled by the state of congestion of the blood vessels in the inferior turbinate and anterior nasal septum. Other factors that 51

may influence nasal patency are mucosal oedema, cellular infiltration, and luminal secretions. Several factors also influence resistance to airflow[ ] (see Chapter 47 ). Although nasal blockage is a common symptom, the subjective sensation of nasal resistance to airflow can be very misleading. Several methods are used to measure nasal patency: nasal peak expiratory flow rate, nasal peak inspiratory flow rate, nasal spirometry, active and passive anterior rhinomanometry, posterior 52] [53]

rhinomanometry, balloon method, and oscillometry.[

The nasal volume is measured by acoustic rhinometry (see later discussion). 51]

To obtain objective measurement of nasal airway resistance, it is necessary to measure nasal pressure and flow parameters and then calculate resistance. [ peak flow measurements are easy to perform and inexpensive and correlate well with rhinomanometry, but reproducibility seems to be

Nasal

52 53 54 poor.[ ] [ ] [ ]

Rhinomanometry (rhinometry) measures the nasal work of breathing (and indirectly some inference of nasal patency) using the physics of airflow. Air will only flow from high pressure to low pressure through the nose when there is a difference in pressure in either end of the nose. To display pressure-flow curves, a computer processes the signals from transducers after digitization by an analog-to-digital converter (ADC). Curves can then be displayed on the computer monitor screen. Rhinomanometry can be performed by active (posterior, anterior) and passive (anterior) methods ( Table 39-2 ). Active Posterior Rhinomanometry

Active posterior rhinomanometry measures resistance or conductance across both nasal cavities simultaneously. The pressure in the pharynx is monitored with an oral tube. Airflow through both nasal cavities is measured by a face mask and pneumotachometer. This method records pressure, volume, and flow rates during tidal breathing. Display of pressure-flow curves in real time is essential because of pressure artifacts caused by malposition of the pharynx and tongue, a problem that prevents measurements in a small proportion of patients. Other artifacts are caused by air leaks around the edge of the face mask or obstruction of the mouth tube. Active posterior rhinomanometry therefore is not suitable for children, patients who cannot tolerate close-fitting masks, and those who are unable to coordinate nasal, palatal, and breathing maneuvers.[ Anterior Rhinomanometry

73]

In passive anterior rhinomanometry, all measurements are obtained from the exterior surface of the nares, so an oropharyngeal tube or mask is unnecessary. Each nostril is measured separately. A pneumotachometer for measuring flow is fitted with a plastic nosepiece. The plastic piece is then placed against the skin of one nostril (the nostril being measured). Slight pressure is applied to prevent air leaks, but the nose must not be distorted by the pressure. As the patient breathes, air is free to move from this nostril through the pneumotachometer, and a pressure tap measures external nares pressure. A similar plastic tube connected to a pressure transducer is placed against the contralateral external nares. Because the transducer is a closed device and air cannot move through it

Figure 39-2 Methods used to obtain NPT outcome measurements.

TABLE 39-2 -- Comparison of Active Forms of Rhinomanometry Anterior

Posterior

Flow

Nostril

Face mask

Pressure

Contralateral nostril

Mouthpiece

High

Slightly higher

Measurement Sites

Major Features Sensitivity

Subject coordination

Small

Large

Species applicability

Human/primate

Human only

Inaccurate if nostril obstructed

Yes

No

Applicable for rapid screening

Yes

No

or the nostril, it records the pressure in the nostril. This pressure is the same as retronasal pressure. With this method, changes in pressure and volume may be measured continuously throughout the respiratory cycle. The plastic tubes are then merely switched to measure the contralateral nostril. Passive anterior rhinomanometry is suitable for measuring air pressure and air flow during breath holding and is useful in children or patients with troublesome dental prostheses or an exaggerated gag reflex.[

74]

On the other hand, this technique does not truly represent the physiologic breathing process.

The most widely used technique is active anterior rhinomanometry, which evaluates nasal pressure-volume or pressure-flow relationships during tidal breathing. The main limitation of this technique results from variability in measurements due to active inspirations. Because this method requires the insertion of nasal adapter inside the nostril, it can cause significant changes in the nasal valve. Active anterior rhinomanometry is not useful in patients who are fragile or have localized nasal pathology (e.g., nasal bleed, polyp, septal deviation, turbinate hypertrophy, rhinorrhea) or other localized obstructive lesions. Methodologic Limitations

With rhinomanometry, changes in resistance are difficult to interpret, and pathologic and physiologic changes may have little impact on resistance. The correlation is unsatisfactory between the subjective perception of nasal stuffiness and the objective measures used for the nasal passage. Rhinomanometry is technically complicated, and some subjects may not be able to perform the necessary maneuvers. Also, the investigator must have considerable skill to obtain accurate results. Acoustic Rhinometry Acoustic rhinometry involves generation of a spark using a trigger module discharged between two electrodes placed in the end of the wave tube. This creates an acoustic pulse, which propagates down the wave tube. It passes the microphone and enters the nasal cavity through a 7-cm-long brass nosepiece inserted into a widening in the end of the wave tube. The free end of the nose tube is inserted a few millimetres into the nostril. The nose tube must be tightly fitting to obtain accurate results. The sound is reflected by changes in the local acoustic impedance resulting from changes in the cross-sectional area of the nasal cavity. The analog signal from the microphone is amplified, low-pass filtered, and digitized. The data are then converted to an area-distance function by software, with area plotted on either log or linear scale. Cross-sectional areas are determined for a distance up to 20•cm. 75

Because acoustic rhinometry measures cross-sectional area of the nasal cavity,[ ] it characterizes the geometry of the nasal cavity. The technique is relatively new and requires little cooperation from the subject. It does not require any flow through the nose, so acoustic rhinometry can be used even when the nose is totally occluded. It is therefore useful to help with the diagnosis of nasal tumors and polyps and to localize septal deviation. This method also has been used to monitor

76]

response after nasal surgical intervention and to assess nasal patency after allergen provocation.[

Studies have shown that acoustic rhinometry is as specific and

[77]

sensitive as nasal inspiratory flow. Acoustic rhinometry is also useful in differentiating aspirin-intolerant from non-aspirin-intolerant asthmatic subjects.[ Potential disadvantages are that pressure and flow are not measured, and the area beyond a severe constriction may not be accurately estimated.

78]

Other Nasal Measures: Uncommon Methods 79

Laser-Doppler velocimetry evaluates capillary blood flow in the nasal mucosa.[ ] This technique requires expensive technology and direct vision using head mirror to place the Doppler probe inside the nostril. The probe itself may cause local irritant effects, and reproducibility has not been studied well. Other uncommon or 80]

rarely used methods include radioactive xenon washout,[ plethesmography,[

82] [83]

H2 clearance into exhaled nasal air, colorimetric evaluation of mucosal erythema,[

81]

photoelectric

and thermography.

Symptom Recording Symptom recording is an important part of NPT and used in most nasal challenge models. Clinical symptoms of nasal blockage, sneezing, itching, and rhinorrhea are usually evaluated at baseline before nasal provocation with saline. These symptoms are then recorded at certain intervals after nasal challenge with the stimulant. Three common methods are used for nasal symptom recording.[

50] [84]

Method 1.

Severity of each nasal symptom is recorded on a 10-cm linear visual analog scale. The severity is then evaluated based on the score (mild 1–3•cm; moderate 4–7•cm; severe 8–10•cm) at the dose that caused a positive reaction. Method 2.

This validated method is often used in both clinical and scientific research studies ( Table 39-3 ). The end point is considered the amount of stimulant that produces a total symptom score of 5 from a maximum score of 13 points. Method 3.

This modified version method 2 is used with nasal flow to calculate the end point (nasal secretions: 0 = mild,

652

TABLE 39-3 -- Thirteen-Point Symptom Score Method Nasal symptom

Point score

Sneezing 0 to 2 sneezes

0

3 or 4 sneezes

1

5 or more sneezes

3

Pruritus Nose

1

Palate

1

Ear

1

Rhinorrhea

0 to 3

Nasal Blockage

1 to 3

Ocular Symptoms

1

1 = moderate, 3 = severe; sneezing: 0 = ≤2 sneezes; 1 point = 3–5 sneezes; 2 points = >5 sneezes). Other symptoms include itching or tearing (1 point) and 84]

conjunctivitis, cough, urticaria, or dyspnea (2 points).[ End Point

End point should be set before nasal challenge and depends on nasal model used, type of measurement, and aim of study. In general, end points take account of both changes in nasal patency and symptoms. If method 1 is used, an end point is the amount of allergen/stimulant required producing a total symptom score of 5. If method 2 is used, a positive response results when the patient has more than 3 symptom points and a reduction in nasal flow of more than 40%.[

50] [84]

Another scoring system considers a positive response when

greater than 20% reduction occurs in nasal expiratory flow rate with nasal symptoms (nasal blockage, sneezes, itching, rhinorrhea).[

85]

Changes in nasal temperature

86 87

and pH of nasal secretions have also served as end points, [ ] [ ] but these methods are not widely used. Unlike in bronchial provocation tests, end points for nasal provocation tests are not standardized. End points vary considerably, and their reproducibility has not been studied in large-scale studies. Investigators should also address false-positive and false-negative results in NPT studies ( Box 39-6 ). Protocols should be standardized and a checklist used to assess

inclusion and exclusion criteria for individual studies. Appropriate diluents should be used to minimize nonspecific irritant effects, and NPT should be undertaken in room temperature. Solutions with extreme pH should be discarded, and iso-osmolar dilution should be used. Collection of Nasal Secretions Four methods are typically used to collect nasal secretions, each with limitations and benefits, as discussed earlier. The amount, viscosity, and spinability of the nasal mucous discharge and the quantity of the lavage cytokines, mediators, proteins, and cells in lavage fluids vary in different forms of rhinitis and in response to different provocation agents and Box 39-6. Causes of False-Positive and False-Negative NPT Results

False-Positive Results Use of preservatives (e.g., phenol, glycerol, benzalkonium chloride) Changes in temperature Changes in osmolarity Recent airway illness (e.g., rhinitis) Allergen extract concentration greater than 1:500•w/v

False-Negative Results Use of medicines contraindicated for NPT Atrophic rhinitis Nasal polyps Recent nasal surgery Chronic sinus disease

method of collection. Interpretation of the techniques may become difficult if the collected secretion is thick and viscous or incomplete or if the volumes collected are small. The main limitations of repeated nasal lavage are dilution of lavage samples, alterations in fluid flux of the mucosal barrier, and removal of certain target cells during initial provocation. These techniques sample large surface areas of nasal mucosa and can quantify vascular, glandular, neural, and cellular components simultaneously. Earlier NPT studies did not take into account dilutional effect. Addition of radiolabeled albumin or lithium to the lavage fluid helps in calculating the percentage of fluid recovery in each study. Measurement of urea in blood and lavage helps to estimate the volume of epithelial fluid sampled.[

88] [89]

Cellular Sampling Techniques Nasal challenge studies may be necessary to study specific cellular changes. The cellular sampling methods differ in the compartments of the nasal mucosa that they sample.[

90]

Depending on the method, biochemical, histochemical, immunochemical, and electron microscopic techniques can be applied.

Nasal Smear.

A nasal smear is obtained by collection of cells using cotton wool swabs moved along the nasal mucosa from the anterior to the posterior part of the nasal cavity. The swab is then smeared over a glass slide and fixed and stained. This technique samples cells from both the nasal fluid and the superficial layer of the nasal mucosa. 90

Although the technique seems much simpler to perform than others, the cellular yield is usually small and has poor reproducibility.[ ] Extreme care is therefore required to interpret the results. Nasal smear is a useful technique to determine the presence or absence of a specific cell type as a relative proportion of total cells. Blown Nasal Secretions.

This method is undertaken by blowing nasal secretions onto wax paper or a plastic film and

653

later placing the secretions on a glass slide. This technique does not sample the nasal epithelium or other layers of nasal mucosa, and therefore the cells obtained are only those present in nasal secretions. Also, patients with inadequate nasal secretions will not be able to provide adequate specimens for analysis. Nasal Scrapings.

A plastic curette is used to scrape the nasal mucosa, usually from the medial surface of the inferior turbinate, under direct vision. This technique samples both nasal secretions and nasal surface epithelium. The sample is then smeared on to a slide, fixed, and stained. The advantages are that the technique (1) is easy to perform without the use of anesthesia, (2) is well tolerated by subjects, and (3) can be repeated several times and is therefore useful in time-course studies. The main disadvantage is that nasal scraping does not provide information about deeper mucosal layers.

Nasal Imprints.

Thin, small plastic strips coated with albumin are introduced into the nasal cavity under direct vision and are pressed gently over the nasal mucosa. The strips are then fixed and stained. This technique usually provides a reasonable amount of cells obtained from both the nasal epithelium and secretions. The main disadvantage is that mucus from the secretions may complicate interpretation of cellular elements. Moreover, it also requires the operator to be manually dextrous. Nasal Brush.

This technique uses a small brush of plastic-coated steel wire with nylon bristles. Using direct vision the brush is introduced into the nasal cavity between the nasal septum and the inferior turbinate. It is then rotated while being removed. Brushing allows cells to be collected from nasal fluid and epithelium. The brush is then dipped into a buffer solution and shaken carefully to move the harvested cells in suspension. The total number of cells is then determined by using a hemocytometer. The suspension is centrifuged on slides and stained. Multiple reagents can be used to enumerate different cell types. Because the total number of cells is known, the clinician should be able to find the number of individual cells. The main advantages of this technique are sampling of different sites of the nasal cavity and useful for time-course studies. The technique causes some nasal discomfort, however, and the number of cell obtained is limited by the capacity of the brush used. Furthermore, nasal brushing does not allow study of the deeper layer of the nasal mucosa. Nasal Lavage.

As mentioned, the volume of the collected specimen is recorded and then spun, and supernatant is used for the assay of different mediators and cytokines. The cell pellet is then resuspended, and the cytospun cells are dried, fixed, and stained to allow enumeration of different type of cells. The percentage of each cell type is counted, and because the total number of cells is known, the total number of each cell type can be determined. This technique is easy to perform and can be repeated several times such that kinetics of cellular changes can be studied. It only allows sampling of the cells in nasal secretions, however, and not sampling of the mucosal layer. Furthermore, lavage does not allow localization of the obtained specimen because the lavage samples both nasal cavities and nasopharynx. Nasal Biopsy.

This invasive procedure requires strong local anesthesia and expertise in this technique using a punch biopsy forceps. The biopsy is usually taken from the anteroinferior edge of the inferior turbinate using direct vision. The major advantage is that it samples all the layers of the nasal mucosa, including layers containing blood vasculature. Disadvantages include discomfort to the patient, need for anaesthesia, limitation of the number of specimens that can be obtained for the same subject, and other complications, including postbiopsy bleeding, pain, and synechiae. Because the lining of the nasal mucosa changes from squamous in the most anterior aspect to pseudostratified epithelium in the middle and posterior part of the nasal cavity, it is important that the biopsies are obtained from similar anatomic sites to make comparisons in the same individual or different subjects.

REPRODUCIBILITY OF METHODS AND MEASURES Only a limited number of studies have specifically addressed reproducibility. Although a number of individual studies have undertaken reproducibility of NPT outcome measures, large-scale studies are still indicated to assess the validity and reproducibility of methods used. However, within the confinement of the

91 92

laboratory setup, most authors have found that some NPT models were reproducible for symptomatic and physiologic responses.[ ] [ ] At present, it is rather difficult to compare nasal patency data obtained from one laboratory to other. However, the reproducibility of nasal patency measurements seems to be acceptable 15] [20] [68] [69]

within the confinements of each laboratory.[

In contrast to bronchial provocation and bronchoalveolar lavage (BAL) studies, little information is available on the reproducibility of cell counts and fluid-phase measurements in nasal fluid obtained by different nasal lavage methods. Most studies have used only a limited number of subjects, so it is difficult to study reproducibility. However, many authors have found that most of the lavage models were reproducible within the limitations of their laboratory setup. A recent study comparing two nasal lavage methods found that both gave poor reproducibility in lavage differential cell count and eosinophilic cationic protein (ECP) in normal 93]

subjects. [

On the other hand, reproducibility seemed to be better in rhinitic subjects. Furthermore, the Grieff method[

count and ECP levels than the Naclerio achieve wider clinical use.

46 approach.[ ]

48]

gave higher and more repeatable total cell

However, more studies involving larger number of subjects clearly are needed for the lavage methods to

CONCLUSIONS Despite being used in for research and some clinical purposes, NPT methods have not been well standardized for general use. To date, NPT has been used primarily as a research tool for the investigation of rhinitis with the wide variety of techniques. NPT will continue to provide useful information about the pathogenesis of rhinitis, and it has already led to an improved understanding of the events that occur after nasal provocation with wide variety of stimulants. NPT has also allowed the establishment of efficacy for many treatments now available for rhinitis. Furthermore, NPT has allowed

654

investigators to study the mechanisms of actions of many therapeutic agents and compare treatment strategies. NPT has provided invaluable insight in the pathophysiology and management of rhinitis. It is anticipated that with further development and refinement through standardization of the methodologies used, NPT can be used to a greater extent in the diagnosis and management of nasal diseases.

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mite, Ann Allergy Asthma Immunol 79:427, 1997. 3. Lavasa S, Kumar L, Kaushal SC, Ganguli NK: Wheat threshing dust: a “new allergen” in April-May nasobronchial allergy, Indian Paedatr 33:566, 1996. 4. Stenius-Aarniala BS, Malmberg CH, Holopainen EE: Relationship [among] the results of bronchial, nasal, and conjunctival provocation tests in patients with asthma, Clin Allergy 8:403, 1978. 5. Hyotenen M, Sala E: Nasal provocation in the diagnostics of occupational allergic rhinitis, Rhinology 34:86, 1996. 6. Gorski P, Krakowiak A, Pazdrak K, et al: Nasal challenge test in the diagnosis of allergic respiratory diseases in subjects occupationally exposed to a high molecular allergen (flour), Occup Med (Lond) 48:91, 1998. 7. Raphel G, Raphel MH, Kaliner M: Gustatory rhinitis: a syndrome of food-induced rhinorrhoea, J Allergy Clin Immunol 83:110, 1989. 8. Palczynski C, Walusiak J, Ruta U, et al: Nasal provocation test in the diagnosis of natural rubber latex allergy, Allergy 55:34, 2000. 9. Hargreave FE, Ryan G, Thomson NC, et al: Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance, Eur Respir J 121(suppl):79, 1982. 10. Borum P: Nasal methacholine challenge, J Allergy Clin Immunol 63:253, 1979. 11. McLean JA, Mathews KP, Solomon WR, et al: Effect of histamine and methacholine on nasal airways resistance in atopic and non-atopic subjects, J Allergy Clin Immunol 59:165, 1977. 12. Guercio J, Sakethhoo K, Birch S, et al: Effect of nasal provocation with histamine, ragweed pollen and ragweed aerosol in normal and allergic rhinitic subjects, Am Rev Respir Dis 119(suppl):69, 1979. 13. Mullens RJ, Olson LG, Sutherland DC: Nasal histamine challenges in symptomatic allergic rhinitis, J Allergy Clin Immunol 83:955, 1989. 14. Hallen H, Juto JE: A test for objective diagnosis of nasal hyperreactivity, Rhinology 31:23, 1993. 15. Rajakulasingam K, Ducey J, Lau LCK, et al: Nasal effect of bradykinin in perennial rhinitic and normal subjects, Clin Exp Allergy 23:77, 1993. 16. Passalacqua G, Albano M, Pronzato C, et al: Long term follow up of nasal immunotherapy to Parietaria: clinical and local immunological effects, Clin Exp Allergy 27:904, 1997. 17. Filiaci F, Masieri S, Zambetti G, et al: Nasal hypersensitivity in purulent middle ear effusion, Allergol Immunopathol (Madr) 25:91, 1997. 18. Olive Perez A: Rhinitis and asthma: nasal provocation test in the diagnosis of asthma, J Investig Allergol Clin Immunol 7:397, 1997. 19. Rajakulasingam K, Polosa R, Holgate ST, et al: Comparative nasal effects of bradykinin, kallidin and [des-arg9 ]-bradykinin in atopic rhinitic and normal volunteers, J Physiol 437:577, 1991.

20. Rajakulasingam K, Polosa R, Lau LC, et al: The nasal effects of bradykinin and capsaicin: influence on microvascular leakage and role of sensory nerve fibres, J Appl Physiol 72:1418, 1992. 21. Naclerio RM, Proud D, Togias AG, et al: Inflammatory mediators in late antigen-induced rhinitis, N Engl J Med 313:65, 1985. 22. Gronborg H, Bisgaard H, Romeling F, et al: Early and late nasal symptom response to allergen challenge: the effect of pre-treatment with a glucocorticoid spray, Allergy 48:87, 1993. 23. Rajakulasingam K, Hamid Q, O'Brien F, et al: RANTES in human allergic rhinitis: evidence for synthesis and release after allergen challenge and relationship to tissue eosinophilia, Am J Respir Crit Care Med 155:696, 1997. 24. Rajakulasingam K, Durham SR, Humbert M, et al: Enhanced expression of high affinity IgE receptors (FcepsilonRI) in human allergen-induced rhinitis with colocalisation to mast cells, macrophages, eosinophils and dendritic cells, J Allergy Clin Immunol 100:78, 1997. 25. Pelikan Z: Late and delayed responses of the nasal mucosa to allergen challenge, Ann Allergy 41:37, 1978. 26. Pelikan Z: The late nasal response: its clinical and immunologic features, possible mechanisms and pharmacological modulation, Free University in Amsterdam, 1996 (thesis). 27. Horak F, Toth J, Hirschwehr R, et al: Effect of continuous allergen challenge on clinical symptoms and mediator release in dust-mite allergic patients, Allergy 53:68, 1998. 28. Del Prete GF, De Carli M, D'Elios MM, et al: Allergen exposure induces the activation of allergen-specific Th2 cells in the airway mucosa of patients with allergic respiratory disorders, Eur J Immunol 23:1445, 1993. 29. Andersson M, Grieff L, Svensson C, et al: Various methods for testing nasal responses in vivo: a critical review, Acta Otolaryngol 115:705, 1995. 30. Walden SM, Proud D, Bascom R, et al: Experimentally induced nasal allergic responses, J Allergy Clin Immunol 81:940, 1988. 31. Wang D, Smitz J, Derbe MP, et al: Concentrations of myeloperoxidase in nasal secretions of atopic patients after nasal allergen challenge and during natural allergen exposure, Int Arch Allergy Immunol 110:85, 1996. 32. Blackley CH: Experimental researches on the causes and nature of Catarrhus aestivus, London, 1873, Bailliere, Tindal, Cox. 33. Nilsson G, Hjertson M, Andersson M, et al: Demonstration of mast cell chemotactic activity in nasal lavage fluid: characterisation of one chemotaxin as c-kit ligand, stem cell factor, Allergy 53:874, 1998. 34. KleinJan A, McEuen AR, Dijkdtra MD, et al: Basophil and eosinophil accumulation and mast cell degranulation in the nasal mucosa of patients with hay fever after local allergen provocation, J Allergy Clin Immunol 106:677, 2000. 35. Braunstahl GJ, Overbeek SH, KleinJan A, et al: Nasal allergen provocation induces adhesion molecule expression and tissue eosinophilia in upper and lower airways, J Allergy Clin Immunol 107:469, 2001.

36. Varney VA, Jacobson MR, Sudderick RM, et al: Immunohistology of the nasal mucosa following allergen-induced rhinitis: identification of activated T lymphocytes, eosinophils and neutrophils, Am Rev Respir Dis 146:170, 1992. 37. Gentile DA, Yokitis J, Angelini BL, et al: Effect of intranasal challenge with interleukin-6 on upper airway symptomatology and physiology in allergic and nonallergic patients, Ann Allergy Asthma Immunol 86:531, 2001. 38. Fokkens WJ, Rowe-Jones JM: The management of perennial rhinitis: links [among] the nose, lung, and asthma, Allergy 52(suppl):20, 1997. 39. Adinoff AD, Corren J, Irvin CG: Changes in bronchial responsiveness following nasal provocation with allergen, J Allergy Clin Immunol 89:611, 1992. 40. Persi L, Demoly P, Harris AG, et al: Comparison between nasal provocation tests and skin tests in patients treated with loratadine and cetirizine, J Allergy Clin Immunol 103:591, 1999. 41. Andersson M, Grieff L, Svensson C, et al: Allergic and non-allergic rhinitis. In Busse W, Holgate ST, editors: Asthma and rhinitis, Oxford, 1995, Blackwell, p 145. Contraindications 42. Bousquet J, Michel FB: Diagnostic tests. In Korenblat PE, Wedner HJ, editors: Allergy: theory and practice, ed 2, Philadelphia, 1995, Saunders, p 156. 43. McLean JA, Bacon JR, Mathews KP, et al: Effects of aspirin on nasal responses in atopic subjects, J Allergy Clin Immunol 72:187, 1983. 44. Demoly P, Campbell A, Lebel B, et al: Experimental models in rhinitis, Clin Exp Allergy 29:72, 1999. 45. Toppozada H, Toppozada M, El-Ghazzawi I, et al: The human respiratory nasal mucosa in females using contraceptive pills: an ultramicroscopic and histochemical study, J Laryngol Otol 98:43, 1984. Nasal Provocation Methods 46. Naclerio RM, Meier HL, Kagey-Sobotka A, et al: Mediator release after nasal airway challenge with allergen, Am Rev Respir Dis 128:597, 1983. 47. Raphael GD, Jeney EV, Baraniuk JN: Pathophysiology of rhinitis: lactoferrin and lysozyme in nasal secretions, J Clin Invest 84:1528, 1989. 48. Grieff L, Pipkorn U, Alkner U, et al: The nasal pool device applies controlled concentrations of solutes on human nasal airway mucosa and samples its surface exudations/secretions, Clin Exp Allergy 20:253, 1990. 49. Baroody FM, Wagenmann M, Naclerio RM: A comparison of the secretory response of the nasal mucosa to histamine and methacholine, J Appl Physiol 74:2661, 1993. Nasal Challenge Techniques 50. Bachert C: Nasal provocation test: critical evaluation. In Ring J, Behrendt HD, editors: New trends in allergy, IV, Berlin, 1997, Vieluf Springer-Verlag, p 277.

51. Eccles R: Rhinomanometry and nasal challenge. In Mackay I, editor: Rhinitis: mechanisms and management, London, 1989, Trinity, p 53. 52. Solomon WR: Nasal provocative testing. In Spector SL, editor: Provocation testing in clinical practice, vol 5, New York, 1995, Marcel Dekker, p 647. 53. Malm L, Gerth van Wijk R, Bachert C: Guidelines for nasal provocations with aspects on nasal patency, airflow, and airflow resistance. International Committee on Objective Assessment of the Nasal Airways, International Rhinologic Society, Rhinology 38:1, 2000. 54. Wihl JA: Methodological aspects of nasal allergen challenges based on a three year tree pollen immunotherapy study, Allergy 41:357, 1986. 55. Melillo G, Bonini S, Cocco G, et al: Provocation tests with allergens, Allergy 52(suppl):5, 1997. 56. Baki A, Ucar B: Diagnostic value of the nasal provocation tests with Dermatophagoides pteronyssinus in childhood asthma, Allergy 50:751, 1995. 57. Small P, Barrett D: Evaluation of dust and dust mite nasal provocation, Ann Allergy Asthma Immunol 75:501, 1995. Methods of Intranasal Drug Administration 58. Solomon WR, McLean JA: Nasal provocation testing. In Spector SI, editor: Provocative challenge: bronchial, oral, nasal and exercise procedures, Boca Raton, Fla, 1983, CRC Press. 59. Pelikan Z, Pelikan-Filipek M: Role of nasal allergy in chronic maxillary sinusitis: diagnostic value of nasal challenge with allergen, J Allergy Clin Immunol 86: 484, 1990. 60. Karlsson G, Rundcrantz H: Peroral chromones: a new way to treat allergic rhinitis? Acta Otolaryngol Suppl 360:27, 1979.

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61. Mygind N, Vesterhauge S: Aerosol distribution in the nose, Rhinology 16:131, 1978. 62. Stankiewicz J, Ruta U, Gjorski P: Latex allergy, Int J Occup Med Environ Health 8:139, 1995. 63. Connell JT: Quantitative intranasal pollen challenge. III. The priming effect in allergic rhinitis, J Allergy 43:33, 1969. 64. Sicherer S, Wood RA, Eggleston PA: Determinants of airway responses to cat allergen: comparison of environmental challenge to quantitative nasal and bronchial allergen challenge, J Allergy Clin Immunol 99:798, 1997. 65. Saito H, Asakura K, Ogasawara H: Topical antigen provocation increases the number of immunoreactive IL-4, IL-5 and IL-6 positive cells in the nasal mucosa of

patients with perennial allergic rhinitis, Int Arch Allergy Immunol 114:81, 1997. 66. Phillips MJ, Ollier S, Davies RJ: Use of anterior rhinomanometry in nasal provocation challenges with allergen and evaluation of the effects of ketotifen, clemastine and sodium cromoglycate on these responses, Respiration 39(suppl):26, 1980. 67. McLean JA, Bacon JR, Mathews KP, et al: Distribution and clearance of radioactive aerosol on the nasal mucosa, Rhinology 22:65, 1984. 68. Rajakulasingam K, Polosa R, Church MK, et al: Comparative nasal effects of bradykinin and histamine on nasal symptoms of rhinitis and plasma vascular leakage, Thorax 48:324, 1993. 69. Rajakulasingam K, Polosa R, Lau LC, et al: Repeatability and reversibility of bradykinin induced nasal symptoms and plasma leakage, Thorax 47:232P, 1992. 70. Druce HM: Nasal provocation challenge: strategies for experimental design, Ann Allergy 60:191, 1988. 71. Okuda M: Basic study of nasal provocative test. First report. Side, site of nose, size of site and allergen amount, Arch Otorhinolaryngol 214:241, 1977. 72. Connell JT: Quantitative intranasal pollen challenges. I. Apparatus, design and technique, J Allergy 39:358, 1967. Outcome Measurements 73. Clement PA: Committee report on standardisation of rhinomanometry, Rhinology 22:151, 1984. 74. Davies RJ, Corrado OJ: Diagnostic tests–challenge tests: oral, nasal and bronchial. In Lessof MH, editor: Allergy: immunological and clinical aspects, London, 1984, Wiley & Sons, p 83. 75. Lenders H, Pirsig W: Diagnostic value of acoustic rhinometry: patients with allergic and vasomotor rhinitis compared to normal controls, Rhinology 22:151, 1984. 76. Miyahara Y, Ukai K, Yamagiwa M, et al: Nasal passage patency in patients with allergic rhinitis measured by acoustic rhinometry: nasal responses after allergen and histamine provocation, Auris Nasus Larynx 25:261, 1998. 77. Ganslmayer M, Spertini F, Rahm F, et al: Evaluation of acoustic rhinometry in nasal provocation test with allergen, Allergy 54:974, 1999. 78. Casadevall J, Ventura P, Mullol J, et al: Intranasal challenge with aspirin in the diagnosis of aspirin intolerant asthma: evaluation of nasal response by acoustic rhinometry, Thorax 55:921, 2000. 79. Druce HM, Bonner RF, Patow C, et al: Response of nasal blood flow to neurohormones as measured by laser-Doppler velocimetry, J Appl Physiol 57:1276, 1984. 80. Olson P: A comparison between the 133 Xe washout and laser-Doppler techniques for estimation of nasal mucosal blood flow in humans, Acta Otolaryngol 102: 106, 1986. 81. Konno A, Togawa K, Nishira S: Participation of vascular reflexes in mucosal swelling in nasal allergy, Acta Otolaryngol 94:131, 1982.

82. Drettner B, Aust R: Plethysmographic studies of the nasal flow in the mucosa of the human maxillary sinus, Acta Otolaryngol (Stock) 78:259, 1974. 83. Davis DL, Hertzman AB: The analysis of vascular reactions in the nasal mucosa with the photoelectric plethysmograph, Ann Otolaryngol 66:622, 1957. 84. Linder A: Symptom scores as measures of the severity of rhinitis, Clin Allergy 18:29, 1988. 85. Bachert C, Gonsior E, Berdel D, et al: Richtlinien fur die Durchfuhrung von nasalen provokationstests mit Allergenen Bei Erkrankungen der oberen luftwege, Allergologie 13:53, 1969. 86. Phipatanakul CS, Slavin RG: Use of thermography in clinical allergy, J Allergy Clin Immunol 50:264, 1972. 87. Jankowski W, Bednarski W, Mikulewicz W: Measurement of the pH of the mucosa in allergic rhinitis in children, Otolaryngol Pol 23:493, 1969. 88. Bisgaard H, Krogsgaard OW, Mygind N: Measurement of secretion in nasal lavage, Clin Sci (Colch) 73:217, 1987. 89. Linder A, Strandberg K, Deuschl H: Histamine concentrations in nasal secretion and secretory activity in allergic rhinitis, Allergy 42:126, 1987. 90. Baroody FM: Mucosal cytology. In McCaffrey TV, editor: Rhinology and sinusology: rhinologic diagnosis and treatment, New York, 1997, Thieme, p 175. 91. Doyle WJ, Skoner DP, Seroky JT, et al: Reproducibility of the effects of intranasal ragweed challenges in allergic subjects, Ann Allergy Asthma Immunol 74:171, 1995. 92. Gerth vanWijk R, Mulder PGH, Dieges PH: Nasal provocation with histamine in allergic rhinitis patients: clinical significance and reproducibility, Clin Exp Allergy 19:293, 1989. 93. Belda J, Parameswaran K, Keith PK, et al: Repeatability and validity of cell and fluid-phase measurements in nasal fluid: a comparison of two methods of nasal lavage, Clin Exp Allergy 31:1111, 2001.

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657

Chapter 40 - Bronchial Challenge Testing

James E. Fish Stephen P. Peters

Bronchial inhalation challenge tests have been used extensively for several decades as a means of modeling asthma for investigative purposes. Such models have contributed significantly to the understanding of the pathophysiology of asthma and to the understanding of mechanisms of antiasthmatic medication therapy. Substantial experience with these tests also furnished a basis for reasonable judgments concerning the uses and interpretation of challenge tests for clinical purposes. Bronchial challenge tests may be used to assess airway reactions to specific allergens and other sensitizing agents and to quantify nonallergic airway responsiveness to pharmacologic agents such as methacholine or histamine. This chapter considers challenge tests of both allergic and nonallergic airway responses and focuses on the technical aspects of bronchoprovocation, the characteristics of airway reactions induced by controlled provocation, and the uses and significance of these tests.

TESTS OF ALLERGIC AIRWAY RESPONSIVENESS A variety of specific sensitizing agents have been used in bronchial challenge testing for investigative and clinical purposes. Although many agents are well-defined aeroallergens, others are either poorly characterized or unusual in that they may not involve immunoglobulin E (IgE)–dependent mechanisms, such as organic 1 2

antigens causing hypersensitivity pneumonitis (HP) and various occupational chemicals causing asthma.[ ] [ ] Bronchial challenge techniques can vary greatly according to the particular agent used, especially occupational agents. This discussion considers the general principles of bronchial challenge and focuses on the use of common aeroallergens. Aeroallergens Almost any allergen capable of penetrating the lower respiratory tract can be used for airway provocation. Lower respiratory tract deposition requires that the 3

allergenic substance be contained within particles of respirable dimensions, usually smaller than 10••m (•) in diameter.[ ] As a rule, allergens used for bronchial challenge should also be suitable for skin testing because the allergen dose administered to the airways is most conveniently determined by the skin sensitivity of the patient. Aqueous allergenic extracts are thus ideal; not only are they suitable for skin testing, but they can also be nebulized in small aerosol particles, optimizing 4

deposition below the larynx. Aerosolized powders and pollen fragments can also be used, provided that they are administered in sufficiently small particle size.[ ] Ideally, allergens standardized for potency and purity should be used, although it is recognized that few allergens are so characterized. The allergen extract must be free of bacterial lipopolysaccharide.[

5]

Most commercially available allergens are obtained as lyophilized extracts or as concentrated solutions. When stored at −20° C, these preparations retain potency for an indefinite period. When reconstituted, however, extracts tend to degrade with time. The rate of degradation can be slowed by using stabilizers (e.g., human serum 6

albumin) and storing the solutions at 4° or −20° C.[ ] Serial dilutions are made with diluent containing 0.5% sodium chloride, 0.275% sodium bicarbonate, and 0.4% phenol. Dilutions greater than 1:20 weight/ volume (w/v) should be used within 7 days so that they do not lose their potency. Differences in potency and purity of

commercially available allergen extracts mandate the use of single-lot preparations if responses will be compared on different days or among different subjects. The most common challenge method uses a graded dosing in which incremental allergen concentrations are given sequentially until the desired pulmonary response is achieved. For safety and expediency, proper selection of the initial allergen concentration is crucial. Initial concentrations that are too high may lead to severe reactions, whereas those that are too low may lead to an inordinate prolongation of the challenge procedure. Because of variation in patient sensitivity, as well as differences in potency and purity among different allergens, no simple or uniform guidelines exist for recommending starting concentrations. In general the selection of an initial concentration is guided by the skin test sensitivity of the patient. Previously published guidelines have recommended a starting concentration that 7 8

produces a 2+ reaction (larger than 5-mm wheal) after intradermal injection.[ ] [ ] Although safe, this approach may result in an unnecessarily prolonged test in some patients, often taking as long as 2 to 3 hours to provoke a mild asthmatic response. The response to inhaled allergen is determined not only by the level of allergic sensitivity (as estimated by skin sensitivity) but also by the level of airway 9 10

responsiveness to inhaled methacholine or histamine.[ ] [ ] Thus the response to allergen is viewed as a product of the quantity of mediators liberated, as well as the airway responsiveness to mediators. Subjects with relatively low levels of methacholine responsiveness

658

(e.g., nonasthmatic subjects with allergic rhinitis) may therefore respond to inhaled allergen, provided that they are highly allergic and a sufficiently high dose of allergen is administered. Conversely, subjects with exaggerated methacholine responsiveness (e.g., asthmatic patients requiring medications for control of symptoms) can also demonstrate reactions, although they possess only a modest degree of allergic sensitivity. Accordingly, selection of an initial allergen concentration for challenge testing should be based on the measured airway responsiveness to histamine or methacholine, as well as skin sensitivity.[

11]

The complexities of selecting an initial allergen concentration are illustrated by study results in ragweed-sensitive subjects. For a given level of skin sensitivity, the provocative concentration of ragweed allergen producing a 20% fall (PC20 ) in forced expiratory volume in 1 second (FEV1 ) is generally less in subjects with the greatest methacholine responsiveness ( Figure 40-1 ). Most notable, however, is the large interindividual variability in allergen responsiveness. Indeed, there is a striking overlap in allergen sensitivity among groups with widely divergent methacholine responsiveness, as well as among groups with different levels of skin sensitivity. These data also reveal that the relationship between airway and skin sensitivity to allergen is not the same at all levels of skin sensitivity, even in subjects with comparable methacholine responsiveness. For example, methacholine-reactive subjects with a 2+ skin reaction at a ragweed concentration of 0.0005••g/ml of Amb a 1 (antigen E) required on average (geometric mean) 0.20••g/ml to provoke a 20% fall in FEV1 , reflecting a 400-fold difference in concentrations to produce respiratory and cutaneous end points. In contrast, subjects with a 2+ skin reaction at 0.05••g/ml required 0.95••g/ml to provoke the same pulmonary response, reflecting only a 19-fold difference in concentrations to produce respiratory and cutaneous end points. These data suggest that an atopic population has a much narrower range of airway allergen sensitivity than range for skin sensitivity. The reasons for this are unknown. Nevertheless, such data can be used to predict safe and expedient dosing regimens in most subjects undergoing ragweed challenge. For example, the ideal dosing regimen for allergen challenge testing should lead to a mild asthmatic reaction (20% to 30% fall in FEV1 ) within 4 or 5 twofold concentration increments

above the initial concentration. An analysis of the data for methacholine-reactive subjects (see Figure 40-1 ) suggests that, in relation to skin sensitivity, optimal initial challenge concentrations would be as follows: (1) fiftyfold higher than the skin titration end point concentration (i.e., 0.025••g/ml Amb a 1) in subjects with 2+ skin reaction at 0.0005••g/ml Amb a 1; (2) tenfold greater (or 0.05••g/ml) in subjects with skin reactivities at 0.005••g/ml; and (3) equal to the skin titration end point concentration in subjects responding at 0.05••g/ml. The variability in responsiveness (see Figure 40-1 ) clearly indicates that such guidelines are inexact and do not preclude the occurrence of severe reactions at the initial allergen concentration or tediously long procedures in some individuals. It is not certain whether similar relationships between skin and respiratory tract sensitivity exist for different allergens or for different commercial preparations of ragweed allergen, although the same approaches have been successfully used to determine initial doses of cat, pollen, and house-dust mite allergens.[

12]

Aerosol Delivery Systems A variety of aerosol delivery systems are suitable for allergen challenge testing. Although different systems vary in type of equipment and pattern of aerosol inhalation, each is designed to optimize reproducible delivery to the lung so that an inhaled allergen dose can be estimated. In general the dose delivered to the lower 3 13

respiratory tract depends on the output characteristics of the nebulizer and several physiologic factors (e.g., pattern of inhalation, airway patency).[ ] [ ] Nebulizers generating aerosols with a particle size range (median mass aerodynamic diameter) of 1 to 5••m are used to minimize deposition of large particles in the mouthpiece assembly, oropharynx, and upper airway, as well as loss of aerosol resulting from exhalation of particles less than 1••m. The distribution of aerosols within the lung is influenced by inspiratory volume and flow rate, lung volume at the start of aerosol inhalation, and breath-holding time. Inhalation at high lung volumes or during slow deep breaths with end-inspiratory breath-holding facilitates particle deposition in the lung periphery. In contrast, rapid shallow breathing facilitates deposition in central airways (see Chapter 46 ). Two methods of aerosol delivery are typically used and provide remarkably similar results.[ inspiration

14]

One method involves the intermittent generation of aerosols during

Figure 40-1 Comparison of respiratory and skin sensitivity to ragweed allergen in subjects with varied methacholine responsiveness. Respiratory sensitivity (ordinate) is represented as ragweed (RW) allergen concentration (expressed in •g/ml of RW Amb a 1), causing a 20% fall in FEV1 (PC20 ). Skin sensitivity (abscissa) is expressed as allergen concentration (•g/ml of RW Amb a 1) injected intradermally, causing a 2+ reaction (8-mm wheal). Methacholine-reactive (R) subjects include subjects demonstrating methacholine PC20 of 4•mg/ml or less; methacholine-nonreactive (NR) subjects include those demonstrating methacholine PC20 greater than 4•mg/ml.

Figure 40-2 Early airway response (EAR) and late airway response (LAR) to inhaled ragweed allergen. EAR develops within 10 to 20 minutes and resolves spontaneously within 1½ to 3 hours. LAR occurs 3 to 8 hours after challenge and may abate within 24 hours, or it may persist for several weeks after challenge.

Figure 40-3 Comparison of responses to inhaled histamine (PC20 H) and methacholine (PC20 M). Open circles, Normal subjects; closed circles, asthmatic subjects. Diagonal line intersecting coordinate axes is the line of identity. Shaded area represents the region of single twofold concentration difference. (From Juniper EF, Frith PA, Dunnett C, et al: Thorax 33:705, 1978.)

(From Juniper EF, Frith PA, Dunnett C, et al: Thorax 33:705, 1978.) described for allergen. The dose of agonist is expressed on the logarithmic abscissa in one of several ways: (1) as the concentration inhaled (milligrams per milliliter), (2) as the cumulative amount of agonist (in micromoles) delivered from the nebulizer, or (3) as cumulative inhalation breath units. An inhalation breath unit has been 7

defined as the equivalent of one breath of a concentration containing 1•mg/ml.[ ] The most common variable used to express nonspecific responsiveness is PC20 or PD20 (dose), as determined by interpolation between points on the dose-response curve ( Figure 40-4 ). Depending on the expression of dose, this variable is presented as the PC20 -FEV1 (milligrams per milliliters), PD20 -FEV1 (micromoles), or PD20 -FEV1 (breath units). These variables can be compared using established protocols ( Table 40-1 ). Because the bronchospastic effects of histamine and methacholine are relatively short-lived, the pulmonary function response is not truly cumulative. Peak responses 158 159

to these agonists generally occur within 1 to 3 minutes after inhalation, with a return toward baseline within 30 to 60 minutes.[ ] [ ] Therefore, it is recommended that pulmonary function measurements be made 3 to 5 minutes after aerosol administration, and that the interval between doses be approximately 5 minutes. The FEV1 remains the most useful test for monitoring challenge responses.[

160] [161]

162]

Similar challenge techniques have been applied while testing children older than 6 years.[

Although earlier studies suggested that responsiveness was not

reproducible in children,[

163]

Weiss et al[

164]

have devised a medication-withholding protocol that gives both short-term and long-term reproducibility for

methacholine challenge testing in children. Responses can be measured using either the FEV1 or the PEFR, although FEV1 is preferable.[

165]

Younger children may

be more responsive to methacholine than older children and

Figure 40-4 Dose-response curves to inhaled bronchoconstrictor agonists in a normal subject and in asthmatic subjects with mildly and severely increased airway hyperresponsiveness (AHR). Responses are measured as the fall in FEV1 from baseline values, in these examples expressed as the provocative concentration of agonist causing a 20% fall in FEV1 (PC20 ). Both mildly asthmatic patient and normal subject demonstrate a plateau response, which is lost in severely asthmatic subject.

TABLE 40-1 -- Comparison of Methacholine Responsiveness Using Three Established Methods Level of Responsiveness Method PC20 (mg/ml)

*

(noncumulative)

Severe

Moderate

Mild

Normal

< 0.25

0.25–2.0

2.0–8.0

> 8.0

PD20 (•mol)

†

< 0.125

0.125–1.0

1.0–4.0

> 4.0

< 2.5

2.5–20.0

20–80

>80

(cumulative) 20]

PD[

(breath

‡

units) (cumulative) Modified from Woolcock AJ: In Hargreave FE, Woolcock AJ, editors: Airway responsiveness: measurement and interpretation, Mississauga, Ontario, Canada, 1985, Astra, p 84. * According to method of Juniper et al.[ † According to method of Yan et al.[

151]

‡ According to method of Chai et al.[

adults,[

166] [167] [168]

16]

7]

perhaps because of the larger agonist doses delivered in younger children relative to body size. However, methacholine responsiveness seems

more closely correlated with baseline spirometric values (FEF25–75 , FVC, and FEV1 /FVC) than with age or height.[

169]

Methacholine challenge testing also has been applied in children younger than 6 years of age. For the study of infants and toddlers, apparati have been developed to produce a sudden compressive pressure to the thorax and abdomen at end-tidal inspiration, during which forced partial expiratory flow rates can be measured and recorded.[

170]

Other investigators have used transcutaneous oxygen tension readings to monitor response of infants to methacholine challenge.[

171]

To investigate

172 al[ ]

alternate response parameters, Springer et performed methacholine challenge testing in two groups of asthmatic children. In the older group (mean age 11.9 years), response to sequential methacholine was monitored by conventional spirometry, whereas in the younger group (mean age 4.2 years), chest and tracheal auscultation for wheezing was substituted for the spirometry. A positive response using the auscultation method was observed in 95.9% of the younger children, suggesting that the chest auscultation method used was effective and safe. Responses to pharmacologic agonists may be altered by the same factors that influence allergen responses: medications, baseline airway caliber, respiratory infections, and exposure to allergens and chemical sensitizers.[

34] [35] [42] [43] [44] [45] [46] [47] [48] [49]

responses and should be withheld before testing for the duration of their action.[ [175] [176] [177]

173] [174]

In particular, bronchodilators and antihistamines may inhibit

Calcium channel blockers also may alter response to pharmacologic agonists

and thus should also be withheld when possible. Under ideal conditions, reproducibility of pharmacologic challenge tests is similar to that of allergen

challenge tests; PC20 values vary within a twofold concentration range in most subjects. [

13]

Studies of both children and adults with asthma document marked

circadian rhythmicity in the airway response to bronchial challenges with histamine and methacholine. Hyperreactivity to these agents is more profound and prolonged after evening and overnight tests compared with tests in the midday and afternoon. Conversely, BHR/AHR to hyperventilation of cold, dry air is greatest

178]

in the afternoon.[

Uses and Interpretation Besides use in epidemiologic and clinical research studies, pharmacologic challenge tests have been used most widely as diagnostic tests for asthma. Although the concept of abnormal airway responsiveness originated within the context of the understanding of asthma and its associated airway lability, it is debatable whether abnormal responsiveness and asthma are equivalent entities. Studies of the distribution characteristics of responsiveness between normal and abnormal populations have provided a basis for examining the value of pharmacologic challenge testing for diagnosing asthma. Challenge studies in asthmatic populations indicate that an abnormal histamine or methacholine response is a sensitive test with a high negative predictive value (i.e., low false-negative rate). Moreover, the degree of methacholine or histamine responsiveness correlates with asthma severity, as measured by pulmonary function alterations, symptom scores, and treatment required to control symptoms.[ well as pulmonary function responses to inhaled bronchodilator 92]

In a clinical survey, Cockcroft et al[ al,[

181]

128] [179]

The level of responsiveness also correlates with diurnal fluctuations in PEFR, as

180 medications.[ ]

found that 100% of patients with current asthmatic symptoms demonstrated a PC20 of 8•mg/ml or less for histamine. Hopp et

on the other hand, reported a sensitivity rate of approximately 85% at a comparable PC20 threshold for methacholine. The discordance in sensitivity rates

found in these studies most likely reflects differences in the definition of “current” symptoms of asthma. Whereas Cockcroft et al[

92]

defined current asthmatic

[181]

patients as those with active disease at testing, Hopp defined this group as those having at least three wheezing episodes in the preceding year. Studies have shown that pharmacologic challenge tests can give false-negative results in patients who experience symptoms only at times of relevant exposure and who are 181

asymptomatic at testing.[ ] This is particularly relevant in atopic patients with seasonal respiratory tract symptoms who are tested out of season. False-negative responses with methacholine and histamine have also been reported in some patients with asthma symptoms when exposed to occupational allergens and sensitizing chemicals.[

182] [183] [184] [185]

185

Although an overall false-negative response rate is difficult to estimate because of differences in definition of current asthma, Banks et al[ ] found that 16% of results were false negative in toluene diisocyanatesensitive workers with asthma symptoms. Thus a false-negative rate as high as 16% can reasonably be expected under certain conditions, and clinicians must be prepared to repeat challenges during symptomatic intervals in patients in whom suspicion of disease remains high. The specificity of pharmacologic challenge tests in the diagnosis of asthma is not known because of a lack of appropriate random population studies. Estimates of 181

specificity can nevertheless be derived from studies of selected populations of normal subjects. An analysis of the study of Hopp et al[ ] in normal nonatopic children reveals a specificity of approximately 95% when a PC20 value of 8•mg/ml or greater is used to define abnormal responsiveness. Using the same cutoff point 92

of 8•mg/ml, Cockcroft et al[ ] reported a 93% specificity rate. In a survey of 154 nonatopic normal individuals aged 19 to 42 years with no prior history of respiratory disease, a specificity rate of 91% was found; that is, 9% of normal

667

individuals demonstrated a 20% fall in FEV1 at concentrations at or below 8•mg/ml.[

186]

Moreover, 21% of normal young adults had PC20 values at concentrations

of 25•mg/ml or less, whereas in other studies, a 29% incidence of PC20 values at or below 25•mg/ml were reported in nonatopic normals.[

187]

The latter observations

7

suggest that earlier provocation guidelines published by Chai et al,[ ] in which PC20 values of 25•mg/ml or less constituted a “positive” response, offer a measure of sensitivity but are lacking as a diagnostic test for asthma in terms of specificity and positive predictive values. Use of the term “false positive” in reference to normal subjects demonstrating PC20 values of 8•mg/ml or less must be viewed with caution. Indeed, such subjects are classified as “normal” only by virtue of their lack of respiratory tract symptoms and their normal spirometry results. The significance of exaggerated responsiveness in asymptomatic individuals is a subject of current interest in epidemiologic investigations. In a prospective, longitudinal study of 912 middle-aged and older men, methacholine responsiveness correlated significantly with the subsequent rate of annual decline in FEV1 and FEV1 /forced vital capacity (FVC).[

188]

Although this and previous studies suggest that such individuals with methacholine hyperresponsiveness demonstrate a

greater decline in lung function with age,[

189]

abnormal reactivity could constitute a risk factor for the development of chronic airway disease. In a study of 9-year190

old children who denied previous wheezing episodes, 41 of 547 (7.5%) showed AHR to methacholine.[ ] When subsequently studied on three occasions until age 26, these 41 individuals were more likely to develop asthma or wheezing, positive skin tests to inhalant allergens, blood eosinophilia, and elevated serum IgE. The authors suggested that rather than considering AHR as a marker of asthma, AHR should be regarded as a parallel pathologic process that may lead to subsequent symptoms and clinical evidence of asthma. Several studies have demonstrated an association between abnormal airway responsiveness and other respiratory conditions, including cystic fibrosis, [ respiratory infections, chronic bronchitis).

[44] [45] [46]

exposures to allergens

[129] [130] [191]

[34] [35]

and oxidizing

47 48 49 pollutants,[ ] [ ] [ ]

131]

viral

and diseases characterized by chronic airflow limitations (e.g.,

In general, changes in responsiveness that occur with acute inflammatory conditions tend to be transient, and in the case of viral 45]

respiratory infections, the response may return to normal within several weeks.[

Abnormal responsiveness associated with chronic bronchitis, on the other hand,

130 191 192 alteration.[ ] [ ] [ ]

tends to be fixed and correlates with the level of pulmonary function For this reason, pharmacologic challenge tests cannot be used to discriminate between asthma and diseases characterized by nonreversible airflow limitation in patients with abnormal spirometry results. In the presence of airway obstruction, the spirometric response to an inhaled bronchodilator provides the best assessment of the “reactivity” or reversibility of the pathologic process. In 128]

asthmatic patients, however, increased responsiveness is usually demonstrable even when spirometry results are normal.[ used in the evaluation of patients with normal spirometry results who have unexplained respiratory tract symptoms.

Thus, challenge testing is most gainfully

Although most asthmatic patients experience classic symptoms of chest tightness, wheezing, and shortness of breath, wheezing may be absent in some individuals, and the sole manifestation of disease may be cough or dyspnea.[

193] [194]

Many studies have shown the poor predictive value of the clinical history alone in

92 195 196

establishing a diagnosis of asthma in patients with atypical symptoms of asthma. [ ] [ ] [ ] In such patients, provocation testing may be useful in defining the underlying cause of symptoms and establishing an appropriate treatment regimen. However, challenge testing provides information regarding the state of airway responsiveness only. Abnormal responsiveness or AHR in a patient with the sole symptom of cough may suggest a diagnosis of asthma or may reflect the transient pathology that occurs with self-limited inflammatory conditions of the airways. Airway responsiveness can vary in an individual, and thus measurements must be viewed as relevant to the current status of the patient, not to remote or future events. Methacholine and histamine challenge tests have not achieved their expected status as “gold standards” in the diagnosis of asthma, largely because of problems in defining “asthma.” Certainly asthma is a heterogenous disorder not only in etiology, but also in pathology and clinical manifestations.

CONCLUSIONS For now, bronchial challenge tests seem best used as adjunctive diagnostic measures with results interpreted in the context of the clinical findings. Because of a reasonably low false-negative rate, challenge procedures appear most useful for excluding a diagnosis of asthma. Despite a high false-positive rate found in the general population, the positive predictive value of challenge testing should increase to a degree when symptomatic patients are evaluated. The demonstration of hyperresponsiveness (AHR, BHR) in these patients should at least suggest a causal relation between symptoms and responsiveness and furnish an objective basis for antiasthmatic therapy. Challenge tests for nonspecific responsiveness also have a special use in the evaluation of patients at risk for the development of occupational asthma as a result of sensitizers in the workplace. Serial challenges may detect changes in responsiveness related to workplace exposure, thus allowing early removal from the risk-related 197]

environment.[

Although some have suggested that methacholine and histamine challenge tests are useful for defining the severity and prognosis of asthma, as 198

well as for guiding intensity of therapy,[ ] these applications are unfounded at present. The severity of asthma at any point in time is best determined on the basis of symptoms and physiologic impairment, not on the basis of non-specific airway responsiveness. Whether serial assessment of responsiveness can effectively be used to guide treatment of airway inflammation and improve long-term prognosis remains to be proved. Repeated methacholine challenges are often performed in 199]

individuals participating in asthma clinical trials, and the data unique to these challenges have recently been summarized.[

Pharmacologic challenge tests are generally safe, and the authors are not aware of deaths or significant morbidity occurring when tests are performed using prescribed guidelines. Severe symptomatic reactions, however, do occur in some patients with unexpectedly high levels of agonist sensitivity. For this reason, experienced physicians trained in provocative testing should perform and interpret airway challenge procedures.

668

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153:899, 1996. Tests of Nonallergic Airway Responsiveness 126. Fish JE, Shaver JR, Peters SP: Airway hyperresponsiveness in asthma: is it unique? Chest 107:154S, 1995. 127. Sterk PJ: Bronchial hyperresponsiveness today, Respir Med 87(suppl B):27, 1993. 128. Hargreave FE, Ryan G, Thomson N, et al: Bronchial responsiveness to histamine or methacholine in asthma: measurement and clinical significance, J Allergy Clin Immunol 68:347, 1981.

670

129. Parker CD, Bilbo RE, Reed CE: Methacholine aerosol as a test for bronchial asthma, Arch Intern Med 115:452, 1965. 130. Tashkin DP, Altose MD, Bleecker ER, et al: The Lung Health Study: airway responsiveness to inhaled methacholine in smokers with mild to moderate airflow limitation, Am Rev Respir Dis 145:301, 1992. 131. Mellis CM, Levison H: Bronchial reactivity in cystic fibrosis, Pediatrics 61:446, 1978. 132. Hardy CC, Robinson S, Tattersfield AE, et al: The bronchoconstrictor effect of inhaled prostaglandin D2 in normal and asthmatic men, N Engl J Med 311:209, 1984. 133. Thomson NC, Roberts R, Bandouvakis J, et al: Comparison of bronchial responses to prostaglandin F2 alpha and methacholine, J Allergy Clin Immunol 68:392, 1981. 134. Griffin M, Weiss JW, Leitch AG, et al: Effects of leukotriene D on the airways in asthma, N Engl J Med 308:436, 1983. 135. Smith LJ, Greenberger PA, Patterson R, et al: The effect of inhaled leukotriene D4 in humans, Am Rev Respir Dis 131:368, 1985. 136. Okayama M, Yafuso N, Nogami H, et al: A new method of inhalation challenge with propranolol: comparison with methacholine-induced bronchoconstriction and role of vagal nerve activity, J Allergy Clin Immunol 80:291, 1987. 137. Roisman GL, Lacronique JG, Desmazes-Dufeu N, et al: Airway responsiveness to bradykinin is related to eosinophilic inflammation in asthma, Am J Respir Crit Care Med 153:381, 1996. 138. DeMeer G, Heederik D, Postma DS: Bronchial responsiveness to adenosine 5′-monophosphate (AMP) and methacholine differ in their relationship with airway

allergy and baseline FEV1 , Am J Respir Crit Care Med 165:327, 2002. 139. Van den Berge M, Kerstjens HA, Meijer RJ, et al: Corticosteroid-induced improvement in the PC20 of adenosine monophosphate is more closely associated with reduction in airway inflammation than improvement in the PC20 of methacholine, Am J Respir Crit Care Med 164:1127, 2001. 140. McFadden ER Jr, Gilbert IA: Exercise-induced asthma, N Engl J Med 330:1362, 1994. 141. Bar-Or O, Newman I, Dotan R: Effects of dry and humid climates on exercise-induced asthma in children and preadolescents, J Allergy Clin Immunol 60:163, 1977. 142. Strauss RH, McFadden ER Jr, Ingram RH Jr, et al: Enhancement of exercise-induced asthma by cold air, N Engl J Med 297:743, 1979. 143. McFadden ER Jr, Ingram RH Jr: Exercise-induced asthma: observations on the initiating stimulus, N Engl J Med 301:763, 1979. 144. Sterk PJ, Fabbri LM, Quanjer PH, et al: Airway responsiveness: standardized challenge testing with pharmacological, physical and sensitizing stimuli in adults, Eur Respir J Suppl 16:53, 1993. 145. Aquilina AT: Comparison of airway reactivity induced by histamine, methacholine, and isocapnic hyperventilation in normal and asthmatic subjects, Thorax 38:766, 1983. 146. O'Bryne PM, Ryan G, Morris M, et al: Asthma induced by cold air and its relation to nonspecific bronchial responsiveness to methacholine, Am Rev Respir Dis 125:281, 1982. 147. Lee TH, Nagakura T, Cromwell O, et al: Neutrophil chemotactic activity (NCA) and histamine in atopic and nonatopic individuals after exercise-induced asthma, Am Rev Respir Dis 129:409, 1984. 148. Godfrey S: Exercise-induced asthma, Allergy 33:229, 1978. 149. Chatham M, Bleecker ER, Smith PL, et al: A comparison of histamine, methacholine and exercise airways reactivity in normal and asthmatic subjects, Am Rev Respir Dis 126:235, 1982. 150. Crapo RO, Casaburi R, Coates AL, et al: Guidelines for methacholine and exercise challenge testing--1999, Am J Respir Crit Care Med 161:309, 2000. 151. Yan K, Salome C, Woolcock AJ: Rapid method for measurement of bronchial responsiveness, Thorax 38:760, 1983. 152. Chatham M, Bleecker ER, Norman R, et al: A screening test for airways reactivity, Chest 82:15, 1982. 153. Rosenfeld MM, Juniper EF, Hargreave FE: Gas chromatographic determination of methacholine chloride in pharmaceutical preparation, J Chromatogr 287:433, 1984. 154. Rosenfeld MM, Juniper EF, Hargreave FE: Stability of histamine acid phosphate solutions, J Allergy Clin Immunol 73S:151A, 1984.

155. Pratter MR, Woodman TF, Irwin RS, et al: Stability of stored methacholine chloride solutions, Am Rev Respir Dis 126:717, 1983. 156. Martinez-Garcia MA, Perpina-Tordera M, Vila V, et al: Analysis of the stability of stored adenosine 5′-monophosphate used for bronchoprovocation, Pulmonol Pharmacol Ther 15:157, 2002. 157. Asmus MJ, Vaughan LM, Hill MR, et al: Stability of frozen methacholine solutions in unit-dose syringes for bronchoprovocation, Chest 121:1634, 2002. 158. Cartier A, Malo JL, Begin P, et al: Time course of the bronchoconstriction induced by inhaled histamine and methacholine, J Appl Physiol 54:821, 1983. 159. Fish JE, Jameson JS, Albright A, et al: Modulation of the bronchomotor effects of chemical mediators by prostaglandin F2 alpha in asthmatic subjects, Am Rev Respir Dis 130:571, 1984. 160. Fish JE, Kelly JF: Measurements of responsiveness in bronchoprovocation testing, J Allergy Clin Immunol 64:592, 1979. 161. Fish JE, Ankin MG, Kelly JF, et al: Regulation of bronchomotor tone by lung inflation in asthmatic and nonasthmatic subjects, J Appl Physiol 50:1079, 1981. 162. Shapiro GG, Furukawa CT, Pierson WE, et al: Methacholine bronchial challenge in children, J Allergy Clin Immunol 69:365, 1982. 163. Hariparsad D, Wilson N, Dixon C, et al: Reproducibility of histamine challenge tests in asthmatic children, Thorax 38:258, 1983. 164. Weiss ME, Wheeler B, Eggleston P, et al: A protocol for performing reproducible methacholine inhalation tests in children with moderate to severe asthma, Am Rev Respir Dis 139:67, 1989. 165. Williams PV: Inhalation bronchoprovocation in children, Immunol Allergy Clin North Am 18:149, 1998. 166. Peat JK, Salome CM, Xuan W: On adjusting measurements of airway responsiveness for lung size and airway caliber, Am J Respir Crit Care Med 154:870, 1996. 167. Hopp RJ, Bewtra AK, Nair NM, et al: Methacholine inhalation challenge studies in a selected pediatric population, Am Rev Respir Dis 134:994, 1986. 168. Forastiere F, Corbo GM, Dell'Orco V, et al: A longitudinal evaluation of bronchial responsiveness to methacholine in children: role of baseline lung function, gender, and change in atopic status, Am J Respir Crit Care Med 153:1098, 1996. 169. Ownby DR, Peterson EL, Johnson CC: Factors related to methacholine airway responsiveness in children, Am J Respir Crit Care Med 161:1578, 2000. 170. LeSouëf PN, Castile R, Turner DJ, et al: Forced expiratory maneuvers. In Stocks J, Sly PD, Tepper RS, et al, editors: Infant respiratory function testing, New York, 1996, Wiley-Liss, p 375. 171. Delacourt C, Benoist MR, Waernessyckles S, et al: Relationship between bronchial responsiveness and clinical evolution in infants who wheeze: a four-year prospective study, Am J Respir Crit Care Med 164:1382, 2001.

172. Springer C, Godfrey S, Picard E, et al: Efficacy and safety of methacholine bronchial challenge performed by auscultation in young asthmatic children, Am J Respir Crit Care Med 162:857, 2000. 173. Cockcroft DW, Killian DN, Mellon JJA, et al: Protective effect of drugs on histamine-induced asthma, Thorax 32:429, 1977. 174. Popa VT: Bronchodilating activity of an H1 blocker, chlorpheniramine, J Allergy Clin Immunol 59:54, 1977. 175. Malik S, O'Reilly J, Sudlow MF: Effects of sublingual nifedipine on inhaled histamine and methacholine-induced bronchoconstriction in atopic subjects, Thorax 37:230, 1982. 176. Williams DO, Barnes PJ, Vickers HP, et al: Effect of nifedipine on bronchomotor tone and histamine reactivity in asthma, Br Med J 283:348, 1981. 177. Fish JE, Norman PS: Effects of the calcium channel blocker verapamil on asthmatic airway responses to muscarinic, histaminergic, and allergenic stimuli, Am Rev Respir Dis 133:730, 1986. 178. Jarjour NN: Circadian variation in allergen and nonspecific bronchial responsiveness in asthma, Chronobiol Int 16:631, 1999. 179. Murray AB, Ferguson AC, Morrison B: Airway responsiveness to histamine as a test for overall severity of asthma in children, J Allergy Clin Immunol 68:119, 1981. 180. Ryan G, Latimer KM, Dolovich J, et al: Bronchial responsiveness to histamine: relationship to diurnal variation of peak flow rate, improvement after bronchodilator and airway caliber, Thorax 37:423, 1982. 181. Hopp RJ, Bewtra AK, Nair NM, et al: Specificity and sensitivity of methacholine inhalation challenge in normal and asthmatic children, J Allergy Clin Immunol 74:154, 1984. 182. O'Brien IM, Newman-Taylor AJ, Burge PS, et al: Toluene diisocyanate-induced asthma. II. Inhalation challenge tests and bronchial reactivity studies, Clin Allergy 9:7, 1979. 183. Hargreave FE, Ramsdale EH, Pugsley SO: Occupational asthma without bronchial hyperresponsiveness, Am Rev Respir Dis 130:513, 1984. 184. Levy DA, Mak H, Fish JE: Dander-induced asthma associated with normal airway reactivity, J Allergy Clin Immunol 69(suppl):158, 1982 (abstract). 185. Banks DE, Sastre J, Butcher BT, et al: Role of inhalation challenge testing in the diagnosis of isocyanate-induced asthma, Chest 95:414, 1989. 186. Fish JE: Unpublished observations. 187. Casale TB, Rhodes BJ, Donnelly AL, et al: Airway reactivity to methacholine in nonatopic asymptomatic adults, J Appl Physiol 64:2558, 1988. 188. O'Connor GT, Sparrow D, Weiss ST: A prospective, longitudinal study of methacholine airway responsiveness as a predictor of pulmonary function decline. Normative Aging Study, Am J Respir Crit Care Med 152:87, 1995.

189. Barter CE, Campbell AH: Relationship of constitutional factors and cigarette smoking to decrease in 1-second forced expiratory volume, Am Rev Respir Dis 113:305, 1976. 190. Rasmussen F, Taylor DR, Flannery EM, et al: Outcome in adulthood of asymptomatic airway hyperresponsiveness in childhood: a longitudinal population study, Pediatr Pulmonol 34:164, 2002. 191. Ramsdale EH, Morris MM, Roberts R, et al: Bronchial responsiveness to methacholine in chronic bronchitis: relationship to airflow obstruction and cold air responsiveness, Thorax 39:912, 1984. 192. Bahous J, Cartier A, Ouimet G, et al: Nonallergic bronchial hyperexcitability in chronic bronchitis, Am Rev Respir Dis 129:216, 1984. 193. McFadden ER Jr: Exertional dyspnea and cough as preludes to acute attacks of bronchial asthma, N Engl J Med 292:555, 1975. 194. Corrao WM, Braman SS, Irwin RS: Chronic cough as the sole presenting manifestations of bronchial asthma, N Engl J Med 300:633, 1979. 195. Pratter MR, Irwin RS: The clinical value of pharmacologic bronchoprovocation challenge, Chest 85:260, 1984. 196. Enarson DA, Chan-Yeung M, Tabona M, et al: Predictors of bronchial hyperexcitability in grain handlers, Chest 87:452, 1985. 197. Cartier A, Pineau L, Malo JL: Monitoring of maximum expiratory peak flow rates and histamine inhalation tests in the investigation of occupational asthma, Clin Allergy 14:193, 1984. 198. Woolcock AJ, Jenkins CR: Assessment of bronchial responsiveness as a guide to prognosis and therapy in asthma, Med Clin North Am 74:753, 1990. 199. Mauger EA, Mauger DT, Fish JE, et al: Summarizing methacholine challenges in clinical research, Control Clin Trials 22:2445, 2001.

Section C - Physiology

671

Chapter 41 - Structural and Functional Cutaneous Immunology

Douglas A. Plager

Kristin M. Leiferman Mark R. Pittelkow

The skin is the largest organ of the human body—in the average adult, it covers an area of 1.5 to 2.0•m2 . It comprises two principal layers, the epidermis and the dermis ( Color Plate 4 ). The epidermal cells are modified epithelial cells and rest on a basement membrane that separates them from the underlying mesenchymal layer or dermis. Vital interactions occur between the cells of the epidermis, dermis, and peripheral blood[

1] [2]

( Table 41-1 ) as a mechanism for maintaining the

3

skin's structure and homeostatic and protective functions. [ ] In addition, blood vessels, lymphatics, nerve tissue, and specialized epidermal appendages (i.e., hair follicles and sweat and sebaceous glands) maintain various skin functions (see Color Plate 4 ). Recognized activities include thermoregulation, sensation, metabolism (e.g., vitamin D synthesis), physical barrier function (e.g., prevention of desiccation; protection from ultraviolet light, mechanical trauma, and chemical irritants or toxins), and immunologic barrier function. Although each of these functions is critical to an individual's health, the main focus of this chapter is on the structural and cellular aspects of the skin as they relate to cutaneous immune function.

CELLS AND STRUCTURE OF THE SKIN Cells of the Epidermis The epidermis is composed primarily of cells; it is approximately 150••m thick and is schematically represented in Color Plate 5 . The projections of the epidermis into the underlying papillary dermis are referred to as rete pegs. The most abundant epidermal cell type is the keratinocyte (approximately 90% of cells). Keratinocytes are continually renewing cells that are roughly divided into four types: basal (stratum germinativum), spinous (stratum spinosum), granular (stratum granulosum), and cornified (stratum corneum) keratinocytes. [ acidic (type I) and basic-neutral (type II) keratin

3] [4] [5]

6 7 proteins.[ ] [ ]

The keratinocytes of the different epidermal layers characteristically express pairs of various

Several elements control keratinocyte proliferation and differentiation, including growth factors, 5

cytokines, neuropeptides, calcium, and cell-cell and cell-matrix interactions.[ ] Some of the critical signals regulating keratinocyte development originate from the 8 9

dermis.[ ] [ ] Most importantly, keratinocytes are key participants in host defense against environmental exposures via both physical and immunologic barrier functions. Nonmigrating basal keratinocytes rest on the basement membrane, a structure that divides the epidermis from the dermis. They are tethered to the basement 10 11 12

membrane by protein structures called hemidesmosomes; known proteins in hemidesmosomes are listed in Color Plate 6 . [ ] [ ] [ ] Basal keratinocytes form a single layer of columnar-shaped cells expressing keratins K5 (basic-neutral) and K14 (acidic), and their three recognized subtypes are distinguished by mitotic activity.[

5] [13]

One subtype includes the so-called epidermal stem cells that constitute approximately 5% to 10% of basal keratinocytes. These cells retain 14

radiolabeled thymidine because of infrequent cell division, and they also express high levels of markers such as β1-integrin.[ ] Transient amplifying cells are a second subtype and constitute the majority of basal keratinocytes. These cells divide to produce the third subtype, postmitotic keratinocytes, which progressively differentiate and migrate toward the surface of the skin. During migration, postmitotic keratinocytes gradually flatten, lose their cellular organelles, dehydrate, and

modify their protein and lipid composition toward a more impermeable, cornified composition.[ the stratum corneum and another 14 days before desquamation from the surface of healthy

15]

The postmitotic basal keratinocyte requires 14 days to migrate to

16 17 18 skin.[ ] [ ] [ ]

Suprabasal spinous keratinocytes are named for their abundant spinelike desmosomes. Desmosomes serve to attach adjacent keratinocytes to one another—in contrast to hemidesmosomes, which attach keratinocytes to the basement membrane—and to provide resistance to mechanical stress. Known proteins in desmosomes 10 11 12

19 20

21 22

23 24

are listed in Color Plate 6 .[ ] [ ] [ ] Adherens junctions,[ ] [ ] gap junctions,[ ] [ ] and tight junctions[ ] [ ] further mediate interkeratinocyte attachment and communication. Changes in keratin composition also accompany the conversion to a spinous cell. For example, K1 (basic-neutral) and K10 (acidic) keratins are newly synthesized in spinous keratinocytes.[ spinous cells as

15 well.[ ]

6] [7]

Lamellar granules containing precursors of stratum corneum lipids are first evident in the cytoplasm of upper 4

These granules contain glucosylceramides that are the precursors for ceramides, the principal lipid of stratum corneum.[ ] Lamellar granules 4

also contain a variety of other lipids, including sterols and triglycerides, [ ] glycoproteins, and enzymes, including lipases, glycosidases, proteases, acid hydrolases, and phosphatases.[

5]

Granular keratinocytes are identified by their basophilic keratohyalin granules, which contain mostly profilaggrin,

672

TABLE 41-1 -- Cells of Human Skin Resident Cells of Healthy Skin Epidermis (% of Epidermal Cells)

Dermis

Specialized Structure

Recruited Cells

Kertinocyte (≅90%)

Fibroblast

Endothelial cell

Neutrophil

Langerhans'cell (≅5%)

Mast cell

Pericyte and smooth muscle cell

Eosinophil

Melanocyte (≅3%)

Macrophage

Schwann cell and nerve axon

Basophil

Merkel cell

Dermal dendritic cell

Hair follicle cell

T lymphocyte

*

†

Epidermal T lymphocyte

Dermal T lymphocyte

Sebocyte Eccrine gland cell Apocrine gland cell

B lymphocyte (?) Natural killer cell (?) Natural killer T cell (?) Monocyte

†

Blood dendritic cell Mast cell precursor

†

†

(?), Recruitment of this cell type to skin remains uncertain. * All nonmacrophage dendritic cells normally present in the dermis. † Corresponds to a resident cell type.

5

loricrin, and keratin intermediate filaments.[ ] Posttranslational modification of K1 and K10 keratin to K2 and K11 keratin, respectively, occurs in granular keratinocytes.[

6] [7]

Granular keratinocytes are the penultimate differentiation stage before cornification. Their lamellar granule contents are released into the

intercellular space between the stratum granulosum and stratum corneum.[ to form the multilamellar lipid matrix of the stratum

26 corneum.[ ]

25]

A recently proposed model challenges the concept that discrete lamellar granules fuse

Nonetheless, the ultimate process forms a hydrophobic lipid seal at the interface between the 27

granular and cornified layers that impedes transepidermal water loss. [ ] Proper formation of this lipid barrier requires a variety of enzymatic proteins and host intake of essential fatty acids. The programmed conversion of granular keratinocytes to cornified keratinocytes also involves numerous degradative enzymes that break down cell organelles and nuclei; these enzymes include DNases, RNases, proteases, phosphatases, esterases, acid hydrolases, plasminogen activator, and, possibly, predesquamin.[

3]

Development into a cornified keratinocyte includes profilaggrin proteolysis to filaggrin (filament aggregation protein) monomeric subunits. Filaggrin appears to 28

serve as the matrix protein that embeds and promotes aggregation and disulfide bonding of keratin filaments.[ ] The cornified cell envelope, a proteinaceous layer just below the plasma membrane, also develops during cornification and involves interprotein disulfide formation and N-(gamma-glutamyl)lysine cross-linking by 15] [29]

transglutaminase.[

Proteins detected in the cornified cell envelope include loricrin (75% of protein mass of the cornified cell envelope), involucrin 29

(transglutaminase cross-linked), keratolinin, small proline-rich proteins, elafin (a serine protease inhibitor), and envoplakin.[ ] Ultimately, the flat, polyhedral cornified keratinocytes of the stratum corneum lose their nuclei, and eventually their desmosomal connections, and form a barrier via their high molecular weight, intermolecular disulfide-bonded keratins (up to 80%), and cornified cell envelopes interlayered with lipid lamellae (primarily ceramides, cholesterol, and free fatty 4] [18] [29]

acids) derived from lamellar granules.[

The thickness of the stratum corneum can vary from as low as 15 to 25 cornified cell layers (approximately 15••m

thick) on many body areas to hundreds of cornified cell layers on the palms and soles.[

30]

A second epidermal cell type interspersed among the living keratinocytes of the substratum corneum layers is the Langerhans' cell (see Color Plate 5 and Table 411 ). [

31] [32]

These bone marrow–derived cells comprise approximately 5% of epidermal cells and are a major histocompatibility class II (MHC II)–bearing dendritic

cell subset[

33]

that most likely develop from a dermal CD14+ cell population.[

protein (MIP)-3α (i.e., CCL20)–directed

35 chemotaxis,[ ]

34]

Their epidermal localization may, in part, result from macrophage inflammatory

precursor cutaneous lymphocyte-associated antigen (CLA) expression,[

36] [37]

and E-cadherin mediated

38

39 40 41

adhesion to keratinocytes.[ ] However, the role for MIP-3α in basal recruitment of the Langerhans' cell to the epidermis has recently been questioned.[ ] [ ] [ ] Their highly dendritic structure allows efficient antigen sampling of virtually the entire surface of the skin. The density of Langerhans' cells varies somewhat over the 42] [43]

surface of the body, with the face being most heavily populated at 600 to 1000 Langerhans' cells per square millimeter.[

Langerhans' cells can be recognized

by electron microscopy, which demonstrates their unique rod- and racket-shaped cytoplasmic Birbeck granules (Lag antibody reactive [ immunohistochemical staining with antibodies recognizing adenosine triphosphatase (ATPase) or

46 47 48 CD1a.[ ] [ ] [ ]

49]

cells may retain their phagocytic activity after activation in

43] [45]

),[

and by

They are phagocytic cells, and the calcium-

dependent (C-type) lectin, langerin, functions in antigen capture and subsequent induction of Birbeck granule formation.[ 50 vivo.[ ]

44]

Contrary to in vitro studies, Langerhans'

Notably, with respect to allergic disease, a unique high-affinity immunoglobulin E (IgE) 51]

receptor, Fc epsilon receptor type I (FcepsilonRI), with an αγ2 subunit composition[

is expressed on Langerhans' cells[

52] [53]

and can greatly “facilitate”

presentation of IgE-bound antigen. The antigen-presentation capabilities of Langerhans' cells are only revealed after interleukin-1β (IL-1β)- and tumor necrosis factor-α (TNF-α)-induced migration toward skin-draining lymph nodes.[ T cells in the lymph node and initiate antigen-specific

54] [55] [56] [57] [58]

After this cytokine-induced migration, Langerhans' cells can prime naïve

673

T cell immunity, as well as possibly present antigen intracutaneously to previously activated effector or memory T cells.[

43] [59]

Intraepidermal T lymphocytes within normal human skin predominantly express αβ T cell receptors (TCRs) and a restricted Vα and Vβ TCR repetoire.[

43] [60] [61]

60

They are irregularly distributed,[ ] constitute fewer than 1% of epidermal cells and only about 2% of all normal skin T cells, and reside within the basal and suprabasal layers of the epidermis. In contrast, mouse skin contains greater than 1% intraepidermal T lymphocytes expressing primarily γδ TCRs and exhibiting a 60

43 60 61

much more dendritic morphology. [ ] A majority of intraepidermal T cells in normal human skin are CLA+ and CD8+/CD45RO+.[ ] [ ] [ ] CLA and CD45RO expression are indicative of prior activation via cutaneous antigen exposure and a memory cell phenotype, respectively, suggesting that they are not naïve T cells; however, the precise functions of these and other intraepidermal T cells of normal skin remain unclear. 3 5 62 63

Melanocytes and Merkel cells make up the remaining cell types of the normal epidermis, and they reside primarily in the basal layer.[ ] [ ] [ ] [ ] As with the dermal mast cell (discussed later), melanocyte development and survival depend heavily on the cell surface tyrosine kinase receptor, c-kit, and its ligand, stem cell factor. However, melanocytes arise from embryonic neural crest cells, whereas mast cells originate from bone marrow CD34+ stem cells. Melanosomes are the distinctive cytoplasmic organelles of melanocytes and are the site of melanin formation, part of the tanning response to ultraviolet radiation exposure. Each minimally proliferating melanocyte interacts with approximately 36 basal or suprabasal keratinocytes via its dendritic extensions; however, no direct junctions form between these two cell types. Still, by an incompletely understood mechanism, the melanocyte transfers pigment to its associated keratinocytes. Similar to Langerhans' cells,

melanocytes utilize E-cadherins to adhere to keratinocytes. 3 5

Merkel cells perform a very different function than melanocytes, apparently acting as slow-adapting, type I mechanoreceptors.[ ] [ ] They are closely associated with nerves via synaptic contacts (i.e., the Merkel cell–axon complex) and are located in sites of high tactile sensitivity, including the so-called tactile discs or touch domes. These neuroendocrine epithelial cells also contain neurotransmitter-like substances in cytoplasmic granules, and these granules often localize in proximity to adjacent unmyelinated axons that innervate the dermis and epidermis. Cytokeratin 20 and villin are reliable markers for the Merkel cell. Interestingly, Merkel cells appear to develop directly from precursor epidermal cells, possibly keratinocytes, and may reside in the dermis of adult skin as well.[

3] [5]

Dermal-Epidermal Junction The dermal-epidermal junction (DEJ) functions to join the epidermis to the dermis. It can be subdivided into three layers visible by electron microscopy after 3 64 65

glutaraldehyde fixation: hemidesmosome-anchoring filament (including the lamina lucida), basement membrane (lamina densa), and anchoring fibril layers[ ] [ ] [ ] (see Color Plate 6 ). The DEJ is composed mainly of basal keratinocyte products, with a minor contribution from dermal fibroblasts. In addition to connecting the epidermis and dermis, it functions to protect against mechanical shear, to orientate cell growth, and to serve as a semipermeable barrier. The various proteins present 10 11 12 65

in each of these layers are indicated in Color Plate 6 . [ ] [ ] [ ] [ ] Within the hemidesmosomes are several macromolecules that attach the plasma membrane of the basal keratinocyte to the basement membrane. These include antigens initially recognized by serum from patients with bullous pemphigoid (BP) and designated BPAg230 (BPAg1) and BPAg180 (BPAg2 or type XVII collagen). BPAg230 resides within the basal keratinocyte but is exposed to the extracellular environment after trauma or ultraviolet radiation exposure. BPAg180 is a transmembrane protein, with the major portion extracellular and located within the lamina lucida. Another recently described antigen, P200, also appears to be expressed in basal keratinocytes and to function in hemidesmosome formation and stability. Additional proteins include plectin and α6β4- and α3β1-integrins. The lamina lucida is the layer most easily disrupted, as demonstrated by its susceptibility to separation by heat, suction, saline solutions, proteolytic enzymes, and autoimmune disease. The lamina lucida is composed of laminin 1 and laminin 5–cruciform, noncollagenous glycoproteins composed of a large α-chain intertwined with a β- and a γ-chain. Laminin 6 (K-laminin) is a Y-shaped isoform composed of β- and γ-chains that complexes with laminin 5 by disulfide bonding. BPAg180 also is located within the lamina lucida. The basement membrane proper, or lamina densa, is composed primarily of type IV collagen, which, along with heparan sulfate proteoglycan and entactin, is present in both DEJ and dermal blood vessel basement membranes. The lamina densa restricts passage of cationic molecules, facilitated by associated anionic sulfated proteoglycans, and molecules with a molecular mass greater than 40•kD. However, various cell types and neurites penetrate the lamina densa, most likely by use of cellular type IV collagenase and metalloproteases. Type IV collagen also makes up the anchoring plaques to which anchoring fibrils, comprised of relatively skin-restricted type VII collagen, attach. Instead of attaching to an anchoring plaque, anchoring fibrils can also attach to another location on the lamina densa, forming a loop through which dermal collagen fibers may traverse. Extracellular Matrix and Cells of the Dermis Unlike the epidermis, the normal dermis is relatively acellular. It is divided into the papillary dermis and the reticular dermis (see Color Plate 5 ). The papillary dermis is about twice the depth of the epidermis, approximately 300••m, and contains dermal papillae that interdigitate with epidermal rete pegs. The papillary dermis contains relatively fine extracellular matrix fibers and extends from the lamina densa to the upper (subpapillary) vascular plexus within the dermis. The

remaining dermis, approximately 2500••m in depth, contains thicker extracellular matrix fibers and is called the reticular dermis. 66]

The relatively few cells within the dermis are interspersed in an extracellular matrix that is composed mainly of collagen, approximately 72% of dry weight.[ 67

There are currently 15 recognized “types” of cutaneous collagen; type I and type III collagens are the most abundant in adult dermis.[ ] Collagen “molecules,” each composed of three individual polypeptides containing the characteristic repeating Gly-X-Y amino acid triplets, are arranged in various configurations–designated staggered, chicken wirelike, and antiparallel–that are

674

intermolecularly cross-linked to produce collagen “fibers.” These fibers provide mechanical strength to the dermis. 68 69

A successive network of elastic fibers are also part of the extracellular matrix of the dermis, approximately 4% of dry weight.[ ] [ ] There are three main “types” of elastic fibers: oxytalan fibers, elaunin fibers, and mature elastic fibers. They contain as much as 90% elastin, with lesser amounts of various other proteins including fibrillin, vitronectin, decay accelerating factor, and fibronectin. The elastic fiber network extends from the lamina densa through the dermis, with oxytalan fibers extending vertically from the DEJ to the interface between the papillary and reticular dermis, then converting to horizontally distributed elaunin fibers and, finally, to 3] [69]

mature elastic fibers in the reticular dermis.[

The elastic fiber network allows the skin to return to its original shape after stretching or deformation.

Dermal collagen fibers and elastic fibers are embedded in a hydroscopic “ground substance” formed by large proteoglycans of approximately 100 to 2500•kD that 3 70

71 72 73

account for up to 0.2% of the dry weight of the dermis. [ ] [ ] Each proteoglycan has a core protein (e.g., versican and decorin[ ] [ ] [ ] ), with one or more covalently attached glycosaminoglycan (GAG) polysaccharides, such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate or hyaluronic acid as a free GAG. Proteoglycan nomenclature depends on both the core protein amino acid sequence and the identity of the disaccharide subunits forming the attached 70]

GAG or GAGs.[

Proteoglycans influence dermal volume and compressibility through their substantial capacity to bind water. They also influence dermal cell

activity by binding growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF),[

74] [75] [76]

their receptors,[

77]

and

[78]

various cytokines. Besides their presence in the extracellular matrix, proteoglycans can be found intracellularly (e.g., a serglycin core protein–containing proteoglycan in mast cell secretory granules) and on membrane surfaces (e.g., a syndecan-2 core protein–containing proteoglycan on fibroblasts) of dermal cells. 70

Proteoglycans are expressed in the DEJ and epidermis of the skin as well.[ ] Fibronectin, thrombospondin, vitronectin, and tenascin are dermal glycoproteins (i.e., proteins glycosylated with non-GAG polysaccharides) that also influence dermal cell functions such as cell activation, migration, and differentiation. For example, Langerhans' cell migration involves the interaction of cellular β1-integrin with basement membrane laminin and extracellular matrix fibronectin.[

79]

The sparsely distributed cells of the dermis are relatively more abundant in the papillary dermis than the reticular dermis of normal skin. Mesenchyme-derived 3

dermal fibroblasts (see Table 41-1 ) synthesize and degrade extracellular matrix proteins, including collagen, elastin, proteoglycans, and fibronectin.[ ] Their activity is increased during wound healing. However, fibroblasts also secrete various soluble mediators involved in an immune response when stimulated by cytokines. [

80]

[81]

For example, eotaxin is produced by fibroblasts in response to IL-4 stimulation. [

cutaneous mast cell development.

82]

Stem cell factor expression by fibroblasts may also contribute to normal

[83]

Mast cells are present in subepithelial connective tissues throughout the body.[

3] [84] [85] [86]

Mast cells occur in normal skin at a density of approximately 7000 to

10,000/mm3 and are often found in close proximity to cutaneous appendages, blood vessels, and nerves.[ 84]

avidin conjugates are used to identify human mast cells in sectioned tissues.[ tryptase and chymase (designated MCTC

89 ).[ ]

84] [87] [88]

Metachromatically staining cationic dyes and

Cutaneous and intestinal submucosal mast cells have granules containing both 89]

In contrast, lung and intestine mucosal mast cells primarily express only tryptase (designated MCT ),[

populations that express only chymase (designated MCC ) have also been reported.[

84]

and mast cell

By electron microscopy, human mast cells are identified by their villous cell

3

surface projections and numerous dark-staining cytoplasmic granules.[ ] Latticelike and scroll-like structures within granules predominate in MCTC and MCT , 86]

respectively.[

Additional functional heterogeneity of mast cells also exists.[

86]

Their development in peripheral tissues from bone marrow– derived stem cells

depends primarily on c-kit receptor and its ligand, stem cell factor (also known as c-kit ligand), as well as IL-3.[

84] [86]

Mast cell involvement in the immediate

allergic reaction is well documented and is exerted via its stores of histamine, various other mediators, and surface FcepsilonRI-bound IgE.[

90]

Interestingly, mast 91

cells may be directly linked to induction of IgE synthesis by B cells; however, this may not be relevant in skin because B cells are typically absent.[ ] In addition to numerous preformed mediators stored in their granules, mast cells synthesize and release growth factors, cytokines including TNF-α (also preformed) and IL-4, and 84] [86]

lipid mediators such as leukotrienes, prostaglandins, and platelet-activating factor.[ parasites and

92 93 bacteria,[ ] [ ]

Furthermore, mast cells probably participate in microbial defense against

in control of vascular tone and permeability via histamine and leukotriene release, in tissue repair and angiogenesis,[

sensation and response to a variety of immunologic (C5a, TNF-α, and IgE) and nonimmunologic (physical and chemical)

macrophages in skin are numerous and include effector phase processing and presentation of activity, and general phagocytic and secretory activities. lauryl sulfate) cutaneous

101 inflammation.[ ]

[3] [100]

wound healing

99 activities,[ ]

3] [96]

The functions of

microbicidal/tumoricidal

Furthermore, CD64+ macrophage-like cells appear capable of mediating irritant-induced (sodium

The CD68 surface marker distinguishes and identifies the macrophage within skin.

Dermal dendritic cells, which include dermal dendrocytes,[

102]

3] [96]

represent a fourth, heterogeneous cell population of the normal dermis.[

evaluation allows distinctions to be made among macrophages, dermal dendrocytes, and the other dermal dendritic

3 96 cells[ ] [ ]

103]

104 cells[ ]

Careful histologic

; however, distinguishing these cell

types can be difficult in the presence of an inflammatory reaction because of increased heterogeneity and cellular activity among them.[ are present in close proximity to mast

and in

84 86 stimuli.[ ] [ ]

Dermal macrophages are bone marrow–derived, phagocytic cells that differentiate from blood monocytes after entering peripheral tissues.[ 97 98 antigen,[ ] [ ]

94] [95]

Dermal dendrocytes often

and blood vessels and, like other dermal dendritic cells, express markers common to antigen-presenting cells. The

precise functions of the various dermal dendritic cells and their relationships to cutaneous macrophages are still being elucidated. [

31] [34] [96] [100] [105]

Dermal T lymphocytes, located near postcapillary venules, comprise the vast majority, approximately 90%, of T cells present in normal dermis.[

3] [60]

An estimated

106]

70,000 lymphocytes are present in a 4-mm punch biopsy of normal skin; [ [107]

B lymphocytes and natural killer lymphocytes are almost absent from normal skin.[

60]

Dermal T lymphocytes are typically CD45RO+ with approximately equal numbers representing

675

CD4+ helper and CD8+ cytotoxic cells. As with their epidermal counterparts, however, little is known about the roles of dermal T lymphocytes in cutaneous homeostasis and immunity. Specialized Structures and Associated Cells 108 109 110 111

Beyond the normal cellular constituents that have been elaborated, the skin includes an extensive network of blood and lymphatic vessels[ ] [ ] [ ] [ ] (see Color Plate 4 ). The myriad of cutaneous blood vessels, consisting of arterioles, capillaries, venules, and anastomoses, is indicative of the skin's important thermoregulatory role. Blood and lymph vessels are typically present together, but they never anastomose. Both are arranged into an upper and a lower plexus, and capillary loops and deeper open-ended lymphatic vessels extend into the dermal papillae (see Color Plate 5 ). Vessels extending vertically connect the upper and lower plexus vessels of both the blood and the lymphatic vasculature. Each day, half of the total circulating protein escapes from blood vessels, and the lymphatic vessels return the extravasated fluid and macromolecules into the bloodstream, thus maintaining plasma volume and preventing increased tissue pressure.[ lymphatics and blood vessels have a continuous endothelial cell

109 lining[ ]

111]

Both

(see Color Plate 5 ), although the respective endothelial cells exhibit distinct phenotypes.

[111] [112]

In general, lymphatic vessels tend to be less densely distributed, have wider and more irregular lumens, and have thinner vessel walls. Perivascular veil cells of unknown function and smooth muscle cells (bordering arterioles) or pericytes (bordering capillaries and venules) are intimately associated with dermal blood vessels[

109]

108] [109]

(see Color Plate 5 ). Both smooth muscle cells and pericytes have contractile function, [

while fine cytoplasmic and extracellular anchoring 111

filaments serve a contractile function for separating overlapping lymphatic endothelial cells in response to increased interstitial fluid pressure.[ ] Integration of elastic fibers into the lymphatic vessel wall also facilitates fluid collection by shuttling interstitial fluid toward the vessels. Finally, tight and adherens junctions are more frequently seen in blood vessels than in lymphatics. Notably, higher-molecular-weight molecules and particles, such as fluorescein-5-isothiocyanate-labeled 111]

dextran and cell debris, more readily enter lymphatic vessels than blood vessels. [

Blood vessel endothelial cells participate in inflammation and coagulation regulation via their expression of such molecules as cytokines, prostaglandins, von 110 113

Willebrand factor, and tissue factor.[ ] [ ] They also allow entry of cells and molecules into the dermis (typically at postcapillary venules) (see Color Plate 5 ). Cellular entry involves the expression of endothelial cell surface adhesion molecules such as P-selectin (also sequestered in endothelial cell Weibel-Palade bodies), Eselectin, intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and chemokines.[

59] [114] [115] [116] [117] [118]

their counterligands on the surface of circulating leukocytes (e.g., E-selectin binding to T cell CLA) to allow the extravasation of these cells. 111 124

These interact with

[119] [120] [121] [122] [123]

However, adhesion molecules do not appear to be expressed on lymphatic endothelial cells. [ ] [ ] Therefore, it is unclear whether the exit of cells (e.g., activated Langerhans' cells) from the skin via open-ended lymphatic vessels is actively mediated by similar ligand-counterligand interactions. Immunostaining readily

distinguishes blood vessels from lymph vessels based on expression of several mutually exclusive endothelial cell markers, such as PAL-E (blood vessels) and 111]

LYVE-1 (lymph vessels).[

3 125 126 127 128 129

Skin is also extensively innervated[ ] [ ] [ ] [ ] [ ] [ ] ( Color Plate 7 ; see Color Plate 4 ). Cutaneous innervation consists of two classes of nerve fibers: afferent sensory and efferent autonomic axons. Sensory and postganglionic autonomic nerve fibers are codistributed by large cutaneous branches of musculocutaneous nerves that arise segmentally from spinal nerves or, for the face, from branches of the trigeminal cranial nerve.[

125] [126]

The main subcutaneous

nerve trunks branch to form a deep nerve plexus at the subcutaneous-dermal junction and a superficial nerve plexus in the papillary dermis.[ 125]

innervating nonfacial skin, as many as 1000/cm2 , [

125]

Sensory nerve fibers

travel as individual continuous axons bundled within peripheral nerves that extend from a single dorsal root 126]

ganglion of the spinal column. This leads to segmental sensory innervation of the skin (dermatomes). [

In contrast, the autonomic nervous system typically uses 126 127

two synaptically coupled neurons, the preganglionic and the postganglionic neurons, to connect the central nervous system with peripheral organs.[ ] [ ] This is the case for skin where, consistent with their functional classification, autonomic nerves innervate involuntary vascular smooth muscle, arrector pili muscles of hair follicles, sweat glands, and sebaceous glands.[

125]

Individual nerve axons in the skin can be either myelinated (i.e., Schwann cell membrane repeatedly wrapped

around the axon) or nonmyelinated (i.e., axon sheathed by fenestrated invagination of the Schwann cell membrane).[ include myelinated A-β and A-δ fibers and nonmyelinated

125 126 C-fibers.[ ] [ ]

126]

In general, these cutaneous nerve axons

Nerve impulse conduction velocity, fiber diameter, sensitivity to anesthetics, and 126]

function (e.g., sensing touch, vibration, heat, or itch) are additional variables used to distinguish the various cutaneous nerve fibers.[

Autonomic activities in skin appear to be mediated by nonmyelinated postganglionic C-fibers, although other C-fibers also have an important role in sensory function. [125] [126]

These autonomic fibers are classified as adrenergic (containing catecholamines), cholinergic (containing acetylcholine), or purinergic (containing ATP or

related purines).[

125]

Notably, all three subclasses of autonomic C-fibers innervate the microcirculation, with adrenergic fibers mediating vasoconstriction and 125]

cholinergic fibers mediating vasodilation. [

Afferent sensory nerves perceive the external environment. An important terminal sensory nerve structure is the so-called “free nerve ending” of A-δ and C-fibers. [125] [126] [125]

These nerve fibers are ensheathed by Schwann cells and a basal lamina. They are present in the dermis and are particularly abundant in the papillary dermis. 125]

However, they lose much of their protective sheath upon penetrating into the epidermis, [

polymodal C-fiber free nerve endings innervate all vital layers of the

130 epidermis[ ]

thus acquiring the designation of free nerve endings. A-δ and 131] [132]

and appear capable of sensing pain, itch,[

125 126 pressure.[ ] [ ]

noxious stimuli, temperature,

and Specific peripheral C-fibers within skin convey the sensation of itch to the lamina I spinothalamic tract neurons within the spinal cord.[ Free nerve endings can also innervate specialized skin structures (e.g., hair follicles). Other terminal nerve structures innervated primarily by A-β nerve fibers include Merkel cell–associated “touch spots” and Meissner's, Pacinian, and

676

132]

125 126

Ruffini's corpuscles, which are believed to sense different modes of deformation of the skin—touch, vibration, and stretch, respectively.[ ] [ ] Efferent activities of sensory nerves in cutaneous inflammation and wound healing are also recognized; for example, sensory nerves release neuropeptide, which leads to vasodilation 128] [133] [134]

and increased vasopermeability.[

Similarly, autonomic nerve fibers produce substances capable of inducing vasoconstriction and vasodilation, such as

3 peptide.[ ]

neuropeptide Y and antinatriuretic Neuropeptides also activate non-neuronal cell types residing in or entering the skin. For example, calcitonin generelated peptide (CGRP) and substance P released by sensory nerves probably modulate Langerhans' cell activity and endothelial cell adhesion molecule expression, 135]

respectively.[

Hair follicles,[

128]

Substance P also induces mast cell TNF-α synthesis and release.[

136] [137]

sebaceous glands,[

follicle's role in cutaneous immunology is 143 dermcidin,[ ]

138] [139]

and apocrine and eccrine sweat glands[

142 limited.[ ]

140] [141]

are composed of differentiated cell types. Knowledge of the hair

However, secretions from sebaceous glands (e.g., free fatty acids) and from sweat glands (lactic acid,

and immunoglobulin) contribute to innate and acquired immunity (see later discussion).

CUTANEOUS IMMUNOLOGY Innate Immunity A vital function of the cutaneous immune system[

43] [144] [145] [146] [147]

is to defend against pathogenic organisms (bacteria, viruses, fungi, and parasites). Passage 148

of a pathogenic organism beyond the skin's surface can occur after physical, chemical, radiant energy, or direct pathogen insult to healthy skin ( Color Plate 8 ).[ ] The first line defense against such an insult is the innate immune system. Innate immunity of the skin can be roughly divided into two separate categories: constitutive innate immunity, involving an anatomic and physiologic barrier, and inducible innate immunity, involving an acute inflammation and cellular infiltration barrier. Neither constitutive nor inducible innate immunity of the skin demonstrates acquired specificity or memory for an invading pathogen. Therefore the immune protection provided by these two functional barriers is essentially unchanged regardless of the number of previous encounters with a particular pathogen. Cutaneous constitutive innate immunity consists of (1) normal skin flora, (2) cornified keratinocytes, (3) constitutively expressed antimicrobial polypeptides and lipids, (4) low pH, and (5) normal body temperature (see Color Plate 8 ). The currently recognized normal flora of the skin includes various bacteria (primarily coryneforms and staphylococci) and, to a much lesser extent, fungi (primarily Malassezia). [ [145] [150]

149]

These microorganisms assist the host by competing with other, more

3 keratinocytes[ ]

pathogenic organisms for resident status. Interlocking cornified form a relatively impenetrable surface, and the outward growth and shedding of cornified keratinocytes help to eliminate superficially bound pathogens. The reduced water content of these cells and the underlying lipid layers of the stratum corneum reduce the relative humidity at the skin surface, providing a less-than favorable environment for pathogenic organisms. The constitutive presence of protective antimicrobial polypeptides includes β-defensin-1 and -2,[ [153] [154]

151]

dermcidin,[

143]

152]

iron-binding proteins,[

lysozyme,[

152]

RNases, DNases, and natural IgM

on the skin from sweat and from keratinocytes entering their final stages of cornification. Similarly, some epidermal lipids,[

sebum-derived fatty acids, exhibit antibacterial activity via their ability to reduce skin surface skin surface pH, and normal body temperature inhibits the growth of some pathogens.

145 156 157 pH.[ ] [ ] [ ]

155]

including keratinocyte- and

Lactic acid excreted in eccrine sweat also lowers

A key initiator of cutaneous inducible innate immunity and a distinguishing feature of skin is the abundant preformed IL-1α stored in the cytoplasm of keratinocytes. [158] [159]

160

If skin integrity is disturbed with concurrent disruption or stimulation of epidermal keratinocytes, IL-1α is directly liberated into the skin.[ ] Even mechanical deformation appears sufficient to induce IL-1α liberation and this may contribute to the itch/scratch-associated changes that often accompany allergic skin disease.[

160]

Release of IL-1α (a so-called “primary” cytokine, along with TNF-α, based on their early and broad inflammatory and immunologic activities) 59] [159]

appears to be key in initiating a cascade of events (see Color Plate 8 ), in part mediated by cellular nuclear factor-κB (NF-κB).[ classic signs of acute inflammation: redness, heat, swelling, and

161 pain.[ ]

These events contribute to the

Important among these events is the induced expression of inflammatory cytokines,

*

chemokines, mediators, and adhesion molecules (see Color Plate 8 ). For example, although the precise molecular response to different antigens and pathogens varies,[

169] [170] [171] [172] [173] [174] [175]

the principal IL-1α-induced molecules of a typical cutaneous inflammatory response include additional IL-1α, IL-1β, TNF-

α, IL-8 (i.e., CXCL8), nitrous oxide synthase, and prostaglandin-producing cyclooxygenase.[ 118 159 E-selectin.[ ] [ ]

159]

IL-1α also induces postcapillary venule endothelial cell expression 159

of ICAM-1, VCAM-1, and Moreover, IL-1α and the other induced molecules activate most cell types of the skin,[ ] alerting and preparing them for further host defense functions including additional cytokine and chemokine secretion, wound repair, release of antimicrobial products, phagocytosis, and 145 159 176

initiation of acquired immune responses.[ ] [ ] [ ] Thus, IL-1α is an important contributor to the inducible acute inflammation barrier of cutaneous innate immunity. Together with other triggers such as plasma-derived kinins, neuropeptides, and mast cell–derived histamine and eicosanoids, the cutaneous inflammatory response ultimately leads to vasodilation and increased vasopermeability, which allow serum protein exudate and leukocytes to enter the injured tissue (see Color 134] [176] [177]

Plate 8 ).[

Under certain circumstances, cutaneous injury occurs without overtly exceeding the inherent capacity of constitutive immunity, such that inflammation followed by tissue repair is sufficient to manage the damage. However, if disruption of the constitutive skin barrier allows significant penetration of commensal microorganisms or pathogens, innate immunity molecules with broad specificity and relatively targeted activity against intruding microbes prevent dissemination of infection and, when necessary, guide acquired immunity (see later discussion). These pathogen-targeted molecules can be broadly divided into soluble molecules (i.e., secreted pattern-recognition receptors [PRRs]) and cell surface molecules (i.e., endocytic and signaling PRRs), both of which are discussed later. Each represents a component of inducible innate immunity, as do early infiltrating

* References [

1] [118] [128] [133] [145] [159] [162] [163] [164] [165] [166] [167] [168]

.

677

leukocytes that express some of these molecules (see Color Plate 8 ). Important inducible pathogen-targeted soluble innate immunity molecules of the skin include (1) inducible antimicrobial polypeptides, (2) complement-activating and/or opsonin proteins, and (3) complement proteins. Included among the inducible antimicrobial polypeptides are the so-called “antimicrobial peptides,” βdefensin-2 and cathelicidin LL-37, that are distinct to epithelial surfaces and are produced by keratinocytes in skin. Their expression greatly increases beyond their

constitutive levels during an inflammatory response.[

178] [179] [180] [181] [182] [183] [184]

properties, such as chemotactic activity for various immune

185 cells.[ ]

Interestingly, these antimicrobial peptides also demonstrate other biologic

Interferon-α (IFN-α) and IFN-β are members of another class of antimicrobial polypeptides

172 186

that are important in antiviral defense.[ ] [ ] Unlike the antimicrobial peptides, the protective activities of IFN-α and -β are apparently related to antiviral effects on host cells rather than direct toxicity toward a pathogen. The complement-activating/opsonin molecules of the innate immune system include members of the acute phase proteins (C-reactive protein, serum amyloid protein, 187 188

189

and mannose-binding protein),[ ] [ ] the C3b fragment of complement component C3,[ ] and possibly natural IgM. The acute phase proteins are produced by the liver and supplied to the skin via the blood. Substantial (1000-fold) increases in serum concentrations of acute phase proteins occur in response to IL-1, IL-6, and TNF-α from activated macrophages at inflammatory sites.[

176]

Initial availability of the C3 complement component, as well as other complement proteins, is 189

provided by the minor but significant quantities produced by resident cells of the skin.[ ] These complement proteins are rapidly supplemented by an influx of additional complement proteins from blood, following vasodilation and increased vasopermeability resulting from the acute inflammatory response. Ultimately, complement activation in skin, as in other parts of the body, leads to generation of the opsonin C3b (which targets a pathogen for phagocytosis via binding to complement receptor-1), the anaphylatoxins C3a and C5a (which have relevant chemoattractant and mast cell degranulating activities), and the membrane attack 189

complex composed of C5b, C6, C7, C8, and C9 (which assembles to form a lethal pore in a target pathogen's external membrane)[ ] ; the complement system is discussed in Chapter 6 ). Pathogen-targeted molecules, such as antimicrobial peptides and complement-related proteins, represent soluble components of the inducible innate immunity barrier that function in skin to counter pathogen invasion. 187 188

Acute phase proteins have been categorized to a group of innate immunity molecules called pattern-recognition receptors (PRRs).[ ] [ ] These are host molecules that recognize pathogen-associated molecular patterns (PAMPs)—that is, molecules that are distinct to certain pathogens. PAMPs include unmethylated CpGs of bacterial DNA, double-stranded RNA (e.g., of influenza), mannans (on a wide range of microorganisms), gram-positive bacterial lipoteichoic acids, gram-negative bacterial lipopolysaccharide (LPS), bacterial peptidoglycan and N-terminal formyl-methionine, parasitic phosphoglycans, and fungal glucans/zymosan.[

169] [190] [191]

190

PRRs have been subcatergorized further as secreted, endocytic, or signaling type. [ ] Secreted PRRs include the soluble pathogen-targeted acute phase proteins; the endocytic and signaling PRRs are cell surface pathogen-targeted innate immunity molecules. The macrophage mannose receptor (present on dendritic cells and 192]

human dermal microvascular endothelial cells) [ uptake and lysosomal

187 degradation.[ ]

and the macrophage scavenger receptor are examples of endocytic PRRs that function to enhance microorganism

The LPS-binding CD14 molecule and members of the Toll-like receptor (TLR) family, which currently comprises 10 distinct

molecules, are vitally important and evolutionarily conserved signaling PRRs that lead to host cell activation after recognition of various pathogens.[

190] [191] [193]

Together, these secreted, endocytic, and signaling PRRs allow host cells to respond more effectively and with enhanced specificity to invading pathogens.[

169]

Resident macrophages and dendritic cells, activated during a cutaneous inflammatory response, are among the first phagocytic host cells capable of utilizing their cell surface PRRs (e.g., the mannose receptor) to rapidly ingest and destroy intruding pathogens. Moreover, inflammation-associated increases in chemotactic factors (e. g., chemokines) and postcapillary venule endothelial cell adhesion molecules (P- and E-selectin, ICAM-1, and VCAM-1) synergistically mediate integrin-dependent extravasation of PRR-expressing leukocytes[

117] [118]

(see Color Plate 8 ). In a typical cutaneous inflammatory response, neutrophils are the earliest infiltrating 145 171 176

leukocytes, as expected, due to early expression of neutrophil active CXC chemokines such as IL-8.[ ] [ ] [ ] Later, monocytes begin to extravasate into the inflammatory site (see Color Plate 8 ). After entering the tissue, opsonizing PRRs and cell surface PRRs on neutrophils and monocytes/ macrophages assist in

176 177 194]

phagocytosis and intracellular destruction of pathogens with cell-derived products such as lysozyme, defensins, and reactive oxygen intermediates.[ ] [ ] [ CD14 is a classic PRR expressed by neutrophils and tissue macrophages that mediates LPS-sensitive killing. However, different pathogens and antigens lead to 169 170 171 172 173 174 175

different patterns of host gene expression and influence the degree and composition of immune cell infiltration.[ ] [ ] [ ] [ ] [ ] [ ] [ ] For example, effective elimination of pathogens that are not easily phagocytosed, such as fungi and parasites, may require mechanisms that target excretion of toxic leukocyte 145

products directly onto the pathogen. Thus, signals generated by “frustrated” phagocytes,[ ] and presumably by PRRs recognizing PAMPs of pathogens that are difficult to phagocytose, direct the immune response toward more effective pathogen clearance. Functional differences between eosinophils and neutrophils may be relevant here. Although eosinophils are less phagocytic than neutrophils, they produce reactive oxygen species at the plasma membrane surface, not intracellularly, [195]

196

and they have been shown to readily degranulate and deposit toxic cationic proteins onto the surface of parasites.[ ] Therefore an immune response favoring eosinophil infiltration over neutrophil infiltration may be a more effective defense against parasitic infection. Furthermore, specific cell types other than neutrophils and monocytes and/or increased numbers of a particular cell type can preferentially enter skin under circumstances of chronic inflammation or disease (see Table 41197]

1 ). These include eosinophils,[

basophils,[

198] [199]

blood dendritic cell precursors,[ 60] [107]

later discussion), but rarely B cells or natural killer cells.[ accessibility of blood components and leukocytes

200]

201]

mast cells,[

202]

natural killer—like T (NK-T) cells,[

and T cells (see

Overall, the acute inflammatory response, leading to resident cell activation and increased

678

to the site of pathogenic incursion, together with various subclasses of PRRs, works synergistically to enhance direct killing and phagocytic pathogen clearance as part of inducible innate immunity in skin. Acquired Immunity Acquired immunity has often been considered distinctly separate from innate immunity. However, more recent studies are elucidating the impact that components of 159 188 190

203

204

the innate immune system have on acquired immunity.[ ] [ ] [ ] Some examples include the chemotactic activity of β-defensin-2[ ] and C5a[ ] for dendritic cells and/or T cells of the acquired immune system. In addition, engagement of PRRs (e.g., LPS receptor, mannose binding receptor, TLRs, and CD1a), expressed on cutaneous antigen-presenting cells such as Langerhans' cells, dermal dendritic cells, and macrophages,[ molecule expression (e.g., CD80 and CD86

[190] [207]

) and modulate cytokine expression

macrophages induces IL-12 secretion that favors type 1 helper T lymphocyte (Th1)

205] [206] [207]

205 patterns.[ ]

188 development.[ ]

most likely promote increased costimulatory

For instance, LPS binding to cell surface CD14 of

Likewise, mast cell–produced cytokines, such as IL-4[

209 histamine,[ ]

208]

55 TNF-α,[ ]

and

direct subsequent acquired immune responses toward a Th2 profile. Furthermore, IL-1, as well as are important in activating Langerhans' cells to migrate to lymphatic vessels within the papillary dermis, while other inflammation-associated molecules such as keratinocyte-produced MIP-3α (i.e., CCL20) lead to influx of additional Langerhans' cells into skin via interaction with Langerhans' cell CCR6.[

200] [210]

Therefore the potential for cutaneous innate

immunity to direct acquired immunity, such as generating a polarized Th1 or Th2 cytokine profile, is an investigative area of great interest.[

188] [109] [193] [211]

Migrating Langerhans' cells that have endocytosed antigen and have been activated by the inducible innate immune response exit the skin via lymph vessels (see Color Plate 8 ) and proceed to the draining lymph node. There they process and present antigenic peptide to naïve T cells in an initial step to develop acquired 57 59 210

immunity.[ ] [ ] [ ] Langerhans' cell antigen presentation can presumably involve either interaction of MHC class II-antigenic peptide with CD4+ T cell TCR (typically extracellular antigen presented) or MHC class I-antigenic peptide with CD8+ T cell TCR (typically intracellular antigen presented), as well as antigen 43] [59] [206] [212]

presentation via CD1a molecules (nonpeptide microbial antigens presented).[

The resulting activated antigen-specific effector or memory T cells

(either CD4+ or CD8+) expressing CLA (a modified form of P-selectin glycoprotein ligand-1) [

121]

are targeted to and infiltrate the inflammatory site, in part via the

interaction of CLA with E-selectin (CD62E) that is up-regulated during cutaneous inflammation (see Color Plate 8 ).[ expressed on most epidermal Langerhans' cells and some other cell

107 215 216 types.[ ] [ ] [ ]

59] [120] [213] [214]

Notably, CLA is also

Chemokines, such as TARC/CCL17 and CTACK/CCL27, and their 59 115 116 117

receptors, CCR4 and CCR10, respectively, also are critical to extravasation of effector or memory T cells into the skin.[ ] [ ] [ ] [ ] Under these circumstances, an effector or memory T cell infiltrates an inflammatory site and is reactivated to perform its particular function (e.g., cytokine secretion, cell killing) via intracutaneous presentation of its specific antigenic peptide. [

43] [59]

However, whether Langerhans' cells or other antigen-presenting cells of the dermis (e.g., dermal

dendritic cells, macrophages) represent the principal antigen-presenting cells in this subsequent elicitation phase remains debatable.[

96] [97] [101] [217]

145]

intraepidermal T cells that express the putative memory marker, CD45RO, may also contribute to the cutaneous immune response.[ (both CD4+ or CD8+ and effector or memory) are critical in cutaneous acquired immunity and

Similarly,

Therefore, CLA+ T cells

59 218 disease.[ ] [ ]

In addition to antigen-presenting cells and T cells, B cells are the other key cells of acquired immunity. Differentiated B cell (plasma cell)–derived antigen-specific antibody, particularly that of the IgE isotype, has a strong association with allergic diseases of the skin. B cells recognize relatively intact antigens, in contrast to T cells, whose activation involves antigenic peptide recognition. Thymus-dependent protein antigens do not induce antibody responses in the absence of T cells.[

176]

176

This is in contrast to thymus-independent polysaccharide and lipid antigens, which do induce antibody production with little or no T cell help.[ ] Antigen recognition occurs via surface immunoglobulins, all with identical specificity on a given B cell. In general, plasma cell precursor and memory B cell development, immunoglobulin class switching (from IgM or IgD to the other classes of IgA, IgE, or IgG), and somatic hypermutation all occur in secondary lymphoid germinal 207

centers such as skin-draining lymph nodes.[ ] Thus the apparent absence of cutaneous B cells and the necessity for relatively intact antigen suggests a need for antigens that enter the skin to be transported to B cells, as opposed to B cells' migrating to antigen in the skin. One possibility is that a relatively intact antigen enters the lymph fluid for transport to an antigen-specific B cell residing in a skin-draining lymph node; however, the exact mechanism of B cell activation induced by an 154]

antigen entering through the skin remains unclear. Nonetheless, all five classes of immunoglobulin have been detected in normal human sweat.[ secretory IgA (sIgA), which appears to arise from secretory epithelia of eccrine

154 glands.[ ]

Sebum may also contain

154 IgA.[ ]

This includes

However, unlike gut mucosa, antibody-

[60]

producing B cells (plasma cells) do not appear to reside in the skin, so the exact source of cutaneously secreted immunoglobulins remains unresolved. One known source is from the circulation; antigen-specific antibody can enter a cutaneous inflammatory site as part of the serum protein exudate (see Color Plate 8 ). Perhaps periglandular plasma cells are a source of cutaneous immunoglobulin as well. Antigen-specific immunoglobulins of the IgG1 and IgA isotypes appear capable of contributing to clearance of pathogenic organisms from the skin via typical Fcγ 176 207

154

receptor-mediated immune mechanisms (e.g., antibody-dependent cell cytotoxicity and complement fixation)[ ] [ ] and sIgA-coating of bacteria.[ ] However, individuals with selective IgA deficiency do not have an inordinate number of cutaneous infections, although reduced sIgA on the skin of atopic dermatitis patients,

219]

who often present with cutaneous infections, has been reported.[ attributable to compensatory protection via secretory

The relative lack of cutaneous infections associated with selective IgA deficiency may be

220 IgM.[ ]

Other immunoglobulin isotypes such as IgE and IgG4 have long been associated with allergic disease, and the so-called type 2 cytokines, IL-4 and IL-13, appear critical for class switching to IgE and IgG4 in humans. Furthermore, like other epithelial surfaces, the skin appears biased toward mounting a Th2 response to epicutaneous administration of intact protein

679

221 222

antigens.[ ] [ ] Therefore, unless tolerance develops or Th1-skewing signals are effectively transmitted during an immune response, Th2 costimulatory “help” to B cells appears to be the default response and will lead to IgE production. Subsequently, antigen-specific IgE, presumably supplied via the blood (although for 52 53

respiratory mucosa, local IgE production appears possible), can bind to cutaneous FcepsilonRI-bearing cells (i.e., Langerhans' cells,[ ] [ ] mast cells, and infiltrating basophils) and contribute to various forms of allergic skin disease. Antibodies specific for FcepsilonRI receptor may contribute to cutaneous diseases such as urticaria; antibodies against desmosomal and hemidesmosomal proteins may provoke immunobullous disease. Thus, like other aspects of the immune response B cell–derived antibodies typically maintain health but, when inappropriately directed, produce disease. As mentioned previously, neurotransmitters/neuropeptides can contribute to cutaneous inflammation. A dynamic dialog exists between the nervous system and the immune system.[ Langerhans'

125] [128] [223]

225 cells[ ]

227 degranulation[ ]

224]

For instance, immune cells liberate factors influencing neural tissue (e.g., neurotrophic factors produced by eosinophils[

) in concert with neural tissue influencing immune cells (e.g., neuropeptide regulation of Langerhans' cell

226 activity[ ]

and

and mast cell

). Clinically, stress can adversely influence symptoms of several cutaneous inflammatory diseases. This includes atopic dermatitis, in which stress228 229

induced changes in immune cells have been identified.[ ] [ ] Therefore the nervous and immune systems reciprocally balance and modulate one another, implicating neural input as an important component of cutaneous immunology that is only beginning to be understood. Overall, unlike the rapid mobilization of innate immunity, a “primary” acquired immune response after the encounter of an antigen for the first time requires several 145 146

days for fully functional antigen-specific T and B cells to develop.[ ] [ ] A “secondary” response (i.e., an immune response to a previously encountered antigen) begins more quickly, with full mobilization of memory T cells in approximately 1 day, and it is more robust, with substantially increased antigen-specific antibody titers produced by memory B cells. In toto, the cutaneous innate immune system controls an initial pathogenic assault while directing the more specific recognition and destruction of the pathogen via the acquired immune system. Modulation of skin immunity or disease via pathogen insult to organs other than the skin (e.g., gut, airways) is an additional level of complexity that is not well understood but warrants consideration. Resolution of Immune Reaction To control an infection, a sufficiently vigorous immune response involving innate and, if necessary, acquired immune mechanisms is required. However, after

clearance of the pathogen and reestablishment of the cutaneous barrier function (e.g., via hemostasis, granulation tissue formation, and re-epithelization), the immune reaction must be down-regulated to avoid continued, untoward damage to host cells and to allow complete wound repair (see Color Plate 8 ). The waning levels of inflammatory cytokines (e.g., IL-1α, TNF-α) and pathogen-derived antigens are principal pathways toward resolution of the inflammatory immune response; that is, with no antigen, there is no triggering of innate immune system PRRs or activation of acquired immune system cells. A proportional increase in antiinflammatory molecules such as IL-1ra (from keratinocytes) and TGF-β (from macrophages phagocytosing apoptotic cells and from regulatory T cells) also contributes to down145 230 231 232

233

234

235

regulation of the inflammatory response.[ ] [ ] [ ] [ ] Similarly, accumulation of extracellular adenosine,[ ] lipoxins,[ ] and stress proteins [ ] in inflamed tissue are implicated in down-regulation of an inflammatory response. Reciprocal regulation of Th1 and Th2 responses, as exemplified by IFN-γ inhibition of a Th2 230

236 237

response and IL-10 inhibition of a Th1 response (possibly via induction of increased TGF-β [ ] ), has also been demonstrated. [ ] [ ] Therefore, effective removal of the pathogen and a shift from a proinflammatory to an antiinflammatory molecular milieu are important components of immune reaction resolution. Apoptosis also contributes to immune reaction resolution—for example, through controlled elimination of activated peripheral T cells.[

238] [239]

In general, apoptotic 240

cell death occurs when a cell exceeds its natural lifespan, but it can be induced via specific cell-cell interactions such as cell surface Fas and Fas ligand binding.[ ] A cell-cell interaction that may have particular relevance to resolution of a cutaneous inflammatory immune response is the killing of activated macrophages by γδ T 241]

cells.[

However, as mentioned previously, unlike in the mouse, γδ T cells have not been observed in human skin. The nervous system may also help regulate 242]

inflammation, as suggested by delayed wound healing of denervated skin.[

Ideally, after exposure to a pathogen and resolution of the inflammatory immune response, the host will have developed appropriate and effective acquired immunity to the invading pathogen. Under these circumstances, a second exposure will result in more rapid and effective elimination of the offending pathogen. Memory T and B cells mediate this secondary response. However, an exaggerated inflammatory immune response is undesirable if it is directed against a commonly encountered and relatively innocuous antigen or autoantigen. In this instance, beyond thymic clonal deletion, peripheral anergic and suppressive mechanisms may be critical for 230] [243] [244] [245] [246]

avoiding atopic and/or autoimmune responses.[

For example, induction of T cell anergy (i.e., unresponsiveness) via antigen presentation by an 246

“amateur” antigen-presenting cell such as a keratinocyte is suggested as a method to control allergic contact dermatitis.[ ] Alternatively, IL-10–producing pulmonary dendritic cells after intranasal ovalbumin administration provoke development of regulatory (i.e., suppressor) CD4+ T cells that lead to immune tolerance 244]

of subsequent respired ovalbumin.[

247]

Similar IL-10–producing regulatory T cells also are important in moderating allergic contact dermatitis,[

play a beneficial role in effective specific

248 249 immunotherapy.[ ] [ ]

and IL-10 may

Therefore, mechanisms leading to immune tolerance toward noninfectious agents also appear to 250 251 252 253

be important in avoiding, and possibly treating, atopy and autoimmunity. [ ] [ ] [ ] [ ] Clinically, immunosuppression of cutaneous immune reactions is a mainstay of dermatology and includes the use of topical corticosteroids, tacrolimus, pimecrolimus, and ultraviolet radiation; however, much remains to be learned about the body's natural mechanisms for terminating or controlling both desirable and undesirable cutaneous inflammatory immune reactions.[ Dysregulation and Disease The skin has remarkable generative, homeostatic, and reparative properties. However, disturbances in these highly regulated

254]

680

states occur, and skin disease results. Under certain circumstances, acquired hypersensitivity develops to a substance that does not normally cause a reaction. In its broadest definition, this acquired hypersensitivity is referred to as allergy, and the offending substance is termed the allergen. A different definition of allergy derives from the pathophysiologic response: an allergic reaction involves histamine release, eosinophilia, and elevated IgE as a consequence of Th2 reactivity. Allergens may be proteins or other biologic antigens. They include substances such as autoantigens (FcepsilonRI), animal products, infectious agents, foods, drugs, chemical contactants, and physical agents. The pathologic consequences of allergy are varied and dependent on the organs involved. The Gell and Coombs classification has 176

been a commonly used means of categorizing hypersensitivity reactions,[ ] but many diseases do not fit the classification well. For example, atopic dermatitis appears to be a combination of type I and type IV reactivity. Other cutaneous diseases cross hypersensitivity types or are not able to be categorized into any specific type. 236

The more recent delineation of T helper cell types into Th1 and Th2 provides another way to define cutaneous immunologic disease.[ ] Classic cell-mediated reactivity (Gell and Coombs type IV), as found in contact dermatitis, graft rejection, granulomatous diseases such as sarcoidosis, and intracellular infections such as tuberculosis, is associated with Th1 activation. On the other hand, atopic dermatitis, urticaria, and angioedema are the result of Th2 activation and the production of cytokines that lead to IgE production and/or other antibody elaboration. Drug reactions (reviewed in Chapters 90 and 92 ), insect bites and stings (reviewed in Chapter 81 ), eosinophil-associated skin diseases (reviewed in Chapter 63 ), and immunobullous diseases are also in this group. The immunobullous diseases are 255

autoreactive diseases characterized by antibody production to various skin components, as summarized in Table 41-2 .[ ] Immunofluorescence tests of both tissue (biopsy specimens) and serum can be particularly helpful diagnostically in these disorders. Other autoreactive diseases include the various types of lupus erythematosus, cutaneous vasculitis, and the so-called connective tissue diseases in which antibodies to self-antigens (nuclear antigens) most likely play a pathogenic role, including scleroderma, dermatomyositis, and connective tissue disease overlap syndromes. Indirect immunofluorescence and serologic testing to detect 256 257 258

circulating antibodies is diagnostically important ( Table 41-3 ).[ ] [ ] [ ] The pathomechanisms leading to the development of these diseases are complex, and the molecular distinctions and similarities among these diseases are under intense investigation. Deviations in the skin's ability to generate and maintain its structural and compositional integrity also result in cutaneous disease. Here, the best examples are malignancies of the skin. Basal cell and squamous cell carcinomas result from dysregulated malignant growth of basaloid-follicular cells or of squamous cells of the epidermis, respectively. Less commonly, melanoma and, very rarely, Merkel cell tumors result from dysregulated malignant growth of melanocytes and Merkel cells in skin, respectively. Langerhans' cell histiocytosis is a complex disorder with several different presentations resulting from neoplastic growth of Langerhans' cells. Mastocytosis (reviewed in Chapter 84 ) also shows many different presentations. Virtually every cutaneous compartment may proliferate excessively, lose proliferative activity and atrophy, or overproduce or underproduce cell products that result in disease. Examples include various types of vascular proliferations, keratodermas, lymphedemas, and dermal mucinoses. As noted, any change in cutaneous structural or immunologic homeostasis may result in skin disease, and aberration in structure or immunology triggers a compensatory response. For example, cutaneous malignancies (basal cell carcinoma, squamous cell carcinoma, and melanoma) are often associated with an immunologic response that may, in some circumstances, control the malignant state and form the basis for new vaccine strategies and immunotherapies. Conversely, chronic antigenic exposure may lead to cutaneous T cell lymphoproliferative disorders. Lymphomas may involve the skin, including B cell, T cell and NK cell types. T cells expressing CLA are found in cutaneous T cell lymphomas, which usually are limited to skin for long periods. Ultraviolet exposure, both acute and chronic, induces changes in skin composition and immunology that may provoke a variety of photodermatoses, in addition to the acute inflammation associated with sunburn.

Inherited or spontaneous genetic mutations also give rise to skin diseases. Genodermatoses are those cutaneous diseases that result from genetic alterations affecting skin composition. A group of inherited blistering diseases called epidermolysis bullosa (distinct from epidermolysis bullosa acquisita, which is an acquired 259

autoimmune disorder with some similar features to the inherited forms) is a good example.[ ] Various mutations in type VII collagen result in dystrophic epidermolysis bullosa, which may be inherited as an autosomal dominant or recessive disease. Patients with epidermolysis bullosa simplex, a distinct type of mechanobullous disease, show severe intraepidermal blistering and ultrastructural evidence for tonofilament clumping. Dominant forms of epidermolysis bullosa simplex have mutations in keratin genes 5 and 14. Mutations in genes that code for proteins involved in the insertion of keratin filaments into hemidesmosomes may also be present in epidermolysis bullosa simplex, but these mutations have not yet been delineated. Mutations in genes coding for all three subunits of laminin-5 are associated with the lethal form of junctional epidermolysis bullosa. Mutations of the gene coding for the hemidesmosomal component, BPAg2 or BP180, have been demonstrated in individuals with the less severe generalized atrophic benign form of junctional epidermolysis bullosa. Interestingly, structural mutations of LAMB3, one of the genes encoding laminin-5, have also been demonstrated in individuals with this less severe form. Mutations of the gene coding for the β4-integrin subunit have been demonstrated in a form of junctional epidermolysis bullosa associated with pyloric atresia. Many parallels exist between these inherited genetic diseases that lead to cutaneous blistering and acquired immunobullous diseases in which autoantibody production to the same skin components results in blistering. The mutations resulting in epidermolysis bullosa are only a small sample of many genodermatoses. Much of skin structure has been defined in the pursuit of explaining cutaneous dysfunction, either through analyses of what inherited mutations mediate as genetic diseases or in determining what targets antibodies are directed toward in various autoimmune skin diseases.

SUMMARY The skin provides protective immunity against pathogenic organisms. To perform this function, the outermost layer of

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TABLE 41-2 -- Immunobullous Skin Diseases Immunobullous Disease

Clinical Presentation

Serum Autoantibodies

Targeted Protein/Structure

Tissue Immunofluorescence *

Pemphigus Pemphigus vulgaris

Flaccid bullae on noninflamed skin, crusting, Nikolsky, sign present, commonly affects scalp, chest, intertriginous areas, and oral mucosa

†

Immunoglobulin G (IgG) epithelial cell surface; correlate with disease activity

Desmoglein 3, also desmoglein 1/ desmosome, and other antigens mediating cell-cell adhesion

Epidermal IgG and C3 cell surface (intercellular substance) staining

Pemphigus foliaceous

Superficial bullae, erosions, and scale with crusting,

IgG epithelial cellsurface; correlate with disease activity

Desmoglein 1/desmosome

Epidermal IgG and C3 cell surface staining

†

Nikolsky, sign present Paraneoplastic pemphigus

Flaccid bullae, lichenoid or erythema multiforme-like, usually involves mucosa, often extensively including esophageal and respiratory tissues

IgG epithelial cell surface and basement membrane zone (staining on rodent bladder epithelium is characteristic); correlate with disease activity

Desmoglein 3, desmoplakin 1, desmoplakin 2, BP230, envoplakin, periplakin, other/ desmosome and hemidesmosome

Epidermal IgG and C3 cell surface and basement membrane zone staining

IgA pemphigus

Flaccid bullae, similar to pemphigus vulgaris

IgA epithelial cell surface; correlate with disease activity

Desmocollin 1/desmosome

Epidermal IgA cell surface staining

Bullous pemphigoid

Tense bullae, often on urticarial base, prominent pruritus

IgG basement membrane zone, epidermal

BP180, BP230/ hemidesmosome and lamina lucida

Linear basement membrane zone IgG and C3

Cicatricial pemphigoid

Tense bullae and erosions, scarring sequelae

IgG basement membrane zone, epidermal

BP180, laminin V/ hemidesmosome and lamina lucida

Linear basement membrane zone IgG and C3

Herpes gestationis

Tense bullae, similar to bullous pemphigoid, onset during or immediately after pregnancy

Complement fixing, basement membrane zone, epidermal

BP180, BP230/ hemidesmosome and lamina lucida

Linear basement membrane zone C3

Epidermolysis bullosa acquisita

Tense bullae, commonly occur in areas of trauma and in oral mucosa

IgG (rarely IgA) basement membrane zone, dermal

Type VII collagen/ anchoring fibrils

Linear basement membrane zone IgG and C3, may show linear IgA and IgM

Linear IgA bullous dermatosis and chronic bullous disease of childhood

Tense bullae, similar to bullous pemphigoid; oral involvement common in adult disease

IgA basement membrane 97•kD portion of BP180/ zone, epidermal (rarely dermal) hemidesmosome and lamina lucida

Dermatitis herpetiformis

Small bullae on extensor surfaces (elbows and knees); markedly pruritic; associated with intestinal gluten sensitivity

IgA endomysial and transglutaminase antibodies; correlate with disease activity and compliance with glutenfree diet

Pemphigoid

Epidermal transglutaminase

Linear basement membrane zone IgA

Granular basement membrane zone IgA with stippling in dermal papillae

Bullous lupus erythematosus (see Table 41-3 for information on other connective tissue diseases)

Tense bullae, photodistributed

IgG basement membrane zone, dermal

Type VII collagen/ anchoring fibrils

Linear basement membrane zone IgG, also may show granular IgM and C3 basement membrane zone as in lupus band

* In all suspected immunobullous disease, it is best to obtain biopsy for diagnosis from perilesional tissue because immunoreactants may not be present in lesional tissue; perilesional is defined as an area of skin immediately adjacent to but not involving a lesion. Serum studies are also essential to distinguish diseases. † Nikolsky's sign is the ability to split the epidermis by pulling a remnant of ruptured blister of by shearing pressure applied on normal-appearing skin or at the edgeof an existing blister. BP230, BP Ag1; BP180, BP Ag2; cell surface, intercellular substance; epidermal and dermal refer to localization of antibodies on humansplit skin by indirect immunofluorescence of serum.

682

TABLE 41-3 -- Autoimmune Cutaneous Connective Tissue Diseases

Autoimmune Disease

Indirect Immunofluorescence Pattern of Antinuclear Antibody Staining in Serum

*

Nuclear Antigens to Which Autoantibodies Are Directed

Direct Immunofluorescence of Tissue

Lupus erythematosus (LE) Systemic LE

Peripheral (rim), homogeneous, nucleolar, speckled

nDNA or double-stranded DNA, single-stranded DNA, histones, nucleolar RNA, various ribonucleoproteins, cardiolipin, Sm (Smith), U1-snRNP, HMG-17

Two or more granular immunoglobulin and complement deposits at BMZ, IgG, IgM, and/or IgA with C3 in involved and uninvolved skin (lupus band); lichenoid changes with numerous cytoids and shaggy fibrinogen staining of BMZ also found

Discoid LE

Usually no circulating antibodies

Usually none detected

Two or more granular immunoglobulin and complement deposits at BMZ, IgG, IgM, and/or IgA with C3 in involved skin; lichenoid changes with numerous cytoids and shaggy fibrinogen staining of BMZ also found

Subacute cutaneous LE

Fine speckled or speckled, may be negative

SS-A/Ro, SS-B/La

Particulate intercellular staining with or without granular immune deposits at BMZ or lichenoid changes

Neonatal LE

Fine speckled or speckled, may be negative

SS-A/Ro, SS-B/La

Granular IgG (tranplacental) at BMZ

Drug-induced LE

Peripheral, homogeneous

Histones

Granular immune deposits at the BMZ

Peripheral

Scl-70, SS-A/Ro, SS-B/La

No characteristic changes; vascular staining may be observed

Scleroderma Cutaneous scleroderma (localized and generalized morphea)

Limited disease (acrosclerosis, CREST) Centromere

Centromere, Scl-70, U1-snRNP, HMG- No characteristic changes; vascular 17 staining may be observed

Diffuse disease (systemic sclerosis)

Nucleolar, speckled

Scl-70, U1- and U3-snRNP, RNA pol I, II, and III

No characteristic changes; vascular staining may be observed

Dermatomyositis, polymyositis

Speckled, nucleolar

Jo-1, PM-Scl, Mi-2, U1-snRNP, SS-A/ Ro

No characteristic changes; lichenoid features and vascular staining may be observed

Sjögren's syndrome

Fine speckled, nucleolar

SS-A/Ro, SS-B/La, histones, U1snRNP

No characteristic changes; vascular staining may be observed

Mixed connective tissue disease

Speckled

U1-snRNP, PM-scl

No characteristic changes; granular immune deposits at the BMZ lichenoid features and vascular staining may be observed

Overlap and undifferentiated connective tissue disease

Any single or combination pattern

Any one or multiple, PM-Scl

May show granular immune complex deposition at BMZ (lupus band), vascular staining, and/or lichenoid changes

BMZ, Basement membrane zone; CREST, calcinosis, Raynaud's syndrome, esophageal dysmotility, sclerodactyly, telangiectasia; Ig, immunoglobulin; Rim, peripheral staining pattern; Scl-70, DNA topoisomerase I. * Indirect immunofluorescence on substrates with nucleated cells was the original means of testing for circulating antibodies in these diseases; with the identification of the antigens to which antibodies are directed, immunoassay has replaced indirect immunofluorescence in most diagnostic laboratories.

the skin (i.e., the stratum corneum) forms a “brick and mortar”–like structure of cornified keratinocytes within layers of lipids as a physical barrier to prevent penetration of potential pathogens. Underlying this outer layer are developing keratinocytes (containing large quantities of preformed IL-1α) and nerve fibers that act together as an early warning system to signal disruption of the overlying barrier. In response to the cascade of signals indicating barrier disruption, other resident cells including Langerhans' cells, macrophages, dermal dendritic cells, mast cells, and fibroblasts are activated to express antimicrobial polypeptides, to phagocytose invading pathogens, to produce additional warning signals, and/or to stimulate vasodilatation and vasopermeability, allowing effective recruitment of soluble PRRs and leukocytes expressing cell surface PRRs from the highly vascularized dermis. If needed to contain pathogens, the immune response persists with Langerhans' cell migration via draining lymphatic vessels to establish antigen-specific acquired immunity involving both T cells and B cells and antibody production. T cell surface CLA and expression of relatively skin-specific chemokines such as CCL17 and CCL27 appear particularly critical in T cell homing to the skin. Ideally, this

683

response completely eliminates an invading pathogen, then subsides and remains poised to respond to a future re-encounter with that pathogen. The normally fine-tuned processes of skin function can become misregulated and lead to skin disease. This results from underproduction or overproduction of cell products, production of defective cell products, and/or excessive or deficient proliferation of skin cells including malignant proliferation. Abnormal immunologic reactivity leads to the inappropriate recognition of and response to otherwise benign environmental allergens or autoallergens. Additional mechanisms and individual patient complexities probably contribute to the clinical manifestations of allergic skin disease as well.

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199. Irani AM, Huang C, Xia HZ, et al: Immunohistochemical detection of human basophils in late-phase skin reactions, J Allergy Clin Immunol 101:354, 1998. 200. Dieu-Nosjean MC, Massacrier C, Homey B, et al: Macrophage inflammatory protein 3alpha is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors, J Exp Med 192: 705, 2000. 201. Damsgaard TE, Olesen AB, Sorensen FB, et al: Mast cells and atopic dermatitis: stereological quantification of mast cells in atopic dermatitis and normal

human skin, Arch Dermatol Res 289:256, 1997. 202. Nickoloff BJ: Skin innate immune system in psoriasis: friend or foe? J Clin Invest 104:1161, 1999. Acquired Immunity 203. Yang D, Chertov O, Bykovskaia SN, et al: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6, Science 286:525, 1999. 204. Tsuji RF, Kawikova I, Ramabhadran R, et al: Early local generation of C5a initiates the elicitation of contact sensitivity by leading to early T cell recruitment, J Immunol 165:1588, 2000. 205. Kadowaki N, Ho S, Antonenko S, et al: Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens, J Exp Med 194:863, 2001. 206. Ulrichs T, Porcelli SA: CD1 proteins: targets of T cell recognition in innate and adaptive immunity, Rev Immunogenet 2:416, 2000. 207. Delves PJ, Roitt IM: The immune system: first of two parts, N Engl J Med 343: 37, 2000. 208. Henz BM, Maurer M, Lippert U, et al: Mast cells as initiators of immunity and host defense, Exp Dermatol 10:1, 2001. 209. Mazzoni A, Young HA, Spitzer JH, et al: Histamine regulates cytokine production in maturing dendritic cells, resulting in altered T cell polarization, J Clin Invest 108:1865, 2001. 210. Kimber I, Cumberbatch M, Dearman RJ, et al: Cytokines and chemokines in the initiation and regulation of epidermal Langerhans cell mobilization, Br J Dermatol 142:401, 2000. 211. Jankovic D, Liu Z, Gause WC: Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways, Trends Immunol 22:450, 2001. 212. Mueller SM, Jones CM, Smith CM, et al: Rapid cytotoxic T lymphocyte activation occurs in the draining lymph nodes after cutaneous herpes simplex virus infection as a result of early antigen presentation and not the presence of virus, J Exp Med 195:651, 2002. 213. Picker LJ, Treer JR, Ferguson-Darnell B, et al: Control of lymphocyte recirculation in man: II. Differential regulation of the cutaneous lymphocyte-associated antigen, a tissue-selective homing receptor for skin-homing T cells, J Immunol 150:1122, 1993. 214. Groves RW, Ross E, Barker JN, et al: Effect of in vivo interleukin-1 on adhesion molecule expression in normal human skin, J Invest Dermatol 98:384, 1992. 215. Kieffer JD, Fuhlbrigge RC, Armerding D, et al.: Neutrophils, monocytes, and dendritic cells express the same specialized form of PSGL-1 as do skin-homing memory T cells: cutaneous lymphocyte antigen, Biochem Biophys Res Commun 285:577, 2001. 216. Rosner K, Ross C, Karlsmark T, et al: Role of LFA-1/ICAM-1, CLA/E-selectin and VLA-4/VCAM-1 pathways in recruiting leukocytes to the various regions of the chronic leg ulcer, Acta Derm Venereol 81:334, 2001. 217. Katou F, Ohtani H, Nakayama T, et al: Macrophage-derived chemokine (MDC/CCL22) and CCR4 are involved in the formation of T lymphocyte-dendritic cell

clusters in human inflamed skin and secondary lymphoid tissue, Am J Pathol 158:1263, 2001. 218. Santamaria Babi LF, Picker LJ, Perez Soler MT, et al: Circulating allergen-reactive T cells from patients with atopic dermatitis and allergic contact dermatitis express the skin-selective homing receptor, the cutaneous lymphocyte-associated antigen, J Exp Med 181:1935, 1995. 219. Imayama S, Shimozono Y, Hoashi M, et al: Reduced secretion of IgA to skin surface of patients with atopic dermatitis, J Allergy Clin Immunol 94:195, 1994. 220. Norhagen G, Engstrom PE, Hammarstrom L, et al: Immunoglobulin levels in saliva in individuals with selective IgA deficiency: compensatory IgM secretion and its correlation with HLA and susceptibility to infections, J Clin Immunol 9:279, 1989. 221. Bellinghausen I, Enk AH, Mohamadzadeh M, et al: Epidermal cells enhance interleukin 4 and immunoglobulin E production after stimulation with protein allergen, J Invest Dermatol 107:582, 1996. 222. Herrick CA, MacLeod H, Glusac E, et al: Th2 responses induced by epicutaneous or inhalational protein exposure are differentially dependent on IL-4, J Clin Invest 105:765, 2000. 223. Lambrecht BN: Immunologists getting nervous: neuropeptides, dendritic cells and T cell activation, Respir Res 2:133, 2001. 224. Kobayashi H, Gleich GJ, Butterfield JH, et al: Human eosinophils produce neurotrophins and secrete nerve growth factor on immunologic stimuli, Blood 99:2214, 2002. 225. Torii H, Yan Z, Hosoi J, et al: Expression of neurotrophic factors and neuropeptide receptors by Langerhans cells and the Langerhans cell-like cell line XS52: further support for a functional relationship between Langerhans cells and epidermal nerves, J Invest Dermatol 109:586, 1997. 226. Torii H, Hosoi J, Asahina A, et al: Calcitonin gene-related peptide and Langerhans cell function, J Invest Dermatol Symp Proc 2:82, 1997. 227. Church MK, el-Lati S, Caulfield JP: Neuropeptide-induced secretion from human skin mast cells, Int Arch Allergy Appl Immunol 94:310, 1991. 228. Schmid-Ott G, Jaeger B, Adamek C, et al: Levels of circulating CD8(+) T lymphocytes, natural killer cells, and eosinophils increase upon acute psychosocial stress in patients with atopic dermatitis, J Allergy Clin Immunol 107:171, 2001. 229. Schmid-Ott G, Jaeger B, Meyer S, et al: Different expression of cytokine and membrane molecules by circulating lymphocytes on acute mental stress in patients with atopic dermatitis in comparison with healthy controls, J Allergy Clin Immunol 108:455, 2001. Resolution of Immune Reaction 230. Terui T, Sano K, Shirota H, et al: TGF-beta-producing CD4+ mediastinal lymph node cells obtained from mice tracheally tolerized to ovalbumin (OVA) suppress both Th1- and Th2-induced cutaneous inflammatory responses to OVA by different mechanisms, J Immunol 167:3661, 2001. 231. Henson PM, Bratton DL, Fadok VA: Apoptotic cell removal, Curr Biol 11: R795, 2001. 232. Lawrence T, Gilroy DW, Colville-Nash PR, et al: Possible new role for NF-kappaB in the resolution of inflammation, Nat Med 7:1291, 2001.

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Chapter 42 - Immunologic and Nonimmunologic Lung Defense Mechanisms

Kenneth S. Knox Homer L. Twigg III

The normally functioning respiratory system employs a number of important defense mechanisms to maintain sterile lung parenchyma. The first line of defense is the upper airway filter. Other mechanical defenses include coordinated glottic function, cough, and a functional mucociliary apparatus. Nonspecific defense mechanisms at the level of the alveolus include surfactant, bacteriostatic proteins, and resident alveolar macrophages. These nonspecific defenses filter foreign debris and “mop up” the constant low-level pathogen burden that penetrates to the lung parenchyma nightly through microaspiration. Specific immunologic defenses that protect the

lung include antibody-mediated (IgG, IgA, IgE) and cell-mediated (inflammatory and immune effector cells) processes. Immunologic defenses, however, may harm the local environment if inflammation proceeds unchecked. Thus an appropriate balance must exist between the helpful immune response and the potentially harmful inflammatory response. In this age of aggressive immunosuppressive therapy for malignancies, organ transplantation, and collagen vascular disease, the likelihood that a physician will encounter an immunocompromised patient is greatly increased. In addition, the growing prevalence of acquired immunodeficiency syndrome (AIDS) also contributes significantly to this patient population. The lung is especially susceptible to infectious and noninfectious diseases in immunocompromised patients. In patients with congenital immunodeficiency states or lymphoproliferative neoplasms and in those who have undergone organ transplantation, the cumulative incidence of 1] [2]

pulmonary infections exceeds 50%. [

Subjects infected with the human immunodeficiency virus (HIV) have an 80% chance of developing a pulmonary

complication at some point during their disease. [

3] [4]

The importance of respiratory infection is seen in the high mortality for immunocompromised patients with 5 6

any form of lung disease, which approaches 40% to 50%. [ ] [ ] Therefore, knowledge about the potential etiologies of impaired pulmonary host defenses, the appropriate evaluation of such impairment, and subsequent therapy is critically important. This chapter focuses on normal defense mechanisms of the lower respiratory tract.

NORMAL HOST DEFENSE It is important to understand how various alterations in pulmonary host defense may increase the risk of lung disease ( Box 42-1 ).

Box 42-1. Pulmonary Host Defense Mechanisms

Mechanical Mechanisms Air filtration in upper airway Sneezing, coughing Mucociliary clearance

Phagocytic Mechanisms Airway and alveolar macrophages Neutrophil recruitment

Release of toxic phagocyte products with antimicrobial properties • Lysozymes • Proteases • Lactoferrin • Reactive oxygen metabolites Specific Immune Responses Cellular immune response mediated by T lymphocytes CD4 T cells • Delayed type hypersensitivity (granulomatous responses) • “Help” for cytotoxic CD8 T cells and antibody-producing B cells CD8 T cells • Cytotoxic responses (antiviral responses, tumor surveillance, allograft rejection) • “Suppress” immune response Natural killer cells • Cytotoxic responses Hormonal immune response mediated by B cells Antibody production • Opsonins • Agglutination of particles • Neutralization of bacterial toxins and certain viruses

Mechanical Host Defense Mechanisms The first line of defense is mechanical and involves filtration of air as it passes through the upper airway. Other mechanical defense mechanisms include sneezing, coughing, and

688

mucociliary clearance. Defects in this host defense can be seen in diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, and the immotile cilia syndrome. The second major line of lung defense is nonspecific phagocytosis. Through this defense mechanism, particles and bacteria that reach the lower respiratory tract are ingested by phagocytes, such as alveolar macrophages, neutrophils, and occasionally eosinophils. The third lung host defense mechanism involves specific humoral and cell-mediated immune responses. Of the three major host defense mechanisms, this third mechanism is the only one that is antigen specific. The alveolar spaces are normally a sterile environment where gas exchange occurs. The alveolar epithelium consists almost entirely of type I and type II alveolar 7

epithelial cells.[ ] End-mitotic, type I cells cover the majority of the alveolar space. When type I cells are injured and replaced, new type I cells are derived from dividing type II cells. In contrast to type I cells, type II cells are neatly situated in the corners of alveoli, can undergo mitosis to become either type I or II cells, and 8

are the source of the phospholipid-rich surfactant in the alveolar spaces.[ ] Maintenance of the normal functioning of these epithelial cells is critical to health because any process that jeopardizes the integrity of the normal cellular constituents of the alveoli can significantly impede gas exchange and result in clinically apparent disease. The terminally differentiated alveolar macrophage is the predominant cell located within the alveolus. This situation is in contrast to the blood compartment, where the predominant inflammatory cell is the circulating neutrophil. As such, macrophages are the most abundant cell found in bronchoalveolar lavage (BAL) fluid. Most of the remaining cells are lymphocytes ( Table 42-1 ). Under normal conditions the alveolar spaces are free of inflammatory cells such as neutrophils and eosinophils. In certain diseases or after various environmental exposures (including cigarette smoke), however, the normal cellular components in BAL fluid are altered. These alterations can manifest as impairment of existing resident cell populations (macrophages, lymphocytes) or expansion of cell populations not normally found in the alveolar space (neutrophils). In either case, the lung becomes a potential target for disease. Mechanical host defense begins in the upper airway with filtration of large particles and ends in the terminal bronchioles at the furthermost reaches of the 9

mucociliary tree. Clearance by mechanical means primarily depends on the physical properties of invading organisms and particles.[ ] Particles larger than 10••m are filtered in the upper airways by nasal hairs and the tortuous nasopharynx. Thus, mouth TABLE 42-1 -- Normal Cellular Constituents of Bronchoalveolar Lavage (BAL) Fluid Cells

Percent

N × 106

Alveolar macrophages

90%–95%

4–14

Lymphocytes

5%–10%

0.5–1.5

Neutrophils

0%

0

*

Eosinophils

0%

TOTAL

0

100%

5–15

* Results from typical BAL with 100•ml of fluid instilled and 40 to 60•ml recovered in a nonsmoking, healthy individual.

10

breathing is associated with an increased particulate exposure to the lower respiratory tract.[ ] Particles 5 to 10••m in diameter may gain access to the tracheobronchial tree but are cleared by the mucociliary escalator after colliding with the walls of the conducting airways. Only particles smaller than 5••m in diameter can reach the alveolar space. This means that bacteria (0.5 to 2.0••m), mycobacteria (0.4 by 1.0 to 10••m), and aerodynamic asbestos fibers can easily gain access to the normally sterile alveolar compartment. The mucociliary tree is perhaps the most important mechanical host defense in the lung. Normally the mucociliary tree transports about 10•ml of mucus cephalad a 11]

day.[

This volume can be increased 20 to 30 times in the presence of chronic bronchitis. The liquid involved in the mucociliary tree consists of two layers; the cilia

can beat freely in the watery sol layer and thus propel the more viscous gel layer on top of it.[

12]

Efficient ciliary function is critical in effectively clearing the

overlying mucus secretions. Cholinergic and β-adrenergic stimulation increase ciliary beat frequency. [

13] [14]

A single puff of tobacco smoke can paralyze cilia.

Impairment of ciliary beat frequency and thus an increased risk of pulmonary infection can be caused by such diverse agents as reactive oxygen species,[ products,

[16]

and acute viral

closely regulated by the 22 autonomous.[ ]

17 infections.[ ]

18 cholinergic,[ ]

15]

bacterial

Mucus is predominantly produced by goblet cells and submucosal glands. Mucus secretion by submucosal glands is

adrenergic,[

19] [20]

21]

and noncholinergic-nonadrenergic nervous systems.[

Mucus secretion by goblet cells appears to be

Generation of the sol layer, a result of water and electrolyte transport across the apical membrane of respiratory epithelial cells, is under β-adrenergic

[23]

24 25

and possibly noncholinergic-nonadrenergic control.[ ] [ ] Thus the drugs that affect the autonomic nervous system may also affect function of the mucociliary tree. Also, the fluidity of mucus secretion and its ease of being expectorated depend on its water content. As such, a dehydrated patient often has difficulty clearing thick, tenacious secretions until fluids are administered. Phagocytic Host Defense (Nonspecific)

Cellular Components

Phagocytic functions in the lung are carried out by mononuclear (monocytes, macrophages) and polymorphonuclear (neutrophils, eosinophils) phagocytes. Mononuclear phagocytes have especially important phagocytic, microbicidal, and secretory functions and play a critical role in lung immunity by regulating immune and inflammatory responses. One of their most important functions is to clear foreign material that has gained access to the lower respiratory tract. The most important phagocytes may be alveolar macrophages because they represent the first line of defense against invading microorganisms or toxic substances that successfully reach alveolar or bronchiolar structures. Failure of the alveolar macrophages to clear potentially hazardous organisms often represents the first step in lung disease.

Controversy surrounds whether the alveolar macrophage population is maintained through continued recruitment of blood monocytes into the alveolar space or through in situ proliferation. Investigators have shown that alveolar macrophages can proliferate.[ macrophages initially arise from a precursor population located in the bone marrow.

[27]

26]

However, compelling evidence suggests that alveolar

In addition,

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TABLE 42-2 -- Functional Differences among Blood Monocytes, Alveolar Macrophages, and Dendritic Cells Dendritic Cells Process/Activity

Monocytes

Macrophages

Immature

Mature

Phagocytosis

Poor

Good

Good

Poor

Respiratory burst

Good

Fair

—

—

Accessory cell function

Good

Poor

Poor

Excellent

Cytotoxic activity

Good

Poor

—

—

Cytokine production

Good

Variable

Fair

Good

Arachidonic acid metabolism

Cyclooxygenase products

Lipoxygenase products

—

—

blood monocytes are recruited to the lung during acute inflammatory processes. A recent study has shown that CD14 is up-regulated on monocytes recruited to the 28

lung and that these cells are primed to liberate tumor necrosis factor (TNF).[ ] It is likely that both recruitment and in situ proliferation are important in maintaining the alveolar macrophage population. This is of more than academic interest because blood monocytes and alveolar macrophages have substantially different functional capabilities ( Table 42-2 ). In the normal setting, alveolar macrophages have an augmented ability to respond nonspecifically to foreign antigens through phagocytosis but a limited ability to initiate specific immune responses. 29

Secretory functions of alveolar macrophages may be as important as phagocytic and microbicidal functions[ ] ( Box 42-2 ). Because phagocytosis is a major stimulus of secretory activity, the functions are clearly related. Perpetuation and amplification of inflammatory responses may occur through the generation of leukocyte chemoattractants, which results in the accumulation of inflammatory cells within the alveolar space. Alveolar macrophage–derived leukocyte chemotaxins 30]

include complement components,[

fibrinolytic fragments,[

31]

and arachidonic acid metabolites, specifically leukotriene B4 (LTB4 ). [

secrete cytokines with chemotactic activity (chemokines), including interleukin-8 (IL-8)[

33]

32]

Alveolar macrophages also

and macrophage inflammatory protein-1 alpha (MIP-1α).[

34]

In addition to chemoattractant agents, alveolar macrophages secrete a variety of cytokines with immunomodulatory functions. These include proinflammatory factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF),[ [40]

35]

TNF-α, [

36]

37]

interferon (IFN),[

38]

IL-12,[

39]

and the T cell growth factors IL-1[

and IL-6.

Alveolar macrophages also secrete cytokines capable of down-regulating immune and inflammatory responses, including transforming growth factor beta (TGF-

41 β),[ ]

42

43

IL-10,[ ] and IL-1 receptor antagonist.[ ] Thus, alveolar macrophages function as both inflammatory and immune effector cells within the lower respiratory tract. In an attempt to minimize unwanted inflammation in the lower respiratory tract, alveolar macrophages are normally quiescent cells. The net effect of a resting lung macrophage may be immunosuppressive. However, they can become activated under the influence of numerous environmental stimuli (e.g., lipopolysaccharide) 44 45 46

or secreted cellular factors (e.g., IFN-γ, GM-CSF).[ ] [ ] [ ] Activation of alveolar macrophages results in an enhanced ability to perform all their basic functions, including phagocytosis, cytokine secretion, and stimulation of specific immune responses. Much of this discussion has focused on alveolar macrophages because they are the most accessible lung cells available for study. The lung consists of several different compartments, however, and macrophages within these compartments may behave differently.[ characteristics have been described in the

48 airway,[ ]

49 vascular,[ ]

50 pleural,[ ]

[51]

and interstitial

47]

Macrophages with different phenotypical and functional

compartments.

Unlike alveolar macrophages and lymphocytes, polymorphonuclear leukocytes such as neutrophils or eosinophils are never found in the lower respiratory tract under normal conditions. Neutrophils and eosinophils are potent inflammatory cells recruited by alveolar macrophages through specific chemoattractants and participate in the destruction and clearance of invading organisms that have reached the alveolar spaces. Despite having good phagocytic capabilities, alveolar macrophages kill intracellular organisms poorly compared with neutrophils and monocytes. The main role of alveolar macrophages apparently is to serve as sentinels against invading organisms and foreign particles, predominantly through phagocytosis. If inefficient killing results or the offending load is too great, macrophages secrete cytokines that recruit more potent phagocytes, as well as cells capable of generating specific immune responses. The recruitment of inflammatory cells to the alveolar spaces, however, represents a “two-edged sword.” Inflammatory cells are clearly necessary for the inflammatory or immune response when an infection is developing in the alveolar spaces. However, the release of potent oxidants and toxic proteins by these inflammatory cells can injure the alveolar structures and interstitium of the lung. A balance must be carefully maintained within the lung to ensure normal function by keeping the gas exchange units pristine. Phagocytic Process

Phagocytosis can be broken down to four basic components: chemotaxis, adherence, ingestion, and digestion ( Figure 42-1 ). The initial step in the phagocytic process involves migration of resident phagocytic cells toward the invading organism or particle. Macrophages can migrate toward a foreign particle in response to a variety of chemotactic stimuli, including C5a, microbial cell wall products, neutrophil-derived chemotactic factors, and lymphokines.[ is greatly facilitated by surfactant protein A

54 (SP-A).[ ]

690

52] [53]

Macrophage migration

Box 42-2. Major Secretory Products of BAL Alveolar Macrophages

Enzymes Collagenase Elastase Cathepsins

Complement C1, C4, C2, C3, C5 Factor B, factor D, factor P (properdin)

Toxic Oxygen Species Superoxide anion (O2 − ) Hydrogen peroxide (H2 O2 ) Hydroxyl radicals (OH• ) Hypochlorous acid (HClO)

Leukocyte Chemotaxins Leukotriene B4 (LTB4 ) Complement C5a Platelet-activating factor (PAF)

Cytokines/Chemokines Tumor necrosis factor (TNF) Macrophage inflammatory protein-1 alpha (MIP-1α) Transforming growth factor beta (TGF-β) Interleukin-1 (IL-1), IL-6, IL-8, IL-10, IL-12 IL-1 receptor antagonist Granulocyte-macrophage colony-stimulating factor (GM-CSF)

Fibroblast Regulatory Mediators Fibronectin Platelet-derived growth factor (PDGF) Prostaglandin E2 (PGE2 )

Arachidonic Acid Metabolites Prostaglandin I2 (PGI2 ), PGE2 PAF LTB4 , LTC4 , LTD4

Coagulation Factors Factor VII

Tissue plasminogen activator (tPA) Platelet activation inhibitor

Once the macrophage has migrated into the vicinity of the material to be phagocytized, binding must occur between the particle and cell surface. Numerous 55 56 57 58

macrophage receptors that bind foreign material have been identified.[ ] [ ] [ ] [ ] Because many organisms or particles are taken up by a specific receptor-ligand interaction, the presence or absence of specific macrophage receptors likely determines the efficiency of phagocytosis. For example, scavenger receptors are important in the uptake of charged environmental particles.[

59]

Alveolar macrophages are not activated when particles are taken up this way, thus minimizing

potential injurious inflammatory responses. Other organisms can bind to macrophages through several receptors. SP-A binds avidly to Pneumocystis carinii[ thus may be important in macrophage uptake of this organism. However, uptake of P. carinii is also mediated by the macrophage mannose SP-A and macrophage mannose receptor expression are altered in HIV infection, pathogenesis of Pneumocystis pneumonia in these patients.

[62] [63]

61 receptor.[ ]

60]

and

Because both

abnormalities in macrophage–P. carinii adherence may be important in the

64 65 66

Ingestion of particles bound to the cell surface occurs primarily through two distinct processes.[ ] [ ] [ ] Phagocytosis is an actin-dependent and adenosine triphosphate (ATP)–dependent uptake process. Ingested material is generally greater than 1••m in diameter. In contrast, endocytosis is not actin or ATP dependent and occurs in portions of the plasma membrane called clathrin-coated pits. This process is also receptor mediated and is associated with receptor recycling. Once ingested, foreign material is localized in cytoplasmic vacuoles called phagosomes. Alternatively, accessory cells are able to take up antigen by fluid-phase pinocytosis or macropinocytosis, which is receptor independent. The final step in phagocytosis is the destruction of ingested material through a variety of cellular digestive mechanisms. This occurs through the fusion of phagosomes with lysosomes containing abundant digestive enzymes, reactive oxygen species, and reactive nitrogen intermediates (RNIs). Alveolar macrophages can generate toxic oxygen species, specifically superoxide anion (O2 − ), hydrogen peroxide (H2 O2 ), and hydroxyl radicals (OH• ), all of which assist in the destruction 67]

of microorganisms in the lower respiratory tract.[

Activated monocyte-derived macrophages generate RNIs such as nitrates and nitrites, adding to their potent

68 activities.[ ]

69

armamentarium of microbicidal However, it remains controversial whether human alveolar macrophages generate RNIs. [ ] Alveolar macrophages also contain pre-formed proteases, such as collagenase, elastase, and cathepsins, which in addition to a potentially lethal effect on invading infectious agents, degrade components of the extracellular matrix, thus amplifying inflammatory responses. In the clinical setting in which the responses of activated alveolar macrophage are unchecked, proteases and toxic oxygen radicals may disrupt epithelial and endothelial cell integrity. As with alveolar macrophages, neutrophils and eosinophils also produce toxic oxygen-derived species, such as O2 − , H2 O2 , OH• , and the myeloperoxidase-derived hypohalite ion, all of which can kill microorganisms.[

70] [71] [72] [73] [74]

Furthermore, eosinophils and neutrophils possess specific proteases that can destroy 72

bacterial cell walls and other barriers to eliminate potentially infectious agents. [ ] For example, leukocyte elastase can digest elastin, collagen, laminin, proteoglycans, and fibronectin. Another protease, cathepsin G, can amplify the effects of elastase and collagenase and digest insoluble collagen. Deficiencies in

antiproteases are associated with lung disease. [ parasites.

[74]

72] [75]

Eosinophils also possess major basic protein, a substance highly toxic to certain microorganisms such as

Recruitment of neutrophils to the lung is discussed further in the section on adult respiratory distress syndrome (ARDS).

If all steps in the phagocytic process are operational, efficient removal of foreign material occurs. However, in contrast to rapid mucociliary clearance mechanisms (minutes

691

Figure 42-1 Four basic components of phagocytosis. For efficient clearance of foreign material or microbes all four components (chemotaxis, adherence, ingestion, digestion) must be functional. ECM, extracellular matrix; Ig, immunoglobulin; FcR, Fc receptor; DR, HLA-DR; SPA, surfactant protein A.

Figure 42-2 Model for generation of specific, primary immune responses in the lung. The accessory cells (alveolar macrophage or dendritic cell) are essential in initiating the cellular immune response and optimizing the humoral immune response. An accessory cell can present antigen to a naive CD4+ Th0 cell in the proper major histocompatibility complex (MHC) context and promote differentiation to either CD8+ or CD4+ T lymphocyte populations (A). Depending on the accessory cell's response to antigen and the prevailing cytokine environment, these lymphocyte populations (A) can be polarized to either a Th1 or a Th2 cytokine-secreting profile (B). These complex processes occur simultaneously but are pictured separately. IL, Interleukin; IFN-γ, gamma interferon; IG, immunoglobulin; TGF-β, transforming growth factor beta; TNF-α, tumor necrosis factor alpha; Ag, antigen; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte-

macrophage colony-stimulating factor; (+), positive feedback; (−), negative feedback.

Figure 42-3 Sites of pulmonary immune responses. 1, Antigen enters the lower respiratory tract, where it is either taken up by phagocytes acting as accessory cells, which then migrate to regional lymph nodes or by direct diffusion into regional lymphoid tissue. 2, Primary immune response occurs in regional lymphoid tissue. 3, Lymphocytes traffic back to the lung through the systemic circulation and take up residence in the lung interstitium. 4, Amplification of the immune response occurs

back in the lung.

TABLE 42-3 -- Selected Human Chemokines Chemokine

Receptor

Predominant Effect

Interleukin-8

CXCR-1, CXCR-2

Inflammatory

RANTES

CCR-1, CCR-4, CCR-5

Inflammatory

MIP-1α, MIP-1β

CCR-1, CCR-5

Inflammatory

MCP-1, MCP-2, -3, -4

CCR-2

Inflammatory

TARC

CCR-4

Lymphoid

ELC

CCR-7

Lymphoid

MDC

CCR-4

Lymphoid > Inflammatory

IP-10

CXCR-3

Inflammatory > Lymphoid

Lungkine

CXCR-?

Inflammatory > Lymphoid

Eotaxin-1, eotaxin-2

CCR-3

Inflammatory

BCA-1

CXCR-5

Lymphoid

RANTES, Regulated on activation, normal T cell expressed and secreted; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein; TARC, thymus- and activation-regulated chemokine; ELC, EBI1 ligand chemokine; MDC, macrophage-derived chemokine; BCA, B cell–attracting chemokine; IP-10, interferon (IFN)-inducible protein 10.

695

times of inflammation. The chemokine lungkine is unique in this regard because it is thought to be present only in the lung. With regard to the pulmonary immune response, a new paradigm emerges: DCs can mediate both antigen-specific inflammation and antigen-specific tolerance. Under steady-state conditions, lung DCs remain immature and produce a weak, baseline Th2 response that is likely antagonized by Th1 tendencies of resident alveolar macrophages. [ [119]

118]

Recent studies suggest that IL-10 produced by lung DCs also plays a pivotal role in the induction of tolerance to inhaled antigenic stimuli.

Under certain circumstances or under the influence of cytokines/chemokines, however, DCs migrate to regional bronchiolar lymph nodes and mature. With 120 121

maturation comes the ability to produce IL-12 and the potential to foster a Th1 inflammatory response in the lymph node.[ ] [ ] If inflammation provoked by inhaled particles or pathogens yields production of antiinflammatory cytokines locally, DCs can mature and travel to lymph nodes in the absence of IL-12 production and can favor Th2 responses. One murine model suggests that DCs are critical for pulmonary allergic inflammation.[ role in perpetuating the asthmatic response. [

122]

Studies suggest that DCs play an important

92]

Down-Regulation of Pulmonary Immune Response

As discussed, it is necessary to employ mechanisms that curtail inflammation in order to preserve the gas exchange surface. Many pathways are involved in downregulating an immune response. Alveolar macrophages may exert an overall antiinflammatory effect. DCs allow for tolerance to certain antigenic exposures. The binding of co-stimulatory molecules on the surface of accessory cells (e.g., CD 80/86) by specific ligands (e.g., CTLA-4) can induce anergy. Antiinflammatory 41] [42] [43]

cytokines[

can suppress the immune response, as can the lipid and protein components of alveolar lining fluid. Alternatively, CD8+ “suppressor T cells”

can interact with activated CD4+ lymphocytes and curtail immune responses.[

123]

Perhaps the most intriguing mechanism employed to turn off the immune response involves the elimination of immunocompetent cells by the process of apoptosis.

Apoptosis minimizes the release of toxic cellular products as occurs in lytic cell death. Phagocytic cells also efficiently remove apoptotic cells. In a murine model of lung inflammation, Milik et al[

124]

demonstrated that CD4– and CD8– lung lymphocytes undergo apoptosis in response to particulate antigen in a CD95-dependent

124 manner.[ ]

They postulate that in situ lymphocyte apoptosis contributes to the termination of immune responses and tolerance to repeated antigenic exposure. This mechanism may be perturbed in diseases (e.g., sarcoidosis) associated with active T cell alveolitis. 125

Neutrophils recruited to the lung undergo apoptosis and are cleared efficiently by macrophages.[ ] DCs are also capable of ingesting apoptotic cells. DCs are able to induce MHC class I–restricted cytotoxic T cells on phagocytosis of apoptotic cells, whereas alveolar macrophages typically do not liberate a proinflammatory 92] [125]

response after ingesting these cells.[

Moreover, phagocytosis of apoptotic neutrophils by alveolar macrophages can liberate growth factors directly related to 126

repair of lung epithelium and regeneration of type II pneumocytes.[ ] If these mechanisms of repair are altered, chronic inflammation and excessive fibrosis may be favored over resolution to normal lung architecture. Traditional thinking about inflammation was that exuberant proinflammatory responses could lead to bystander epithelial and endothelial injury. However, inefficiencies or defects in the arm of immunity that controls down-regulating and repair mechanisms may be involved as well. This may be particularly important in diseases (e.g., bacterial pneumonia, ARDS) associated with intense neutrophil recruitment to the lung. Other Host Defense Mediators Antiproteases

The lung is constantly exposed to degradative enzymes released by neutrophils, macrophages, and connective tissue cells in response to inflammatory stimuli. To limit damage to normal tissue, the lung contains numerous antiproteases that can inactivate harmful enzymes ( Table 42-4 ). Whereas many antiproteases are carried or diffuse to the lung through the vascular compartment, others are produced locally by inflammatory cells and connective tissue. Human serum contains several antiproteases: α1 -antitrypsin, also called α1 -proteinase inhibitor (α1 -PI); α2 -macroglobulin, and α1 -antichymotrypsin, all of which prevent tissue damage from the toxic effects of leukocyte-derived proteases, especially neutrophil elastase (human leukocyte elastase). α1 -PI, a 52,000-D glycoprotein synthesized predominantly in the liver, is the major plasma antiprotease.[ The deficiency state of this protein causes premature emphysema.[

73]

72]

α1 -PI accounts for 90% of the α1 peak on a serum protein electrophoresis.

Easily detected in BAL fluid, α1 -PI is probably the major antiprotease responsible for

controlling proteolysis in the alveolar spaces.

α2 -Macroglobulin, a large molecule of approximately 725,000•D, is synthesized in the liver and interacts with leukocyte elastase. α2 -Macroglobulin is the major inhibitor of neutrophil collagenase, an enzyme of potential importance in the pathogenesis of certain fibrosing lung disorders. Unlike α1 -PI, α2 -macroglobulin does not have access to the alveolar space in normal subjects and affords protection within the vascular and interstitial compartments of the lung. α1 -Antichymotrypsin, a 68,000-D protein, is also synthesized in the liver and is important in the inactivation of cathepsin G.

Several locally produced protease inhibitors that inactivate neutrophil serine proteases have been described. Human monocyte/neutrophil elastase inhibitor (HEI), a 42,000-D protein produced by neutrophils and macrophages, inhibits the activity of neutrophil elastase, cathepsin G, and proteinase 3 by forming a covalent proteaseprotease inhibitor complex.[

127]

An 11,700-D antiprotease secreted by airway mucosal cells has been named secretory leukocyte protease (leukoprotease) inhibitor 128]

(SLPI). This inhibitor inactivates neutrophil elastase and cathepsin G but is inactive against proteinase 3.[

129

A series of metalloproteinases, including collagenase, gelatinase, and stromelysin, can be produced in the lung and cause tissue damage.[ ] Metalloproteinases are produced by neutrophils, macrophages, and connective tissue cells. Although metalloproteinases are inhibited by α2 -macroglobulin, this antiprotease is rarely found in the lung. Fortunately, lung connective tissue cells that secrete metalloproteinases simultaneously produce tissue inhibitors of metalloproteinases

696

TABLE 42-4 -- Types of Proteases and Antiproteases in Lower Respiratory Tract Protease

Source

Antiprotease

Source

Neutrophil elastase

Neutrophils

α1 -Proteinase inhibitor

Liver

α2 -Macroglobulin

Liver

HEI

Neutrophils Macrophages

Cathepsin G

Neutrophils

SLPI

Airway mucosal cells

α1 -Antichymotrypsin

Liver

HEI

Neutrophils Macrophages

Proteinase-3

Neutrophils

SLPI

Airway mucosal cells

α1 -Proteinase inhibitor

Liver

HEI

Neutrophils Macrophages

Metalloproteinases Collagenase

Gelatinase

Stromelysin

Neutrophils

TIMPs

Connective tissue cells

Macrophages, connective tissue cells

α2 -Macroglobulin

Liver

Neutrophils

TIMPs

Connective tissue cells

Macrophages, connective tissue cells

α2 -Macroglobulin

Liver

Macrophages, connective tissue cells

TIMPs

Connective tissue cells

α2 -Macroglobulin

Liver

HEI, Human monocyte/neutrophil elastase inhibitor; SLPI, secretory leukocyte protease inhibitor; TIMPs, tissue inhibitors of metalloproteinases. capable of inactivating these enzymes.[

130]

Type II pneumocytes can also produce collagenase.

The original interest in antiproteases emerged when neutrophils and the α1 -antitrypsin–deficient state were implicated in the development of precocious emphysema. [75]

It is now believed that emphysema is also a CD8+ T cell–mediated disease. [

their importance in mediating acute lung injury and 42-4 ).

132 ARDS.[ ]

131]

Current study of proteases (e.g., leukocyte elastase) in lung disease centers on

A balance must exist between proteases and antiproteases to facilitate normal lung function ( Figure

Complement

The complement system is another major mediator of inflammatory responses. BAL fluid contains a wide variety of complement components, many of which are synthesized by both alveolar macrophages and type II epithelial cells. Complement components C3, C1, C1q, C4, C6, and C5 and factor B have all been 133 134

demonstrated in BAL fluid, although their concentrations are quite low.[ ] [ ] Complement activation is critically important in the recruitment of inflammatory cells to the lung (C5a) and in opsonizing foreign material for optimal phagocytosis (C3b). More recently, complement has been shown to play an important role in antibody responses and a broader role in the inflammatory response.[

135]

Surfactant

Lipids and proteins in the lower respiratory tract clearly have a vital role in the lung's defense against external challenges. Surfactant, the protein-lipid complex that reduces surface tension in the alveolar space, has an enhancing effect on some pulmonary immune responses. SP-A, the major protein component of surfactant, 54]

facilitates chemotaxis of alveolar macrophages[ 136 macrophages.[ ]

and enhances Fc receptor (FcR)–mediated and complement receptor (CR1)–mediated phagocytosis by

SP-A appears to act as an activation ligand, although it can also serve as an opsonin, forming a bridge between certain infectious agents and

137

138

macrophages. SP-A enhances macrophage phagocytosis of Mycobacterium tuberculosis [ ] and P. carinii[ ] in this manner. However, not all surfactant properties enhance immunologic responses. SP-A adsorbed to the surface of alveolar macrophages appears to be responsible for the inhibitory effect of these cells on lymphoproliferation. In a recent study, human SP-A purified from patients with alveolar proteinosis suppressed murine alveolar inflammation and attenuated the severity of lung injury by inhibiting allogeneic donor T cell immune responses.[

139]

Evidence suggests that both SP-A and complement can up-regulate the innate

immune response and down-regulate the adaptive immune response, permitting “cross-talk” between these two arms of the immune response.[

135] [140]

DISEASES ASSOCIATED WITH IMPAIRED HOST DEFENSE When specific components of the host defense mechanism become impaired, it is frequently possible to predict the infectious complications to which the patient will be susceptible ( Table 42-5 ), because many organisms tend to be

697

Figure 42-4 Proposed historical scheme of the balance between neutrophils–neutrophil products and lung antiproteases–antioxidants in health and disease. Lung injury may be associated with a marked influx of neutrophils and depletion of normal antiproteases or oxidants.

TABLE 42-5 -- Causes and Infectious Complications of Major Types of Impaired Host Defense Type of Disorders

Inherited Defects

Acquired Defects

Infectious Complications

Mechanical defense

Immotile cilia syndrome Cystic fibrosis

Chronic obstructive pulmonary disease Ethanol Tobacco smoke

Gram-negative bacteria, especially Pseudomonas Staphylococcus Haemophilus influenzae

Phagocytosis

Chronic granulomatous disease

Catalase-positive bacteria (staphylococci, gram-negative bacteria)

Chédiak-Higashi syndrome

Cyclophosphamide therapy Acute myelocytic leukemia Any cause of neutropenia Viral infections Ethanol Hypoxia Tobacco smoke

Aspergillus Candida

Congenital X-linked hypogammaglobulinemia Common variable immunodeficiency Immunoglobulin G (IgG) deficiency (especially IgG2 and IgG3)

Corticosteroid therapy Antimetabolite therapy Multiple myeloma Chronic lymphocytic leukemia

Pneumococci Other encapsulated bacteria (H. influenzae, Neisseria, Staphylococcus aureus)

IgA deficiency

—

Usually asymptomatic

Cellular immunity

DiGeorge's syndrome Wiskott-Aldrich Syndrome Ataxia-telangiectasia

Corticosteroid therapy Cyclosporine therapy Antithymocyte globulin therapy Hodgkin's disease Acquired immunodeficiency syndrome

Mycobacteria Nocardia Pneumocystis Candida Legionella Parasites Viruses (especially herpesviruses)

Complement

C2 deficiency

—

Autoimmune diseases

C3 deficiency

—

Pneumococcus, H. influenzae Klebsiella, Neisseria

C5-C9 deficiencies

—

Gram-negative organisms

Humoral immunity

* Unless associated with IgG2 and IgG3 deficiency.

*

698

predominantly cleared by a particular immune response. Impairment in host defense may result from an inherited immunodeficiency or may be acquired as a result of disease processes or treatment with immunosuppressive drugs. For example, disorders of phagocytosis (as manifested by neutropenia) are probably the most common immunodeficiencies now encountered as a result of aggressive therapies for malignancies and collagen vascular diseases. Disorders of cellular immunity are common in patients undergoing immunosuppression after organ transplantation, as well as in those with lymphoproliferative diseases (e.g., Hodgkin's disease) and AIDS. Disorders of humoral immunity frequently occur in patients taking corticosteroids or in those with multiple myeloma or chronic lymphocytic leukemia. Knowledge of the underlying immunodeficiency suggests rational therapies for the infectious complications so common in these patients. Disorders of Phagocytosis Disorders of phagocytosis can be manifested either by the inability of neutrophils to kill ingested organisms (e.g., chronic granulomatous disease, Chédiak-Higashi syndrome) or by an absolute decrease in the number of phagocytes (e.g., neutropenia). Patients with chronic granulomatous disease and Chédiak-Higashi syndrome are especially susceptible to catalase-positive organisms because these organisms can destroy endogenous H2 O2 and thereby further impair the antimicrobial defense 141]

system of phagocytes.[

Patients with chronic granulomatous disease have a defect in the nicotinamide adenine dinucleotide phosphate-oxidase system, leading to 142

impaired production of the reactive oxygen species important for intracellular killing.[ ] Administration of subcutaneous IFN-γ is an effective measure in preventing serious bacterial infections, presumably by augmenting oxygen-independent antimicrobial pathways in phagocytes or improving the function of other 143

components of the immune system.[ ] Chédiak-Higashi syndrome, another disorder characterized by impaired phagocytic intracellular killing, is characterized by abnormal granule function. Phagocytic granules, which contain toxic materials and enzymes important in intracellular killing, bind poorly to phagosomes containing organisms recently ingested.[

144]

Profound neutropenia greatly predisposes individuals to numerous bacterial and fungal pulmonary complications. This risk is related to the degree and duration of 145

neutropenia. The risk of infection begins at a granulocyte count less than 500/mm3 and increases dramatically up to 100/mm3 .[ ] Neutropenia persisting more than 2 weeks greatly increases the risk of infection. The rate at which neutropenia develops is also important. Patients rapidly rendered neutropenic in preparation for bone marrow transplantation are more susceptible to infections than those with collagen vascular disease whose neutrophil counts drop slowly over months secondary to treatment with immunosuppressive agents. Although gram-negative infections remain the most common cause of infectious complications in neutropenic patients, the most devastating infection is invasive aspergillosis.[

146]

The well-recognized risk of neutropenia has led to the development of novel therapies with colony-stimulating factors to shorten the degree and

duration of neutropenia in patients receiving chemotherapeutic regimens.[

147]

Chronic treatment with granulocyte colony-stimulating factor (G-CSF) has also been

148]

used to prevent infections in individuals with idiopathic neutropenia[

and cyclic neutropenia.[

149]

Alveolar macrophage dysfunction and thus impairment of phagocytosis can also be seen in viral infections, ethanol abuse, uremia, metabolic acidosis, ozone exposure, chronic hypoxia, radiation exposure, and cigarette smoking. Disorders of Humoral Immunity Defects in humoral immunity are mediated by either decreased immunoglobulin levels or the inability to form specific antibodies in response to exposure to new antigens. These patients are particularly susceptible to infections of the upper and lower respiratory tracts with encapsulated organisms such as Haemophilus influenzae, Streptococcus pneumoniae, Staphylococcus aureus, and Neisseria species. IgA deficiency is a serum IgA level less than 5•mg/dl in the presence of normal levels of other immunoglobulins and normal cell-mediated immunity. IgA deficiency 150

is the most prevalent primary immunodeficiency in humans, occurring in approximately 1 in 500 individuals.[ ] In these patients, IgM replaces IgA as the predominant immunoglobulin in respiratory secretions. Many patients with selective IgA deficiency have chronic illnesses associated with their condition, and autoimmune diseases occur in a third of cases. A third of IgA-deficient patients also exhibit recurring infections of the respiratory and gastrointestinal tracts. Despite this, less than 5% develop bronchiectasis secondary to recurrent respiratory tract infection. Deficiencies of IgG subclasses, particularly IgG2 and IgG3, often coexist with IgA deficiencies. Patients with IgG2 and IgG3 deficiencies exhibit frequent sinus and respiratory tract infections regardless of whether the deficiency is associated with IgA deficiency or is an isolated defect.[

151]

In addition, measurement of IgG 152

subclasses may not be sufficient; documentation of an immunologic response after vaccination is required to establish a definitive diagnosis. [ ] Bronchiectasis has developed in some of these patients, but prospective studies have not been completed to elucidate fully a definitive cause-and-effect relationship. Nevertheless, analysis of IgG subclasses is an important component in the clinical evaluation of patients with bronchiectasis of unknown cause. IgE deficiency has also been associated with sinopulmonary disease. [

153]

In addition to selective immunoglobulin defects, some congenital immunodeficiencies are associated with a generalized defect in antibody production. Patients with 154]

X-linked agammaglobulinemia produce negligible amounts of all immunoglobulin classes.[ pulmonary hypertension with cor pulmonale as a result of recurrent pulmonary

These patients develop bronchiectasis, pulmonary fibrosis, and

154 155 infections.[ ] [ ]

Patients with common variable immunodeficiency typically have

156 production.[ ]

greater defects in IgG production than in IgA or IgM These patients are also prone to developing recurrent sinopulmonary infections.[ these patients are treated chronically with intravenous immunoglobulin. Disorders of Cellular Immunity A variety of genetic and acquired deficiency states are associated with loss of cell-mediated immunity. The most important

157]

Many of

699

clinical examples of impaired immunity relate to the use of immunosuppressive therapeutic agents (e.g., corticosteroids) and cytotoxic therapy and in recent years to AIDS. The major risk of widespread immunodeficiency is the development of a life-threatening opportunistic pulmonary infection. When an individual with a deficiency in cellular immunity develops pulmonary infiltrates, it must be assumed the patient has an infection until proved otherwise. Patients are particularly susceptible to organisms such as mycobacteria, viruses, Pneumocystis species, and parasites. These infections are particularly prominent in organ transplant recipients, because all these patients have iatrogenically induced defects in cellular immunity resulting from agents such as cyclosporine, corticosteroids, and antithymocyte globulin. Human Immunodeficiency Virus

HIV infection represents the modern paradigm for cellular immune deficiency. In patients with AIDS the profound loss of immunity results in opportunistic infection or neoplasm and leads invariably to the death of the patient. Early studies focused on CD4+ lymphocyte infection by the virus. HIV preferentially infects memory CD4+ T cells, thus explaining the early loss of antigen-specific delayed-type hypersensitivity reactions in these individuals.[

158] [159]

However, more recent studies have focused on infection of monocytes and macrophages and the resultant perturbations in immune function. Alveolar macrophages and macrophages in bronchiole-associated lymphoid tissues can clearly be infected with HIV.[ chemokine co-receptor, CCR5, to permit entry of many primary HIV macrophages.

[163] [164]

167 168 169 170 cytokines[ ] [ ] [ ] [ ]

Lung macrophages appear to express the appropriate

HIV infection does not appear to impair the phagocytic function of alveolar

Alveolar macrophages in HIV-infected individuals are highly activated.[

secrete large amounts of inflammatory [171]

162 isolates.[ ]

160] [161]

165] [166]

As a result, alveolar macrophages from these patients

and have an enhanced ability to act as accessory cells in the induction of T cell proliferation.

This finding may explain the phenomenon of lymphocytic alveolitis seen in patients with HIV, even in the asymptomatic stages of the disease. Lymphocytic 172]

alveolitis portends a poor prognosis and is believed to be an appropriate immunologic response to the local HIV viral burden in the lung.[ response is mediated by

173 IFN-γ[ ]

and CXC

The lymphocytic

174 chemokines.[ ]

Although these effects might be expected to enhance immune responses to invading organisms, the inflammatory reaction to pathogens is often overly exuberant, resulting in damage to lung tissue and viral propagation. Furthermore, antigen-specific responses may be lost as infected accessory cells interact with uninfected T cells, leading to their infection and destruction. Evidence suggests that alveolar macrophages infected with HIV have an impaired oxidative burst response that contributes to the pathogenesis of Pneumocystis pneumonia.[

175]

Not all the cytokines released by alveolar macrophages may be proinflammatory in nature. For example, secretion of enhanced TGF-β by alveolar macrophages may explain the reduced amount of opsonizing IgG in BAL fluid of asymptomatic HIV-infected patients.[

176]

Inconsistent results after measuring immunoglobulins in

177]

BAL[

may result from the study of patients in different stages of disease. Evidence is lacking for a direct cause-and-effect relationship between decreased BAL 178

IgA or IgG levels and lung infection in AIDS patients. IgG3 may be the most important immunoglobulin in neutralizing HIV.[ ] Alternatively, the HIV-induced loss of the CD4+ lymphocyte population may impair regulation of B cell function and ultimately result in the inability of B cells to participate effectively in the immune response. This impairment in antigen-specific antibody responses may explain the increased incidence of infection with Streptococcus pneumoniae and other encapsulated bacteria in HIV-infected subjects.[

179] [180]

Thus, virtually all aspects of immunity and all cell types are either infected by or affected by HIV, leading to devastating clinical results as the disease progresses. As such, HIV infection can also predispose patients to pulmonary hypertension and precocious emphysema.[

181]

Disorders of Complement Deficiencies of various complement components can predispose patients to pulmonary infections. The most common is C2 deficiency, associated with an increased incidence of various autoimmune diseases.[

182]

Patients with C3 deficiency demonstrate increased respiratory tract infections, especially those caused by

183 bacteria,[ ]

encapsulated resulting from the loss of complement as an important opsonin for optimal phagocytosis of these organisms. Deficiencies in any component of the terminal membrane attack complex (C5 to C9) predisposes individuals to infections caused by gram-negative bacteria, especially Neisseria species. [184]

Activation of the complement cascade leads to a series of events resulting in the hallmark findings of inflammation: heat, erythema, edema, pain, and loss of function. These events are normally kept in check by complement inhibitors. Several disorders of complement are not characterized by deficiencies of complement but rather are associated with defects in the inhibitors of complement. Hereditary angioneurotic edema, a disease characterized by episodic acute subcutaneous or mucosal swelling that can be fatal if the upper respiratory tract is involved, results from a quantitative or functional defect of C1 inhibitor, leading to uncontrolled 185] [186]

activation of the classic pathway of complement.[

Familial Mediterranean fever, characterized by recurrent inflammatory attacks of serosal surfaces,

including the pleura, has been associated with a deficiency of C5a inhibitor.[

187]

DISEASES ASSOCIATED WITH UNCHECKED IMMUNE RESPONSE Mechanisms operative in lung defense are important in the pathogenesis of many lung disorders. This fact is most obvious in deficiency states in which the absence of functioning inflammatory or immune effector cells is associated with an increased risk of infection. However, many of these defense mechanisms may be amplified and perpetuated in certain lung disorders. For example, granulomatous disorders of unknown etiologies (e.g., sarcoidosis) are mediated by activated Th cells and monocyte/macrophages within the interstitium and alveolar spaces of the lung. Sarcoidosis, a potentially fatal lung disorder, is likely caused by an abnormal immune response to an unidentified antigen. Thus, lung disorders may result from either deficient or excessive immune responses. The key to understanding a specific lung disease is to determine the etiology and the

700

pathogenetic scheme propagating the disorder and, if possible, to develop a therapeutic strategy to reverse the underlying mechanisms of the disease process. 96 103

The use of BAL was initially popularized in the study of specific interstitial lung disorders such as idiopathic pulmonary fibrosis (IPF) and sarcoidosis. [ ] [ ] For the first time, these studies suggested that inflammation of the alveolar spaces, or “alveolitis,” was a critical step in the pathogenesis of these disorders. Clearly, many 188 189

lung disorders can be classified as either a polymorphonuclear leukocyte-predominant or lymphocyte-predominant alveolitis[ ] [ ] ( Box 42-3 ). The recognition that an abnormal immune response may be compartmentalized within the lung (and not reflected by peripheral blood studies) is in large part related to investigative studies using BAL to probe the underlying mechanisms. The use of BAL to provide new information regarding the pathogenesis of these lung disorders is not questioned. The controversy regarding the use of BAL relates to its clinical diagnostic or prognostic value, a subject well beyond the scope of this chapter. Current concepts of the evolution of fibrosis and interstitial lung disorders postulate a central role for infiltration of inflammatory cells into the alveolar compartment. The activation of alveolar macrophages and newly recruited lung phagocytes (e.g., neutrophils, eosinophils) within the lower respiratory tract has been implicated in the pathogenesis of Box 42-3. Types of Alveolitis in Common, Noninfectious Diffuse Lung Disease

Neutrophil Predominant Acute interstitial pneumonia (AIP, Hamman-Rich syndrome) Usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) Rheumatoid interstitial lung disease (RILD) Scleroderma Asbestosis Drug reactions Adult respiratory distress syndrome (ARDS)

Eosinophil Predominant Chronic eosinophilic pneumonia Pulmonary infiltrates with eosinophilia Drug reactions UIP/IPF

Lymphocyte Predominant Sarcoidosis Hypersensitivity pneumonitis Silicosis Sjögren's syndrome Berylliosis Drug reactions Nonspecific interstitial pneumonia (NSIP) Bronchiolitis obliterans organizing pneumonia (BOOP)

inflammatory lung disorders, including ARDS, rheumatoid interstitial lung disease (RILD), and IPF, now known as usual interstitial pneumonia (UIP). Other lung disorders, particularly granulomatous disorders, are associated with abnormal recruitment and replication of T cells and monocytic cells within the lower respiratory tract. The best-studied lung disorders with these features include sarcoidosis and hypersensitivity pneumonitis. Adult Respiratory Distress Syndrome ARDS, an acute lung injury syndrome with a high mortality rate (approximately 50%), is a common sequela to a variety of pulmonary (pneumonia) and nonpulmonary (sepsis, trauma, pancreatitis) insults. ARDS results in the accumulation of a protein-rich edema fluid within the alveolar compartment. This flooding

of the airways, a consequence of enhanced permeability of the alveolar capillary membrane, results in severe hypoxemia, diffuse radiographic infiltrates, and the frequent need for mechanical ventilation because of hypoxemic respiratory failure. The pathologic hallmark of ARDS is diffuse alveolar damage (DAD). Neutrophils are more abundant in the pulmonary vasculature than in any other area of the body. Once neutrophils are sequestered in the pulmonary capillaries, they may adhere to the endothelial surface and traverse the pulmonary interstitium to gain access to the alveolar space. Although many aspects of ARDS are still unclear, BAL studies have demonstrated increased numbers of polymorphonuclear neutrophils (PMNs) in the lower respiratory tract, suggesting that these potent effector 190] [191]

cells are involved in the vascular and alveolar injury observed in these patients. [

Alveolar macrophages are important in the pathogenesis of ARDS because

they are responsible for secreting the “early-response cytokines” IL-1 and TNF. These cytokines can initiate and potentiate the inflammatory response.[ of ARDS patients appear to continually recruit monocytes to the lung due to persistent up-regulation of monocyte chemotactic protein-1 chemokine may prove to have prognostic utility as may the measurement of IL-8 complexes or surfactant levels in BAL the most well studied being IL-8, are present in BAL samples from patients with collagenase, are present in BAL samples from patients with through its function as a neutrophil chemoattractant.[

132 197 ARDS.[ ] [ ]

194 196 ARDS.[ ] [ ]

193 (MCP-1).[ ]

193 194 195 fluids.[ ] [ ] [ ]

192]

A subset

This

PMN chemoattractants,

Additionally, PMN secretory products, such as elastase and

ARDS may be mediated by activation of the complement cascade, either directly[

198]

or

199]

When these cells release or generate toxic substances in the microcirculation, a highly permeable vascular state may be produced through direct damage to the endothelium. Morphologic data in animal studies suggest that PMN sequestration in the pulmonary microcirculation occurs in areas contiguous with endothelial cell 200

201 202

injury and may be causally related.[ ] Furthermore, PMN sequestration can be triggered by experimentally induced endotoxemia.[ ] [ ] Detailed electron microscopic examinations have shown PMNs penetrating the vascular endothelium in proximity to injured endothelial cells. In some animal models of acute lung 203]

injury, neutrophil depletion before infusion of a variety of vasoactive substances greatly attenuates edema formation and lung injury.[ neutrophils is noted in lung injury and ARDS. Efficient clearance of these cells is important

Apoptosis of lung

701

126] [204]

in regulating inflammation[ secreted by apoptotic

because bystander lung epithelial cells may themselves undergo apoptosis in the presence of the soluble Fas ligand that is

205 neutrophils.[ ]

Despite this large volume of data implicating leukocyte activation in ARDS, the finding that ARDS develops in neutropenic patients and in certain animal models 206 207

despite neutrophil depletion suggests that noninflammatory cell-mediated pathways for acute lung injury also exist.[ ] [ ] The pathogenesis of ARDS is complex and involves a variety of cellular components, inflammatory cells, and inflammatory mediators. Considerable data suggest that neutrophils are important participants in the development of the acute lung injury in ARDS patients, but the exact mechanisms by which neutrophils are recruited to lung tissue and produce these effects remain incompletely understood.

Although survival of patients with ARDS has improved, it is generally attributed to advances in supportive care in the intensive care unit. Studies aimed at inhibiting inflammation in the lung have not been successful, and administration of surfactant has not been proven as clinically useful to date. However, two therapies deserve mention. First, late (7 to 10 days after lung injury) and prolonged administration of corticosteroids may be beneficial and alter the course of the disease.[ mechanical ventilation at lower tidal volumes improves survival of ARDS patients.

208]

Second,

[209]

Pulmonary Fibrosis Many chronic, noninfectious lung disorders have similar pathogenic mechanisms with the end result being fibrosis. Patients who survive a bout of pneumonia and ARDS typically recover normal lung architecture despite the intense inflammation described in these processes. This is not the case for the chronic, fibrotic lung disorders. The prototypic human fibrotic lung disease is idiopathic pulmonary fibrosis (IPF). Other causes of chronic interstitial lung disease that may have similar pathogenic mechanisms to IPF include RILD, drug reactions (bleomycin), occupational or environmental exposures (silicosis, asbestosis, tobacco smoke), and lung allograft rejection. “Cryptogenic fibrosing alveolitis,” or IPF, is now known as usual interstitial pneumonia (UIP). This change in terminology reflects an evolution in the clinical course and pathologic findings of these diseases. Entities that were previously lumped into the broad category of IPF are now separated into UIP, nonspecific interstitial 210]

pneumonia (NSIP), desquamative interstitial pneumonia (DIP), and others.[

UIP is a relatively common and ultimately fatal disorder, with a 5-year survival of 30%. Increased alveolar macrophages, neutrophils, and eosinophils are present in BAL fluid from patients with IPF.[ and

212 elastase[ ]

96] [103] [211]

BAL fluid shows evidence of neutrophil activation and increased levels of neutrophil products such as collagenase

in many patients with pulmonary fibrosis. Additionally, many cytokines, including TGF-β, have been associated with lung inflammation and

pulmonary fibrosis.[

213]

214]

Patients who demonstrate objective responses to steroid therapy have a decline in the number of neutrophils in subsequent lavages.[

Other lung inflammatory cells may also play critical roles in the pathogenesis of UIP (IPF) and interstitial lung disease. For example, eosinophils have been detected 96 103 188 189

in BAL fluid from patients with IPF.[ ] [ ] [ ] [ ] In selected patients with IPF or RILD, the number of eosinophils may exceed the number of neutrophils recovered from BAL. BAL fluid eosinophilia correlates with functional abnormalities (reduced diffusion and lung volumes) and predicts functional outcome and 215]

disease progression. For example, Peterson et al[

have shown that the presence of eosinophils in BAL fluid predicts worsening pulmonary function and decreased

capacity for responses to pharmacologic therapy. This may result from the release of toxic products from activated eosinophils (e.g., eosinophil cationic protein[ or from the interaction of major basic protein with primed

216]

)

217 fibroblasts.[ ]

Lung lymphocytes may also participate in the development of lower respiratory tract abnormalities in UIP. Lymphocytes may accumulate in the lung before the 218]

development of marked fibrosis.[

103] [219]

The finding of increased numbers of immunoglobulin-releasing cells[

in BAL fluid and altered T cell subpopulations is

96 level.[ ]

further evidence of abnormal immunoregulation at the alveolar In a lung fibrosis model using influenza antigen, it appears that CD8+ T lymphocytes interact with alveolar epithelial cells in an antigen-specific manner. However, rather than inducing cell death, these lymphocytes actually cause activation of alveolar cells to produce MCP-1 and recruit cells to the alveolar space.[

220]

This action may have important implications in perpetuating the immune response and pulmonary

fibrosis. Recent studies in pulmonary fibrosis have used a murine model of bleomycin-induced injury. The different strains of mice used lends variability to the conclusions, but it is clear that T lymphocytes and associated cytokines are important mediators/modulators of fibrosis.[

213] [221] [222]

The formation of immune complexes may account for the alveolar macrophage activation and subsequent inflammatory response in these disorders. Immunoglobulin, growth factors, complement, and immune complex components have been demonstrated in BAL fluid and histologically in the alveolar walls of patients with UIP and RILD. Furthermore, pulmonary fibrosis can be induced in an animal model by the intratracheal administration of haptens, low-molecularweight compounds that bind to host cellular proteins and render them immunogenic.[ alveolar macrophage activation in UIP and perhaps other interstitial lung disorders.

223]

Thus, evidence indicates that immune complexes may be the stimuli for

A pathogenetic scheme emerges: fibroblast foci are the pathologic hallmark of UIP. The initial event in UIP/IPF is the presence of an inhaled foreign protein (possibly a viral protein), which results in an immune response, accumulation of stimulated lymphocytes, subsequent immunoglobulin production, and the formation of immune complexes within the lung. In cases of circulating autoantibody, immune complexes may be deposited within alveolar structures. Response to these 213

stimuli could create macrophage production of leukocyte chemotaxins and the soluble factors (predominantly Th2 cytokines)[ ] that modulate fibroblastproliferative activities in order to “wall off” these repeated acute insults. Immune complex–stimulated neutrophils and eosinophils within the lung structures could also contribute to alveolar inflammation and fibrosis. This paradigm is highlighted by repeated, successive acute insults with aberrant repair[ progressive fibrosis and differs from the “one-hit”

225 hypothesis[ ]

224]

that cause

of lung injury in ARDS ( Figure 42-5 ).

702

Figure 42-5 In adult respiratory distress syndrome (ARDS) a “single hit” is necessary and sufficient to begin the cascade of acute inflammation. The host response is perpetuated in a “big hit”; acute lung injury is excessive, and fibrosis with end-stage lung disease and death can occur. In contrast, a “little hit” can cause initial injury with subsequent resolution and minimal lung scarring. In patients with classic idiopathic pulmonary fibrosis (IPF), now called usual interstitial pneumonia (UIP), multiple repeated hits are required to perpetuate lung injury. If the healing process is abnormal, each acute insult, depending on genetic and environmental factors, can cause fibrotic scars and eventually end-stage lung disease.

TABLE 42-6 -- Radiographic Patterns of Pulmonary Infiltrates in Immunocompromised Patients Pattern

Infectious

Noninfectious

Focal

Cryptococcus Aspergillus Mucor

Pulmonary infarct NSIP

Nodular/cavitary

Cryptococcus Nocardia Aspergillus Bacterial lung abscess Septic emboli

Neoplastic disease

Diffuse

Pneumocystis carinii Cytomegalovirus Cryptococcus Aspergillus Candida

Pulmonary edema Drug-induced disease BOOP Alveolar hemorrhage NSIP

NSIP, Nonspecific interstitial pneumonitis; BOOP, bronchiolitis obliterans organizing pneumonia.

TABLE 42-7 -- Patient Evaluation for Suspected Immunodeficiency Test

Disease Process

General Tests Complete blood cell count Culture of sterile site Sweat chloride test Lymph node biopsy Bone marrow examination

Neutropenia, lymphopenia Specific infection Cystic fibrosis Specific pathogens, neoplasm Specific pathogens, neoplasm, marrow failure

Human immunodeficiency virus (HIV) testing

Acquired immunodeficiency syndrome

Specific Tests C3, C4, C5, C6, C7, C8, C9, CH50

Complement deficiency

Delayed-type hypersensitivity skin tests, mitogen/antigen proliferation assays, T cell subsets

Cellular immune deficiency

Quantitative immunoglobulins, IgG subclasses response to vaccination

Humoral immune deficiency

Nitroblue tetrazolium test

Phagocytosis

Nasal mucosa biopsy

Ciliary dysfunction

or biopsy ( Table 42-7 ). If specific immunologic defects are suspected, the evaluation can be tailored along those lines. For example, quantitative immunoglobulin levels, including measurement of IgG subclasses, are used to evaluate the humoral immune system. Skin testing with three to five antigens and measurement of T cell subsets are used to evaluate the cellular immune system. If suspected, the initial evaluation should also include a measurement of the α1 -PI level. Other tests include the nitroblue tetrazolium test to evaluate the oxidative capacity of neutrophils, specific complement levels, and possibly, nasal biopsies to evaluate ciliary function and structure. Finally, most patients eventually undergo bronchoscopy to obtain materials from the lower respiratory tract for diagnostic study. Understanding of the normal events in alveolar and bronchiolar tissue is largely derived from lung biopsy and BAL immunohistochemistry. Each method has advantages and disadvantages. Although histopathologic methods often represent the “gold standard” for cell and tissue pattern identification of the alveolar spaces and interstitium, these tests are often limited to a single sampling and do not easily permit quantification of the observed changes. In contrast, BAL performed during flexible fiberoptic bronchoscopy permits easy sampling of fluid from the lower respiratory tract and allows the investigator to quantify changes in the cellular and 96] [237] [252]

noncellular constituents of the alveolar lining fluid.[

BAL is rarely as diagnostic as standard tissue biopsies; however, much recent information regarding the pathogenesis of lung disease is from BAL examination of lower respiratory tract fluids in healthy and unhealthy research subjects. BAL is

705

performed during bronchoscopy using specific aliquots of fluid (e.g., 5 × 20•ml of normal saline), which are instilled into a fifth-order or sixth-order subsegmental bronchus with immediate return suction after each aliquot. Typically, 40% to 60% of instilled fluid is recovered. Because no worldwide-standardized procedure for BAL exists, the results vary significantly. The authors use the method originally developed at the National Heart, Blood, and Lung Institute in the 1970s, instilling 5 × 20•ml of aliquots at each site in the lung and typically doing two or three sites per BAL procedure. Improper “wedging” of the bronchoscope, suction of purulent secretions, and significant bleeding during the procedure reduce the validity of the results. BAL can be used to determine cell differentials (using cytocentrifuge preparation smears or filter method) with a wide number of special stains to determine noncellular constituents (e.g., proteins, immunoglobulin levels, drug levels, asbestos bodies) or to store aliquots at −85° C or in liquid nitrogen for future use. BAL can also be used as a diagnostic test for various pulmonary infections. In the past, BAL by itself had been considered adequate to diagnose most pulmonary infectious processes in immunosuppressed patients with pulmonary infiltrates. However, with the widespread use of prophylactic medications against common opportunistic pathogens (e.g., trimethoprim-sulfamethoxazole for Pneumocystis carinii prophylaxis), the spectrum of diseases has shifted in immunocompromised patients. Thus, many clinicians now believe that to obtain adequate diagnostic sensitivity and specificity through bronchoscopy, transbronchial biopsies are required in addition to BAL.[

253]

Considering the vast number of lung disorders associated with an “unchecked” or exuberant immune response, only a very broad recommendation can be made for

evaluation of these patients. Ultimately, the clinician should choose a method of evaluation that provides the best diagnostic information in a timely and costeffective manner using the least invasive methods. Many tests, such as pulmonary function tests, may not be diagnostic but may provide important information regarding the activity of disease or the response to therapy. However, it is controversial whether a characteristic high-resolution chest computed tomography (HRCT) 224

scan is diagnostic in UIP/IPF.[ ] More invasive procedures such as bronchoscopy or open lung biopsy should be reserved for persons with progressive lung disorders in whom routine testing has failed to establish a diagnosis and for whom the hope of effective therapeutic intervention exists. The importance of an efficient evaluation of patients with progressive lung disease cannot be overstated. This provides the best chance for an early diagnosis and reversal of the disease process before end-stage lung damage and fibrosis occur. Because of the long waiting period for patients, early referral for lung transplantation is recommended.

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Cell Mol Biol 25:362, 2001. 221. Chen ES, Greenlee BM, Wilks-Karp M, et al: Attenuation of lung inflammation and fibrosis in interferon gamma–deficient mice after intratracheal bleomycin, Am J Respir Cell Mol Biol 24:545, 2001. 222. Keane MP, Belperio JA, Burdick MD, et al: IL-12 attenuates bleomycin-induced pulmonary fibrosis,Am J Physiol 281(Lung Cell Mol Physiol):L92, 2001. 223. Stein-Streilein J, Lipscomb MF, Fisch H, et al: Pulmonary interstitial fibrosis induced in hapten-immune hamsters, Am Rev Respir Dis 136:119, 1987. 224. Gross TJ, Hunninghake GW: Idiopathic pulmonary fibrosis, N Engl J Med 345:517, 2001. 225. Meduri GU: Levels of evidence for the pharmacologic effectiveness of prolonged methylprednisolone treatment in unresolving ARDS, Chest 116:116S, 1999. 226. Striz I, Wang YM, Kalaycioglu O, et al: Expression of alveolar macrophage adhesion molecules in pulmonary sarcoidosis, Chest 102:882, 1992. 227. Pinkston P, Bitterman PB, Crystal RG: Spontaneous release of interleukin-2 by lung T lymphocytes in active pulmonary sarcoidosis, N Engl J Med 308:793, 1983. 228. Muller-Quernheim J, Saltine C, Sondermeyer P, et al: Compartmentalized activation of the interleukin 2 gene by lung T lymphocytes in active pulmonary sarcoidosis, J Immunol 137:3475, 1986. 229. Hunninghake GW, Crystal RG: Mechanisms of hypergammaglobulinemia in pulmonary sarcoidosis: site of increased antibody production and role of T lymphocytes, J Clin Invest 67:86, 1981. 230. Kishimoto T: B-cell stimulatory factors (BSFs): molecular structure, biological function, and regulation of expression, J Clin Immunol 7:343, 1987. 231. Rohrbach MS, Veek-Pavlovic Z, Martin WJ, et al: Induction of angiotensin converting enzyme in cultured monocytes by a factor in the bronchoalveolar lavage fluid of sarcoidosis patients, Sarcoidosis 5:17, 1988. 232. Seitzer U, Swider C, Stuber F, et al: TNF-α promotor gene polymorphisms in sarcoidosis, Cytokine 9:787, 1997. 233. Maliarik MJ, Rybicki BA, Malvitz E, et al: ACE gene polymorphism and risk of sarcoidosis, Am J Respir Crit Care Med 158:1566, 1998. 234. American Thoracic Society: Statement on sarcoidosis, Am J Respir Crit Care Med 160:736, 1999. 235. Baughman RP, Ohmichi M, Lower EE: Combination therapy for sarcoidosis,Sarcoidosis Vasc Diffuse Lung Dis 18:133, 2001. 236. Trentin L, Migone N, Zambello R, et al: Mechanisms accounting for lymphocytic alveolitis in hypersensitivity pneumonitis, J Immunol 145:2147, 1990. 237. Costabel U, Guzman J: Bronchoalveolar lavage in interstitial lung disease, Curr Opin Pulm Med 7:255, 2001. Occupational Lung Disorders: Pneumoconioses

238. Struhar D, Harbeck RJ, Mason RJ: Lymphocyte populations in lung tissue, bronchoalveolar lavage fluid, and peripheral blood in rats at various times during the development of silicosis, Am Rev Respir Dis 139:28, 1989. 239. Christman JW, Emerson RJ, Graham WCB, et al: Mineral dust and cell recovery from the bronchoalveolar lavage of healthy Vermont granite workers, Am Rev Respir Dis 132:393, 1985.

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240. Rom WN: Relationship of inflammatory cell cytokines to disease severity in individuals with occupational dust exposure, Am J Ind Med 19:15, 1991. 241. Begin R, Cantin A, Masse S: Recent advances in the pathogenesis and clinical assessment of mineral dust pneumoconioses: asbestosis, silicosis and coal pneumoconiosis, Eur Respir J 2:988, 1989. 242. Ghio AJ, Kennedy TP, Schapira RM, et al: Hypothesis: is lung disease after silicate inhalation caused by oxidant generation? Lancet 336:967, 1990. 243. Lesur O, Veldhuizen RR, Whitsett JA, et al: Surfactant associated proteins (SP-A, SP-D) are increased proportionally to alveolar phospholipids in sheep silicosis, Lung 171:63, 1993. 244. Robinson BWS, Rose AH, James A, et al: Alveolitis of pulmonary asbestosis: bronchoalveolar lavage studies in crocidolite- and chrysotile-exposed individuals, Chest 90:396, 1986. 245. Garcia JGN, Griffith DE, Cohen AB, et al: Alveolar macrophages from patients with asbestos exposure release increased levels of leukotriene B4 , Am Rev Respir Dis 139:1494, 1989. 246. Wallace JM, Oishi JS, Barbers RG, et al: Bronchoalveolar lavage cell and lymphocyte phenotype profiles in healthy asbestos-exposed shipyard workers, Am Rev Respir Dis 139:33, 1989. 247. Cullen MR, Cherniack MG, Kominsky JR: Chronic beryllium disease in the United States, Semin Respir Med 1:203, 1986. 248. Newman LS, Kreiss K, King TE Jr, et al: Pathologic and immunologic alterations in early stages of beryllium disease: re-examination of disease definition and natural history, Am Rev Respir Dis 139:1479, 1989. 249. Kriebel D, Brain JD, Sprince NL, et al: The pulmonary toxicity of beryllium, Am Rev Respir Dis 137:464, 1988. 250. Saltini C, Winestock K, Kirby M, et al: Maintenance of alveolitis in patients with chronic beryllium disease by beryllium-specific helper T cells, N Engl J Med 320:1103, 1989. 251. Kreiss K, Newman LS, Mroz MM, et al: Screening blood test identified subclinical beryllium disease, J Occup Med 31:603, 1989.

Clinical Evaluation 252. Martin WJ II, Williams DE, Dines DE, et al: Interstitial lung disease: assessment by bronchoalveolar lavage, Mayo Clin Proc 58:751, 1983. 253. Cazzadori A, Di Perri G, Todeschini G, et al: Transbronchial biopsy in the diagnosis of pulmonary infiltrates in immunocompromised patients, Chest 107:101, 1995.

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711

Chapter 43 - Airway Smooth Muscle and Related Extracellular Matrix in Normal and Asthmatic Lung

R. Robert Schellenberg Chun Y. Seow

From a clinical perspective, research in structure and function of airway smooth muscle is sustained by the interest in pathologic conditions demonstrating exaggerated airway narrowing caused, at least in part, by airway smooth muscle (ASM) contraction. The reversible airway narrowing seen in asthma is believed to be the result of ASM contraction.[ 4] [5]

lung when stimulated.[

1] [2] [3]

In vitro studies of ASM mechanics suggest that airway smooth muscle has the potential to close all airways completely in the

Although complete closure can occur in some maximally stimulated airways,[

6] [7]

most airways remain open in a healthy lung even

[8] [9] [10]

when the muscle is maximally activated. Answers to why most of the airways remain open during maximal bronchochallenge in healthy subjects may provide insights necessary to understand the pathogenesis of asthma and other obstructive lung diseases. Understanding of airway narrowing has been based on a static balance of forces. The airway should stop narrowing when the force generated by the encircling 11] [12]

smooth muscle cells is exactly counterbalanced by the tethering force of the lung parenchyma,[ 13 14 15 compression,[ ] [ ] [ ]

the axial and radial constraints of the extracellular

16 17 matrix,[ ] [ ]

the force necessary to accomplish mucosal folding and

and the force arising from the transluminal pressure difference. This static

model has been challenged by the recent reports that the dynamic environment of the breathing lung plays an important role in modulating ASM contractility. [ [20]

Although useful in elucidating several important mechanisms in determining airway caliber,[

21] [22]

18] [19]

the static model failed to predict accurately the extent of

8 10

airway narrowing during bronchoprovocation in normal subjects.[ ] [ ] The missing link appears to be the oscillatory stress and strain imposed on the ASM cells by the constant tidal action of spontaneous breathing interposed by deep inspirations. The effect of load fluctuation on the mechanical performance of airway smooth muscle has been investigated.[

9] [23] [24] [25] [26]

The negative inotropic effect of load fluctuation on airway smooth muscle may be explained by (1) a mechanism in

which the static equilibrium of myosin cross-bridge binding to actin filament is disrupted, resulting in “premature” detachment of some of the cross-bridges,[ (2) a less understood mechanism in which organization of the contractile filaments is disrupted, resulting in a diminished ability to generate Both mechanisms likely are involved in producing the bronchodilating effect of oscillatory strains.

19]

and

18 20 27 28 force.[ ] [ ] [ ] [ ]

The cytoskeleton of airway smooth muscle and the extracellular matrix in which the muscle cells are embedded have increasingly been viewed as dynamic structures that have an actively supportive role in force generation. In altering their mechanical properties, these structures also modulate the load against which the muscles shorten and therefore regulate the degree of airway narrowing caused by ASM contraction.

MECHANISM OF SMOOTH MUSCLE CONTRACTION 29 30 31

The mechanism of contraction at the cross-bridge level in smooth muscle is believed to be fundamentally the same as that in striated muscle[ ] [ ] [ ] because of the striking ultrastructural, biochemical, and biophysical similarities between the contractile and cytoskeletal proteins found in both smooth and striated muscles. Studies of molecular mechanics of myosin have revealed that, in terms of the ability to produce force and displacement, smooth and striated muscle myosins behave similarly.[

32] [33] [34]

35] [36]

In terms of function at the cellular level, the hyperbolic force-velocity relationship characterizing striated muscle contractility[

found to exist in single smooth muscle

is also

37 cells.[ ]

The structural similarity between smooth and striated muscles found at the cross-bridge level, however, largely disappears at the higher level of intracellular organization. The ultrastructure of the contractile apparatus in smooth muscle appears to be less organized than that in striated muscle. No regularly spaced filament arrays are found in smooth muscle. Some studies suggest that plastic rearrangement of the contractile and cytoskeletal elements in smooth muscle occurs whenever the muscle is subjected to a large change in length; this is thought to enable the muscle to adapt to changes in cell geometry and retain optimal contractile function. [38] [39]

The need to constantly reconfigure its contractile apparatus may be the underlying cause for the muscle to adopt a less organized and more malleable structure. Contractile Unit Although the structure of a contractile unit in smooth muscle has never been clearly delineated, it likely consists of three basic components: a myosin thick filament, actin thin filaments, and dense bodies, or dense plaques on the cell membrane. These elements can be seen to occupy most of the cytoplasmic space in airway smooth 40] [41]

muscle ( Figure 43-1 ). The thick filaments in smooth muscle are believed to be side-polar. [ with a thin filament possessing the “right”

The cross-bridges on one side of a thick filament can interact only

712

Figure 43-1 Electron micrograph of transverse section of airway smooth muscle showing myosin thick filaments (arrow), surrounded by actin thin filaments, and dense bodies (arrowhead), surrounded by intermediate filaments. Inset, Magnified area outlined by square, showing a dense body and intermediate filaments. Calibration bar represents 200•nm.

Figure 43-2 Contractile units of smooth (A) and striated (B) muscles showing basic components and how side-polar and bipolar thick filaments may influence contractile unit design. Double arrows indicate direction of thin filament sliding relative to thick filament.

Figure 43-3 Reaction scheme of adenosine triphosphate (ATP) hydrolysis in cross-bridge cycle. A, Actin; M, myosin; D, MgADP; P, inorganic phosphate; T, MgATP. Asterisk (*) represents an isomerization state.

Figure 43-4 A, Effect of thick filament lengthening. Force increases due to increase in number of cross-bridges acting in parallel; maximal sliding velocity remains the same. B, Series-to-parallel transition (top to bottom) that sacrifices higher velocity (top) for greater force (bottom) while maintaining the same power output.

Figure 43-5 Hypothetic models of smooth muscle cell at different lengths. Each contractile unit produces unit force (F) and shortening velocity (V) when cell is activated.

Figure 43-6 Smooth muscle length adaptation. Number of contractile units in series is varied to preserve optimal overlap of contractile filaments.

TABLE 43-1 -- Mechanical Properties of Asthmatic versus Nonasthmatic Airway Smooth Muscle Preparations Asthmatic Property

Patient 1

Patient 2

Nonasthmatic

Maximal shortening (percent of starting length)

25%

31%

4.4% to 15%

Stress (kg/cm2 )

0.9

1.2

0.2 to 0.6

720

The finding of unique mechanical properties in human airway preparations should be emphasized. Under the restraints of the normal surrounding tissue in larger 162

163]

airways, muscle shortening is very limited, with maximal shortening averaging about 10%.[ ] This compares with shortening of 80% for pig tracheal muscle.[ comparison of ASM responses from different species revealed that the degree of shortening is inversely correlated with the amount of ECM present in the tissue 164

A

preparation. [ ] These differences highlight the importance of external loads in influencing the muscle contractile response. For this reason (and the finding that we could not explain increased muscle shortening on the basis of increased amount of muscle in asthmatic tissues), we hypothesized that decreasing the load imposed on 151]

the smooth muscle by ECM elements could induce the changes noted in asthma[

( Figure 43-7 ). 165 166

A host of studies have evaluated the proliferation and properties of isolated ASM cells[ ] [ ] (see Chapter 25 ). Such studies are helpful in defining intracellular signaling mechanisms and cell surface determinants but have limitations in terms of modulating factors present in the normal tissue milieu. This restriction results 167] [168]

from the rapid differentiation of cultured cells to a noncontractile phenotype, with loss of muscle myosin and α-smooth muscle actin.[ occur in asthma to increase muscle mass, these cells would have to redifferentiate later to a contractile phenotype.

Although this might

ECM elements may limit smooth muscle proliferation, and degradation of these elements would then allow increases in muscle. A recent study demonstrated that 169

MMP-1, but not MMP-2, -3, or -9, was increased in asthmatic smooth muscle by immunocytochemistry and biologic activity.[ ] In addition to cleaving collagen, which surrounds smooth muscle cells in vivo, MMP-1 has proteolytic activity for insulin-like growth factor (IGF)–binding proteins that inhibit cell growth. In isolated smooth muscle cell cultures, leukotriene D4 is

Figure 43-7 Decrease in stiffness of extracellular elements composing parallel elastic elements leads to increased smooth muscle shortening without a requirement for change in the muscle itself. (Modified from Bramley AM, Thomson RJ, Roberts CR, et al: Eur Respir J 7:337, 1994.)

(Modified from Bramley AM, Thomson RJ, Roberts CR, et al: Eur Respir J 7:337, 1994.) 170

171

mitogenic[ ] and modulates the IGF axis by inducing MMP-1.[ ] Therefore the increased MMP-1 activity could lead to increased amount of smooth muscle. Whether this occurs in vivo remains to be demonstrated. In studies we have performed with airway explants, factors shown to induce proliferation of isolated airway cells (including leukotrienes) have had no effect on the amount of muscle measured morphometrically or by BrdU incorporation, and increases in muscle force generation also had no effect. [ well as function.

172]

This emphasizes the importance of the normal cellular milieu, including ECM elements, in modulating myocyte proliferation as

Proteoglycans 173 174

Matrix proteoglycans influence tissue mechanics, ECM assembly, cell growth, and cell maturation and affect the biologic activities of growth factors. [ ] [ ] Increased amounts of a number of proteoglycans are found in human asthmatic airways. Deposition of versican, biglycan, and hyaluronan was greater in autopsy samples from fatal asthma cases,[ with the degree of

175 AHR.[ ]

173]

and versican, biglycan, and lumican were increased in the submucosa of asthmatic airway biopsies, with the amount correlating 173] [176]

Loss of proteoglycan (primarily aggrecan) from airway cartilage in fatal asthma has been described.[

Perichondrial thickening

in asthma and COPD was found to correlate with the degree of eosinophilia,[

177]

in keeping with the previously mentioned studies of TGF-β1 . This study also

evaluated the proportion of degenerated cartilage that was increased in airways of asthmatic patients and those with chronic bronchitis, and this parameter correlated with the number of neutrophils in the airway.[ asthma who are refractory to steroid

177]

149 therapy,[ ]

This finding is of specific interest in light of the demonstration of increased neutrophils in patients with severe consistent

721

with neutrophilic proteases degrading matrix elements and eosinophil TGF-β1 inducing collagen production. Matrix-Degrading Enzymes A major interest in asthma has been the role played by MMPs and tissue inhibitors of metalloproteinases (TIMPs). The regulation of these enzymes, which are secreted as zymogens, is complex, and measurements of activity likely provide limited information. Therefore, some of the data are conflicting. The most striking elevations in MMP-9 (tenfold to 160-fold increases) were noted in bronchoalveolar lavage (BAL) fluid from ventilated subjects with status asthmaticus who also had elevated MMP-3 levels.[

178]

No increases in these MMPs were determined in BAL fluid of patients with mild asthma. Another study found higher TIMP-1 levels in

untreated asthmatic subjects that correlated with interleukin-6 (IL-6) levels and numbers of alveolar macrophages.[ lower in asthmatic patients, correlating with FEV1 , neutrophils, and corticosteroids in a group of severely asthmatic subjects.[

180 macrophages,[ ]

179]

Sputum MMP-9/TIMP-1 molar ratios were

and serum MMP-9/TIMP-1 ratio correlated with a response to oral

181]

182 183

Mast cells are increased in the smooth muscle layer in asthmatic airways[ ] [ ] and contain a number of proteases, such as tryptase, chymase, and cathepsin G. Tryptase has striking effects on the ECM of human airways. In preliminary experiments in our laboratory, the addition of mast cell tryptase to human airway explants leads to a threefold increase in the shortening ability of the smooth muscle, comparable to the changes found in asthmatic tissues. Smaller changes have been reported with shorter incubations with guinea pig tissue.[

184]

Using the selective tryptase inhibitor 1,5-bis-{4-[(3-carbamimidoyl-benzenesulfonylamino)-methyl]-

185 al[ ]

phenoxyl}-pentane, Wright et demonstrated inhibition of allergen-induced early-phase and late-phase bronchoconstriction as well as AHR in sheep. This inhibitor has more selective activity on tryptase than the naturally produced secretory leukocyte protease inhibitor (SLPI), which is a potent inhibitor of cathepsin G, elastase, chymotrypsin, and trypsin, as well as mast cell tryptase. SLPI's broader action may be preferable in light of evidence for neutrophil-mediated changes in asthmatic airways. Mast cell tryptase may also influence collagen deposition in airways, as suggested by Cairns and Walls, [ collagen synthesis in a fibroblast cell line. Whether this effect is seen in whole airway tissue remains to be determined.

186]

who showed that tryptase stimulates

These studies highlight the difficulty in determining primary versus secondary events in a pathophysiologic process involving simultaneous inflammatory injury and tissue repair. Although most studies have concentrated on the deposition of new collagen as a detrimental component of remodeling, largely because this is a

relatively easy parameter to evaluate with bronchial biopsies, the mechanical consequences of this are unknown. The relative contributions of geometric factors, quality of matrix, and structural ECM changes to increased airway narrowing are as yet difficult to assess.

IMPLICATIONS FOR ASTHMA THERAPY Alterations in ECM components in allergic diseases such as asthma involve both degradation and new synthesis. Both processes are ongoing simultaneously by the time the disease state is recognized and treatments with agents such as corticosteroids may alter many different elements, some limiting ECM degradation and some limiting repair. If the initial injury in allergic disease causing structural changes is the release of proteolytic enzymes from mast cells and recruited inflammatory cells, inhibition or release and inhibition of enzymatic activities would be important therapeutic goals. Inhibition of protease release also occurs through agents that inhibit mast cell mediator release or immunoglobulin E (IgE) induction of release (see Chapters 13 and 16 ). 187

188

Tumor necrosis factor alpha (TNF-α) is synthesized and released from isolated mast cells[ ] and from alveolar macrophages after antigenic challenge.[ ] Because TNF-α stimulates the release of numerous proteolytic enzymes from inflammatory cells, it will be interesting to determine if inhibitors of its action, such as the TNF receptor fusion protein[

189]

or monoclonal chimeric antibody,[

190]

inflammatory arthritic conditions that respond to these therapies,

have effects on airway changes leading to AHR. Based on many similarities of ECM alterations in

[191] [192]

these therapeutic agents warrant investigation.

Inhibitors of MMPs may also be of value in limiting the initial injury. A number of specific inhibitors have been developed and await testing in animal models and human allergic disease. Although eosinophil transmigration across membranes appears to depend on MMP-9, this is not the case for basophils or neutrophils. Thus, specific inhibitors of MMP-9 may selectively block eosinophil infiltration, which could be beneficial in allergic disease. However, recent studies with anti-IL-5 suggest that inhibiting eosinophil infiltration does not alter late-phase allergic reactions or AHR. Whether preventing eosinophilia decreases collagen deposition (e.g., secondary to TGF-β production) remains to be seen. Beta-adrenergic agonists may modulate ECM as well as inhibit mast cell mediator release. Increasing cyclic adenosine monophosphate (cAMP) in fibroblasts downregulates collagen synthesis induced by TGF-β through its effect on connective tissue growth factor.[ elements in addition to the known action on smooth muscle.

193]

Therefore, long-acting β-agonists have effects on ECM

Therapy for remodeling processes in chronic inflammatory lung disease will likely continue to center on corticosteroids, despite these agents affecting multiple components of airway inflammation, injury, and repair. More specific agents that antagonize cytokines and growth factors generated in the inflammatory process are in development, as are agents that may have repress mesenchymal cell proliferation or matrix synthesis. Long-term suppression of ECM remodeling will be central to future therapeutic approaches to chronic inflammatory lung diseases.

SUMMARY AND CONCLUSIONS The dominant factor contributing to airway narrowing is force generated by the muscle cross-bridges. The force constricting the airways is opposed extracellularly by tethers that connect the force generators (muscle cells) to the extracellular matrix and intracellularly by the cytoskeleton. This network of protein filaments provides elastic loads and radial constraints on shortened cells. Tidal breathing and deep inspiration reduce the force generated by the cross-bridges by disrupting the

equilibrium of cross-bridge binding and assembly of contractile

722

filaments. This disruption is possible only if mechanical perturbation associated with the lung volume change can be transmitted to the muscle cells; that is, it requires effective tethering between muscle cells and their dynamic physical environment. Enzymes and mediators that disrupt mechanical coupling between muscle cells and extracellular matrix therefore constitute limiting factors to muscle shortening. As defined by a conventional length-tension relationship, the ability of smooth muscle to generate force is reduced when the muscle shortens beyond its optimal length; under normal circumstances this provides a brake on muscle shortening. Adaptation of the shortened muscle to its new length will occur, however, if the muscle is allowed to remain at the short length for a prolonged time; the ability of the shortened muscle to generate force will increase, and the braking effect will be offset. It appears that muscle adaptation to short length should be avoided if airway patency is to be maintained. We propose a unifying hypothesis: altered mechanical restraints and muscle plasticity induce excessive airway smooth muscle shortening in asthma. The initial insult to the airway allowing excessive muscle shortening is proteolytic damage to extracellular matrix caused by inflammatory cells. Once the muscle is allowed to reach a shorter length, its innate ability to optimize force generation at this new, shorter length (plasticity) accentuates airway narrowing. Acknowledgment This chapter was supported by grants from the British Columbia Lung Association and Canadian Institutes for Health Research.

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169. Rajah R, Nachajon RV, Collins MH, et al: Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle, Am J Respir Cell Mol Biol 20:199, 1999. 170. Cohen P, Bhala A, Herrick D, et al: The effects of leukotriene D4 on the proliferation of airway smooth muscle cells are mediated by modulation of the IGF axis, Am J Physiol 269:L151, 1995. 171. Rajah R, Nunn S, Herrick D, et al: LTD-4 induces matrix metalloproteinase-1, which functions as an IGFBP protease in airway smooth muscle cells, Am J Physiol 271:L1014, 1996. 172. Loewen DAJ, Pare PD, Schellenberg RR: Prolonged effects of epithelial injury and repair on the mechanical properties and proliferation of airway smooth muscle (ASM), Am J Respir Crit Care Med 155:A369, 1997. 173. Roberts CR: Is asthma a fibrotic disease? Chest 107:111S, 1995. 174. Iozzo RV: Matrix proteoglycans: from molecular design to cellular function, Ann Rev Biochem 67:609, 1998. 175. Huang J, Olivenstein R, Taha R, et al: Enhanced proteoglycan deposition in the airway wall of atopic asthmatics, Am J Respir Crit Care Med 160:725, 1999. 176. Roberts CR, Okazawa M, Wiggs BR, Pare PD: Airway wall thickening. In Barnes PJ, Grunstein MM, Leff AR, Woolcock AJ, editors: Asthma, New York, 1997, Lippincott-Raven, p 925. 177. Haraguchi M, Shimura S, Shirato K: Morphometric analysis of bronchial cartilage in chronic obstructive pulmonary disease and bronchial asthma, Am J Respir Crit Care Med 159:1005, 1999. 178. Lemjabbar H, Gosset P, Lamblin C, et al: Contribution of 92•kDa gelatinase/type IV collagenase in bronchial inflammation during status asthmaticus, Am J Respir Crit Care Med 159:1298, 1999. 179. Mautino G, Oliver N, Chanez P, et al: Increased release of matrix metalloproteinase-9 in bronchoalveolar lavage fluid and by alveolar macrophages of asthmatics, Am J Respir Cell Mol Biol 17:583, 1997. 180. Vignola AM, Riccobono L, Mirabella A, et al: Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis, Am J Respir Crit Care Med 158:1945, 1998. 181. Bosse M, Chakir J, Rouabhia M, et al: Serum matrix metalloproteinase-9:tissue inhibitor of metalloproteinase-1 ratio correlates with steroid responsiveness in moderate to severe asthma, Am J Respir Crit Care Med 159:596, 1999. 182. Carroll NG, Mutavdzic S, James AL: Distribution and degranulation of airway mast cells in normal and asthmatic subjects, Eur Respir J 19:879, 2002. 183. Brightling CE, Bradding P, Symon FA, et al: Mast-cell infiltration of airway smooth muscle in asthma, N Engl J Med 346:1699, 2002. 184. Barrios VE, Middleton SC, Kashem MA, et al: Tryptase mediates hyperresponsiveness in isolated guinea pig bronchi, Life Sci 63:2295, 1998.

185. Wright CD, Havill AM, Middleton SC, et al: Inhibition of allergen-induced pulmonary responses by the selective tryptase inhibitor 1.5-bis{4-[(3carbamimidoyl-benzenesulfonylamino)-methyl]}-phenoxyl-pentane (AMG-126737), Biochem Pharmacol 58:1989, 1999.

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186. Cairns JA, Walls AF: Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts, J Clin Invest 99:1313, 1997. Implications for Asthma Therapy 187. Gordon JR, Galli SJ: Mast cells as source of both preformed and immunologically inducible TNFα/cachectin, Nature 346:274, 1990. 188. Gosset P, Tsicopoulos A, Wallaert B, et al: Increased secretion of tumor necrosis factor α and interleukin-6 by alveolar macrophages consecutively to the late asthmatic reaction after bronchial allergen challenge, J Allergy Clin Immunol 88:561, 1991. 189. Mohler KM, Sleath PR, Fitzner JN, et al: Protection against a lethal dose of endotoxin by an inhibitor of tumor necrosis factor processing, Nature 370:218, 1994. 190. Brennan FM, Chantry D, Jackson A, et al: Inhibitory effect of TNFα antibodies on synovial cell interleukin-1 production in rheumatoid arthritis, Lancet 2:244, 1989. 191. Moreland LW, Baumgartner SW, Schiff MH, et al: Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein, N Engl J Med 337:141, 1997. 192. Maini R, St Clair EW, Breedveld E, et al: Infliximab (chimeric anti-tumor necrosis factor α monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving concomitant methotrexate: a randomized phase III trial, Lancet 354:1932, 1999. 193. Duncan MR, Frazier KS, Abramson S, et al: Connective tissue growth factor mediates transforming growth factor β-induced collagen synthesis: downregulation by cAMP, FASEB J 13:1774, 1999.

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Chapter 44 - Development, Structure, and Physiology in Normal and Asthmatic Lung

Scott S. Wagers Elizabeth F. Jaffe Charles G. Irvin

Alterations in structure of the asthmatic lung are central in the clinical syndrome of asthma, which includes dyspnea, wheezing, cough, excess mucus production, airflow limitation, and airway hyperresponsiveness (hyperreactivity). A similar clinical syndrome is seen in diseases with an entirely different cytokine milieu, such 1

2

3

as sarcoidosis,[ ] which is predominantly a T helper type 1 (Th1) cytokine disease[ ] ; asthma, however, is dominated by T helper type 2 (Th2) cytokines.[ ] If a similar clinical syndrome can be developed with an entirely different set of cytokines, one could postulate the existence of a redundant mechanism, but the fact that airways are affected in sarcoidosis argues that the clinical syndrome must be at least partly caused by structural alterations. Epidemiology of asthma shows that the 4

greatest incidence is in childhood,[ ] when the lungs are structurally incomplete, which suggests that structural changes are central to the pathophysiology of asthma. This chapter reviews current knowledge about development of the lung, lung structure, and how development and structure contribute to and are impacted by asthma.

LUNG DEVELOPMENT The development of the lung occurs rapidly; in a term infant the lung volume doubles by 6 months and triples by 1 year, a phenomenal rate of growth. This rapid 5

growth is likely to predispose the lung to damage from inflammatory insults. [ ] Other factors implicated in the alteration of developmental lung structure and 6]

function include fetal nutrition, maternal smoking during pregnancy, environmental allergens, respiratory infections during infancy, and genetics.[

The lung develops in a nonuniform manner that has been termed dysanaptic growth. Airway branching and development are essentially completed by the sixteenth 7

week of gestation,[ ] but airways continue to increase in size and complexity. Parenchymal development proceeds at a different time course because the alveoli do 7

not begin to develop until 28 weeks of gestation, with most alveoli developing postnatally.[ ] Accordingly, airways are relatively large in relation to lung volume in fetal life and infancy. Thus, airway edema and mucus secretion associated with lower respiratory infections are more likely to produce wheezing in the lung of a child. In addition, the infant chest wall is highly compliant, which leads to an increased tendency for peripheral airways to close during tidal breathing.[

8]

The lung develops in a series of defined stages. The embryonic stage begins approximately day 24 to 26 after fertilization and is complete by 7 weeks' gestation. Lung sacs develop from the ventral wall of the esophagus, which accounts for some of the common features between the two structures, such as patterns of anatomic innervation. The major airways and lobes are formed by a series of successive buddings. The pseudoglandular stage occurs during the fifteenth to seventeenth week of gestation. The bronchial tree first develops as solid tubes, then primitive acini appear. Arteries and veins develop in parallel with the airways. In the canalicular

stage, 16 to 26 weeks' gestation, lumens begin to form in the bronchial tree, and at this point the bronchioles are produced. The peripheral epithelium differentiates into type I and II pneumatocytes. Also during this stage, capillaries arrange into sleeves around the bronchioles. The clinical significance of the canalicular stage is that it marks the beginning of gas exchange units and surfactant production. The saccal stage, 24 to 38 weeks' gestation, is characterized by the formation of alveolar ducts and air sacs. Growth that occurs in first year of life is accompanied by other developments in respiratory physiology, including changes in shape and compliance of the rib cage. Gender differences in airway development and lung anatomy have been proposed as the explanation for the increased prevalence of asthma in prepubertal boys 9

compared to girls.[ ] Longitudinal studies of airway parenchymal size in 555 asymptomatic or mildly symptomatic boys and girls between 12 and 19 years of age 10

demonstrated that the airways of boys are smaller than those of girls.[ ] Smaller airways are more likely to develop critical airway narrowing that results in airflow limitation and wheezing. Over time, boys demonstrate more pronounced airway growth compared with girls; as a result, airway/lung volume ratios in males are equal to or greater than ratios in females after puberty. This finding may explain why symptomatic improvement is observed with puberty. Whereas the process of lung development results in susceptibility to insults that alter lung structure, the alterations in lung structure themselves result in changes in lung function. These changes in lung function have implications not only for investigating the etiology of asthma and new therapies, but an understanding of these changes is helpful in the interpretation of clinical measures of lung function and the effects of therapy.

728

AIRWAY REMODELING Remodeling of the airway has become an area of intense focus in contemporary asthma research, but what defines “airway remodeling” is controversial. Most agree, however, that airway remodeling is a structural change that is evident in asthmatic airways compared with normal airways. Asthma is characterized by variable airflow limitation, but most patients show little evidence of persistent abnormalities in lung function. However, a subset of patients has persistent airflow limitation, presumably caused by significant structural changes in their airways. [ health care dollars are spent on this

11] [12] [13] [14]

Although this is a limited subset of all patients with asthma, the majority of

15 16 group.[ ] [ ]

Figure 44-1 Airway of lung in patient who died from asthma (status asthmaticus), showing profound changes in structure of the airways and lungs (“remodeling”). Airway wall is thickened by cellular infiltration, extracellular matrix deposition, and expansion of airway smooth muscle, with pronounced neovascularization. Epithelium is clearly friable and disintegrating, and a mucus plug occupies the airway lumen. (×100; hematoxylin-eosin stain.)

Figure 44-2 Model of airflow limitation. A, Simple model consists of an airway-alveolus unit with a box (thorax). Before maximal expiration, pressure within the box is zero, and there is no pressure gradient between the alveolus (Palv ) and the atmosphere (Patm ). Pel , Elastic recoil pressure. B, When diaphragm contracts, pressure (pleural pressure) increases outside the alveolus and airways. Because of the additional effect of elastic recoil, pressure in alveolus is higher than pleural pressure, which creates a gradient of pressure (shown by size of P) from the alveolus to the atmosphere. Because of energy loss from resistive forces and Bernoulli effect, at some point the pressure outside equals the pressure within the airway, the equal pressure point (EPP). Beyond this point the airways narrow and airflow is limited. Rus , Resistance of upstream segment.

Figure 44-3 Basic flow-volume relationships. A, Flow plotted against volume during forced expiration results in a characteristic flow volume “loop,” the forced expiratory volume in 1 second (FEV1 ). FEV1 occurs in a normal person at approximately 80% of the forced vital capacity (FVC). RV, Residual volume; TLC, total lung capacity; PEFR, peak expiratory flow rate; lps, liters per second. B, When expiratory maneuvers are performed at different TLCs or on effort, a “family” of loops is created. Whereas the inspiratory loops are all effort dependent, the expiratory loops are clearly flow limited, even when expiratory rate is less than maximal (effort independent) or when a lower TLC is achieved. C, Effect of failing to inspire maximally to TLC on the measurement of FEV1 . When the patient inhales to a volume that is 0.5 L less than TLC, the FEV1 falls by 0.4 L, or 12.7%. Solid line, Maximal effort; dotted line, submaximal effort.

Figure 44-4 Characteristic flow-volume loops. A, Flow-volume relationship characteristic of airway collapse, and illustration of negative effort dependence. First (higher flow-volume) loop occurs when the patient makes a submaximal effort. Second loop occurs when the patient exhales maximally; that is, flow falls with increased effort or negative effort dependence. Pred, Predicted flow-volume relationship. B, Patient showing consistent truncation of the inspiratory loop (solid line), indicative of large airway obstruction or extrathoracic lesion. C, Partial truncation of the type usually associated with vocal cord dysfunction (solid line). Initially there is preservation of flow, then flow truncation as the vocal cords appose. D, Bronchodilator responses can vary. This patient shows marked improvement in airflow and an increase in forced vital capacity. However, the flow-volume relationship is still not normal, suggesting further treatment is warranted. Solid line, Before bronchodilation; dashed lines, after bronchodilation.

Figure 44-5 Lung volumes and capacities. A, Spirogram of maximal lung volume excursion, showing four lung volumes (IRV, VT, ERV, RV) and four lung capacities (TLC, IC, FRC, VC). A lung capacity is made up of more than one lung volume. B, Lung volumes of various patients. Restrictive lung disease is, by definition, a reduction in lung volumes. In asthma or other obstructive lung disorders, lung volume(s) rises. In mild asthma the first change is a rise in residual volume (RV); more severe cases show a rise in functional reserve capacity (FRC) and total lung volume (TLC). ERV, Expiratory reserve volume; VT, tidal volume; IRV, inspiratory reserve volume; VC, vital capacity; IC, inspiratory capacity.

Figure 44-6 High-resolution computed tomography (HRCT) scan through upper thorax during inspiration (A) and expiration (B) of patient with air trapping. On expiration, scan should demonstrate homogenous increase in radiodensity in the lung parenchyma; instead, areas of persistent radiolucency are visible (arrows), demonstrating retention of trapped gas behind obstructed small airways. (Image courtesy Jeffery Klein.)

Figure 44-7 Determination of airway resistance (Raw ) and specific conductance (sGaw ) by body plethysmograph. A, Patient (lung within the box) pants against closed shutter to obtain the mouth (alveolar) pressure/box volume relationship. B, Next, shutter opens, and patient continues to pant. The box volume and airflow relationships are obtained. Raw is calculated as shown, then “corrected” for lung volume to obtain sGaw .

Figure 44-8 Bronchial responsiveness is determined by measuring lung function, such as the forced expiratory volume in 1 second (FEV1 ), as a function of the concentration of an inhaled bronchoactive agonist, either histamine or more often methacholine. FEV1 is expressed as a percentage of control, either baseline or after saline inhalation, and is plotted for normal subject (solid line) and asthmatic patient (dashed line). Airway responsiveness is determined as the interpolated dose of agonist (provocative concentration) that causes the FEV1 to fall 20% (PC20 ). Asthmatic patient responds early and at a lower dose than normal subject (left-shift dose-response curve).

Figure 44-9 Parenchyma-airway interdependence. A, Stained histologic section of lung, with airway and parenchyma readily apparent. B, Attachments of the alveolar septae to the outer portion of the airway wall (arrows) function as tethers. C, Tethers apply outward tension (arrows) when a normal (nonremodeled) airway

constricts. D, When an airway is remodeled (shaded area), the tethers are effectively although not literally broken, allowing the airway to constrict more than with a nonremodeled airway.

Figure 44-10 Effects of deep inspiration. Ten subjects with asthma and 11 healthy controls undergoing two methacholine bronchoprovocations. On one occasion, conventional spirometry is performed (full expiratory maneuvers, from total lung capacity to residual volume), which involves deep inspirations. The slope of the reduction in the forced expiratory volume in 1 second/forced vital capacity (FEV1 /FVC) ratio is much steeper in the asthmatic patients compared with the healthy subjects, who received up to 75•mg/ml of methacholine with minimal effect. On second occasion, methacholine provocation is performed in the absence of deep inspirations (partial expiratory maneuvers, from end-tidal inspiration to residual volume). The slope of the reduction in the partial FEV1 /FVC ratio is similar in both groups because, in the absence of deep inspirations, healthy controls demonstrate significant loss of lung function at concentrations of methacholine that are within the asthmatic range (less than 7.5•mg/ml). In contrast, the presence or absence of deep inspirations does not appear to influence the airway responsiveness of the asthmatic subjects. If the two groups are compared with respect to the slope of the reduction in FEV1 /FVC, in the absence of deep inspirations the difference is very small. The challenge does not progress to higher doses of methacholine in the asthma group because baseline lung function is already low (data not shown), and the reduction induced by the methacholine drops lung function to a point that is the cutoff point for safety concerns. (Modified from Skloot G, Permutts S, Togias A: J Clin Invest 96:2393, 1995; data courtesy Alkis Togias, Johns Hopkins University.)

Figure 44-11 Ventilation defects. Magnetic resonance scan of healthy individual who inhaled polarized helium shows homogenous distribution of the gas. In asthma the polarized helium distribution is heterogenous, demonstrating multiple ventilation defects (arrowheads), presumably from airway closure. (Courtesy John P. Mugler III, Center for In-vivo Hyperpolarized Gas MR Imaging, University of Virginia.)

Box 44-1. Questions Addressed in Interpretation of Pulmonary Function Tests (PFTs) 1. Was patient effort acceptable? What was overall test quality? 2. In assessing patient demographics, how useful are reference equations in this specific case? 3. In reviewing flow-volume loops and spirograms, are there characteristic findings (e.g., Figure 44-4 )? 4. In assessing FEV1 , FVC, and then FEV1 /FVC ratio, what is the degree of airflow limitation? 5. Is there a significant bronchodilator response, and is there residual obstruction? 6. Is the TLC low (restriction), normal, or elevated (obstruction)? Is FRC low, normal, or high? Is the RV elevated? Are the lung volumes consistent with asthma and its severity (see Figure 44-5 )? 7. Is the DLco elevated? Is VA low, or similar to TLC? 8. Is the PC20 below 8•mg/ml? 9. Do the patterns of PFT results confirm the clinical impression, or are

they at odds?

volumes, flow-volume loops, diffusion capacity, and an acute response to bronchodilator. The additional tests to be considered are a methacholine challenge or an exercise challenge. Other studies, however, such as pressure-volume curves or cardiopulmonary stress tests, could and, if indicated, should be considered in the individual patient as indicated by the clinical picture. Quality of Results and Concepts of Normalcy The first step in assessing lung function is to determine test quality. Although tests of poor quality have limited usefulness, even poor tests often can be helpful.[ [118]

70] [86] [118] [119] [120]

The guidelines for test quality are well known and are not repeated here.[

65]

However, some key elements to examine are reproducibility and

121 ±200•ml,[ ]

good patient performance. Spirometry should be reproducible within a range of whereas good patient performance is best assessed by direct observation or at least by careful annotations by the examiner. When poor test performance is suspected, increased reliance should be placed on the endpoints that are less effort dependent, such as functional residual capacity, specific conductance, lung diffusing capacity of carbon monoxide, and evidence of a flow-volume envelope, even if it occurs at the point of midrange vital capacity flow (see Figure 44-3, B ). The next step is to assess the applicability of the “predicted equations” for the specific patient in question. For some patients, such as a 30-year-old white male 5 feet 10 inches in height, the equations for FEV1 and FVC are probably a good estimate of normalcy. For others, such as a very short Asian boy age 13 years, the “predictions” are much less accurate because fewer Asian-specific equations are available. Short stature leads to false-positive results, and persons during growth 70

spurts have more variable lung function. Nevertheless, age, gender, race, and height explain only 70% of the variance,[ ] which means that 30% of the variance in spirometry is, for example, simple biology. Therefore, some prefer to use term reference equations rather than predicted equations. Many equations for FEV1 /FVC exist, but it is recommended that the NHANES III study be used because currently it provides the most complete equations, covering a large age range and including 122

race-specific information.[ ] The lack of current reference equations for other endpoints, such as lung volumes, makes reliance on the “percent predicted” for these values dubious. Accordingly, the examiner should also consider how the values change for a given individual over time or in response to therapy. Although most interpretations simply comment on the presented values for FEV1 and FVC, it is recommended first to review each of the flow-volume curves, looking for reproducibility and characteristic patterns (see Figure 44-4 ). It has been documented that although physicians in practice can correctly identify airflow 123

limitation and a significant response to bronchodilation, many fail to observe a fixed, or variable, intra/extrathoracic lesion.[ ] Mixed disease patterns are particularly problematic. Some common variants are the shape of the inspiratory flow-volume loop caused by vocal cord dysfunction (VCD), a known comorbid condition of asthma.[

124]

A probable diagnosis of VCD can be made only if the shape of the inspiratory limb is abnormal and not limited to just one or two loops.

Airflow Limitation and Bronchodilator Responses

Response to bronchodilators is a critical addition to PF testing for the asthmatic patient (see Figure 44-4, D ). The current recommendations state that only a 12% or 200-ml change is considered clinically significant.[ for the current 12% and 200-ml index.

70]

125] [126]

A change of 8% or 150•ml may be caused by inherent variability of testing [

and forms the rationale

However, failure to achieve this magnitude of a bronchodilator response does not preclude the prescribing of bronchodilators because the response will vary with the activity of the disease. Moreover, bronchodilator responses are often taken as a measure of underlying inflammation.[ warranted, since this measure is notably different from flow-volume airway

81 disease.[ ]

127 determinants[ ]

79]

The addition of specific conductance is also

and certainly is different in a subset of patients presenting with reversible 79] [80]

Incomplete reversal of airflow limitation is also an indication of lung remodeling and the need for additional therapy.[

Lung Volumes: Restriction versus Obstruction Measurement of lung volumes promises critical insight into the function of the lung because the size of the lung determines the overall gas exchange capacity. FVC is the major predictor of morbidity and mortality,[ units, placing the patient at risk.

65] [128] [129]

so it is reasonable to assume that a temporal decrease in FVC represents the loss of gas exchange

The measurements of TLC, FRC, and RV are the most useful indices of lung volume to obtain (see Figure 44-5 ). Falls in TLC (120% of predicted) are indicative of obstruction, as observed in more chronic or persistent asthma. The rise in TLC is greatest in the most severe

738

cases[

76] [78]

and, if not readily reversed with acute treatment with a bronchodilator, would indicate that remodeling of the lung has occurred. Elevations in RV

(>150% of predicted) are usually the first alteration in lung volume and are also a sensitive measure of small airways function[

54]

because RV is a function of airway

59 closure.[ ]

Instead of RV measurements, the difference between slow vital capacity (SVC) and the forced vital capacity (FVC) may provide an alternative estimate of airway closure and gas trapping. Patient weight must be considered because obesity alone results in reduced TLC when the body weight (kg)/height (cm) ratio is 130

equal to 1.0.[ ] Lastly, it is important to note that the equations most often used for predicted lung volumes were made from studies years ago, and people in general are now taller, so that the predictions for lung volume are generally about 10% low. Classification of Airflow Limitation with Carbon Monoxide Diffusing Capacity Measurement of the lung diffusing capacity of carbon monoxide (DLco) can be useful in two areas.[ In the patient with emphysema, DLco is reduced in proportion to the loss of lung

120 131 tissue.[ ] [ ]

120]

First, DLco contributes to the assessment of airway disease.

This finding is important because patients who smoke often present

with a self-diagnosis of asthma, a disease they know to be more socially acceptable and, more importantly, treatable. In COPD patients, DLco is diminished, whereas in asthmatic patients, DLco is normal or elevated. The elevated DLco suggests active inflammation and the uptake of CO by red blood cells known to be present in the bronchoalveolar lavage fluid of asthmatic patients; thus an elevated DLco will be found in asthma that is uncontrolled. Another useful aspect of the DLco is that testing typically determines the alveolar volume (Va), which is determined by a single breath measurement of a gas dilution and thus measures gas volume that is communicating with the airway opening. In normal subjects the Va should equal TLC (±200•ml), but in the setting of air trapping the Va will be less than the TLC because the trapped air is not communicating with the airway opening. Therefore the VA-TLC difference can be used to detect the presence of air trapping. Lastly, it is important to note that DLco has little correlation to gas exchange (e.g., PaO2 ), and that DLco is in essence a measurement of the potentially available lung for gas exchange.[

120]

Bronchial Challenge 86

Details of bronchial challenge are found in several publications [ ] and elsewhere in this text. Probably the most important aspect for interpretation is that bronchial challenge is sensitive but not specific for the diagnosis of asthma. Patients with many other diseases, such as COPD, adult respiratory distress syndrome, and cystic fibrosis, will present with bronchial hyperresponsiveness. However, if the PC20 is less than 1•mg/ml, the specificity of the test increases considerably. One guideline 86]

suggests that the PC20 can also be used to stage the severity of the disease.[

As with the response to acute treatment with bronchodilation, changes in the measures

43

of bronchial challenge can be used to assess inflammatory activity.[ ] Most importantly, bronchial challenge results must be linked to current symptoms to be useful. In addition, the PC20 falls and then resolves very quickly in some patients (e.g., after antigen challenge), making the connection to current clinical presentation 132]

difficult.[

Assessment of bronchial response to exercise is less often indicated [

86]

but can be useful to assess airway responsiveness in the child or athlete. It is important to 133

understand that there is not a complete overlap in hyperresponsiveness in patients who are methacholine and exercise positive.[ ] This finding suggests that the mechanisms involved may not be the same, and, in some patients, both tests may need to be performed for a diagnosis to be made.

SUMMARY Knowledge of the physiology and structure of the lung is clinically useful. Asthma is the most significant disease in this regard because the syndrome is defined by a pattern of lung dysfunction that characterizes the disorder. In fact, asthma is the only lung disease that is diagnosed solely on the basis of clinical presentation and PFTs. Interpretation of PFTs requires an in-depth knowledge of both the structures and the function of the lung. Interpretation of a battery of tests should include elements of test quality, lung volume considerations, presence of airflow limitation, and the response to bronchodilation. DLco and bronchial challenge provide further clarification of the nature of the airflow limitation present. In addition to diagnosis, lung function tests provide an invaluable guide to the progress of the disease or its resolution to successful treatment. Acknowledgement

We would like to acknowledge the support of the National Heart, Lung, and Blood Institute (HL 62746) and the National Center for Research Resources (Centers of Biomedical Research Excellence P20RR15557). We would also like to thank Dr. Alkis Togias and Dr. John P. Mugler III for contributions to the chapter's figures.

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66. Dawson SV, Elliott EA: Wave-speed limitation on expiratory flow: a unifying concept, J Appl Physiol 43:498, 1977. 67. Mead J, Turner JM, Macklem PT, et al: Significance of the relationship between lung recoil and maximum expiratory flow, J Appl Physiol 22:95, 1967. 68. Enright PL, Johnson LR, Connett JE, et al: Spirometry in the Lung Health Study. 1. Methods and quality control, Am Rev Respir Dis 143:1215, 1991. 69. Cockcroft DW, Berscheid BA: Volume adjustment of maximal midexpiratory flow: importance of changes in total lung capacity, Chest 78:595, 1980. 70. American Thoracic Society: Lung function testing: selection of reference values and interpretative strategies, Am Rev Respir Dis 144:1202, 1991. 71. Troyanov S, Ghezzo H, Cartier A, et al: Comparison of circadian variations using FEV1 and peak expiratory flow rates among normal and asthmatic subjects, Thorax 49:775, 1994. 72. Paggiaro PL, Moscato G, Giannini D, et al: Relationship between peak expiratory flow (PEF) and FEV1 , Eur Respir J Suppl 24:39S, 1997. 73. Mellisant CF, Van Noord JA, Van de Woestijne KP, et al: Comparison of dynamic lung function indices during forced and quiet breathing in upper airway obstruction, asthma, and emphysema, Chest 98:77, 1990. 74. Clausen JL, Coates AL, Quanjer PH: Measurement of lung volumes in humans: review and recommendations from an ATS/ERS workshop, Eur Respir J 10:1205, 1997. 75. Dykstra BJ, Scanlon PD, Kester MM, et al: Lung volumes in 4,774 patients with obstructive lung disease, Chest 115:68, 1999. 76. Woolcock AJ, Read J: Lung volumes in exacerbations of asthma, Am J Med 41:259, 1966. 77. Goldin JG, McNitt-Gray MF, Sorenson SM, et al: Airway hyperreactivity: assessment with helical thin-section CT, Radiology 208:321, 1998. 78. Corbridge TC, Hall JB: The assessment and management of adults with status asthmaticus, Am J Respir Crit Care Med 151:1296, 1995. 79. Enright PL, Lebowitz MD, Cockroft DW: Physiologic measures: pulmonary function tests—asthma outcome, Am J Respir Crit Care Med 149:S9, S19, 1994. 80. Childhood Asthma Management Program Research Group: Long-term effects of budesonide or nedocromil in children with asthma, N Engl J Med 343: 1054, 2000. 81. Smith HR, Irvin CG, Cherniack RM: The utility of spirometry in the diagnosis of reversible airways obstruction, Chest 101:1577, 1992. 82. Virchow JC Jr, Holscher U, Virchow C: Sputum ECP levels correlate with parameters of airflow obstruction, Am Rev Respir Dis 146:604, 1992. Airway Hyperresponsiveness 83. Wagers SS, Irvin CG: The pharmacology of aerosolized airway challenge, Respir Care Clin North Am 5:633, 1999.

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84. Juniper EF, Frith PA, Hargreave FE: Airway responsiveness to histamine and methacholine: relationship to minimum treatment to control symptoms of asthma, Thorax 36:575, 1981. 85. Murray AB, Ferguson AC, Morrison B: Airway responsiveness to histamine as a test for overall severity of asthma in children, J Allergy Clin Immunol 68:119, 1981. 86. Popa V: ATS guidelines for methacholine and exercise challenge testing, Am J Respir Crit Care Med 163:292, 2001. 87. Vathenen AS, Knox AJ, Wisniewski A, et al: Time course of change in bronchial reactivity with an inhaled corticosteroid in asthma, Am Rev Respir Dis 143:1317, 1991. 88. Moreno RH, Hogg JC, Pare PD: Mechanics of airway narrowing, Am Rev Respir Dis 133:1171, 1986. 89. O'Byrne PM, Dolovich M, Dirks R, et al: Lung epithelial permeability: relation to nonspecific airway responsiveness, J Appl Physiol 57:77, 1984. 90. Armour CL, Black JL, Berend N, et al: The relationship between bronchial hyperresponsiveness to methacholine and airway smooth muscle structure and reactivity, Respir Physiol 58:223, 1984. 91. Armour CL, Lazar NM, Schellenberg RR, et al: A comparison of in vivo and in vitro human airway reactivity to histamine, Am Rev Respir Dis 129:907, 1984. 92. Roberts JA, Raeburn D, Rodger IW, et al: Comparison of in vivo airway responsiveness and in vitro smooth muscle sensitivity to methacholine in man, Thorax 39:837, 1984. 93. Vincenc KS, Black JL, Yan K, et al: Comparison of in vivo and in vitro responses to histamine in human airways, Am Rev Respir Dis 128:875, 1983. Lung Parenchyma 94. Kraft M, Djukanovic R, Wilson S, et al: Alveolar tissue inflammation in asthma, Am J Respir Crit Care Med 154:1505, 1996. 95. Kaminsky DA, Irvin CG, Gurka DA, et al: Peripheral airways responsiveness to cool, dry air in normal and asthmatic individuals, Am J Respir Crit Care Med 152:1784, 1995. 96. Tomioka S, Bates JH, Irvin CG: Airway and tissue mechanics in a murine model of asthma: alveolar capsule vs. forced oscillations, J Appl Physiol 93:263, 2002. 97. Gelb AF, Zamel N: Unsuspected pseudophysiologic emphysema in chronic persistent asthma, Am J Respir Crit Care Med 162:1778, 2000. 98. Gold WM, Kaufman HS, Nadel JA: Elastic recoil of the lungs in chronic asthmatic patients before and after therapy, J Appl Physiol 23:433, 1967.

99. Woolcock AJ, Read J: The static elastic properties of the lungs in asthma, Am Rev Respir Dis 98:788, 1968. 100. Martin RJ: Therapeutic significance of distal airway inflammation in asthma, J Allergy Clin Immunol 109:S447, 2002. 101. Juniper EF, Price DB, Stampone PA, et al: Clinically important improvements in asthma-specific quality of life, but no difference in conventional clinical indexes in patients changed from conventional beclomethasone dipropionate to approximately half the dose of extrafine beclomethasone dipropionate, Chest 121:1824, 2002. 102. Macklem PT: A theoretical analysis of the effect of airway smooth muscle load on airway narrowing, Am J Respir Crit Care Med 153:83, 1996. 103. Skloot G, Permutt S, Togias A: Airway hyperresponsiveness in asthma: a problem of limited smooth muscle relaxation with inspiration, J Clin Invest 96:2393, 1995. 104. Irvin CG, Pak J, Martin RJ: Airway-parenchyma uncoupling in nocturnal asthma, Am J Respir Crit Care Med 161:50, 2000. 105. Fredberg JJ: Frozen objects: small airways, big breaths, and asthma, J Allergy Clin Immunol 106:615, 2000. 106. Irvin CG, Dempsey JA: Role of H1 and H2 receptors in increased small airways resistance in the dog, Respir Physiol 35:161, 1978. 107. Wiggs BR, Bosken C, Pare PD, et al: A model of airway narrowing in asthma and in chronic obstructive pulmonary disease, Am Rev Respir Dis 145:1251, 1992. Airway Closure 108. in 't Veen JC, Beekman AJ, Bel EH, et al: Recurrent exacerbations in severe asthma are associated with enhanced airway closure during stable episodes, Am J Respir Crit Care Med 161:1902, 2000. 109. King GG, Eberl S, Salome CM, et al: Differences in airway closure between normal and asthmatic subjects measured with single-photon emission computed tomography and Technegas, Am J Respir Crit Care Med 158:1900, 1998. 110. Siegler D, Fukuchi Y, Engel L: Influence of bronchomotor tone on ventilation distribution and airway closure in asymptomatic asthma, Am Rev Respir Dis 114:123, 1976. 111. Sergysels R, Scano G, Vrebos J, et al: Effect of fenoterol on small airways and regional lung function in asymptomatic asthma, Eur J Clin Pharmacol 24:429, 1983. 112. Bates JH, Schuessler TF, Dolman C, et al: Temporal dynamics of acute isovolume bronchoconstriction in the rat, J Appl Physiol 82:55, 1997. 113. Gillis HL, Lutchen KR: How heterogeneous bronchoconstriction affects ventilation distribution in human lungs: a morphometric model, Ann Biomed Eng 27:14, 1999. 114. Altes TA, Powers PL, Knight-Scott J, et al: Hyperpolarized 3He MR lung ventilation imaging in asthmatics: preliminary findings, J Magn Reson Imaging

13:378, 2001. Pulmonary Function Testing 115. Becklake MR: Concepts of normality applied to the measurement of lung function, Am J Med 80:1158, 1986. 116. Crapo RO, Forster RE Jr: Carbon monoxide diffusing capacity, Clin Chest Med 10:187, 1989. 117. Pennock BE, Cottrell JJ, Rogers RM: Pulmonary function testing: what is “normal”? Arch Intern Med 143:2123, 1983. 118. American Thoracic Society: Standardization of spirometry: 1994 update, Am J Respir Crit Care Med 152:1107, 1995. 119. Aldrich TK, Spiro P: Maximal inspiratory pressure: does reproducibility indicate full effort? Thorax 50:40, 1995. 120. Abboud RT, Sansores R: ATS recommendations for DLco, Am J Respir Crit Care Med 154:263, 1996. 121. Hankinson JL, Bang KM: Acceptability and reproducibility criteria of the American Thoracic Society as observed in a sample of the general population, Am Rev Respir Dis 143:516, 1991. 122. Hankinson JL, Odencrantz JR, Fedan KB: Spirometric reference values from a sample of the general U.S. population, Am J Respir Crit Care Med 159:179, 1999. 123. Hnatiuk O, Moores L, Loughney T, et al: Evaluation of internists' spirometric interpretations, J Gen Intern Med 11:204, 1996. 124. Elshami AA, Tino G: Coexistent asthma and functional upper airway obstruction: case reports and review of the literature, Chest 110:1358, 1996. 125. Eliasson O, Degraff AC Jr: The use of criteria for reversibility and obstruction to define patient groups for bronchodilator trials: influence of clinical diagnosis, spirometric, and anthropometric variables, Am Rev Respir Dis 132:858, 1985. 126. Tweeddale PM, Alexander F, McHardy GJ: Short term variability in FEV1 and bronchodilator responsiveness in patients with obstructive ventilatory defects, Thorax 42:487, 1987. 127. Kaminsky DA, Wenzel SE, Carcano C, et al: Hyperpnea-induced changes in parenchymal lung mechanics in normal subjects and in asthmatics, Am J Respir Crit Care Med 155:1260, 1997. 128. Schunemann HJ, Dorn J, Grant BJ, et al: Pulmonary function is a long-term predictor of mortality in the general population: 29-year follow-up of the Buffalo Health Study, Chest 118:656, 2000. 129. Weiss ST, Segal MR, Sparrow D, et al: Relation of FEV1 and peripheral blood leukocyte count to total mortality. The Normative Aging Study, Am J Epidemiol 142:493, 499, 1995. 130. Ray CS, Sue DY, Bray G, et al: Effects of obesity on respiratory function, Am Rev Respir Dis 128:501, 1983.

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Chapter 45 - Airway Mucus and the Mucociliary System

John V. Fahy

Airway mucus has multiple functions ( Box 45-1 ) that include hydration and protection of the airway epithelium and entrapment of foreign substances for their removal. Airway mucus forms two layers. The “sol” layer is a watery mixture that is in direct contact with the airway epithelial cells. The “gel” layer is a more elastic layer that sits on top of the sol, in direct contact with the inhaled air. Cilia on epithelial cells beat freely in the sol layer, constituting an “escalator” that moves trapped material in the gel toward the pharynx. Airway mucus is a complex mixture of water, proteins, carbohydrates, and lipids. The components of mucus that are principally responsible for its viscoelastic properties are the mucin glycoproteins (mucins). The cellular sources of airway mucus include airway submucosal glands, goblet cells, ciliated epithelial cells, Clara cells, and possibly type 2 alveolar cells. Plasma proteins also

Box 45-1. Functions of Respiratory Mucus I. Preserves the Mucous Membrane Lubricates Humidifies Waterproofs Insulates Provides appropriate environment for ciliary action II. Acts as a Barrier Entraps microorganisms, irritants, and cellular debris Provides extracellular surface for immunoglobulin and enzyme actions Neutralizes toxic gases III. Transports Trapped Materials Works in concert with cilia to move trapped materials to nasopharynx

Modified from Kaliner M, Marom Z, Patow C, et al: J Allergy Clin Immunol 73:318–323, 1984.

contribute to airway mucus through processes of transudation or exudation. Changes in the composition of airway mucus or changes in functioning of the mucociliary escalator can cause decreased efficiency of clearance of mucus and can result in cough, sputum production, airway infection, and airway obstruction. Abnormalities in mucus production and mucus clearance are a feature of most airway diseases, including asthma, bronchitis, bronchiectasis, bronchiolitis, and cystic fibrosis (CF). Mucus hypersecretion and abnormal mucus clearance in these diseases result in shared clinical features such as productive cough and airway obstruction; some of the pathophysiologic mechanisms of mucus hypersecretion may also be shared. It is beyond the scope of this chapter to review the clinical features and pathophysiologic mechanisms of mucus hypersecretion and abnormal mucus clearance in every airway disease. Instead, a review is presented of the normal physiology of airway mucus production and mucus clearance, followed by a review of abnormalities of mucus production and clearance in asthma.

CONSTITUENTS OF AIRWAY MUCUS 1

Detailed analysis of normal airway mucus reveals that approximately 90% is water and the remaining 10% is composed of protein, carbohydrate, and lipid.[ ] The nonaqueous constituents may be in the sol phase or gel phase or both, depending on their solubility. The protein constituents of airway mucus include mucin 2

glycoproteins, proteoglycans, and a variety of other proteins that are important in airway host defense ( Box 45-2 ).[ ] Glycoproteins and proteoglycans are distinct molecules characterized by the carbohydrate constituents of their oligosaccharide chains (glycosides), by the nature of the glycosidic bond between the initial 3 4 5

saccharide and the protein backbone, and by the types of amino acids in the protein backbone.[ ] [ ] [ ] For example, mucin glycoproteins are 50% to 90% carbohydrate by weight; they have O-glycosidic linkages between N-acetylactosamine and serine or threonine (or both) in the protein backbone, which is rich in threonine, serine, glycine, and alanine. Proteoglycans are 80% to 95% carbohydrate by weight and have O-glycosidic linkages between xylose and serine in the protein backbone, which is rich in serine and glycine.

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Box 45-2. Principal Constituents of Airway Mucus and Their Sources

Constituent

Source

1. Ions and water

Ciliated epithelial cells and submucosal gland cells

2. Mucin glycoproteins

Goblet cells, mucous cells in submucosal glands

3. Proteoglycans

Serous cells

4. Proteins and peptides ••Secretory IgA

Serous cells

••Lactoferrin

Serous cells, neutrophils

••Lysozyme

Serous cells, macrophages

••Trefoil factor peptides

Goblet cells, submucosal gland cells

5. Antiproteases and antioxidants ••Secretory leukocyte proteinase inhibitor

Serous cells, Clara cells

••α1-Antitrypsin

Transudate, macrophages

••α2-Macroglobulin

Macrophages

••Peroxidases

Serous cells

6. Lipids

Clara cells, type II alveolar cells, all cell membranes

7. Albumin, plasma proteins

Plasma transudate

8. Products of cell lysis ••DNA

All cells, especially inflammatory leukocytes

••Actin

All cells, especially inflammatory leukocytes

Modified and expanded from Jeffery PK: Eur Respir J 71:34–42, 1987.

Mucin Glycoproteins (Mucins) Although the name mucin suggests a specific molecule, mucin is actually a generic term denoting a group of highly glycosylated proteins synthesized by epithelial cells. Mucin glycoproteins are large, complex molecules consisting of a peptide backbone and numerous oligosaccharide side chains, which represent the products of 1

mucin genes and glycosyltransferase genes, respectively.[ ] Mucin molecules are among the largest molecules in nature, ranging in size from 3 to 23 million daltons. [6]

Electron microscopy reveals them to be linear, flexible threads. These threads are composed of a polypeptide backbone onto which multiple oligosaccharide side chains are attached ( Fig. 45-1 ). A total of 12 human mucin (MUC) genes have been identified and numbered in chronologic order of their description: MUC1-4, MUC5AC, MUC5B, MUC6-8, and MUC11-13. Of these, the complete complementary DNA sequence has been published only for six mucins: 1, 2, 4, 5B, 5AC, and 7. The genes for mucins 2, 5AC, 5B, and 6 are located on chromosome 11p15 and code for the major gel-forming secreted mucins. The genes for mucins 1, 3, 4, 12, and 13 code for nonsecreted transmembrane mucin molecules. Although many different mucins are now known to exist, all have been found to contain extended arrays of tandemly repeated peptides that are rich in potential O-glycosylation sites. These tandem repeats make up a central repetitive domain in the mucin molecule (see Fig. 45-1 ). Flanking this central domain are unique sequences of peptides, which are only sparsely glycosylated and which are therefore sensitive to protease degradation (so-called naked 5

regions). [ ] These unique polypeptide sequences are also characterized by numerous cysteine residues that link mucin monomers into mucin oligomers via disulfide bonds. The mucin peptide product of mucin genes is substituted with a large number of oligosaccharides attached via an O-glycosidic linkage between serine or threonine residues and N-acetylgalactosamine (GalNAc). The complex oligosaccharides on proteins have been arbitrarily divided into three regions. The innermost two or three sugars of the glycan chain proximal to the peptide have been called the core region, the region formed by uniform elongation has been called the

Figure 45-1 Schematic representation of a secreted human respiratory mucin. The current conceptual structure is that of a central repetitive domain containing a tandem repeat array of heavily glycosylated sequences, flanked by unique sequences, less heavily glycosylated, that link the monomers together. The oligosaccharide side chain comprises serine or threonine residues in the core molecule (filled circle) linked to N-acetylhexosamine residues (filled square) via O-glycosidic linkages. (Modified from Gum JR: Am J Respir Cell Mol Biol 7:557–564, 1992; and Kaliner M, Marom Z, Patow C, et al: J Allergy Clin Immunol 73:318–323, 1984.)

(Modified from Gum JR: Am J Respir Cell Mol Biol 7:557–564, 1992; and Kaliner M, Marom Z, Patow C, et al: J Allergy Clin Immunol 73:318–323, 1984.)

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backbone region, and the terminal region, which exhibits a high degree of structural complexity, has been termed the peripheral region. The structural variability of O-linked glycans in mucins can occur in all three regions, including the terminal structures, which can vary according to blood group and related antigens (A, B, H, SE, Le). The first step in O-glycan synthesis is the transfer of GalNAc to Ser/Thr residues, by uridine diphosphate (UDP)-GalNAc:polypeptide αGalNAc transferase (GalNAc transferase). A homologous family of UDP-GalNAc transferases initiate O-glycosylation, and three GalNAc transferase genes (GALNT1, GALNT2, GALNT3) have 7

been described.[ ] A wide range of O-linked core structures is possible depending on the initial and subsequent substitutions on the GalNAc residue. For the terminal 8

structures, alterations in Lewis type 1 (Lea and Leb ) and type 2 (Lex and Ley ) structures on mucin side chains have been described for gastric mucins.[ ] The enzymes responsible for Lewis antigen synthesis are the fucosyltransferases, and seven human “FUT” genes have been cloned. Among them, FUT1, FUT2, and FUT3 code for the enzymes that synthesize Lewis type 2 (Galα1,4-GlcNAc) and type 1 (Galα1-3-GlcNac) antigen precursors. In the stomach FUT1 is exclusively detected with MUC6; FUT2 is only detected when MUC5AC is present. Data for the glycosylation patterns of mucins in normal gastric epithelium show that it is

9

dictated by the specific set of fucosyltransferases expressed by surface or gland cells, rather than by the apomucin peptide sequence.[ ] Also, the development of 8

intestinal metaplasia and gastric cancer is associated with the appearance of mucin glycosylation patterns that are absent in normal epithelium. [ ] This suggests that goblet cell hyperplasia (GCH) or submucosal gland hypertrophy in the airway in asthma may be accompanied by abnormal mucin glycosylation patterns. Little is known about this from clinical studies of airway disease, except for the observation that mucin-associated oligosaccharides in CF show increased sulfation.[

10]

The polydiverse carbohydrate components of mucins bind surface adhesins or hemagglutinins on microorganisms, and recognition sites on carbohydrates have been 5 11

described for adhesins on Mycoplasma pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, influenza virus, and Escherichia coli.[ ] [ ] Therefore, mucin-associated carbohydrates may serve as an important host defense mechanism by presenting multiple potential sites for the attachment and clearance of 5

microorganisms.[ ] Another important function may be binding with lysozyme, lactoferrin, or antiproteinases so as to protect these molecules and facilitate their role in host defense. Nonmucin Components of Mucus In addition to mucins, airway mucus contains a variety of other proteins. These include proteoglycans, lactoferrin, lysozyme, secretory immunoglobulin A (IgA), peroxidase, and antiproteases such as α1-protease inhibitor (also known as α1-antitrypsin), α2-macroglobulin, secretory leukocyte proteinase inhibitor (also known as mucous proteinase inhibitor, bronchial inhibitor, or antileukoprotease), and trefoil factor peptides. The proteoglycans in serous cell granules include chondroitin 12

sulfate and dermatan sulfate.[ ] The function of proteoglycans in airway mucus is unknown, but their high anionic charge density is probably important. For example, ionic interactions between cationic proteins in the serous cell granule and proteoglycans may reduce the osmotic activity of the granule contents and 12

thereby exclude water from the granule without the need for energy-dependent pumping mechanisms.[ ] In addition, because of their polyanionic charge, secreted proteoglycans in airway mucus provide a more readily hydrated network than that provided by mucins. The ratio of secreted proteoglycans and mucins may, therefore, determine the water content of mucus. The other nonmucin components of airway mucus have important functions in airway host defense. Lysozyme is a bactericidal cationic protein that hydrolyzes 13 14

12 15 16

components of bacterial cell walls.[ ] [ ] Lactoferrin is a cationic protein with high iron-binding capacity.[ ] [ ] [ ] One possible host defense function for lactoferrin is to reduce the growth of iron-dependent bacteria through iron deprivation; another is prevention of iron-catalyzed degradation of mucins. Secretory IgA 17

is the major form of immunoglobulin present in airway mucus and has important antiviral and antibacterial functions.[ ] IgA plays a key role in host defense by eliminating antigens from mucosal surfaces, thereby preventing infection or inappropriate immune stimulation of mucosal lymphoid tissue. Peroxidases are a family 18

of enzymes that catalyze the reduction of hydrogen peroxide to water by electron donors.[ ] In combination with hydrogen peroxide and either thiocyanate or halide ions, peroxidases are active against bacteria, viruses, fungi, and mycoplasmas. The antiprotease activity of airway mucus is important in preventing protease19

20

21

mediated epithelial injury[ ] and mucus hypersecretion.[ ] The lipids in mucus include free fatty acids, phospholipid, triglyceride, and cholesterol. [ ] The function of airway lipids is uncertain, but possible functions include associating with mucins to change the rheology of airway mucus, altering the physical properties of periciliary fluid so as to facilitate effective ciliary beating, changing the adhesiveness of airway mucus to the airway epithelium, and altering the surface activity of airway secretions to lessen the tendency to collapse of small airways.[

21]

22]

The trefoil factor family (TFF)-domain peptides (TFF1, TFF2, TFF3) are thought to provide protection to the epithelium and to influence cell migration, [ 23 glands.[ ]

and 24

TFF3 has been found to localize mainly to mucous cells of healthy airway submucosal Interestingly, TFF3 peptide increases the viscosity of mucus.[ ] However, the precise nature of the interaction between TFF domains and mucins is not known. Conceivably, overproduction of TFF3 or other TFF peptides could have adverse consequences for the rheology of airway mucus in asthma.

MUCUS-SECRETING CELLS The principal cells in the airway that secrete mucus are ciliated epithelial cells, goblet cells, and Clara cells in the airway surface epithelium and serous cells and mucous cells in the submucosal glands ( Fig. 45-2 ). Ciliated Airway Epithelial Cells In addition to approximately 250 cilia, the apical membranes of these cells contain microvilli and fine cytoplasmic processes. Water movement into and out of these cells is mediated by active ion transport and probably plays a key role

744

Figure 45-2 Schematic representation of the airway epithelium showing goblet cells in the surface epithelium and mucous and serous cells in the submucosal gland. The basement membrane has been omitted for clarity of presentation. The submucosal gland comprises a short ciliated duct that is a continuation of the surface epithelium, a nonciliated collecting duct, and secretory tubules lined by mucous and serous cells. The products of serous cells must pass through tubules lined by mucous cells before reaching the collecting duct. The schematic structure of the normal cilium shows an inner microtubule pair connected by radial spokes to nine outer microtubule doublets that are connected by nexin. The outer doublets each carry an outer and inner dynein arm. (Modified from Basbaum C, Welsh MJ: Mucous secretion and ion transport in airways. In Murray JF, Nadel JA, editors: Textbook of respiratory medicine, vol 1, ed 2, Philadelphia, WB Saunders, 1994, pp 323–344; Lundgren JD: J Allergy Clin Immunol 85: 399–416, 1990; and Eliasson R, Mossberg B, Camner P, et al: N Engl J Med 297:1–6, 1977.)

(Modified from Basbaum C, Welsh MJ: Mucous secretion and ion transport in airways. In Murray JF, Nadel JA, editors: Textbook of respiratory medicine, vol 1, ed 2, Philadelphia, WB Saunders, 1994, pp 323–344; Lundgren JD: J Allergy Clin Immunol 85: 399–416, 1990; and Eliasson R, Mossberg B, Camner P, et al: N Engl J Med 297:1–6, 1977.) 6

in regulating the depth of periciliary fluid in which the cilia beat.[ ] These cells serve the important function of propelling airway secretions toward the mouth in the defense mechanism of mucociliary clearance, but they may also have regulatory roles in mucus secretion, especially in disease states, because ciliated epithelial cells secrete cytokines and other mediators that may influence mucus secretion. Goblet Cells 25 26

Goblet cells take their name from the characteristic (but perhaps artefactual) goblet shape they exhibit in chemically fixed tissue.[ ] [ ] In the normal airway, goblet cells represent approximately 10% of the surface epithelial cells. At the basal part of the goblet cell is a nucleus surrounded by rough endoplasmic reticulum. The apical part of the cell is cup shaped and is filled with granules containing mucins and also a number of nonmucin components of mucus, including endoperoxidases, proteinase inhibitors, and lipids. The Golgi apparatus is located between these two cell compartments. The mucin protein core is translated in the rough endoplasmic reticulum and is glycosylated in the Golgi apparatus before the mucin glycoproteins are packaged in mucin granules in the apical part of the cell (see Fig. 45-2 ). Goblet cells are present at most airway levels, but they decrease in number peripherally, normally disappearing at the terminal bronchioles. The regulation of goblet cell secretion is poorly understood, partly because experimental models in which secretory products and secretory responses can be unequivocally associated with goblet cells (and not with other secretory and nonsecretory cells in the mucosa) are difficult to develop. It appears, however, that goblet cells in human airways do not have direct neuronal innervation. Rather, environmental stimuli (smoke, sulfur dioxide, chlorine, ammonia) and inflammatory 25]

mediators with secretagogue activity ( Box 45-3 ) seem to be the most important stimuli for mucin secretion from goblet cells.[

For example, tachykinins,

leukotrienes, histamine, neutrophil elastase, nonprotease neutrophil products, and hyposmolar media all stimulate goblet cell secretion.[

27] [28] [29] [30] [31] [32]

Goblet

cells may not discharge all of their mucus when stimulated; some studies have shown that stimulation leads to secretion of only 30% of the secretory granules. [ [32] [33]

31]

25]

This may be because loss of intracellular mucus to a critical level is a stimulus for goblet cells to enter the cell cycle.[

GCH and goblet cell metaplasia (GCM) in the airway occur in response to subacute or chronic stimuli to mucus secretion and represent important mechanisms for mucus hypersecretion. GCH involves cell division, whereas GCM involves differentiation of preexisting epithelial cells into goblet cells. GCM is a more typical response in peripheral airways, where goblet cell numbers are few. Increases in goblet cell number in the airway epithelium can occur quickly and are reversible when stimuli to secretion are removed or when treatment is instituted.[

25]

For example, antiinflammatory agents such as indomethacin and corticosteroids attenuate 30] [34]

the GCH associated with tobacco smoke, and dexamethasone attenuates the GCH induced by neutrophil elastase or neutrophil lysates.[ Submucosal Gland Cells

The submucosal gland comprises a short ciliated duct that is a continuation of the surface epithelium, a nonciliated collecting duct, and secretory tubules lined by mucous and serous cells. The products of serous cells must pass through tubules lined by mucous cells before reaching the collecting duct (see Fig. 45-2 ). The body of these seromucous glands is located in the submucosa between the spiral bands of smooth muscle and the cartilaginous plates. They are not found in noncartilaginous, membranous bronchioles. The mucus products of the mucous and serous cells are secreted into the lumen of the submucosal gland and are hydrated by

745

Box 45-3. Potentially Important Mucus Secretagogues in the Lower Airways I. Proteases Chymase Neutrophil elastase Cathepsin G II. Arachidonic Acid Metabolites Prostaglandins Thromboxane A2 5- and 15-Hydroxyeicosatetraenoic acid Leukotrienes C4 and D4 III. Inflammatory Cell Mediators Histamine Eosinophil cationic protein IV. Neurotransmitters Acetylcholine α-Adrenergic agonists Gastrin-releasing peptide Vasoactive intestinal peptide Substance P Bradykinin V. Cytokines Interleukin-6 VI. Nucleotide Triphosphates

Adenine triphosphate (ATP) Uridine triphosphate (UTP)

Modified and expanded from Lundgren JD: J Allergy Clin Immunol 85:399– 416, 1990. (A comprehensive list of mucus secretagogues in the airways has recently been presented by Wanner A, Salathe M, O'Riordan TG: Am J Respir Crit Care Med 154:1868–1902, 1996.)

water from the nonciliated columnar epithelial cells in the gland duct. 12]

Serous cells form crescentic caps, or demilunes, over the ends of the acini of the gland.[

In humans, serous cells exist in the surface epithelium before, but not

[35]

after, birth. After birth they are confined to the submucosal glands, where they comprise approximately 60% of the submucosal gland volume in healthy individuals. In the glands, the cells are pyramidal in shape and have small, round nuclei in the basal region of the cell. Their supranuclear cytoplasm is rich in rough endoplasmic reticulum and Golgi apparatus. The apical portion of the cell contains numerous secretory granules. The granules of serous cells contain proteoglycans, 12

lysozyme, lactoferrin, secretory IgA, peroxidase, antiproteases, and a variety of other proteins.[ ] Although serous cells themselves do not synthesize antibodies, they do synthesize the glycoprotein receptor (secretory component) that binds IgA released by plasma cells in the airway submucosal interstitium. This receptor, which is located on the basolateral surface of the serous cell, mediates the internalization and transport of IgA through the cell. Secretory IgA (the term applied to the IgA-IgA receptor complex) is then secreted into the gland lumen, and from there it is transported to the airway lumen. 36

Mucous cells are the other cell type found in the acinar part of the submucosal gland.[ ] These are columnar cells whose nucleus is flattened against the base of the cell because the cell is packed with granules containing mucin. The free border of the cell is smooth and bulging. In response to injury (e.g., sulfur dioxide, tobacco 12

smoke), mucous cells are thought to form from serous cells by a process of serous cell transdifferentiation.[ ] Therefore, in diseases such as chronic bronchitis, mucous cells rather than serous cells may be the predominant cell in the submucosal gland. This metaplastic response to injury can be expected to affect the type of mucus secreted by the submucosal gland. The relative contributions of goblet cells and submucosal gland cells to the mucin component of airway mucus are uncertain and are likely to vary by airway level 37

and in health and disease. Reid[ ] estimated that the volume of glands in the airway mucosa was 40 times greater than the volume of goblet cells, but this calculation was based on several assumptions about the frequency and distribution of goblet cells and glands in the airway. Using quantitative morphometry to analyze mucins in 38

the surface epithelium and submucosal glands of airways of macaque monkeys, Heidsiek et al[ ] reported twice as much stainable stored mucins in the goblet cells in the epithelium of the tracheobronchial airway as in the submucosal glands. This suggests that in primates, goblet cells—not mucous cells—might be the principal source of airway mucins.

Submucosal gland cells, unlike goblet cells, are neuronally innervated. [

39]

Efferent cholinergic nerves originating in the vagi reach the trachea via the superior

laryngeal nerves and the recurrent laryngeal nerves and cause mucus secretion when stimulated.[

40]

Experimental studies in animal models suggest that the

41

cholinergic nervous regulation of mucus secretion is mediated partly through pulmonary reflexes.[ ] For example, stimulation of mechanoreceptors in the larynx, lung, and stomach; hypoxia; and chemical stimulation of cough receptors, bronchial C fibers, and pulmonary C fibers all reflexly increase glandular secretion via a 39] [42] [43]

cholinergic efferent pathway. [

It is not established, however, that all of these reflexes are present in humans. Postganglionic sympathetic nerves are also 44

45

found in approximation with submucosal gland cells, at least in animals,[ ] and cause mucus secretion when stimulated.[ ] Both sympathetic and parasympathetic ganglia contain cells that produce neuropeptides in addition to the classic neurotransmitters, norepinephrine and acetylcholine. Candidate neurotransmitters for this 39] [46]

nonadrenergic noncholinergic (NANC) stimulation include neuropeptides such as substance P, neurokinin A, vasoactive intestinal peptide, and bradykinin.[ [47]

48] [49]

The NK-1 receptor mediates substance P- and neurokinin A-induced mucus secretion, [

and β-adrenergic receptors mediate bradykinin-induced mucus

47 secretion.[ ]

Clara Cells 35 36 50

Clara cells, also termed nonciliated bronchial secretory cells, are most abundant in terminal bronchioles.[ ] [ ] [ ] They are columnar in shape and contain electrondense granules. Clara cells do not secrete mucins—their main secretory products are lipoproteins and possibly antiproteases. The exact identity

746

of the lipoprotein products of Clara cells, their function, and their relationship to pulmonary surfactant are unknown. The Clara cell may act as a stem cell of small airways where basal and goblet cells are normally sparse; both ciliated and goblet cells may develop from the Clara cell subsequent to its division and differentiation. Mucin Packaging and Discharge from Airway Secretory Cells Whether mucins are secreted constitutively by goblet cells or mucus cells is unknown, but it is likely that they are, because constitutive secretion would ensure minimal but consistent maintenance of the airway mucus layer. Regulated secretion of mucins involves the release of mucins from intracellular granules and can be induced by a variety of neural and humoral stimuli.[

1] [25] [42]

The mechanism for regulated secretion is activation of cell surface receptors by signaling molecules (e. 6

g., neurotransmitters), which results in activation of signal transduction pathways and increases in intracellular calcium.[ ] Constitutive and regulated protein secretion can take place within the same cell, but a mechanism for sorting the correct secretory protein into the correct secretory vesicle is required.[

51]

The mucin peptides destined for secretion differ from membrane-bound mucins such as MUC1 because they have cysteine-rich domains at their amino (N) and hydroxyl (C) termini.[

52]

Mucins are oligomerized in the endoplasmic reticulum by disulfide bonding, an event that follows N-glycosylation,[

53]

and then transferred

to the Golgi apparatus in an energy-dependent step modulated by molecular chaperones.[ elongation of O-oligosaccharide side chains.

52] [54]

In the Golgi complex there is further processing of mucins, especially

[52]

The packaging of the large mucin molecules in the intracellular storage granules requires condensation—a process made difficult by the polyanionic nature of mucins. High concentrations of calcium in the granules function as shielding cations to nullify the repulsive forces within the mucin molecule. During mucin secretion, the outer surface of the granule fuses with the inner surface of the secretory cell to form a channel between the extracellular space and the granule. This channel provides a conduit for calcium to exit and extracellular water to enter the granule. The loss of calcium allows electrostatic repulsion to rapidly expand the mucin macromolecule, which is simultaneously hydrated with incoming water. The large hydrated macromolecule then erupts from the cell like a “jack-in-thebox.”[

26] [52]

Through this mechanism, secreted mucin macromolecules are much greater in volume than the stored mucins in the goblet cell granules.

MUCOCILIARY CLEARANCE 55 56

Airway mucus and the foreign material entrapped in it is normally removed from the tracheobronchial tree by a process known as mucociliary clearance. [ ] [ ] This process depends on the coordinated activity of ciliated airway epithelial cells. Rhythmic coordinated beating of cilia on ciliated cells propels mucus cephalad to clear a total daily volume of mucus of about 10•ml. The continuous motion of cilia results in continuous movement cephalad of lung secretions, and the descriptive term mucus escalator has been applied to this process. The energy for ciliary bending is provided by adenosine triphosphate; the mechanism is most likely tied to its interaction with an intraciliary calcium-calmodulin complex.[

57]

Temperature, pH, viscoelasticity, and volume of the overlying mucus all influence the activity of the ciliated cells. In addition, cholinergic and β58] [59]

adrenergic stimulation increase ciliary activity markedly, so autonomic reflexes may be the most important defensive response to inhaled irritants.[

The rhythmic synchronized beating of cilia on airway epithelial cells is facilitated by the specialized structure of the cilia (see Fig. 45-2 ). The cilium is composed of a shaft anchored to the cell cytoplasm by a basal body from which rootlets extend into the apical part of the cell. The shaft of the cilium has longitudinal fibrils or 60

axonemes, which are composed of nine outer pairs of microtubules (or doublets) and two central mictrotubules.[ ] The microtubules are composed of a contractile protein called tubulin. Dynein arms together with nexin arms join adjacent doublets. Radial spokes link with the central microtubules. The bending of the cilium is caused by an active sliding of the doublet microtubules. The dynein molecule undergoes cyclic changes in shape as it engages with and disengages from the adjacent 61]

doublet. Fine, clawlike projections have been observed on the apical but not the lateral aspect of the cilium tip ( Fig. 45-3 ).[ the overlying mucus and propel it toward the larynx.

These “claws” may function to grip

62

The ciliary beat consists of an effective stroke and a recovery stroke.[ ] During the effective stroke, the cilium is extended and penetrates the mucus. The recovery stroke takes twice as long as the active stroke. The rhythmic beating of cilia, therefore, resembles repetitive cracking of a whip. The effective clearance of mucus from the airway relies on optimal rheologic properties of the mucus and normal functioning of cilia on epithelial cells. The optimal rheologic properties of mucus for maximum transport velocity along the mucociliary escalator are high elastic recoil and intermediate viscosity. The rheologic properties are altered when airway diseases such as asthma, bronchitis, or CF result in the secretion of “pathologic” mucus (i.e., mucus that is abnormal in volume or

composition or both). Primary ciliary dyskinesia, formerly known as the immotile cilia syndrome, describes a genetic disorder in the ultrastructure of the cilium and serves to emphasize the importance of normal ciliary function in the

Figure 45-3 An electron micrograph illustrating the claw-like projections (arrows) at the tip, but not on the lateral surface, of cilia on rat airway epithelial cells (×120,000). (From Jeffery PK, Reid L: J Anat 120:295–320, 1975.)

(From Jeffery PK, Reid L: J Anat 120:295–320, 1975.)

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63 64 65

maintenance of upper and lower airway health.[ ] [ ] [ ] The inborn abnormalities of the ciliary cytoskeleton include lack of dynein arms and defective ciliary spokes. These abnormalities result in disruption of the ciliary motor so that, although the cilia may still move, they do not beat synchronously and therefore do not function optimally. The airway consequences of this disorder include chronic rhinitis, sinusitis, otitis, bronchitis, and bronchiectasis. The nonairway manifestations include situs inversus, male sterility, and corneal abnormalities.

CLINICAL IMPORTANCE OF MUCUS HYPERSECRETION IN ASTHMA 66

Mucus hypersecretion is an important clinical feature of asthma. For example, Oppenshaw and Turner-Warwick[ ] questioned 130 asthmatic patients who presented to two asthma clinics in London. They found that 100 patients (77%) reported a history of sputum production. Maximum sputum production occurred at the height of an asthma attach for 73 patients (56%) and toward the end of an attack for 55 patients (42%). Fifty-six patients (43%) produced some sputum on most days, to fulfill

the Medical Research Council criteria for chronic bronchitis. Although some of these subjects were smokers (38 [29%] of the 130 subjects had a history of smoking cigarettes in amounts exceeding 1 pack-year), 32 (57%) of the 56 subjects who qualified for a diagnosis of chronic bronchitis were nonsmokers. Other studies of sputum production in asthmatic subjects have suggested that sputum volume increases with patient age and asthma severity.[

67]

Bronchorrhea (the expectoration of

67 asthma.[ ]

100•ml or more of sputum daily) can occur in association with Therefore, sputum production is a frequent symptom of asthma and contributes significantly to the chronic morbidity associated with the disease. Although chronic sputum production has been linked to increased mortality in patients with chronic obstructive pulmonary disease (COPD),[

68] [69]

a similar link has not been extensively investigated or proven for asthmatic patients. However, it has been shown that

a history of chronic mucus hypersecretion is associated with an accelerated loss of lung function in asthmatic patients.[

70]

Mucus hypersecretion is a prominent symptom during asthma exacerbations and especially during severe asthma attacks that necessitate treatment in the emergency room or as an inpatient in the hospital. Indeed, abnormalities of gas exchange secondary to severe bronchoconstriction can be further exacerbated by lobar or whole lung collapse secondary to mucus plugs in the large airways ( Fig. 45-4 ). A history of recurrent episodes of lobar collapse or of significant chronic mucus hypersecretion should prompt consideration of a diagnosis of allergic bronchopulmonary aspergillosis (ABPA), because APBA is associated with bronchiectasis and mucus hypersecretion. Mucus plugging of the airways is also a documented feature of the pathophysiology of fatal asthma, and it is likely to be the principal cause of the asphyxiation that 71

leads to death in these patients. For example, Dunnill[ ] described the pathologic features of the lungs of 20 patients who died from acute severe asthma. The findings in all 20 cases were very similar. He noted that “both lungs are acutely distended. They fill the chest, completely covering the pericardium, nearly meeting at the midline, and failing to collapse once the negative pressure has been released.” He also found that “the cut surface of the

Figure 45-4 Chest radiograph of a 20-year-old woman admitted to the intensive care unit for management of acute severe asthma. The radiograph shows collapse of the right upper lobe secondary to mucus impaction. The abnormality resolved completely within 24 hours after initiation of treatment with mechanical ventilation, corticosteroids, and bronchodilators.

Figure 45-5 Expression of nine mucin genes in homogenates of endobronchial biopsy specimens from 8 healthy and 11 asthmatic subjects. Gene expression was measured by real-time reverse transcriptase–polymerase chain reaction (RT-PCR), and the expression of mucin genes is represented as the ratio of the copy number of mucin gene-specific messenger RNA (mRNA) to the copy number of the transferrin receptor (a housekeeping gene). Data are presented as mean ± SD. An asterisk (*) indicates that mRNA expression in asthmatic subjects is significantly different than in healthy subjects, P < 0.005. (From Ordoñez CL, Khashayar R, Wong HH, et al: Am J Respir Crit Care Med 163: 517–523, 2001.)

(From Ordoñez CL, Khashayar R, Wong HH, et al: Am J Respir Crit Care Med 163: 517–523, 2001.)

749

expression was higher than normal in the asthmatic subjects. Although at much lower levels than for MUC5AC, detectable gene expression was also found for MUC1, MUC2, MUC4, MUC5B, and MUC7 in biopsy specimens from the asthmatic subjects. In addition, the expression levels of MUC2 and MUC4 were significantly increased in the asthmatic subjects, and the expression of MUC5B was significantly decreased. The significance of these data is uncertain, because much more needs to be learned about the physical properties of the protein products of various mucin genes and about the effects on the overall physical properties of mucus when changes in mucin gene expression occur with disease. Presently, little is known about the concentrations of secreted products of mucin genes in airway secretions of patients with asthma. Available data from healthy controls and from patients with chronic bronchitis or CF show that mucins 5AC and 5B are the 99] [100]

principal secreted mucins in airway secretions.[ [101]

Although MUC2 protein expression in the airway has been found immunohistochemically in some studies,

[99]

it is not a consistent finding.

99

Goblet cell numbers are 2.5-fold higher than normal in the airway epithelium of subjects with asthma,[ ] and the volume of stored mucin in the epithelium is approximately three times higher than normal ( Fig. 45-6 ). Also, higher stored mucin levels in the epithelium are associated with lower secreted mucin in samples of induced sputum, and vice versa. Because secreted mucin levels are higher in patients with lower 1-second forced expiratory volume (FEV1 ), it is possible that the

pathogenesis of airway obstruction in more severe forms of asthma involves ongoing mucin hypersecretion from goblet cells. In addition, higher levels of stored mucins in goblet cells of patients with mild asthma suggests a mechanism for

Figure 45-6 The volume of stored mucin in goblet cells in the airway epithelium of 13 asthmatic and 12 healthy subjects. Measurements were made using methods of design based stereology and the data are presented as volume of mucin per surface area of basement membrane (Vs muc,bm). Solid bars indicate mean values. An asterisk (*) indicates that mucin volume in asthmatic subjects was significantly greater than in healthy subjects, P < 0.05. (From Ordoñez CL, Khashayar R, Wong HH, et al: Am J Respir Crit Care Med 163: 517–523, 2001.)

(From Ordoñez CL, Khashayar R, Wong HH, et al: Am J Respir Crit Care Med 163: 517–523, 2001.) mucus formation and airway obstruction in these patients during acute exacerbations. Knowledge of the growth factors driving GCH or GCM in allergic airway diseases has advanced greatly as a result of experiments in animal models. Airway allergen 102] [103] [104]

challenge in sensitized animals causes marked GCH,[

and the major effector cell is the Th2 subtype of the CD4+ T cell. The evidence for this

conclusion is compelling. Adoptive transfer of Th2 cells, but not of Th1 cells, into the lungs of naive mice causes GCH,[ cytokines, including IL-4, IL-5, IL-9, and IL-13, leads to

107 108 109 110 111 GCH.[ ] [ ] [ ] [ ] [ ]

105] [106]

and overexpression in mice of Th2

Surprisingly, eosinophils do not appear to mediate allergen- or Th2 cytokine-

induced GCH, because allergen- or Th2 cytokine-induced eosinophilia and GCH can be dissociated. [

112] [113] [114]

This suggests that cytokine growth factors such as

IL-13 and IL-9 have direct effects on epithelial cells, but the mechanism is uncertain. Candidate precursor cells for goblet cells in the airway include basal cells and Clara cells. It is possible that upregulation of MUC5AC in these cells is the critical first event leading to cell differentiation to goblet cells. However, only IL-9, and 115

not IL-4 or IL-13, increases mucin gene expression in human airway epithelial cells.[ ] Alternatively, therefore, MUC5AC expression may simply represent a cellular marker of goblet cells hyperplasia induced by Th2 activation of other intracellular signaling mechanisms. Investigations in this area have led to other surprising findings. For example, it has been found that a calcium-activated chloride channel (gob-5 in the mouse; hCLCA1 in humans) is induced by Th2 cytokine stimulation of airway epithelial cells.[

116]

116] [117]

In addition, overexpression of this channel in cultured airway epithelial cells induces mucus cell metaplasia. [ 118

Interestingly, another ion channel, a sodium potassium chloride cotransporter (NKCC1), is upregulated in goblet cells in human asthma.[ ] Therefore, overexpression of ion channels may be an important downstream effect of Th2 cytokine stimulation of epithelial cells. The mechanisms by which activation of these channels leads to GCH in asthma are unknown. An important role for the epidermal growth factor (EGF) receptor and its ligands in GCH in asthma is also evident. For example, selective inhibitors of EGF receptor tyrosine kinase inhibitors block IL-13-induced mucin gene expression in rat airways[

119]

120]

and epithelial cell proliferation in cultured bronchial epithelial cells.[ 121]

The critical ligand for the EGF receptor in these instances is transforming growth factor-α (TGF-α), which is released from neutrophils[ epithelium

120 itself.[ ]

or from the airway

These data suggest a scheme for GCH in asthma that is summarized in Fig. 45-7 .

Overall, it is clear that the mechanisms of allergen-induced GCH involve CD4+ T cells and Th2 cytokines. However, much remains to be learned about the mechanisms of cytokine-induced GCH, the interaction of Th2 cytokines and the EGF receptor system, and the role of ion channels in goblet cell differentiation. The mechanisms governing resolution of GCH or GCM are poorly understood, but ozone-induced GCM in the rat appear to involve the BCL-2 family of apoptosis 122

proteins.[ ] BCL-2 is antiapoptotic, and overexpression of this protein promotes a metaplastic cell phenotype. Other proteins, including BAX and BAK, are proapoptotic. The roles of these protein families in mucus cell hyperplasia and metaplasia in allergic airway disease are just beginning to be investigated.

750

Figure 45-7 Simplified schematic diagram showing possible mechanisms of GCH in asthma. Th2 cytokines (e.g., IL-9, IL-13) secreted by CD4+ T cells cause increased MUC5AC gene expression, which may be a critical signal for goblet cell differentiation from basal cells or Clara cells. The effect of Th2 cytokines on MUC5AC in vivo may be mediated by a variety of other molecules, including the EGF receptor and its ligand TGFα, and by ion channels such as the calcium activated chloride channel (hCACL1) or the sodium potassium chloride cotransporter (NKCC1). (From Fahy JV: Chest [in press].)

(From Fahy JV: Chest [in press].) Goblet Cell Degranulation The principal functional consequence of increased goblet cell numbers is thought to be hypersecretion of mucin glycoproteins when goblet cells degranulate. The mechanisms of goblet cell degranulation in vivo are separate from those of GCH or GCM. In rodents, goblet cell degranulation and mucus occlusion of the airways 123] [124] [125]

occurs after high-dose allergen challenge in sensitized animals.[ leukotrienes mediate this effect in this eosinophil cationic protein,[

127]

123 model.[ ]

A specific 5-lipoxygenase inhibitor (Zileuton) prevents degranulation, indicating that

Other possible inflammatory mediators of allergen-induced goblet cell degranulation include chymase,[

and neutrophil elastase.[

126]

20]

Extracellular nucleotide triphosphates such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) may have a role in goblet cell degranulation and in 128

mechanisms of metaplasia. In the airways, ATP and UTP are equally potent in causing mucin release from goblet cells,[ ] an effect that may be mediated by the Gprotein-coupled receptor, P2Y2. Recently, the effects of extracellular ATP and UTP on goblet cell mucin production and mucin gene expression were examined in 129

more detail.[ ] It was found that UTP is the only nucleotide that stimulates MUC5AC and MUC5B expression as well as mucin secretion in cultured human airway epithelial cells. Further, in mice, intratracheal instillation of UTP-saline caused similar effects. Preliminary studies with various inhibitors demonstrated that separate signaling pathways are involved in UTP regulation of mucin secretion and MUC expression.

130

The effect of allergen challenge on goblet cell degranulation in vivo in human subjects has recently been examined.[ ] Eight allergic asthmatic subjects were challenged with aeroallergen aerosol in a whole lung challenge protocol. Although eosinophilic inflammation occurred as soon as 1 hour after challenge, there was 130

no evidence of goblet cell degranulation in the airway biopsy specimens or in lavage samples at either 1 or 24 hours after challenge ( Fig. 45-8 ).[ ] This finding suggests that the mechanisms of degranulation in human asthma are complex. Important inhibitory mechanisms must be in place that prevent degranulation of goblet cells even on exposure to allergens, except in specific clinical circumstances. An example of such a circumstance might be allergen exposure coincident with a viral infection of the airway. Knowledge of the mechanisms linking cell surface stimulation with a mucin secretagogue and extracellular release of mucin granule contents is quite rudimentary, but recent advances have been made. Specifically, a role has been discovered for MARCKS protein (myristolylated alanine-rich C kinase substrate), whereby secretagogue-induced activation of protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm.[ In the cytoplasm MARCKS is dephosphorylated, associates with both actin and myosin, and also interacts with mucin granule membranes. In this way, mucin granules are linked to the contractile cytoskeleton, facilitating their movement to the cell periphery and subsequent exocytosis.

131]

As more is learned about the mechanisms of degranulation, it should be possible to develop specific inhibitors of degranulation. Currently no such inhibitors exist, and their development would open up a new treatment strategy for mucus hypersecretion in asthma and other airway diseases.

751

Figure 45-8 Photomicrographs of 3-•m sections of bronchial biopsy specimens from an asthmatic subject before and after aerosolized allergen challenge. The stain is sequential Alcian blue periodic acid Schiff (AB/PAS). Representative biopsy sections are shown in the baseline state (A), 1 hour after challenge (B), and 24 hours after challenge (C). Allergen challenge did not result in a reduction in AB/PAS-stained mucins in the epithelial goblet cells; in fact, stained mucins tended to be increased in the epithelium 24 hours after allergen challenge. (From Hays SR, Woodruff PG, Khashayar R, et al: J Allergy Clin Immunol 108:753–758, 2001.)

(From Hays SR, Woodruff PG, Khashayar R, et al: J Allergy Clin Immunol 108:753–758, 2001.) Mucin Hypersecretion

Higher-than-normal levels of mucin and lactoferrin in airway secretions indicate hypersecretion from goblet cells and submucosal gland cells. At least two possible mechanisms could account for increased secretion from these cells. One is increased mucus secretagogue activity in the airway mucus or in the submucosal interstitium, and the other is hyperplasia or hypertrophy of mucus-secreting cells. Many inflammatory mediators secreted by cells such as mast cells, eosinophils, and neutrophils can stimulate secretion of mucin from goblet cells and submucosal gland cells ( Box 45-3 ). [

132]

20] [126]

Among the most potent mucin secretagogues are neutrophil elastase and chymase.[

important in acute exacerbations of asthma, because free elastase activity is detectable in sputum in this situation.[ bronchiectasis, CF), we have found that most of the secretagogue activity of sputum is due to neutrophil elastase.[ eosinophil cationic protein,[

127]

and leukotrienes C4 and E4 [

In addition, neuropeptides such as substance P,[

33]

136]

Neutrophil elastase may be especially

87] [133]

In suppurative airway diseases (e.g.,

134] [135]

In asthma, mast cell chymase,[

126]

are likely to be important.

vasoactive intestinal peptide,[

46]

47]

and bradykinin[

cause mucin hypersecretion. These peptides may be especially 137]

important when epithelial neutral endopeptidase activity is reduced (e.g., sloughing of the airway epithelium in asthma, damage by injury in viral infection).[ Neutral endopeptidase cleaves neuropeptides, thereby inactivating them.[

138]

Hyperplasia or hypertrophy of goblet cells and submucosal gland cells may occur in asthma and represents another mechanism for mucus hypersecretion. Stimuli that have been shown to induce GCH or GCM include cigarette smoke, sulfur dioxide, nitrogen dioxide, chlorine, adrenoceptor or cholinoceptor agonist drugs, and endotoxin.[

29] [34] [139] [140]

In addition, neutrophil elastase and extracts from purified neutrophils cause GCH. [

25] [141]

GCH occurs in asthma even in mild disease.

[99]

142

Submucosal gland cells may also be hypertrophied in asthma. For example, Dunnill et al,[ ] using a point counting method, calculated the mucous gland volume as a percentage of airway wall volume in 12 patients who died from acute severe asthma and found that the gland volume in asthmatic subjects was twice that of nonasthmatic control subjects. Increased Bronchovascular Permeability 19 63

Higher-than-normal levels of plasma proteins in airway secretions indicate that bronchovascular permeability is abnormally increased in asthma.[ ] [ ] Increased bronchovascular permeability has a number of adverse consequences, including mucosal edema leading to airway narrowing and leakage of plasma proteins into the airway lumen. The mixing of plasma-derived proteins, plasma-derived inflammatory cells, excessive amounts of mucin glycoproteins, and sloughed airway epithelial cells results in abnormal mucus with abnormal viscoelastic properties. Coupled with reduced mucociliary clearance, a consequence of injury to cilia on airway 143

epithelial cells, [ ] this “pathologic” mucus proves difficult to expectorate and tends to form mucus “plugs” that contribute significantly to airway obstruction in asthmatic patients. Interestingly, in acute asthma compared with CF-related lung disease, plasma-derived proteins contribute relatively more than mucin glycoproteins to luminal secretions.[

21]

Plasma proteins in airway mucus, especially albumin, may have important consequences for its physical properties. For 144

example, the incubation of human serum albumin and pig gastric mucin results in a highly viscous solution.[ ] Viscosity enhancement is proportional to the albumin concentration and is considerably greater than the additive or multiplicative viscosity values calculated for albumin or for mucin solutions measured separately. Abnormal Mucociliary Clearance

The reduced clearance of airway mucus from the airways in asthma is multifactorial in etiology. Three of the more important mechanisms are abnormal viscoelastic properties of asthmatic mucus, cilioinhibitory effects of inflammatory mediators in asthmatic sputum, and disruption or destruction of the ciliated epithelial cell “carpet.” First, airway mucus in asthma is abnormal in volume and consistency, which leads to abnormal viscoelastic properties, abnormal interactions with cilia on epithelial cells, and delayed clearance. For example, the ciliary beating frequency of bronchial mucosal explants was significantly decreased when the explants were 145]

incubated with sputum from asthmatic subjects.[ reversible, indicating that it was

This effect was greatest for sputum from asthmatic subjects experiencing an asthma exacerbation and was

752

a functional (not a toxic) phenomenon. The mediators in the sputum that are responsible for this effect are unknown. Many inflammatory mediators in asthmatic 146

sputum, such as the leukotriene and prostaglandin products of arachidonic acid metabolism, are ciliostimulatory,[ ] so direct ciliary impairment may be less important in impairing sputum clearance than the abnormal viscoelastic properties. Second, some inflammatory mediators found in the asthmatic airway decrease 147

19 148

ciliary beat frequency directly; these mediators include eosinophil major basic protein[ ] and neutrophil elastase.[ ] [ ] Finally, impairment of mucociliary clearance can also be expected as a consequence of reduced numbers of ciliated airway epithelial cells in the asthmatic airway. Reduction in ciliated epithelial cell numbers occurs because ciliated epithelial cells are exfoliated by mediators such as eosinophil granule proteins and because the numbers of goblet cells and

squamous cells are increased as a result of metaplasia and hyperplasia in the airway epithelium. These cellular changes result in a decrease in the normal complement of ciliated cells and a consequent decrease in the maximal efficiency of the mucociliary escalator.

TREATMENT OF MUCUS HYPERSECRETION IN ASTHMA The goals of treatment for mucus hypersecretion in asthma should be to reduce mucus volume, to normalize mucus composition, and to improve mucus clearance. However, the effects of currently available antiasthma medications on mucus volume, mucus composition, and mucus clearance are largely unknown, because these outcomes are difficult to quantitate in clinical practice or in clinical research studies. Presently, the pharmacologic agents for mucus hypersecretion in asthma can be divided into bronchodilators, antiinflammatory agents, mucolytic agents, and expectorants ( Box 45-4 ). Bronchodilators Beta agonists and methylxanthines have beneficial effects on mucociliary clearance independent of their bronchodilator activity. For example, beta agonists and methylxanthines increase intracellular cyclic adenosine monophosphate levels, which in turn increase ciliary beat frequency and mucus secretion.[

149]

Subcutaneous 150]

terbutaline, oral metaproterenol, and inhaled albuterol have all been shown to augment radioaerosol clearance in normal volunteers and in asthmatic subjects. [ [151]

Aminophylline acutely increases tracheal mucus velocity in dogs.[

152]

Beta agonists may also beneficially affect mucus volume and composition by decreasing 153]

the plasma protein content of airway mucus through decreased bronchovascular permeability. [

The role of anticholinergic drugs such as ipratropium bromide, oxitropium bromide, or atropine in the treatment of mucus hypersecretion is uncertain, because these drugs have multiple effects on the mucociliary apparatus, some of which are beneficial and some not.[ 42 155 cells,[ ] [ ]

154]

For example, because cholinergic stimuli are potent

secretagogues for mucin and other products of goblet cells and submucosal gland anticholinergic drugs may decrease airway mucus levels. This effect might be beneficial in the treatment of airway mucus hypersecretion so long as the reduction in secretion of mucus components does not render the residual mucus more

Box 45-4. Pharmacotherapy for Mucus Hypersecretion in Asthma I. Bronchodilators Beta agonists Ipratropium bromide Theophylline II. Antiinflammatory Agents Oral corticosteroids Inhaled corticosteroids Sodium cromoglycate Nedocromil sodium III. Mucolytic Agents N-acetylcysteine S-carboxymethyl cysteine Bromhexine IV. Expectorants Guaifenesin Iodinated glycerol Potassium iodide Terpin hydrate V. Newer Agents Drugs that interrupt the leukotriene pathway (LTD4 antagonists, 5-lipoxygenase inhibitors) Recombinant human DNase

difficult to clear due to unfavorable changes in its viscoelastic properties. Atropine decreases tracheal mucus secretion in dogs and changes some of the physical 155]

properties of residual mucus.[

However, ipratropium bromide does not change the physical properties of sputum in patients with chronic bronchitis treatment,[

156]

157

and oxitropium bromide does not change the physical properties of sputum from patients with chronic bronchitis or asthma.[ ] In addition, because cholinergic stimuli increase ciliary beat frequency, anticholinergic drugs may decrease ciliary beat frequency and possibly decrease mucociliary clearance. This effect seems 158

159

157

most marked for atropine,[ ] and it does not occur with ipratropium bromide[ ] or oxitropium bromide.[ ] Finally, the bronchodilator effect of anticholinergic drugs aids mucus clearance. Overall, present data indicate that atropine may have some deleterious effects on mucociliary clearance rate, mucus volume, and the physical properties of mucus; that ipratropium bromide and oxitropium bromide do not seem to share these deleterious effects; and that the bronchodilator effects of these agents are likely to aid mucus clearance. Antiinflammatory Agents Inhaled corticosteroids can be expected to decrease mucus hypersecretion and improve mucociliary clearance by their antiinflammatory actions.[ include reduction

160]

These actions

753

in leukocyte numbers in the airway mucosa, reduction in levels of leukocyte and mast cell mucin secretagogues, restoration of airway epithelial integrity, and reduction in bronchovascular permeability.[

161]

In addition, corticosteroids have some specific effects on mucus-secreting cells. For example, dexamethasone directly 162]

inhibits both basal glycoconjugate secretion and stimulated secretion in feline airway submucosal glands,[

and both dexamethasone and methylprednisolone

produce dose-related suppression of the spontaneous release of radiolabeled mucous glycoproteins from cultured human airways.[

163]

It has also been demonstrated

that dexamethasone decreases steady-state messenger RNA levels of mucin genes (MUC2 and MUC5) in airway mucus-producing cancer cells.[

164]

Corticosteroids represent critically important pharmacotherapy for the treatment of acute severe asthma attacks. The mechanism of action of the beneficial effects of corticosteroids in the management of acute asthma is unknown but may involve steroid-induced reductions in mucus secretion, reductions in adhesiveness and viscosity of mucus plugs, and steroid-induced improvements in mucociliary clearance. Direct proof of these effects of corticosteroids in asthmatic subjects is lacking because of the difficulty of measuring these outcomes during acute severe asthma attacks. In stable asthmatics, inhaled budesonide has been shown to decrease goblet cell numbers in the airway epithelium.[

165]

Mucolytic Agents Use of the cysteine derivatives, N-acetylcysteine and S-carboxymethylcysteine, has been advocated for the treatment of mucus hypersecretion for many decades. These agents break disulfide bonds that form bridges between mucin chains, and in vitro studies have convincingly demonstrated their favorable effects on the

166

viscoelastic properties of asthmatic sputum.[ ] In addition to these “mucolytic” effects, N-acetylcysteine and S-carboxymethylcysteine have antioxidant effects that may have relevance for mucus hypersecretion. For example, N-acetylcysteine or S-carboxymethylcysteine administered concurrently during 2 weeks of cigarette 167

smoke exposure significantly inhibited the development of mucus hypersecretion in the rat.[ ] The mechanism for this effect is probably a direct or indirect antioxidant effect. The direct antioxidant effect may be to increase intracellular stores of reduced glutathione or to block oxidant-induced depletion of glutathione. The indirect effect (relevant only for N-acetylcysteine) may be a direct oxygen-scavenging effect. Despite these favorable in vitro and animal experiments with cysteine derivatives, the clinical experience with N-acetylcysteine in asthma has not been very favorable, mainly because it causes bronchoconstriction when 168]

administered by aerosol.[

Therefore the use of aerosolized or oral preparations of N-acetylcysteine or S-carboxymethyl cysteine cannot be recommended.

Bromhexine is another oral agent classified as a mucolytic (although it does not break disulfide bonds), and it is one of the few mucolytic drugs whose therapeutic efficacy has been examined in a placebo-controlled trial involving patients with acute severe asthma. Unfortunately, bromhexine proved to be no better than placebo in affecting outcomes that included rate of recovery, measures of oxygenation and ventilation, and measurements of peak flow.[

169]

Expectorants The term “expectorant” is derived from the Latin word expectore, which means “from the chest.” Drugs that allow patients to cough up sputum more easily are considered expectorants. Writing about expectorants for the third edition of this textbook in 1983, Hirsch wrote: “Of the areas in modern treatment, none remain more firmly attached to tradition than the use of bitter, sour, salty, and/or sweet medicaments in various preparations and at various temperatures as cough medicines. Each prescription of potassium iodide, ammonium chloride, terpin hydrate, guaifenesin, honey and lemon, or ethyl alcohol associated with antihistamines and cough depressants carries with it the fervent hope of the physician that the prescription will dislodge the phlegm and the cough will subside. It is my conviction that, although these drugs may have some therapeutic action in patients with acute bronchitis, the zeal with which the physician prescribes the drug is as therapeutic as the 170

ingredients.”[ ] Twenty years later, the zeal with which expectorants are prescribed does not seem to have subsided despite the lack of any new body of research supporting their use. Expectorants such as ammonium chloride and members of the terpin hydrate group may have some ciliostimulatory effects, but there are no published reports of their efficacy in clinical trials. Some expectorants such as guaifenesin (glycerol guaiacolate) are emetics and are given in subemetic doses for the theoretic possibility that gastric irritation promotes an increase in mucus secretion by a cholinergic reflex mechanism.[

171]

The clinical data to support the efficacy of guaifenesin are

149 172 173 unimpressive.[ ] [ ] [ ]

conflicting and, on balance, The mechanism of action of the iodide salts (iodinated glycerol and potassium iodide) on the mucociliary system in unknown. They are thought to improve mucus expectoration by improving mucus hydration, perhaps through increased epithelial water secretion or serous cell secretion. In placebo-controlled trials, the administration of iodide salts resulted in improved symptoms in patients with airway disease, although lung function did not improved significantly. For example, iodinated glycerol treatment for 6 months in children with asthma resulted in improved asthma symptom scores, but objective measures of pulmonary function did not change significantly and the treatment was associated with the development of palpable goiter in 15% of patients. [174]

In addition, the administration of iodinated glycerol for 8 weeks to patients with COPD resulted in significant improvements in airway symptom scores, but

pulmonary function, sputum volume, and mucus rheology were not objectively measured.[ Miscellaneous Pharmacologic Agents

175]

Sodium thiophene carboxylate decreased the viscosity and elasticity of pig mucin collected from a tracheal pouch but had no effect on pulmonary function or airway mucus rheology in patients with COPD and asthma.[

176]

Inhaled indomethacin had a significantly greater effect than inhaled placebo in reducing sputum volume in

patients with bronchorrhea secondary to chronic bronchitis.[

67]

Two case reports have indicated that erythromycin reduces sputum volume significantly in asthmatic

177 178 bronchorrhea.[ ] [ ]

patients with This effect of erythromycin may be mediated by a direct effect of erythromycin on mucus secretion rather than an indirect antibacterial effect, because in vitro studies have demonstrated

754

that erythromycin suppresses baseline mucus secretion and stimulated secretion.[

179]

In the last decade, advances have been made in the treatment of mucus hypersecretion in CF, and some of these advances may prove relevant in asthma. For example, 94 95

aerosolized recombinant human DNase improves pulmonary function and reduces CF-related pulmonary exacerbations in patients with CF,[ ] [ ] presumably by liquefying DNA-rich CF sputum and enhancing mucus clearance. In addition, aerosolized amiloride slows the loss of forced vital capacity and improves sputum 180

viscosity and elasticity in patients with CF, presumably by blocking sodium channels in airway epithelial cells and increasing mucus hydration.[ ] Finally, aerosolized uridine 5′-triphosphate (in combination with aerosolized amiloride) improves mucociliary clearance from the periphery of the lungs in patients with CF, [181]

presumably by stimulating airway ciliary beat frequency.

The recent discovery that hCACL1, a calcium-activated chloride channel, is upregulated in the airway epithelium and localizes to goblet cells in asthmatic subjects [182]

provides a novel therapeutic target for GCH in airway disease. An inhibitor of hCACL1 is in the early phases of clinical testing as a specific treatment for airway mucus hypersecretion. Chest Physical Therapy Chest physical therapy is probably most helpful in asthmatic patients with allergic bronchopulmonary aspergillosis, because these patients have bronchiectasis. Chest physical therapy usually is not considered part of the regimen of management for acute severe asthma, although a trial of chest physical therapy should be considered for patients with atelectasis secondary to mucus plugs. Bronchospasm and cough paroxysms are potential side effects of chest physical therapy in asthmatic subjects who are experiencing an acute exacerbation. Bronchoscopy Bronchoscopy and bronchoalveolar lavage with saline or with saline and N-acetylcysteine has been advocated as a treatment for acute severe asthma for more than 30 years.[

183]

No controlled trials of this therapy have been performed, however. The most recent uncontrolled series involved 19 subjects with asthma refractory to

184

at least 48 to 72 hours of aggressive inpatient medical management.[ ] A total of 51 bronchoscopies (1–6 per patient) were performed transnasally with local anesthesia. Mucus visualized in the large airways was removed by suction, and mucus in smaller, more peripheral airways was removed by bronchoalveolar lavage (BAL) with a total of 50 to 300•ml of saline. Airway casts were recovered by BAL in some instances ( Fig. 45-9 ). Complications included three episodes of transient mild hypoxemia and four episodes of bronchospasm responsive to bronchodilator therapy. Mean FEV1 values before bronchoscopy were 41% of predicted; they increased to 60% of predicted within 96 hours after bronchoscopy. Despite these encouraging uncontrolled data, the expense and potential dangers of bronchoscopy in acutely ill asthmatic patients require that a properly controlled, randomized study be completed before BAL can be recommended as a treatment for acute severe asthma refractory to usual therapeutic measures.

Figure 45-9 Airway casts recovered by bronchoalveolar lavage from an asthmatic subject in acute exacerbation. (From Lang DM, Simon RA, Mathison DA, et al: Ann Allergy 67:324–330, 1990.)

(From Lang DM, Simon RA, Mathison DA, et al: Ann Allergy 67:324–330, 1990.)

SUMMARY

In health, airway mucus effectively traps inhaled foreign material and the mucociliary escalator effectively removes this material from the airways. In airway diseases such as asthma, the composition of airway mucus changes. Specifically, there are increases in mucin levels, plasma protein levels, cell numbers, and products of cell lysis. These changes in mucus composition result in changes in viscosity and adhesiveness of mucus that render it more difficult to expectorate. These abnormalities, coupled with abnormalities in ciliary function, result in accumulation of mucus in the airways and airway obstruction. Consequently, mucus hypersecretion in asthma contributes significantly to the morbidity and mortality of the disease. The pathophysiology of mucus hypersecretion in asthma is probably closely linked to that of airway inflammation, because important consequences of the abnormal cellular infiltration and vascular permeability that characterize asthma include higher-than-normal levels of cell-derived and plasma-derived mucus secretagogues in the airway and higher-than-normal levels of mediators that promote GCH and GCM. Therefore, it is not surprising that the most effective therapeutic agents for mucus hypersecretion in asthma are oral and inhaled corticosteroids. The roles of mucolytics and expectorants in the treatment of mucus hypersecretion remain controversial. New therapeutic agents for mucus hypersecretion and mucus impaction are under development, based on improved understanding of the mechanisms of mucus hypersecretion and of the contribution of mucus components such as mucins, albumin, DNA, and actin to the viscoelastic properties and rate of clearance of airway mucus.

755

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Chapter 46 - Aerosols

Thomas G. O'Riordan Gerald C. Smaldone

DEFINITION AND DESCRIPTION OF AN AEROSOL 1 2 3 4 5 6 7 8

An aerosol can be defined as a system of solid particles or liquid droplets that can remain dispersed in a gas, usually air.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] Aerosolized pharmaceutical solutions, suspensions, and dry powders are a mainstay of therapy of allergic diseases of the respiratory tract. Aerosols are also a means by which the 9

allergens[ ] and pollutants are delivered into the respiratory tract, topics that are reviewed elsewhere (see Chapter 34 ). Pharmaceutical aerosols almost always contain a wide range of particle sizes. Because the aerodynamic behavior of an aerosolized particle is critically influenced by its mass, it is important to be able to precisely describe the size distribution of aerosolized particles. In clinical studies, the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (σg) are often used to characterize the dimensions of an aerosol. The MMAD represents the point in the distribution above which 50% of the mass resides, expressed as the diameter of a unit density (1•g/ml) sphere having the same terminal settling velocity as the aerosol particle in question, regardless of its shape and density. The σg is the ratio of the size at 84% (or 16%) to the MMAD and is an indicator of the variability in particle diameters (1 standard deviation). If the particle size varies over a wide range (σg > 1.2), it is described as having a polydisperse particle distribution. If all of the particles in an aerosol are of similar size (σg < 1.2), the particle distribution is described as monodisperse. Monodisperse aerosols are usually only encountered in research studies in which specialized generators are used to create such aerosols.[

10]

AERODYNAMIC BEHAVIOR OF AEROSOLS Mechanisms of Deposition in Human Subjects Therapeutic aerosols with diameters of 0.5 to 10••m deposit in the lung by inertial impaction and gravitational sedimentation. Inertial impaction occurs when an inhaled particle is not able to follow the airstream and impacts into the airway wall, usually at airway bifurcations. Larger particles traveling at high velocities are

more likely to impact in the upper airway than smaller particles at slower velocities. Particles less than 5••m can also settle by gravitational sedimentation in more distal airways. Deposition by sedimentation is critically time dependent with, for example, breath holding significantly increasing the number of particles deposited. [1] [4] [5]

In healthy subjects, both the regional distribution and the absolute number of inhaled particles that deposit in the lung are primarily determined by the size of the particles and by the breathing pattern. Conversely, in disease states, the geometry of the airways and changes in regional ventilation may become the dominant influences.

Particle Size and Deposition 11 12 13 14

The most precise data describing the effects of particle size on deposition were provided by investigators using monodisperse aerosols.[ ] [ ] [ ] [ ] When particles of less than 0.5••m are inhaled tidally by subjects with normal lung function, 90% will be exhaled. Below this size, deposition in the lung periphery can occur by diffusion. From 0.5 to 3.0••m, a progressive increase in deposition in the periphery of the lung occurs by means of gravitational sedimentation. With progressive increases in diameter greater than 4.5••m, there is a rapid reduction in peripheral deposition as particles deposit in central airways and extrapulmonary sites through sedimentation and inertial impaction. With even larger diameters, an increasing proportion of particles deposits in the central airways, oropharynx, and other extrapulmonary sites. Deposition fractions (the fraction of inhaled particles that are not exhaled) of large particles (10••m) can approach 1.0, although few of the particles deposit in the alveoli and peripheral airways. Therapeutic aerosols with MMAD greater than 15••m rarely deposit in the lungs, even when inhaled by mouth. Compared with the amount of published data on solid monodisperse aerosols, the amount of literature on deposition of polydisperse aqueous aerosols is rather scant. 6

Preliminary data suggest that polydisperse aerosols tend to have a more central deposition pattern than monodisperse aerosols with the same MMAD. [ ] Besides the effect of σg, there has been much theoretic discussion as to whether aqueous aerosols become enlarged, stay the same size, or become smaller in their passage through the airway.[

15]

Breathing Pattern and Deposition 16

17

For a given particle size, breathing pattern can affect both the pattern of deposition[ ] and the number of particles deposited.[ ] Three components of the breathing pattern can independently affect deposition: the inspiratory flow rate, the residence time of particles in the airway, and the tidal volume. Rapid inspiratory flow rates facilitate deposition by inertial

760

Figure 46-1 Deposition images following inhalation of 1.5••m particles. Clockwise, upper left, normal subject quiet tidal breathing; normal subject rapid inhalation; patient with severe chronic obstructive pulmonary disease quiet tidal breathing; normal subject quiet tidal inspiration, rapid exhalation. The lung outlines were drawn around a133 Xe equilibrium scan. (From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled

glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp 447–477.)

(From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp 447–477.) impaction, and increasing flow rates have a proportionately greater influence on larger rather than smaller particles. Rapid flow rates increase deposition in the large airways and in the oropharynx. Figure 46-1 shows that, even for normal subjects inhaling relatively small particles with MMAD of 1.5••m, rapid inhalation increases central deposition of the particles (top right) compared with tidal breathing (top left). To facilitate deposition in more peripheral portions of a lung, breath holding can be employed.[ and increases peripheral deposition. Airway Anatomy and Deposition

17]

This increases the time available for gravitational sedimentation

A further influence on deposition of particular importance in disease states is airway caliber. With increasing airway obstruction, the deposition pattern of inhaled 16] [18] [19]

aerosol becomes more central,[

probably as a result of an increase in impaction. In addition, in subjects with more severe airway obstruction, deposition in

central airways can occur during expiration because of turbulence associated with the formation of flow-limiting segments (FLSs). [

20] [21]

GENERATION OF AEROSOLS To generate an aerosol, energy must be applied to solid or liquid material to disperse the material and cause it to be suspended in a carrier gas, which, for the purpose of this chapter, is usually air. Pressurized Metered-Dose Inhalers Pressurized metered-dose inhalers (pMDIs) consist of a pressurized metal can that contains a mixture of propellants (Freon), which, when in equilibrium, are both in 22

liquid and gaseous form at room temperature.[ ] Suspended in the liquid, the agonist usually consists of solid particles that have been milled into an aerodynamically respirable distribution of diameters. In addition to the medication and propellant, many preparations contain a surfactant or dispersal agent such as lecithin or oleic acid because surface tension affects aerosol behavior. In addition, ethanol is used to enhance the solubility of some formulations. This two-phase suspension is evenly distributed by shaking and then releasing a “metered dose” into the atmosphere via the valve system when the canister is compressed and triggered. The metering valve controls the volume of liquid released into the atmosphere. At that point, the Freon instantly evaporates, imparting a relatively high kinetic energy to the solid particles. Thus the metering valve provides precise control of the triggered amount of drug. 22 23 24

The volume of drug released per actuation is a function of the size of the metering valve.[ ] [ ] [ ] As the metering volume is increased, a greater amount of each spray is deposited on the actuator mouthpiece. In addition, however, the proportion of particles depositing in the central airways relative to the lung periphery increases proportionate to metering volume.[

22]

In contrast, warming of the MDI canister has been reported to increase drug deposition in the distal parts of the lung.

[25]

22

An increase in concentration of propellant loaded into the canister elicits a finer aerosol at a higher ejection velocity[ ] (as discussed earlier, a high ejection velocity increases the inertia of the particle with important clinical consequences). The milled size of the drug powder is another limiting factor determining the particle size of the MDI aerosol: the coarser the drug powder, the larger the aerosol. If the pMDI is actuated within 60 seconds of a prior actuation, the dose of drug released may decrease significantly.[

26]

The Autohaler is an automatic device that the patient triggers during inhalation, like a dry powder inhaler. The trigger is controlled by a spring-loaded system, which can be energized electively by the patient using a lever. Therefore, patients with problems of digital manipulation (e.g., arthritis) can use this MDI more conveniently. [27]

Concern about the effects of subtle changes in the for-mulation of pMDI medications on aerosol delivery has led regulatory agencies to demand strict requirements that the manufacturers of generic formulations document clinical “equivalence” with existing products.[

28]

Use of Accessory Devices with pMDIs There are practical difficulties encountered when administering drugs by pMDI. There is a need to coordinate actuation with respiration. Even with the use of optimal inhaler technique, significant extrapulmonary deposition occurs because, even though the particles are of respirable diameters, there is high inertia. Recognition of these problems has led to important attempts to modify the behavior of the

761

Figure 46-2 Influence of intrinsic particle inertia on deposition pattern. On the left, a patient inhaling particles generated by a metered dose inhaler without a spacer. On the right, the same patient inhaling the same aerosol but after modification by a Rondo spacer device. (From Newman SP, Talae N, Clarke SW: Acta Ther 17:49–58, 1991.)

(From Newman SP, Talae N, Clarke SW: Acta Ther 17:49–58, 1991.) 29 30 31 32 33 34

aerosol as it is generated.[ ] [ ] [ ] [ ] [ ] [ ] Spacer devices have a significant influence on drug delivery. As shown in the deposition study of Figure 46-2 , the spacer device absorbs those particles that have high inertia and that would, in the absence of the spacer, be likely to deposit on the pharynx and larynx. Significant losses occur in the spacer as particles impact on the walls, but the available data in human studies indicate that the parenchymal deposition of drug within the lung is similar with and without the spacer, resulting in decreased exposure of the larynx and pharynx to the inhaled agonist. In addition, some spacer devices have been 29

designed to provide coordination control for the patient.[ ] Rather than relying on the patient to inhale at the instant of MDI triggering, the spacer serves as a reservoir from which particles remaining in the gas phase can be inhaled more conveniently. The presence of one-way valves within some spacer devices facilitates this maneuver.

The use of spacer devices reduces extrapulmonary deposition. This is especially important in preventing toxicity when doses of inhaled medications that are in excess of the usual maintenance doses are used. Pharyngeal deposition of high doses of inhaled corticosteroid agents, which are designed to be poorly absorbed, can cause local toxicity such as pharyngitis and Candida infection. Pharyngeal deposition of high doses of inhaled beta-sympathetic agonists can cause systemic toxicity because these agents are swallowed and absorbed through the gastrointestinal tract. Controversy exists as to whether the use of spacer devices improves airway 30 31

deposition of inhaled medications in situations in which optimal inhaler technique is used.[ ] [ ] However, because patients' pMDI techniques in “real world” clinical practice are frequently suboptimal, the use of spacers most likely does result in increased airway drug deposition for many patients. Commercial holding chambers vary in design, size, and construction materials. Some are designed for use with only one drug formulation, but most are marketed to 32 33

be used with most MDIs. The simplest chambers are tubes, which should be at least 10•cm in length and 3•cm in diameter.[ ] [ ] These devices decrease oropharyngeal deposition and compensate in part for delay in inhalation after actuation of the device. If the patient exhales during actuation, simple tube spacers are not effective. Whereas chambers of 150•ml volume are efficient, larger chambers up to 750•ml may provide enhanced drug delivery, albeit at a cost of reduced 29]

portability and convenience.[

The effects of poor patient coordination can be reduced by the addition of one-way valves (valved holding chambers [VHCs]) that prevent patients from exhaling into the device. A variety of valve designs have been patented. For portability, holding chambers may be collapsible. In addition, some devices provide sonic feedback if the patient's inhalation flow rate is too rapid. 34

It has also been noted that electrostatic interactions between aerosol and plastic chambers may reduce drug delivery.[ ] As a result, some manufacturers now include more detailed cleaning instructions (i.e., wash plastic chamber with detergent and leave to dry without wiping) and others have produced metal chambers that have less electrostatic interactions. Reformulation of Metered-Dose Inhalers without Chlorofluorocarbons [

35] [38]

Environmental pressures have led to the development of aerosol dispensers that are free of chlorofluorocarbons (CFCs), the production of which ceased in 1996. (Manufacture of therapeutic aerosols with CFC continues, however, from reserved supplies). Such research has centered on dry powder systems (see later text) and pMDIs that are free of CFCs. For example, 1,1,1,2, tetrafluoroethane (HFA 134a), is a propellant that does not affect the ozone layer and is found in the majority of reformulated pMDIs. All existing CFC MDIs will be replaced by HFA and/or dry powder inhalation (DPI) formulations. As a result of this ruling, clinical trials for the purpose of registering new products no longer involve CFC formulations. In replacing CFC formulations with HFA formulations, the pharmaceutical industry has adopted two strategies. The first replaces existing CFC formulations with HFA products designed to be clinically equivalent to each other. For example, Proventil (albuterol) HFA and Proventil CFC are very similar in terms of their in vitro aerosol characteristics and clinical effects.[

36]

The reformulation of beclomethasone dipropionate (BDP) from CFC to HFA has involved a different strategy; the

development of an aerosol with both different aerosol properties and clinical effects.[

37] [38]

Compared with the CFC formulation, HFA BDP particles are

significantly smaller in size, resulting in reduced extrapulmonary deposition in the oropharynx (which is universally accepted to be an advantage). On the other hand, the penetration of the aerosol into the peripheral regions of the lung is enhanced. The benefits of the latter are more controversial and are discussed later. The pace of introduction of new HFA products and DPIs has been slower in the United States than in Europe, due in part to stricter regulatory requirements in the United States. The U.S. Food and Drug Administration has been reluctant to approve products that do not meet the quality standards that apply to existing CFC formulations in terms of fine particle mass (dose in particles less than 5••m in diameter), dose-to-dose variability, and stability of the formulation over a 12-month 28

storage time.[ ] In addition, extensive clinical programs have been required for regulatory approval of these products because in vitro aerosol measurements are not viewed as acceptable surrogates for clinical data. The changeover from CFC to HFA has therefore been costly.

762

Producing new HFA formulations equivalent in clinical efficacy to CFC formulations is difficult on a cost-of-goods basis when compared with the manufacture of generic CFC formulations. Governments will therefore consider banning the sale of generic CFC products when reliable supplies of the HFA versions of these products become available. Therefore, one effect of the initiative to eliminate CFCs will be increased costs to consumers and insurers because of the extension of patent protection to these new HFA formulations. Dry Powder Inhaler Devices[

39] [40] [41]

Dry powder devices store the drug either as premeasured quantities of powder in capsules or blistered foil strips, from which a single dose of powdered drug is released into an inhalation chamber by opening or puncturing the capsule or foil blister (i.e., Rotahaler or Diskus, respectively). Alternatively, the powdered drug is stored in a multidose reservoir from which single doses are forced into the inhalation chamber (e.g., Turbuhaler, Astra-Zeneca, Lund, Sweden). To suspend the powder in air, the required energy is provided by the patient. This is usually accomplished via a rapid inhalation through the device while turbulence, created by a series of baffles, blends the powder into a respirable distribution that is inhaled from the device. By modifying the design features, there is precise control of the released dose, but these systems are flow dependent to a varying degree and, in a manner similar to the MDI, the inhaled material of necessity leaves the device at relatively high velocity. Some DPIs contain only drug particles (e.g., Pulmicort Turbuhaler), whereas other DPIs (e.g., salmeterol Diskus) also contain large particles of an inert dispersal agent such as lactose. DPIs, because they require the patient's own rapid inhalation to generate the aerosol, are, by definition, coordinated with the patient's breathing. Nevertheless, significant extrapulmonary deposition occurs because of the high inspiratory flow rates needed to disperse the powder. Therefore, even though dry powder devices may be preferable to pMDIs in patients with suboptimal coordination who are unwilling to use a spacer, the high extrapulmonary deposition raises safety considerations in using higher than usual doses of corticosteroids or bronchodilators. A further reservation concerning these devices is that a threshold inspiratory flow rate is required to actuate these devices and patients with acute exacerbations of asthma may not be able to reach the required flow rates. Because of this concern, manufacturers are endeavoring to reduce the required threshold flow rates of their respective devices. An alternative to lowering the required flow rate is to

lower the resistance of the device so that an equivalent flow could be generated with less respiratory effort. Nebulizers Nebulizers generate aerosols usually from liquids. Energy can be transferred to the liquid surface by either a jet as the liquid phase is forced through a narrow orifice 8 42

at high velocity[ ] [ ] or by sheer forces at the surface generated by ultrasonic waves. Usually, the drug is dissolved in the solvent, but it is also possible to nebulize suspensions, although the efficiency of some nebulizers may be less with suspensions than with solvents. In terms of the basics of aerosol generation, the major difference between nebulizers and the metered systems (MDIs and DPIs) is that the velocity of particles leaving the generator is really a function of the patient's method of breathing. Commonly, a patient quietly breathing through a nebulizer inhales particles whose inertia is a function of the aerodynamic diameter and the local convective flows of the physiologic situation defined by the breathing pattern rather than the rapid evaporation of Freon in the MDI or the high flow rates necessary via rapid inhalation in a dry powder device. Therefore, even though particles may have the same aerodynamic characteristics as described by cascade impaction, the nature of the aerosol generator significantly influences the actual aerosol that is inhaled (aerosol being defined as the distribution of particles and the carrier gas, with the carrier gas imparting its own velocity to the particles) and the subsequent deposition patterns are, as described earlier, significantly affected. Whereas MDIs and powder inhalers are approved for use by regulatory agencies in concert with a specific medication and, therefore, are highly regulated, there is much more variability and non-uniformity in the manufacture of nebulizers. Aerosols emitted from nebulizers are primarily modified by baffling systems that are 43]

either internal or external to the device. Nebulizer delivery systems must be assessed with the tubing and mouthpieces with which they are marketed.[ New Developments in Aerosol Delivery Systems

Significant enhancements in drug delivery by nebulizers are possible by coordinating nebulization with inspiration (e.g., “breath actuation”), which essentially turns the nebulizer off during expiration. Another improvement in efficiency is called “breath enhancement,” which uses the patient's inspiratory flow through the 43

nebulizer to increase drug delivery (e.g., LC Star Pari, Germany; Ventstream, Medicaid, Bognor Regis, UK).[ ] Another new nebulizer (Halolite, Profile Therapeutics, Boston, Massachusetts) is not only breath actuated but also monitors the patient's breathing pattern and delivers a predetermined inhaled dose, 44]

adjusting the amount of aerosol that is generated in proportion to changes in the patient's breathing pattern.[

Most inhaled medications have been designed for delivery to the airway to treat asthma or chronic obstructive pulmonary disease. However, considerable resources have been devoted recently to the development of delivery systems that deliver proteins systemically via the lung, with much of the effort devoted to delivery of 45

insulin.[ ] Many of these new devices may yet be adapted for use in asthma. Some represent refinements of dry powder technologies while others use new liquid aerosol generators. Gamma camera images generated using an example of the latter (AERx, Aradigm, Hayward, CA) are shown in Figure 46-3 . The AERx 46

aerosolizes drug formulation that has been prepackaged into a blister pack. [ ] The aerosol is generated early in inspiration by a rapid extrusion of solution through hundreds of small holes in the blister pack into the airstream. Compared with delivery by a jet nebulizer, the delivery of a radiolabeled experimental protein by way of the AER resulted in a more peripheral uniform deposition pattern, a higher lung dose, and greater systemic bioavailability.

PRINCIPLES OF DOSIMETRY OF INHALED MEDICATIONS

Whereas it is necessary to establish the dose-response relationship to assess the efficacy and safety of a medication,

763

Figure 46-3 Deposition images from four asthmatic patients following inhalation via AERx (top panel) and PARI LC STAR (bottom panel). Outlines were drawn from 133 Xe scan. (From Sangwan S, Agosti J, Bauer L, et al: J Aerosol Med 14:185–195, 2001.)

(From Sangwan S, Agosti J, Bauer L, et al: J Aerosol Med 14:185–195, 2001.) the determination of this relationship for inhaled drugs is relatively complex. Simply extrapolating the dose in the lung from the dose contained in the nebulizer, pMDI, or dry powder system is not feasible. Figure 46-4 summarizes the many variables that can complicate the calculation of lung dose from dose placed in the aerosol generator.

Dose Placed in Generator and Aerosolized Dose The dose placed in the generator is often referred to as the nebulizer charge. Some of the charge exits the nebulizer as an aerosol; the rest remains in the nebulizer as a “dead volume” (usually equivalent to about 0.5•ml). Rather than a simple liquid, the dead volume represents impacted material on the internal baffles of the device or as condensate on the walls of the nebulizer cup. Nebulizers vary in the amount of dead volume and internal impaction. However, if nebulizers are charged with very small volume fills (less than 1.5•ml), the dead volume may represent a disproportionate percentage of nebulizer charge. Varying the flow rate of air through the nebulizer can potentially affect the time to dryness and the particle size. Dose Inhaled[

5] [7]

Not all of the aerosol that exits the nebulizer will be inhaled into the mouth. (Most adults inhale aerosols orally; face masks, from which a significant proportion of aerosol may be inhaled nasally, are usually reserved for children.) Many of the larger particles impact on external baffles (sometimes these baffles have been deliberately inserted as a

Figure 46-4 Algorithm of dosimetry of inhaled medications. Dose placed in generator, dose aerosolized, dose inhaled, aerosol not inhaled, dose depositing in a patient, and regional doses including systemic exposure. For metered dose inhalers and dry powder inhalers, the aerosolized dose that exits the device is equal to the nominal dose. For nebulizers, the nominal dose is the nebulizer charge, which is significantly greater than the dose exiting the device as aerosol. “Aerosol not inhaled” is substantial for most nebulizers that produce aerosol throughout the respiratory cycle.

Figure 46-5 Deposition image during quiet tidal breathing in a child inhaling recombinant DNAse. The mass median aerodynamic diameter was 4••m. There is significant upper airway deposition (approximately 50%) as reflected by combined laryngeal and stomach activity. (From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp 447–477.)

(From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp 447–477.) images were obtained from the same individual. The aerosol deposition patterns are superimposed on an outline of the whole lung, which was generated using a 66

xenon (133 Xe) equilibrium scan. 133 Xe is a gas that measures regional lung volume.[ ] Even though these images are of the same subject, without the guidance of the 133 Xe outline, it would be quite difficult to determine whether these patterns were peripheral, indicating deposition in small airways and alveoli, or central, with deposition in large airways. Similarly, if these deposition images were from different individuals, it would be almost impossible to make a precise comparison between scans because there would be no way to know the location of the lung outlines. Figures 46-1 , 46-2 , 46-3 , and 46-5 show planar two-dimensional images of three-dimensional structures. Therefore, simply dividing the planar images into arbitrary regions labeled as central and peripheral regions cannot by itself distinguish between alveolar and airway deposition because the central region would also 66

contain alveoli. Investigators[ ] have tried to overcome this problem by quantifying the regional pattern of deposition of an inhaled aerosol in a manner that normalized for differences in relative lung thickness by dividing the regional aerosol deposition counts by the regional 133 Xe counts, a measure of lung volume. (An example is shown in Figure 46-6 ). Therefore, using regions of interest based on the 133 Xe image, it is possible to facilitate comparison between serial studies in the same subject or make intersubject comparisons of the distribution of deposited particles in the lung and airways. If the distribution of aerosol is proportional to volume, then the aerosol is predominantly in the peripheral airspaces (which account for 95% of the lung volume in normal subjects). Disproportionate deposition of aerosol in the central region (relative to 133 Xe) is an index of increasing central airway deposition.

Figure 46-6 Equilibrium xenon (133 Xe) scan with lung regions outlined (left); schematic of central and peripheral lung outlines and equations that define C/P ratios (right). C/P, Central/peripheral.

(From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp. 447–477; and Smaldone GC, Perry RJ, Bennett WD, et al: J Aerosol Med 1:11–20, 1988.)

(From Smaldone GC: Determinants of dose and response to inhaled therapeutic agents in asthma. In Schleimer RP, Busse WW, O'Byrne PM, editors: Inhaled glucocorticoids in asthma. Mechanisms and clinical actions, New York, 1997, Marcel Dekker, pp. 447–477; and Smaldone GC, Perry RJ, Bennett WD, et al: J Aerosol Med 1:11–20, 1988.)

767

18

Other investigators have used krypton (81•m Kr) to standardize regional deposition patterns.[ ] 81•m Kr is another radioactive gas, but, in contrast to 133 Xe, it has a short half-life (13•sec). Central to peripheral ratios or penetration indices (the reciprocal of central to peripheral ratios), obtained with 81•m Kr are similar but not equivalent to those obtained with 133 Xe. Early studies using simple external gamma counters (as opposed to gamma cameras) endeavored to partition ciliated airway (tracheobronchial) deposition and alveolar deposition by deducting radioactivity remaining after the first 24 hours from the initial deposition counts, based on the assumption that tracheobronchial clearance would be complete within 24 hours.[

67] [68]

However, recent data suggest that this assumption is not necessarily valid in chronic diseases of the airways.[

66]

An alternative approach to describing aerosol images involves superimposition of an outline of the right lung (obtained from a chest radiograph) on a gamma camera 69]

aerosol image. Statistical analysis of the uniformity of radioaerosol deposition is then performed.[

Other approaches include the use of transmission images (interposing the subject's chest between the gamma camera face and a known radioactive “phantom”) to delineate the lung outline before radioaerosol deposition studies.[

57]

Three-Dimensional Radionucleotide Imaging Newer technologies are being investigated to further improve the measurement of regional deposition. Single-photon emission computerized tomography (SPECT) uses a dual head camera to create cross-sectional images of the lung. These studies are more expensive and may involve higher doses of radiation. However, these 70

techniques may enhance the precision of regional deposition measurements.[ ] As with planar imaging, it is necessary to superimpose the radioisotope images on an image of lung anatomy and to calculate attenuation of radiation by tissue. In addition to simple transmission images, computed tomography and nuclear magnetic resonance images have been obtained to delineate anatomy. Mucociliary clearance of radiolabel into central airways is a potential confounding variable in 71]

interpreting SPECT images because of the time required for the acquisition of images in multiple planes.[ minimize acquisition time.

Recognition of this problem has resulted in efforts to

70

Positron-emission tomography (PET) has also been used to measure drug deposition.[ ] In addition to providing greater image resolution, PET has the advantage that the radiolabel is bound directly to the drug as opposed to being mixed with the formulation.

APPLICATION OF PRINCIPLES IN AEROSOL TECHNOLOGY TO TREATMENT STRATEGIES FOR ALLERGIC DISEASE Delivery of Aerosols to Patients with Airway Obstruction The therapy of asthma is discussed in other chapters. However, the principles discussed in this chapter can be applied to the design of logical treatment strategies for acute exacerbations of asthma. In the setting of airway obstruction, aerosols deposit in more proximal airways because impaction is inversely related to airway caliber [16]

20

and because of the turbulence caused by FLSs, which form during exhalation.[ ] Small aerosols 1••m in diameter, which deposit in the alveoli in normal subjects, can deposit in the airways of patients with airway obstruction. Aerosol deposition images are a more sensitive indicator of airway obstruction than spirometry. [

19]

In addition to changing the pattern of regional distribution of aerosols, airway obstruction increases the DF, the fraction of inhaled aerosol retained by the lung as opposed to being exhaled (see earlier text).[

17]

Therefore, aerosol therapy should still be feasible in all but the most severe forms of airway obstruction.

The responsiveness of the airway to aerosolized beta-sympathomimetic agents decreases during acute exacerbations of the disease, and increased doses are required. [72]

This can be accomplished using either a jet nebulizer or a pMDI, in combination with a spacer device in which serial metered doses are administered until a

response is achieved. The development of holding chambers for use with pMDIs has enabled health care providers to reduce the number of prescriptions for jet nebulizer bronchodilator therapy, with potential cost savings. Dry powder systems may not be appropriate for use in acute asthma because the patients may not be capable of generating sufficient inspiratory force to disperse the powder (see earlier text). Optimization Delivery of Inhaled Steroids in Asthma: Drugs and Devices 73 74

Inhaled corticosteroids are a mainstay of the treatment of asthma. [ ] [ ] Over the past decade, the therapeutic options for delivering inhaled corticosteroids have greatly increased as newer compounds, formulations, and delivery systems have been developed. This discussion briefly reviews how principles of aerosol delivery have been applied to development of inhaled corticosteroid formulations, emphasizing the need to match delivery systems with particular drugs and target populations. Concern has been raised about possible effects of long-term systemic exposure related to low doses of inhaled corticosteroids. This question has arisen primarily 75

because of the finding that inhaled steroids cause a dose-related decrease in short-term growth of prepubertal children,[ ] and it has raised fears about longer term effects on bone density and cataract formation. As a result of these concerns, pharmaceutical companies and clinicians have employed a number of strategies to reduce the risk of systemic exposure as summarized in the following paragraphs. Reducing the Oral Bioavailability of the Steroid Molecule

Inhaled steroid particles that deposit on the nasopharynx are swallowed and absorbed by the gastrointestinal tract. The percentage of an oral dose that reaches the 74

systemic circulation is referred to as the oral bioavailability.[ ] With most pMDIs, approximately 4 times more drug is deposited in the oropharynx than in the lung. Thus, oral bioavailability is potentially the most important source of systemic exposure. The oral bioavailability of dexamethasone and methylprednisolone approach 100%; therefore, these agents are not administered by inhalation, which offers little advantage over systemic administration. Fortunately, the majority of inhaled

768

74

steroids when given by a pMDI have relatively low oral bioavailability[ ] (e.g., beclomethasone 15%–20%, budesonide 11%, flunisolide 20%, triamcinolone 23%). Thus the development of agents with an oral bioavailability of less than 25% has made inhaled steroid therapy a feasible and successful undertaking for the past 3 decades. However, the oral bioavailability of some newer agents has decreased even further—to 1% (fluticasone) or less than 1% (mometasone)—because of increased first pass metabolism in the intestinal mucosa or liver. Therefore, with the latter compounds, significant systemic exposure only occurs by absorption from the lung. Figure 46-4 is a schematic summary of the fate of inhaled particles. Reducing Extrapulmonary Deposition by Modifying the Drug Delivery System

Reducing extrapulmonary deposition is desirable not only to reduce local adverse effects (candidiasis, irritation) but also to reduce systemic exposure if the molecule

has a significant oral bioavailability. A simple and commonly applied method is to employ a spacer or holding chamber with a pMDI, onto which nonrespirable highvelocity or large particles deposit; thus preventing their impaction on the oropharynx.[

29]

Reduction of extrapulmonary deposition can also be achieved by modifying the formulation of the drug. Budesonide was reformulated for use as a dry powder with a higher fraction of respirable particles than the budesonide pMDI, thus reducing oropharyngeal deposition.[

39]

Recently, beclomethasone has been reformulated as a 38]

non-fluorocarbon pMDI with an aerosol of smaller aerodynamic diameter that reduces extrapulmonary deposition.[ Targeting Inhaled Medication to Airways Rather Than Alveoli

It is generally accepted that alveolar deposition of inhaled steroids is undesirable because of concerns that medication that deposits in the alveoli will be absorbed and lead to systemic exposure without depositing on and treating the inflamed airways. Even though recent data suggest that the alveoli may have a role in the inflammatory process in asthma, the significance of these findings remains unclear and until further studies are performed, manufacturers of asthma medications are likely to continue to target the ciliated airways. Efforts to reduce the average particle size of a polydisperse aerosol and thereby reduce oropharyngeal deposition are also likely to increase the probability of alveolar deposition. Such alveolar deposition is difficult to quantify and is a matter of considerable controversy for a number of reasons: 1. Current imaging techniques using gamma scintigraphy of radiolabeled drug cannot reliably distinguish between small airways deposition (desirable outcome) and alveolar deposition (probably undesirable). Physiologic measurements of “small airways function” are not reproducible enough for definitive clinical studies. As a result, investigators have begun to look for alternative approaches to assess the effects of reformulation on the small airways (e.g., functional computed tomography). [

38]

2. In patients with obstructive airway disease, airway caliber has a profound influence on regional deposition.[ proximally during inhalation but they will also deposit more proximally on exhalation if FLSs

20 develop.[ ]

16]

Not only will particles deposit more

Therefore the more severe the degree of airway

[76]

obstruction, the less the likelihood of alveolar deposition and vice versa. A recent study compared the systemic exposure of fluticasone delivered by pMDI/spacer in normal volunteers and asthmatic patients with chronic airflow obstruction and found that serum levels of fluticasone were higher in the normal volunteers and that the degree of hypothalamic-pituitary-adrenal axis suppression was also greater in the normal subjects. The most likely explanation for this discrepancy is that particles were more likely to deposit in ciliated airways in the asthmatics where they would be likely to undergo mucociliary clearance and thus not be absorbed systemically. In contrast, in the normal subjects, increased alveolar deposition could result in increased systemic absorption. Even though rates of mucociliary clearance may be decreased in asthmatic subjects, mucociliary mechanisms remain effective at removing particles in stable asthma. Thus, comparisons of the systemic effects of inhaled steroids undertaken in normal subjects should be extrapolated to asthmatic patients with caution. Nevertheless, it is in those patients with minimal or no obstruction that the risk/benefit ratio of inhaled steroids may be considered marginal and, ironically, these are the patients with the greater likelihood of alveolar and thus systemic exposure with smaller particles. 3. There is difficulty in demonstrating therapeutic equivalence or superiority of reformulations of inhaled steroids. The dose–response relationship is harder to establish for inhaled steroids than for beta agonists, the bioequivalence of which can be accurately and readily assessed by spirometry, because the effects of these drugs as measured by spirometry reaches a plateau at relatively low doses and because the effects of therapy develop over days and weeks. As a result, pharmacodynamic studies of systemic effects (e.g., adrenal suppression) are often used to make comparisons between steroids rather than using head-to-head clinical studies.[

77]

Nevertheless, interpretation of comparative data acquired using pharmacodynamic parameters includes a number of caveats: (1) as

discussed in the preceding paragraph, airway obstruction in different groups of subjects can affect deposition and systemic exposure; (2) in comparing reformulations of a drug with significant oral bioavailability, differences in systemic exposure could be due to changes in oropharyngeal deposition or in bronchial-alveolar deposition patterns. Investigators have attempted to distinguish between these effects by reducing gastrointestinal absorption with 62

charcoal.[ ] 4. There are no in vitro tests that can adequately predict regional deposition. Most current knowledge concerning the effects of particle size on regional deposition comes from studies of normal subjects inhaling monodisperse aerosols. Inhaled steroids, in contrast, are polydisperse aerosols and are inhaled by patients who have variable airway narrowing. However, the most important confounding variable may be that particles emitted from pMDIs have high velocities that may have a greater degree of influence on the likelihood of impaction than the diameter of the ambient particle, thus greatly complicating in vitro modeling. In conclusion, the controversy over the ideal size of inhaled steroid particles is likely to persist, but it may afford clinical investigators the opportunity to learn more about variations in airway inflammation in different generations of airways in asthma, the best method of targeting these regions, and the best method of assessing response to treatment.

769

In terms of current knowledge, it seems reasonable to propose that drugs with higher oral bioavailability are more appropriate candidates for reformulation as finer particles than drugs with lower oral bioavailability. Furthermore, the less severe a patient's airway obstruction, the greater should be the concern about systemic absorption through the alveoli. Other Manipulations of Steroid Structure that May Affect Systemic Exposure

Inhaled corticosteroid (ICS) compounds differ in potency, lipophilicity, volume of distribution, rate of absorption from the lung, systemic clearance, and activity of 74 76

metabolites.[ ] [ ] In contrast to manipulations designed to reduce oral bioavailability, which are agreed to be beneficial, other modifications and differences between ICS are more controversial in terms of their impact on systemic exposure and the ratio of local to systemic effects. The possible clinical impact of these 74]

differences has been reviewed elsewhere. [

While the search continues for the ideal method of delivering inhaled steroids to patients with asthma, advances in drug design, formulation, and delivery systems now provide a wider array of options to the clinician. However, no delivery system can be considered to be intrinsically superior to all others. The delivery system should be judged instead by its ability to optimize the pharmacokinetic properties of the drug, most notably oral bioavailability, and by its suitability for the target subpopulation of asthmatics. 78]

Aerosolized Drug Delivery in Infants and Young Children [

78

For children younger than age 4 years, inhaled medications can be delivered by a nebulizer and face mask or using a pMDI with holding chamber and mask.[ ] DPI devices can be used from age 4 years and older. The breath-actuated feature of these devices makes these devices preferable to pMDIs without a holding chamber in children age 4 to 6 years. After age 6 years, children can be taught to use a pMDI without a spacer, although the use of spacers should continue to be encouraged. 79

For young children with asthma, nebulized treatment with a suspension of budesonide is an effective and well-tolerated alternative.[ ] Nebulizers tend to be less effective at aerosolizing suspensions than solutions, and clinicians are advised to prescribe budesonide only with jet nebulizers. Ultrasonic nebulizers should not be used with a suspension. Masks are used with holding chambers and nebulizers because young children breathe nasally and because they may have difficulty holding a mouthpiece. The delivery and deposition of nasally inhaled medications differs from that of orally inhaled medications. In adults and older children, nasal breathing leads to upper airway deposition and decreased lower airway deposition. The effects in infants are less clear because gamma scintigraphy studies are not usually permitted in infants. A selection of face masks that fit to the faces of different populations (e.g., infants, children, adults) is desirable because air leaks affect medication delivery. When holding chambers are used with infants and children, the resistance of the valves may be important in affecting medication delivery and patient acceptability. Furthermore, when medications are used with infants and children, their small breathing volumes complicate the calculation of ideal chamber size. Most adults can empty a 150-ml holding chamber with one breath, whereas young children may have tidal volumes less than 150•ml.[

78]

Aerosol Delivery during Mechanical Ventilation 72]

Aerosolized beta-sympathomimetic agents are essential components of the treatment of status asthmaticus.[ undergoing mechanical ventilation.

[78] [79]

Such delivery is complicated if the patient is

Recent studies, however, have identified ways in which such delivery can be optimized. The use of a pMDI is feasible as

26 met.[ ]

long as certain conditions are For example, it is necessary to use a spacer/holding chamber when using a pMDI in this setting. Not all holding chambers are equivalent in efficiency and different brands are not necessarily interchangeable. In contrast to the treatment of spontaneously breathing patients, delivery during mechanical ventilation must be synchronized with respiration. It is essential that a dose escalation protocol be employed because doses far in excess of those used for maintenance therapy may be needed. Hence, efforts to obtain objective evidence on response to treatment (e.g., peak airway pressure, dynamic compliance) and toxicity (e.g., tachycardia, arrhythmias) should be made. In endeavoring to maximize delivered doses, however, one should remember that the efficiency of pMDI delivery decreases significantly if the interval between serial actuations is less than 1 minute and if the synchronization with the respiratory cycle is suboptimal.[

26]

80 81 82

The efficiency of jet nebulizers in delivering aerosols in the setting of mechanical ventilation is variable and affected by several factors.[ ] [ ] [ ] Different brands of commercial nebulizers vary in terms of their output per minute and the duration of treatment. Breath actuation by mechanical ventilators (nebulization takes place only during inspiration) may prolong treatment times. Humidification may cause “rain out” of aerosol in tubing and reduce aerosol delivery by 50%. The use of helium oxygen mixtures has been shown to affect aerosol delivery from MDIs and jet nebulizers in a model of mechanical ventilation, and could result in either 82

enhanced or impaired delivery, depending on how the system was configured. [ ] In conclusion, once technical factors have been identified and optimized, efficient delivery of aerosolized medications to patients undergoing mechanical ventilation is readily attainable.

Delivery of Therapeutic Aerosols to the Nasal Mucosa

Among an increasing list of aerosolized medications that are being used to treat diseases of the nasal mucosa are corticosteroids, cromolyn, anticholinergics (ipratropium bromide), saline, and decongestants. The particle size of nasal aerosols tends to be larger than pulmonary inhalers. Manual pumps that produce large, relatively low-velocity particles are being used as an alternative to high-velocity Freon-based pMDIs. There are isolated reports of nasal perforation occurring with 83

high-velocity inhalers[ ] and patients should be advised to direct the spray in the direction of the ipsilateral ear and away from the nasal septum. In severe allergic sinusitis, the mucosa may be so congested that a short course of systemic corticosteroids may be needed to allow penetration of aerosolized therapy. Prolonged use of topical decongestant sprays may lead to rebound hyperemia and intractable nasal

770

congestion, and systemic administration of decongestants may be preferable. Intranasal corticosteroids are an effective therapy for seasonal and perennial rhinitis. Whereas regular use of such agents may lead to short-term growth suppression in children,[

75] [84]

it appears that this complication can be prevented by the use of a formulation with a low oral bioavailability (e.g., mometasone).[

85]

AEROSOLS AND THE MEASUREMENT OF AIRWAY HYPERREACTIVITY (METHACHOLINE BRONCHOPROVOCATION) Increased airway responsiveness to stimuli that have little or no effect on normal airways is an integral part of the definition of asthma adopted by the 1997 National 73

86

Asthma Educational Program expert panel.[ ] A variety of bronchoprovocation protocols are used to document and quantify nonspecific hyperresponsiveness.[ ] Common uses of such protocols include aiding in the diagnosis of suspected asthma in patients with normal resting spirometry, in the investigation of suspected occupational asthma, in epidemiologic studies, and in research protocols to determine the efficacy and safety of asthma medications. Airway hyperresponsiveness and its measurement by bronchoprovocation protocols are discussed in detail in Chapter 40 . However, most bronchoprovocation protocols involve the inhalation of aerosols and the determination of a dose–response relationship. As described earlier in this chapter, the determination of dose–response relationships for inhaled agents is complex. This discussion concentrates on methacholine bronchoprovocation, which is probably the best characterized method of measuring airway hyperresponsiveness. Although the clinical results of methacholine bronchoprovocation are not necessarily interchangeable with data from other methods of bronchoprovocation (nonisotonic saline, adenosine, histamine), the principles of aerosol dosimetry discussed here can in general be applied to these other protocols as well. Dose to Lung As discussed earlier, the dose of an inhaled medication depositing in the lung is determined by a number of factors (summarized in Figure 46-4 ). Because the determination of lung dose is complex, lung dose is not measured in routine clinical bronchoprovocation studies. For practical purposes, the amount of methacholine required to induce a change in a specific measure of airway function is expressed as either the provocative concentration (PC) or provocative dose (PD).[

86]

For

example, the concentration or dose required to induce a 20% decrease in forced expiratory volume in 1 second (FEV1 ) is reported as the PC20 and PD20 , respectively. In protocols that report a PC20 , the patient is exposed to serially increasing concentrations of methacholine (usually 0.25•mg/ml to 16•mg/ml, breathing 19]

tidally for 2 minutes for each concentration), until either the FEV1 decreases by 20% or the maximum concentration is reached.[

The PC20 is calculated from a log

dose-response curve. The PC20 is therefore a measure of the concentration to which the patient was exposed, not the dose in the lung. Protocols that report PD20 take into account not only the concentration to which the patient was exposed but also the number of breaths that the patient inhales. In addition, some protocols use 87]

“dosimeters” to synchronize the administration of the aerosol with the patient's respiratory cycle and the patient is instructed to inhale in a standardized manner.[ However, the PD20 is at best a rough estimate of inhaled dose and does not measure the dose depositing in the lung. Variability in the performance of nebulizers

(between different brands and between different batches of the same brand) can also affect dose inhaled per breath. In addition, the fractional retention of aerosols (DF) varies significantly even in individuals with normal lung function and is very sensitive to subtle changes in airway caliber.[

17]

Nevertheless, even though PC20 and PD20 do not accurately predict lung dose, these indices of hyperresponsiveness have been shown to be useful in distinguishing 86 87 88 89

currently symptomatic asthmatic patients from healthy individuals.[ ] [ ] [ ] [ ] The most likely explanation is that symptomatic asthmatic subjects are so sensitive to methacholine compared to normal subjects that technical issues related to nebulizer performance and fractional retention of inhaled dose are not clinically important. For example, using tracer dyes and relatively complex filter techniques, it has been shown that measurable changes in airway function can be observed in asthmatics to approximately one tenth the deposited dose compared with normal subjects.[

90]

Sites of Deposition 1 16

Even though patients with airways obstruction are known to deposit particles in more proximal airways[ ] [ ] and central airways are thought to be more responsive to methacholine than peripheral airways, differences in regional deposition patterns of methacholine do not explain the differences between hyperreactive and normal subjects because both normal and hyper reactive subjects demonstrate increased central deposition of radioaerosols after administration of methacholine ( Figure 467 ).[

19]

Diagnostic Implications of the Uncertainties in Bronchoprovocation Testing

Because hyperresponsiveness is often incorporated in the definition of asthma, its presence can be used as a diagnostic tool. An individual with normal pulmonary function but who reports current symptoms suggestive of reversible airway obstruction or chronic, undiagnosed cough may have asthma, and the clinical diagnosis can be supported by demonstration of bronchial hyperreactivity. For such individuals undergoing methacholine testing during tidal breathing maneuvers, the dose and site of methacholine in the lung do not appear to be important in defining the presence or absence of the disease. Although laboratories should endeavor to standardize their aerosol delivery technique, the utility of bronchoprovocation testing in diagnosis must, in retrospect, reflect the relative unimportance of these variables in establishing the presence of the disease. In other studies, technical issues related to aerosol delivery and deposition may be important. In random population studies (as opposed to clinical evaluation of

symptomatic patients), the sensitivity of bronchoprovocation in detecting asthma decreases because patients (especially children) whose disease has been in remission or whose symptoms are infrequent may not demonstrate significant hyperresponsiveness at the time of the survey. Specificity also

771

Figure 46-7 Distribution of deposited methacholine during aerosolized bronchoprovocation testing in normal subjects and in patients with asthma. Mean ± SE are also shown. sC/P ratios are not different at the beginning of methacholine inhalation. After methacholine inhalation, deposition in both normal subjects and asthmatic patients shifts significantly to central airways. MR, Subjects achieving a PC20 (i.e., methacholine reactive); NMR, subjects not achieving a PC20 , sC/P1 , initial deposition pattern; sC/P2 , post-methacholine deposition pattern. (From O'Riordan TG, Walser L, Smaldone GC: Chest 103:1385–1389, 1993.)

(From O'Riordan TG, Walser L, Smaldone GC: Chest 103:1385–1389, 1993.) decreases, with several investigators reporting that 10% of subjects with a PC20 between 4•mg/ml and 8•mg/ml have no symptoms of asthma.[

86]

Because of the

continuous unimodal distribution of hyperresponsiveness in the general population, precise characterization and standardization of aerosol delivery systems is critical in such studies. Interpretation of Changes in Hyperresponsiveness as a Result of Therapy[89] [90] [91] [92] [93] [94]

In recent years, changes in airway reactivity are often used as an estimate of efficacy to therapeutic agents. The growing dependence of clinical investigators on this technique is related to the fact that many drugs (e.g., aerosolized steroids) often do not result in immediate effects. A major challenge in clinical pharmacology is the

assessment of “the response” to agents that do not have immediate effects on airway caliber. Diaries of peak flow measurements and serial assessment of airway reactivity are, therefore, critical in assessing the response to therapy. However, as the antiinflammatory becomes effective, the degree of obstruction can change over time. Changes in airway caliber may affect the measurement of airway reactivity without affecting “intrinsic” responsiveness. Any drug that affects airway geometry 19

93

will affect sites of deposition[ ] and possibly total deposited dose of methacholine.[ ] In addition, because airway resistance is inversely proportional to the cube of the radius of the airway, small changes in resting airway diameter can have marked effects in airway responsiveness without a significant improvement in the 89 94

inflammatory state of the airway.[ ] [ ] Whereas the diagnosis of bronchial hyperresponsiveness is relatively easy to make because of the large differences in reactivity between asthmatics and normal subjects, the interpretation of changes in hyperresponsiveness as evidenced by subtle changes in reactivity before and after therapy are much more difficult to interpret. It is critical in such studies that the aerosol delivery systems and methods of assessing airway function be rigidly standardized. In particular, coadministration of long-acting bronchodilators may increase baseline airway caliber and thus appear to diminish responsiveness to methacholine. Until studies in which the dose and response of the local airway can be more directly estimated, changes in reactivity, while being indicative of clinical improvement, may not be reflective of changes in bronchial inflammation.

AEROSOLS AND THE MEASUREMENT OF MUCOCILIARY CLEARANCE In health, there are two principal means by which insoluble deposited particles are removed or “cleared” from the lung. Particles located in the ciliated airways are borne proximally by a stream of mucus that is propelled by rhythmic ciliary movement. When the mucus reaches the pharynx, it is swallowed. This process is called 95

mucociliary clearance and in healthy subjects is usually completed within 24 hours of deposition.[ ] The mucous path can be followed using radiolabeled aerosols, and this discussion is confined to the technical aspects of the application of radiolabeled aerosols to measure mucous transport. Particles that deposit on ciliated airways containing99m Tc, bound to a molecule that prevents absorption into the circulation follows the clearing mucus. Particles in the terminal nonciliated airways 96

and alveoli are removed by processes that can take many weeks to complete. These processes are designated as “alveolar clearance” and are reviewed elsewhere.[ ] There is clinical evidence that the clearance of secretions is impaired in many diseases, including asthma, chronic bronchitis, bronchiectasis, and cystic fibrosis, and in patients undergoing prolonged mechanical ventilation. A valuable technique for the measurement of mucociliary clearance in peripheral airways involves measuring the rate of removal of deposited radioactive particles from designated regions of interest. The interpretation of mucociliary clearance studies is complex, and to compare rates of clearance between subjects or even to compare a series of studies in one subject, it is essential to consider a number of important variables. 97

The most important parameter affecting the rate of mucociliary clearance in healthy subjects is the initial pattern of particle deposition.[ ] Factors that determine this pattern and how it can be quantified (e.g., the sC/P ratio) are discussed earlier in this chapter. The relevance of the initial pattern is that particles that are deposited in the central airways will be cleared more rapidly into the pharynx than more distally deposited particles. Therefore, if a subject with a central pattern of deposition is studied, the rate of mucociliary clearance will be faster than if it had been studied using a more peripheral initial deposition pattern. In addition, the

772

Figure 46-8 Quantitative measure of mucociliary clearance in asthma. In serial studies, radioactivity is measured in lung regions drawn in the same manner as in

Figure 46-6 ; measurements are decay corrected and plotted over time. Patients hospitalized with severe asthma (open circles, mean ± SE, n = 5) retain almost all the deposited activity over 2 hours. After discharge, mucociliary clearance in the same patients is much improved. (From O'Riordan TG, Zwang J, Smaldone GC: Am Rev Respir Dis 146: 598–603, 1992.)

(From O'Riordan TG, Zwang J, Smaldone GC: Am Rev Respir Dis 146: 598–603, 1992.) fastest rates of mucous transport in normal subjects is believed to be in the trachea, with a progressive decrease through more distal generations and the most peripherally deposited particles in the alveolar region not accessible to mucociliary clearance. Based on these principles, for data to be compared between studies, the initial pattern of deposition must be standardized. In a given patient, a radioaerosol study may provide important insights as to changes in airway function before and after a given therapeutic regimen. Besides the pattern of deposition, the clearance of radiolabeled test aerosols from the airways may also be an index of overall epithelial function as assessed by the performance 98 99

of the mucociliary apparatus. Mucociliary clearance has been shown to vary depending on the clinical state of patients with the asthmatic syndrome.[ ] [ ] It is likely that this pathophysiologic change is related, in some way, to inflammatory processes seen in airway epithelial biopsy studies. As shown in Figure 46-8 , serial mucociliary clearance measurements during and after treatment of a severe exacerbation of asthma show a significant improvement in clearance. In basic studies of drug efficacy, these techniques may serve to complement more invasive measurements. Beta agonists and theophylline have been shown to increase rates of mucociliary clearance.[

95]

Although corticosteroids do not affect mucociliary clearance acutely, Agnew and colleagues found that peripheral mucociliary clearance

was improved in asthmatics after treatment with oral prednisone for 2 weeks.[

100]

Finally, besides the clearance of test aerosols from the airways, there are few data

regarding the clearance of active drug from various regions of the respiratory tract. Whereas deposition sites of topical agents are intuitively thought to be important for the ultimate effect, it is conceivable that a deposited drug may move from one region of the lung to another via forms of airway clearance. The drug can pass into the bronchial circulation, for example, and regions of the lung may be exposed that were not part of the initial sites of aerosol delivery.

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29. Newman SP, Newhouse MT: Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects, J Aerosol Med 9:55–70, 1996. 30. Wilkes W, Fink J, Dhand R: Selecting an accessory device with a metered dose inhaler: variable influence of accessory devices on fine particle dose, throat deposition and drug delivery with asynchronous actuation from a metered dose inhaler, J Aerosol Med 14:351–360, 2001. 31. Nelson HS, Loffert DT: Comparison of the bronchodilator response to albuterol administered by the OptiHaler, the AeroChamber, or by metered dose inhaler alone, Ann Allergy 72:337–340, 1994. 32. Moren F: Drug deposition of pressurized inhalation aerosols. I. Influence of actuator tube design, Int J Pharmaceutics 1:205–212, 1978. 33. Newman SP, Moren F, Pavia D, et al: Deposition of pressurized suspension aerosols inhaled through extension devices, Am Rev Respir Dis 124:317–320, 1981. 34. Wildhaber JH, Devadason SG, Eber E, et al: Effect of electrostatic charge, flow, delay and multiple actuations on the in vitro delivery of salbutamol from different small volume spacers for infants, Thorax 51:985–988, 1996. 35. Dolovich M: New propellant-free technologies under investigation, J Aerosol Med 12:S9-S17, 1999. 36. Ramsdell JW, Colice GL, Ekholm BP, et al: Cumulative dose response study comparing HFA-134a albuterol sulfate and conventional CFC albuterol in patients with asthma, Ann Allergy Asthma Immunol 81:593–599, 1998. 37. Busse WW, Brazinsky S, Jacobson K, et al: Efficacy response of inhaled beclomethasone dipropionate in asthma is proportional to dose and is improved by formulation with a new propellant, J Allergy Clin Immunol 104:1215–1222, 1999. 38. Goldin JG, Tashkin DP, Kleerup EC, et al: Comparative effects of hydrofluoroalkane and chlorofluorocarbon beclomethasone dipropionate inhalation on small airways: assessment with functional helical thin-section computed tomography, J Allergy Clin Immunol 104:S258-S267, 1999. 39. Thorsson L, Edsbacker S, Conradson TB: Lung deposition of budesonide from Turbuhaler is twice that from a pressurized metered-dose inhaler P-MDI, Eur Respir J 7:1839–1844, 1994. 40. Fuller R: The Diskus. A new multidose dry powder device: efficacy and comparison with Turbuhaler, J Aerosol Med 8:S11-S17, 1998. 41. Bisgaard H, Klug B, Sumby BS, et al: Fine particle mass from the Diskus inhaler and Turbuhaler inhaler in children with asthma, Eur Respir J 11:1111–1115, 1998. 42. Nerbrink O, Dahlback M, Hansson HC: Why do medical nebulizers differ in their output and particle size characteristics? J Aerosol Med 7:239–276, 1994. 43. Devadason SG, Everard ML, Linto JM, et al: Comparison of drug delivery from conventional versus “Venturi” nebulizers, Eur Respir J 10:2479–2483, 1997. 44. Nikander K: Adaptive aerosol deliver: the principles, Eur Respir Rev 7:385–387, 1997.

45. Corkery K: Inhalable drugs for systemic therapy, Respir Care 45:831–835, 2000. 46. Sangwan S, Agosti J, Bauer L, et al: Aerosol protein delivery in asthma: gamma camera analysis of regional deposition and perfusion, J Aerosol Med 14 185– 195, 2001. Principles of Dosimetry of Inhaled Medications 47. Smaldone GC, Fuhrer J, Steigbigel RT, et al: Factors determining pulmonary deposition of aerosolized pentamidine in patients with human immunodeficiency virus infection, Am Rev Respir Dis 143: 727–737, 1991. 48. Newman SP: Can lung deposition data act as a surrogate for the clinical response to inhaled asthma drugs? Br J Clin Pharmacol 49:529–537, 2000. Measurements of Particle Size and Aerosol Delivery 49. Newman SP: Characteristics of radiolabelled versus unlabelled inhaler formulations, J Aerosol Med 9:S37-S47, 1996. 50. Lever S: Technetium and rhenium compounds. In Wagner HN, Szabo Z, Buchanan J, editors: Principles of nuclear medicine, ed 2, Philadelphia, 1995, WB Saunders, pp 220–229. 51. Bennett WD, Ilowite JS: Dual pathway clearance of 99m Tc-DTPA from the bronchial mucosa, Am Rev Respir Dis 139:1132–1138, 1989. 52. O'Riordan TG, Greco MJ, Perry RJ, et al: Nebulizer function during mechanical ventilation, Am Rev Respir Dis 145:1117–1122, 1992. 53. Ruffin RE, Dolovich MB, Wolff RK, et al: The effects of preferential deposition of histamine in the human airway, Am Rev Respir Dis 117:485–492, 1979. 54. Ryan G, Dolovich MB, Roberts RS, et al: Standardization of inhalation provocation tests: two techniques of generation and inhalation compared, Am Rev Respir Dis 123:195–199, 1981. 55. Messina MS, Smaldone GC: Evaluation of quantitative aerosol techniques for use in broncho-provocation studies, J Allergy Clin Immunol 75:252–257, 1985. 56. Itoh H, Smaldone GC, Swift DL, et al: Quantitative measurement of total aerosol deposition: comparison of three different techniques, J Aerosol Sci 16:367–371, 1985. 57. Macey DJ, Marshall R: Absolute quantitation of radiotracer uptake in the lungs using a gamma camera, J Nucl Med 23:731–734, 1982. 58. Smaldone GC, Dickinson G, Marcial E, et al: Deposition of aerosolized pentamidine and failure of Pneumocystis prophylaxis, Chest 101:82–87, 1992. 59. Smaldone GC, Vinciguerra C, Morra L: Urine pentamidine as an indicator of lung pentamidine in patients receiving aerosol therapy, Chest 100:1219–1223, 1991. 60. Le Conte P, Potel G, Peltier P, et al: Lung distribution and pharmakinetics of aerosolized tobramycin, Am Rev Respir Dis 147:1279–1282, 1993. 61. Watterberg KL, Clark AR, Kelly HW, et al: Delivery of aerosolized medication to intubated babies, Pediatr Pulmonol 10:136–141, 1991.

62. Borgstrom L, Derom E, Stahl E, et al: The inhalation device influences lung deposition and bronchodilating effect of terbutaline, Am J Respir Crit Care Med 153:1636–1640, 1996. 63. Weber A, Smith A, Williams-Warren J, et al: Nebulizer delivery of tobramycin to the lower respiratory tract, Pediatr Pulmonol 17:331–339, 1994. 64. Ilowite JS, Gorvoy JD, Smaldone GC: Quantitative deposition of aerosolized gentamicin in cystic fibrosis, Am Rev Respir Dis 136:1445–1449, 1987. 65. Hull FP, Condos R, Rom WN, et al: A novel method for measuring upper airway aerosol deposition, Am J Respir Crit Care Med 163:A163, 2001. 66. Smaldone GC, Perry RJ, Bennett WD, et al: Interpretation of “24 hour lung retention” in studies of mucociliary clearance, J Aerosol Med 1:11–20, 1968. 67. Bateman JR, Pavia D, Sheahan NF, et al: Impaired mucociliary clearance in patients with mild stable asthma, Thorax 38:463–467, 1983. 68. Pavia D, Bateman JR, Sheahan NF, et al: Tracheobronchial mucociliary clearance in asthma: impairment during remission, Thorax 40:171–175, 1985. 69. Garrard CS, Gerrity TR, Schreiner JF, et al: Analysis of aerosol deposition in the healthy human lung, Arch Environ Health 36:184–193, 1981. 70. Fleming JS, Conway JH: Three dimensional imaging of aerosol deposition, J Aerosol Med 14:147–153, 2001. 71. Smaldone GC: Radionucleotide scanning, respiratory physiology and pharmacokinetics, J Aerosol Med 14:135–137, 2001. Application of Principles in Aerosol Technology to Treatment Strategies for Allergic Disease 72. Corbridge T, Hall J: The assessment and management of adults with status asthmaticus, Am J Respir Crit Care Med 151:1296–1316, 1995. 73. National Asthma Education Program Expert Panel Report: Guidelines for the diagnosis and management of asthma. Bethesda, MD, US Department of Health and Human Services, Public Health Service, National Institutes of Health, August 1997. Publication Number: 97-4051. 74. Kelly HW: Potency and clinical efficacy of inhaled steroids, Respir Care Clin North Am 5:537–554, 1999. 75. Allen DB: Do intranasal corticosteroids affect childhood growth? Allergy 55:15–18, 2000. 76. Brutsche MH, Brutsche IC, Munawar M, et al: Comparison of pharmacokinetics and systemic effects of inhaled fluticasone propionate in patients with asthma and healthy volunteers: a randomized crossover study, Lancet 356:556–561, 2000. 77. Derendorf H, Hochhaus G, Meibohm B, et al: Pharmacokinetics and pharmacodynamics of inhaled corticosteroids, J Allergy Clin Immunol 101:S440-S446, 1998. 78. Everard M: Aerosol delivery in infants and young children, J Aerosol Med 9:71–77, 1996. 79. Szefler SJ: Pharmacodynamics and pharmacokinetics of budesonide: a new nebulized corticosteroid, J Allergy Clin Immunol 104:175–183, 1999. 80. Dhand R, Tobin MJ: Inhaled bronchodilator therapy in mechanically ventilated patients. Am J Respir Crit Care Med 156:3–10, 1997.

81. O'Riordan TG, Palmer L, Smaldone GC: Aerosol deposition in mechanically ventilated patients: optimizing nebulizer delivery, Am J Respir Crit Care Med 149:214–219, 1994. 82. Goode ML, Fink JB, Dhand R, et al: Improvement in aerosol delivery with helium–oxygen mixtures during mechanical ventilation, Am J Respir Crit Care Med 163:109–114, 2001. 83. Soderberg-Warner ML: Nasal septal perforation associated with topical corticosteroid therapy, J Pediatr 105:840–841, 1984. 84. Skoner DP, Rachelefsky GS, Meltzer EO, et al: Detection of growth suppression in children during treatment with intranasal beclomethasone dipropionate, Pediatrics 105:E23, 2000. 85. Schenkel EJ, Skoner DP, Bronsky EA, et al: Absence of growth retardation in children with perennial allergic rhinitis after one year of treatment with mometasone furoate aqueous nasal spray, Pediatrics 105:E22, 2000. Aerosols and the Measurement of Airway Hyperreactivity (Methacholine Bronchoprovocation) 86. Spector S: Bronchial inhalation challenges with aerosolized bronchoconstrictive substances. In Spector S, editor: Provocative challenge procedures: bronchial, oral, nasal and exercise, vol 1, Boca Raton, Fla, 1983, CRC Press, pp 137–176. 87. Rosenthal RR: Methodologies of aerosol delivery. In Spector SL, editor: Provocation testing in clinical practice, vol 5, New York, 1995, Marcel Dekker, p 215. 88. Cockcroft DW, Hargreave FE: Airway hyperresponsiveness: relevance of random population data to clinical usefulness, Am Rev Respir Dis 142:497–500, 1990 (editorial). 89. Tattersfield AE: Measurement of bronchial reactivity: a question of interpretation, Thorax 36:561–565, 1981. 90. Donna E, Danta I, Kim CS, et al: Relationship between deposition of and responsiveness to inhaled methacholine in normal and asymptomatic subjects, J Allergy Clin Immunol 83:456–461, 1989. 91. Macklem PT: The interpretation of lung function tests. In Hargreave FE, Woolcock AJ, editors: Airway responsiveness measurement and interpretation, Mississauga, Ontario, Canada, 1985, Astra Pharmaceuticals, p 69. 92. Fish JE, Kelly JF: Measurement of responsiveness in bronchoprovocation testing, J Allergy Clin Immunol 64:592, 1979. 93. Anderson PJ, Garshick E, Blanchard JD, et al: Intersubject variability in particle deposition does not explain variability in responsiveness to methacholine, Am Rev Respir Dis 144:649–654, 1991. 94. Benson MK: Bronchial hyperreactivity, Br J Dis Chest 69:227–239, 1975.

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Aerosols and the Measurement of Mucociliary Clearance 95. Wanner A, Salathe M, O'Riordan TG: State of the art: mucociliary dysfunction in airways disease, Am J Respir Crit Care Med 154:1868–1902, 1996. 96. Jones JG: Clearance of inhaled particles from the alveoli. In Clarke S, Pavia D, editors: Aerosols and the lung: clinical and experimental aspects, London, 1984, Butterworth, pp 49–70. 97. Ilowite JS, Smaldone GC, Perry RJ, et al: Relationship between tracheo-bronchial particle clearance rates and sites of initial deposition in man, Arch Environ Health 44:267–273, 1989. 98. Messina MS, O'Riordan TG, Smaldone GC: Changes in mucociliary clearance during acute exacerbations of asthma, Am Rev Respir Dis 143:993–997, 1991. 99. O'Riordan TG, Zwang J, Smaldone GC: Mucociliary clearance in adult asthma, Am Rev Respir Dis 146:598–603, 1992. 100. Agnew JE, Bateman JR, Pavia D, et al: Peripheral airways mucous clearance in stable asthma is improved by oral glucocorticosteroid therapy, Bull Eur Physiopathol Respir 20:295–301, 1984.

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Chapter 47 - Anatomy and Physiology of the Nose and Control of Nasal Airflow

Ronald Eccles

FUNCTIONS OF THE NOSE The nose is situated at the entrance of the airway and acts as an air conditioner and a chemosensor. The 10,000 to 20,000•L of air we breathe in each day is composed of a mixture of gases and suspended particulate matter. The nose acts as a very efficient filter and gas scrubber, and much of the suspended particulate matter and soluble gases such as sulfur dioxide are deposited there. The nasal epithelium is in direct contact with the external air and is exposed to a continuous threat of irritation, infection, and allergy because the inspired air often contains air pollutants such as ozone, various pathogenic viruses, bacteria, fungal spores, and allergens

derived from pollens, house dust mites, and animal dander. The nose defends the lower airways from the harmful effects of the inspired air by acting as an efficient air conditioner. The nose warms, filters, and humidifies the inspired air so that clean air that is fully saturated with water vapor at a temperature of 37° C is delivered to the lungs. Filtration The nose acts as a very efficient filter of suspended particulate matter, and most suspended matter is either deposited in the nose or breathed in and out without 1

respiratory deposition.[ ] The filtration mechanism of the nose is firstly related to a sudden change in the direction of nasal airflow as the air stream passes upward through the nasal vestibule and then turns through 90 degrees to enter the nasal cavity. The sudden change in the direction of nasal airflow tends to spin suspended matter out of the main air stream and onto the surface of the nasal epithelium. Secondly, there is an acceleration of the airflow as it passes through the nasal valve region, which is the narrowest part of the nose, and then a deceleration of airflow as the air stream enters the nasal cavity. The changes in direction and velocity of the air stream occur over the same area of the nose, at the level of the anterior end of the inferior turbinate, and the slowing of the airflow at this point deposits particulate matter onto the nasal epithelium. Much of the work of breathing is related to nasal filtration, because the nasal valve area forms the narrowest cross-sectional area of the whole airway and presents a considerable resistance to nasal airflow. Nasal airway resistance contributes up to two thirds of the total airway resistance, and the work cost required from the respiratory muscles to move the airflow through the nasal valve area is the price that is paid for filtration of the inspired air. 2

Although nasal airflow may be laminar at very low levels of flow, for the greater part of the respiratory cycle, especially during inspiration, the flow is turbulent.[ ] The twisting course of nasal airflow, with changes in both velocity and direction, ensures that nasal airflow is mainly turbulent, and this is important for proper conditioning and mixing of the inspired air. If nasal airflow were mainly laminar, then only the portion of air directly in contact with the nasal epithelium would be conditioned by exchange of heat and water. The tendency for a particle suspended in the air stream to be deposited in the nose is determined by factors such as its physical size, shape, density, and hygroscopicity. The physical size, shape, and density of a particle can be quantified as its aerodynamic equivalent diameter (AED). Regardless of size, shape, or density, the AED refers to the behavior of a particle in the air stream if it were of unit density, spherical, and of the stated diameter. For example, a particle having an AED of 5••m may actually be smaller than 5••m but of a higher density or of a shape other then spherical. Particles with an AED larger than 180••m are virtually noninspirable; for particles with smaller AEDs, a proportion will be inspired. At AEDs of 30, 10, and 2.5••m, about 50%, 70%, and 90% of the particles, respectively, will be inspired. During nose breathing the majority of particles larger than 15••m AED are deposited in the upper respiratory tract, but with mouth breathing some of these penetrate into the trachea. Particles with AEDs higher than 2.5••m are primarily deposited in the trachea and bronchi, whereas those with lower AEDs penetrate into the gas exchange region of the lungs.[

3]

Humidification of Inspired Air Despite the fact that the upper airways condition the inspired air by increasing its humidity, so that air reaching the lungs is saturated with water, we often feel

uncomfortable when the humidity of the air around us is high. This may be partly related to feeling “hot and sticky,” but it may also be related to the fact that we often have a sensation of “nasal stuffiness” when breathing air of high humidity, because the inspiratory nasal airflow does not provide the same cooling sensation of breathing.

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The humidity of inhaled air may vary from 0.000002% to 4% or 5% by volume, with the lowest values measured at high altitudes or over the Antarctic plateau and 3]

the highest values in the equatorial regions near shallow bodies of water.[

The capacity of the upper airways to humidify the inspired air is very great, and no drying of the nasal epithelium is seen after prolonged exposure to dry air. The inhaled air rapidly reaches saturation with water vapor as it passes through the nose and upper airways, so that by the level of the trachea it is completely saturated. 4

This saturation of the inhaled air is achieved despite a wide range of ambient air temperatures and humidities.[ ] The water required for the humidification of the inhaled air is provided mainly by nasal glandular secretions, although it is unknown whether there are any control mechanisms linking the water requirement for air conditioning and the rate of glandular secretion. It is possible that changes in the osmolarity of the thin layer of nasal fluid control the movement of water across the nasal epithelium and that there is balance between water loss and the replenishment of water in the nasal fluid. The humidification of inspired air may also influence the filtration of particulate matter from the inspired air, because hydration of hygroscopic particulate matter may cause the particles to swell and thereby increase their AED. Water is lost from the surface of the nasal epithelium during inspiration as it contributes to the humidification of the inspired air. The nasal epithelium is cooled as it gives up heat both for warming the inspired air and for evaporation of water. During expiration, warm and fully saturated air passes through the cooler nasal airway, and some water may be reclaimed from the expired air by condensation onto the cooler nasal epithelium. In cold weather this condensed water may drip from the nose. Heat Exchange In most environments, the temperature of the air we breathe is well below body temperature, and therefore the airway is always exposed to a degree of heat loss and 5]

cooling. Even in a temperate climate such as that of London (United Kingdom), air temperature can vary seasonally from −13° C to +34° C.[

The nasal epithelium contains a complex network of blood vessels with a relatively high blood flow. These blood vessels act as a heat exchanger so that, over a wide range of ambient temperatures, the temperature of the inspired air is brought close to body temperature by the time it leaves the nose. In extremely cold climates, 4

there may be further warming of the inspired air along the trachea, so that the air temperature is close to 37° C by the time the air reaches the lungs.[ ] Under usual laboratory conditions, the temperature of the nasal epithelium is approximately 30° C immediately after inspiration and rises to 32° C immediately after expiration, with the temperature of the expired air being close to 32° C.

EMBRYOLOGY AND ANATOMY OF THE NOSE Embryology The external nose and nasal airway are recent developments in the evolution of the respiratory system. Our ancestors developed in a watery environment, and chemoreception in the aqueous medium was well developed at an early stage of evolution. The evolution of the nose is recapitulated in the embryo. The growth of a 6

nasal airway is preceded by the formation of an olfactory placode from ectoderm in the 5-mm embryo.[ ] The olfactory placode sinks to form a nasal sac, connected by the external nares to the exterior. The nasal sac is the primitive nasal airway, which is at first separated from the nasopharynx by an oronasal membrane. Persistence of the oronasal membrane leads to the congenital malformation of choanal atresia. The nasal cavity is divided into two halves by the nasal septum, which grows to separate the nasal cavity in the 19-mm embryo. At the same time as the nasal septum is developing, the maxillary processes grow from each side and fuse in the midline to form the palate. Failure of the maxillary processes to fuse causes a cleft palate. The three bony shelves—nasal turbinates or conchae—that project from the lateral wall of the nasal cavity develop as outgrowths from the lateral wall between the 7th and 12th weeks of embryonic growth. The paranasal sinuses develop in the embryo as outgrowths from the nasal cavity. The ethmoidal, sphenoidal, maxillary, and frontal sinuses are rudimentary at birth and slowly grow in size so that they are well developed by the seventh or eighth year of childhood, with further slow growth until puberty. Anatomy The anatomy of the nose is depicted in Fig. 47-1 and as a coronal computed tomographic scan in Fig. 47-2 . Two

Figure 47-1 Anatomy of the nasal cavity with a view of the lateral wall (top) and a coronal section through the middle of the nasal cavity (bottom). The coronal section is based on the computed tomographic scan illustrated in Fig. 47-2 . A fluid level in the maxillary sinuses is a common finding in patients who have acute rhinitis associated with a viral infection or allergy and does not necessarily indicate sinusitis.

Figure 47-2 A 2-mm-thick coronal computed tomographic scan of the nose in a patient who is lying prone with the neck extended, simulating erect posture. The scan is shown in diagrammatic form in Figure 47-1 . The superior turbinate is not apparent in this scan, and the middle turbinate shows signs of pneumatization with large air spaces. Fluid levels are apparent in the maxillary sinuses. Note the asymmetry in size and degree of congestion of the nasal turbinates caused by the nasal cycle. (Courtesy Rhian Rhys, consultant radiologist at the Royal Glamorgan Hospital, Llantrisant, Wales, UK.)

(Courtesy Rhian Rhys, consultant radiologist at the Royal Glamorgan Hospital, Llantrisant, Wales, UK.) many cells join to form the sinuses. The paranasal sinuses communicate with the nasal cavity via small ostia of 2 to 6•mm in diameter, and this restricted access means that there is only a very slow exchange of air between the sinuses and the nasal cavity. The function of the nasal sinuses has been the source of much 8 9

speculation. [ ] [ ] From their position, wrapped around the nasal airway, the paranasal sinuses may act as air insulators and protect the brain from cooling with inspiration of cold air. A novel role for the paranasal sinuses may be as the source of the gas nitric oxide, which is found in high concentrations in the nasal airway. [10]

Nasal Epithelium The epithelium of the nasal airway comes into direct contact with the inspired air. The physical, chemical, and biologic characteristics of the air have great potential for causing damage and infection. The epithelium of the nose is directly exposed to the external environment; it acts as an air conditioner and as the first line of defense against toxic and infectious agents in the inspired air. The nose is lined by three distinct types of epithelium: a stratified squamous epithelium in the nasal vestibule and nasopharynx, a pseudostratified ciliated columnar epithelium in the main respiratory area of the nasal cavity, and a specialized olfactory epithelium with ciliated receptor cells in the olfactory area.

The anterior portion of the nose forms the nasal vestibule, which is lined with a stratified squamous epithelium similar to that of the facial skin, almost as though a portion of the facial skin had been turned inward to form the vestibule. At the entrance to the nares, the skin is covered with short, stiff hairs which are extremely sensitive to certain kinds of mechanical stimuli that cause itching, tickling, and sneezing. The stiff nasal hairs are not stimulated by deformation resulting from forced inspiration or expiration. The stratified squamous epithelium lining the nasal vestibule has sensory properties similar to that of facial skin. The thermoreceptors that are responsible for the cool sensation on inspiration may be situated in this region.[

11]

The nasal valve area is strategically placed at the entrance of the nasal cavity, and the inspired air velocity is maximal at this point. Any cooling or mechanical stimulus from the inspired air would be greatest at this point, and the valve area may be of major importance as far as the sensation of nasal airflow is concerned. Just past the nasal valve area, the skin gradually changes into a ciliated respiratory epithelium. At the junction between the squamous epithelium of the nasal vestibule and the respiratory epithelium of the nasal cavity lies a 1.5-mm-wide strip covering a region of capillary loops that are unusually wide and long, with 6 12

dermal papillae.[ ] [ ] In this transitional area, sometimes referred to as Kiesselbach's plexus, even mild trauma is a common source of nasal bleeding, particularly in childhood. The long capillary loops in this area may act as a source of plasma transudation and prevent drying of the transitional region between the keratinized skin of the nasal vestibule and the ciliated respiratory epithelium. Alternatively, the dermal papillae may act as sensitive mechanoreceptors or thermoreceptors that detect nasal airflow. The inspiratory airflow is a source of trauma to the nasal epithelium, and this trauma stimulates the formation of a squamous epithelium in the anterior areas of the nose that are

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directly exposed to unconditioned air. The anterior edge of the inferior turbinate has a squamous epithelium, and, with increasing age, the surface area of squamous epithelium in the anterior part of the nose increases at the expense of the respiratory epithelium of the nasal cavity. 13

The typical respiratory epithelium is a pseudostratified ciliated columnar type, resting on a continuous basement membrane.[ ] The epithelial cell types of the respiratory nasal epithelium are basal cells, goblet cells, and ciliated and nonciliated columnar cells. The Clara cells, serous cells, and brush cells found in the tracheobronchial epithelium are not found in the human nose. The ratio of columnar cells to goblet cells is approximately 5:1, and there is a significant increase in the density of goblet cells in the anterior-posterior direction through the nasal cavity. The density of goblet cells in the respiratory epithelium is decreased by airflow 14

trauma and increased by infection.[ ] The goblet cells are not innervated by the autonomic nervous system, and local factors, such as inflammatory mediators or changes in osmolarity, may control the release of mucus precursors from the goblet cells. The movements of cilia on the columnar cells propel the blanket of mucus that covers the nasal epithelium. The majority of columnar cells are covered with cilia 4 to 15]

6••m long, and there are about 100 cilia per cell.[

Each columnar cell is covered by 300 to 400 microvilli with a maximum length of 2••m. Microvilli are not

precursors for cilia, and they do not move. The microvilli may aid transport of fluid and electrolytes between the cells and nasal fluid.[

15]

Nasal Fluid and Mucociliary Clearance Nasal fluid is a mixture of elements derived from glands, goblet cells, capillary plasma transudate, and cell debris. The relative contributions of these different elements to the blanket of mucus overlying the nasal epithelium are not well understood. Nasal fluid acts as an interface between the air we breathe and the delicate epithelium of the nasal cavity. The respiratory epithelium continuously clears secretions through the beating action of cilia, and this is an important respiratory defense mechanism. Nasal fluid is a mixture of elements derived from four sources in the respiratory area of the nasal cavity[ 1. 2. 3. 4.

16]

:

Seromucous glands within the nasal epithelium Goblet cells distributed along the surface of the nasal epithelium Exudation of plasma from capillaries and veins within the nasal epithelium Secretions and cell debris from leukocytes and epithelial cells

Lacrimal gland secretions (via the nasolacrimal duct) and secretions from the specialized Bowman's glands of the olfactory epithelium also contribute to nasal secretions. The glands found deep within the lamina propria of the nasal epithelium are of the compound alveolar type, and both mucous and serous glands may be present in 17

one alveolus.[ ] The secretions from these glands flow onto the surface of the epithelium via large ducts. Some of the anterior nasal glands have long ducts that open in the anterior portion of the nose and produce a serous secretion. Secretions from these anterior nasal glands may be drawn out into a fine spray during inspiration, 18]

and this may contribute to the humidification of the inspired air.[

The seromucous glands within the nasal epithelium have a parasympathetic cholinergic

innervation, and electrical stimulation of these nerve fibers causes a watery nasal secretion.[

19]

Goblet cells are distributed throughout the surface of the respiratory epithelium, and they release mucus directly onto the surface of the ciliated epithelium. The mucous blanket, composed of the secretions of goblet cells and mucous glands in the lamina propria, forms a protective layer overlying the delicate nasal epithelium and prevents the escape of water. There is no evidence that the activity of goblet cells is controlled by the parasympathetic secretory nerves to the nasal epithelium. However, there is some evidence that goblet cell secretion is under the control of capsaicin-sensitive sensory nerves and that the release of neuropeptides from these nerves may induce mucus secretion.[

20]

Nasal fluid is important in respiratory defense, because immunoglobulins such as immunoglobulin A (IgA) are secreted from 21

the nasal glands and act as a first line of defense against infection.[ ] Antioxidants such as uric acid, glutathione, and vitamin C are found in nasal fluid; they protect the nasal epithelium against the oxidizing action of air pollutants such as ozone and against the oxidative damage caused by peroxides released by leukocytes during 22] [23]

periods of nasal inflammation.[

Plasma exudation directly from surface capillaries and small veins [

24]

makes a contribution to nasal fluid, especially during nasal inflammation associated with 25

infectious or allergic rhinitis. It has been proposed that exudation of plasma and plasma-derived mediators is a major respiratory defense mechanism.[ ] In rhinitis, an outpouring of plasma from the subepithelial microcirculation provides mediators and substrates for the inflammatory response and immunoglobulins to defend against infection. On the surface of the respiratory epithelium, the nasal secretions are found in two layers: a watery periciliary fluid in which the cilia beat freely, and an overlying layer of viscous, sticky mucus that is propelled by ciliary action. This mucus does not form a continuous blanket overlying the epithelium but rather consists of a discontinuous sheet or patches.[

26] [27]

The direction of mucociliary clearance in the nose is mainly toward the nasopharynx, where cleared mucus is swallowed. There is, however, some anterior clearance toward the nasal vestibule. The anterior clearance of material deposited in the anterior part of the nose ensures that deposited material is cleared forward toward the nares rather than being spread over the whole posterior part of the nasal epithelium.[

27]

Nasal mucociliary clearance can be measured in humans by placing a particle of saccharin on the anterior end of the inferior turbinate and timing the onset of a sweet taste when the saccharin reaches the pharynx and is swallowed. The rate of mucociliary clearance as measured by saccharin transport or other methods has a wide 27]

normal range, reported as 1 to 20•mm/min[

or as a clearance time of 7 to 11 minutes.[

28]

It is not clear why there should be such a large range of transport times for mucociliary clearance in normal healthy subjects, but this may be related to variation in the previous history of upper respiratory tract infections, which is not always documented in studies on mucociliary clearance. Nasal Blood Vessels The anatomy of the nasal blood vessels is complex but, to simplify matters, the vasculature can be discussed in terms of resistance and capacitance vessels. The resistance vessels are

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mainly small arteries, arterioles, and arteriovenous anastomoses. Blood flow to the nasal epithelium is regulated by constriction and dilation of these vessels.[

29] [30]

31

The resistance vessels have a dense adrenergic innervation from the sympathetic nervous system.[ ] Stimulation of sympathetic nerves to the nose causes a marked vasoconstriction and reduction in blood flow. Under normal conditions, nasal blood flow is probably controlled by local mediators, because there is very little resting sympathetic vasoconstrictor tone to the resistance vessels, and sympathetic nerve section or blockade causes only a slight increase in nasal blood flow.[

32] [33]

34]

Nasal blood flow is increased with nasal inflammation caused by infective rhinitis,[

but it is not clear how blood flow changes with allergic rhinitis. Nasal

challenge with allergen in subjects with allergic rhinitis has been shown to cause both decreases and increases in nasal blood flow.[

35] [36]

A special feature of the vasculature of the nasal epithelium is that it contains large venous sinuses, which are especially well developed over the inferior and middle 31

turbinates and the nasal septum.[ ] The venous sinuses are also referred to as capacitance vessels or venous erectile tissue. The venous erectile tissue of the nasal epithelium is well developed at the anterior end of the inferior turbinate, and swelling of the erectile tissue in this region controls nasal airway resistance. The venous 32]

erectile tissue has a dense adrenergic innervation, and congestion and constriction of the erectile tissue is regulated via the sympathetic nerve supply to the nose.[ [37] [38]

The subepithelial capillaries and veins of the nasal epithelium are unusual in that they are fenestrated, and the large fenestrated areas of the endothelium face toward 24 39

26

the respiratory epithelium.[ ] [ ] Some capillaries penetrate the basement membrane and lie between the cells of the epithelium.[ ] The permeability of the capillaries is very high, and some endothelial cells are wide open, similar to the capillaries of the liver sinusoids, allowing free passage of large particles from the circulation.[

40]

41]

The high permeability of nasal capillaries may also explain why intranasally administered drugs readily enter the bloodstream.[

Nerve Supply and Nasal Reflexes The nose is ideally situated at the entrance to the respiratory tract to sample the inspired airflow and to detect chemical and physical irritants that could damage the airway. The sensory innervation of the nose is mainly supplied by the olfactory and trigeminal nerves. The olfactory nerves enter the nose through the cribriform plate and form a distinct olfactory area. Most of the sensory nerves to the nasal epithelium and nasal vestibule are supplied by two branches of the trigeminal nerve, 42]

the ophthalmic and maxillary nerves.[

The olfactory area acts as a long-distance chemoreceptor, sampling the odorants contained in the inspired air and giving us our appreciation of foods, perfumes, and other substances. The trigeminal nerves provide the sensations of touch, pain, hot, cold, and itch, as well as the sensation of nasal airflow, which is perceived as a cool sensation on inspiration. This sensation not only reassures us that breathing is taking place but may also influence the pattern of respiration and the activity of upper airway accessory respiratory muscles.[ substances, such as menthol, acetone, and

43] [44]

The trigeminal nerves also detect chemicals, such as ammonia and sulfur dioxide, and a range of organic

45 pyridine.[ ]

Chemical or physical stimuli to the nasal epithelium may initiate potent respiratory and cardiovascular reflexes via stimulation of trigeminal nerves, resulting in 42

expiration with apnea, closure of the larynx, and bradycardia. Mild stimuli result in sneezing and nasal hypersecretion.[ ] These reflex responses protect the lower airways from inhalation of physical and chemical irritants. Sneezing may be initiated by a number of factors, such as mechanical stimulation of the nasal epithelium, cooling of the skin, bright light in the eyes, irritation of the scalp near the frontal hairline, challenge with allergen extract, and psychogenic causes.[

42] [46] [47]

The trigeminal nerves in the nasal epithelium initiate the sneezing and hypersecretion associated with upper respiratory tract infection and allergy. They may also be responsible for neurogenic inflammation associated with nasal infection and allergy, because various inflammatory mediators (e.g., substance P, neurokinin A,

48] [49]

calcitonin gene-related peptide) have been found in these nerves.[

The nasal epithelium is innervated by both sympathetic and parasympathetic nerve fibers, with the parasympathetic fibers supplying nasal glands and the sympathetic fibers controlling nasal blood flow and the filling of venous erectile tissue. The parasympathetic nerve supply to the nose originates in the salivatory nuclei of the brain stem and follows the greater superficial petrosal nerve and the nerve of the pterygoid canal (Vidian nerve). The parasympathetic fibers relay in the sphenopalatine ganglion, and postganglionic nerves reach the nasal glands via branches of the posterior nasal nerve. Electrical stimulation of parasympathetic nerves causes a profuse watery nasal secretion, and section of the nerve of the pterygoid canal, which contains parasympathetic fibers, has been used successfully for the treatment of profuse watery rhinorrhea.[

50]

The parasympathetic nerves in the nasal epithelium release acetylcholine as their primary neurotransmitter and they also

release vasoactive intestinal polypeptide, which is a potent vasodilator. [

51]

The activity of the sympathetic nerves to the nose can be influenced by electrical stimulation of areas of the hypothalamus and brain stem.[ stimulation of areas of the hypothalamus in the anesthetized cat causes a pronounced nasal

52 vasoconstriction,[ ]

52] [53]

Electrical

whereas stimulation of areas of the brain stem causes

53 vessels.[ ]

reciprocal changes in sympathetic tone to nasal blood The preganglionic sympathetic nerve fibers originate in the thoracolumbar region of the spinal cord, relay in the superior cervical ganglion, and are distributed to the nasal blood vessels via the nerve of the pterygoid canal and branches of the trigeminal nerve. Electrical stimulation of the sympathetic nerves causes a pronounced nasal vasoconstriction and a reduction in nasal blood flow, accompanied by decongestion of 42

venous erectile tissue and a subsequent decrease in nasal airway resistance.[ ] Norepinephrine is the primary neurotransmitter in the sympathetic nerves supplying the nasal blood vessels, and the vasoconstriction caused by norepinephrine is supplemented by the release of neuropeptide Y, which is also a potent vasoconstrictor. [54]

NASAL AIRFLOW Influence of Nasal Blood Vessels on Nasal Airflow The role of the nasal venous sinuses in the control of nasal airflow is now well recognized, and their ability to swell and

780

31 55

completely obstruct the nasal passage has been reported.[ ] [ ] A network of nasal venous sinuses is found throughout the epithelium of the nose, but they are particularly well developed at the tip of the inferior turbinate and on the nearby anterior end of the nasal septum. The location of nasal venous sinuses at the anterior tip of the inferior turbinate and nasal septum is critical for the control of nasal airflow, and this area of the nose is often referred to as the “nasal valve.” Nasal Valve and Control of Nasal Airflow

55 56 57

The narrowest point of the nasal passage determines the nasal resistance to airflow; this is the area referred to as the nasal valve.[ ] [ ] [ ] However, there is some dispute in the literature as to whether the nasal valve lies in the nasal vestibule or more posteriorly, within the bony cavum of the nose. The anatomic and physiologic evidence indicates that the nasal valve occurs at the entrance of the piriform aperture, with the major site of nasal resistance being just anterior to the tip of the inferior turbinate.[

58]

The nasal airway resistance can be thought of as consisting of three components: the nasal vestibule, the nasal valve, and the turbinated nasal passage. These three components are illustrated diagrammatically in Fig. 47-3 . The compliant nasal vestibule is stiffened by cartilage to which facial muscles such as the alae nasi muscles attach. During inspiration these muscles contract and splint the vestibule to prevent collapse.[

59]

The change in airway resistance along the nasal passage can be determined by passing a pressure-sensing cannula carefully along the passage and determining the relationships between pressure and flow during quiet breathing. Using this

Figure 47-3 Diagram of the nose. The compliant wall of the nasal vestibule is supported by the alae nasi muscles. The nasal valve is at the level of the anterior tip of the inferior turbinate. The degree of congestion of the tip of the inferior turbinate determines the dynamic cross-sectional area of the nasal valve and therefore the nasal airway resistance. The diagram illustrates the normal asymmetry of nasal congestion, with one side of the nose congested as a result of dilation of the venous sinuses in the inferior nasal turbinate and the other side open and decongested (i.e., venous sinuses constricted).

Figure 47-4 Nasal cycle. Spontaneous changes in unilateral nasal airway resistance recorded in one healthy subject by the technique of rhinomanometry. Open symbols (–•–), left nasal airway resistance; closed symbols (–•–), right nasal airway resistance. (Redrawn from Eccles R, Reilly M, Eccles KS: Acta Otolaryngol 116:77, 1996.)

(Redrawn from Eccles R, Reilly M, Eccles KS: Acta Otolaryngol 116:77, 1996.)

782

97

But closer examination of the phenomenon led Gilbert and Rosenwasser[ ] to conclude that there was little empiric support for the nasal cycle as strictly defined— that is, as a reciprocal and rhythmically alternating relation between the two nasal passages. 86

In a study by Flanagan and Eccles[ ] involving 52 healthy volunteers, unilateral nasal airflow was measured every hour over an 8-hour period. A wide range of patterns of airflow was observed, with some volunteers exhibiting regular spontaneous and reciprocal changes in unilateral airflow and others exhibiting irregular changes in airflow. The authors defined a nasal cycle in terms of numeric parameters of correlation and distribution of nasal airflow between the nasal passages and concluded that only 21% (11/52) of the volunteers exhibited airflow patterns that could be defined as a nasal cycle. When present, the reciprocal changes in nasal airflow have been reported to occur over a time course of 0.5 to 3.0 hours.[

91] [92] [93]

The spontaneous, often reciprocal changes in nasal airflow are caused by congestion and decongestion of nasal venous sinuses under the influence of the sympathetic

nervous system.[

42]

Section or local anesthesia of the cervical sympathetic nerves that supply one side of the nose and face causes ipsilateral nasal congestion and 67] [68]

abolition of the spontaneous changes in nasal airflow.[

Understanding of the factors that influence the spontaneous and reciprocal changes in nasal airflow is very limited. The idea that the nasal cycle may act to share the burden of air conditioning between the two nasal passages, so that dominance of airflow alternates over a period of several hours, is an attractive hypothesis. If nasal airflow in some way influenced the control of the nasal cycle, then one would expect the spontaneous changes in nasal venous sinuses not to occur in the absence of nasal airflow. However, there is some evidence that spontaneous congestion and decongestion of nasal blood vessels still occurs in the laryngectomized patient, in the absence of any nasal airflow.[

98] [99]

Some reports in the literature of Yoga state that dominance of nasal airflow may be changed by breathing exercises

100 (Pranayama),[ ]

but studies on various breathing patterns have failed to substantiate any such effect of nasal breathing.[ knowledge does not provide any support for the hypothesis that nasal airflow in some way influences the nasal cycle.

101]

Therefore, the present state of

The spontaneous changes in nasal airflow that are associated with the nasal cycle may be related to oscillations in the activity of autonomic control centers in the central nervous system (discussed later). Central Control of Nasal Airflow The idea that the spontaneous changes in nasal airflow associated with the nasal cycle may be controlled from some center in the brain was first put forward by Stoksted in 1953,[

91]

who speculated that the nasal cycle was “regulated by a central sympathetic centre possibly situated in the hypothalamus.” Studies on 52 102

anesthetized animals [ ] [ ] demonstrated that pronounced nasal vasoconstriction and a reduction in nasal airway resistance may be induced on electric stimulation of areas of the hypothalamus, indicating that this area of the brain does have an influence on nasal vasomotor activity. However, the nasal vasoconstrictor responses may be more related to a generalized vasoconstrictor response than to specific control of the nasal cycle. Hypothalamic stimulation caused bilateral nasal vasoconstrictor responses but was not associated with any reciprocal changes in nasal vasomotor activity, indicating that the hypothalamus may be associated with control of a defense reaction rather than control of the nasal cycle.[

52]

Reciprocal nasal vasomotor responses, with ipsilateral vasoconstriction and contralateral vasodilation, were initiated on electric stimulation of areas of the brainstem 53

in the anesthetized cat.[ ] The reciprocal nasal vasomotor responses were proposed to originate from groups of neurons on either side of the brainstem, which acted as oscillators and controlled the sympathetic tone to each side of the nose. By stimulation of first one side of the brainstem and then the other, the dominance of sympathetic tone to nasal blood vessels could be switched from one side to the other, with a reciprocal relationship between the two halves of the brainstem.[ These switches in nasal sympathetic vasomotor activity could also occur spontaneously and were closely linked to the control of respiration.[

53]

103]

At present, understanding of the central control of nasal airflow is limited to animal experiments, but this knowledge has allowed the development of a model of a control system,[

104]

illustrated in Fig. 47-5 .

Effect of Changes in Posture on Nasal Airflow

The changes in nasal airflow associated with changes in posture may be explained by two separate mechanisms: an increase in central venous pressure on moving from erect to supine, and a reflex change in nasal vasomotor activity on

Figure 47-5 Model illustrating the sympathetic nervous control of nasal venous sinuses. Vasomotor control is proposed to reside in two half-centers in the brainstem. The half-centers have reciprocal connections so that the dominance of activity oscillates over a period of hours, with only one center having dominance at any one time. The sympathetic vasoconstrictor tone exerted by the right and left cervical sympathetic nerves that supply the two halves of the nose is normally asymmetric. The asymmetric sympathetic tone causes decongestion of the venous sinuses on one side of the nose and congestion on the other side. The spontaneous changes in nasal congestion over a period of hours are often referred to as the nasal cycle. –VE, Inhibition of neuronal activity.

Figure 47-6 The effects of ingestion of an 11-mg l-menthol lozenge on subjective sensation of nasal congestion and on nasal resistance to airflow in human volunteers with a common cold. The subjective sensation of nasal congestion was significantly reduced 10 minutes after ingestion of the lozenge, but nasal airway resistance as measured by rhinomanometry was unaffected. Shaded symbols represent mean values for the menthol-treated group; open symbols, mean values for the placebo-treated group. The congestion score represents change in score on a 100-mm visual analog scale. Nasal resistance is given in units of Pa cm3 s. (Data from Eccles R, Jawad MS, Morris S: J Pharm Pharmacol 42:652, 1990.)

(Data from Eccles R, Jawad MS, Morris S: J Pharm Pharmacol 42:652, 1990.) Pharmacology of Nasal Airflow Almost any substance that has an effect on vascular smooth muscle will have some influence on nasal airflow by causing changes in the activity of the smooth muscle of nasal venous sinuses. Similarly, any substance that influences the activity of sympathetic noradrenergic nerve endings is likely to influence nasal airflow by altering the sympathetic vasoconstrictor tone to nasal venous sinuses. Only the pharmacology relevant to effects on nasal airflow is discussed here. Sympathomimetics and Sympatholytics

The nasal blood vessels are extremely sensitive to sympathomimetic medications that mimic the vasoconstrictor effects of norepinephrine and epinephrine and cause

decongestion of nasal venous sinuses and a decrease in nasal resistance to airflow. Nasal decongestant medications are sympathomimetics that act on α1- and α2145]

receptors on nasal venous sinuses. There is some evidence that α1-receptors are the major receptor type on the smooth muscle of nasal venous sinuses.[ pharmacology of sympathomimetics and nasal decongestants was reviewed in 1999. vasodilation and nasal

147 148 congestion[ ] [ ]

[146]

The

Repeated application of topical sympathomimetics can cause a rebound

that may be related to tissue hypoxia cause by pronounced vasoconstriction. Rhinitis

785

medicamentosa can occur after prolonged abuse of topical nasal decongestants[ used in topical nasal decongestants than to the vasoconstriction

149] [150]

; this condition may be related more to chronic exposure to the preservatives

148 itself.[ ]

Sympatholytic medications may cause nasal congestion due to their inhibitory effects on the sympathetic nervous system, and this was a very common side effect associated with medications such as reserpine.[

149]

Although one might expect that the “pharmacologic” decongestion caused by treatment with a topical nasal decongestant sympathomimetic medication would be greater than that seen with “physiologic” or spontaneous decongestion associated with the nasal cycle, in fact the magnitude of decongestion has been shown to be equivalent.[

151]

Histamine and H1 Antihistamines

Histamine is a potent vasodilator mediator that is associated with the inflammatory allergic response. It has effects on nasal sensory nerves and blood vessels, causing sneezing, itching, runny nose, and nasal congestion.[

152]

The vasodilator actions of histamine influence nasal airflow by causing congestion of the nasal venous 72] [153] [154]

sinuses. Histamine challenge is often used as an experimental method to elicit nasal congestion and other symptoms of nasal allergy.[

The effects of histamine on the human nose are mediated by both H1 and H2 receptors. Both of these receptor types are involved in the dilation of venous sinuses, whereas only the H1 receptors are involved in sneezing, itching, and hypersecretion.[

155]

The involvement of both H1 and H2 receptors in nasal congestion may

explain why H1 antihistamines are relatively ineffective in treating the nasal congestion associated with nasal allergy and histamine challenge.[

155] [156]

Bradykinin

The kallikrein enzyme that is responsible for the generation of bradykinin was first shown to be present in the nose in cat nasal secretions.[

157]

Bradykinin is a potent

vasodilator mediator associated with the inflammatory response to acute upper respiratory tract infection.[ sensory nerves, causing nasal congestion, nasal irritation, and runny

159 160 nose.[ ] [ ]

158]

It has effects on both nasal blood vessels and nasal

Although there has been considerable research to discover a bradykinin 161

antagonist that could be useful in the treatment of symptoms associated with acute upper respiratory tract infection, at present no such agent is available.[ ] If a suitable bradykinin antagonist could be discovered, then it would be useful in the treatment of symptoms of the common cold, and would probably play a role similar to that of H1 antihistamines in the treatment of allergic rhinitis. Corticosteroids

Intranasal corticosteroids are widely used for the treatment of allergic rhinitis, but there have been very few studies of the effects of corticosteroids on nasal airflow. Unlike the H1 antihistamines, which have a small effect on nasal congestion, intranasal corticosteroids are generally believed to provide relief for the symptom of nasal congestion associated with allergic rhinitis.[

162]

Topical nasal steroid treatment has been shown to reduce the increase in nasal airway resistance that is 163

associated with nasal challenge with grass pollen in allergic patients.[ ] After dermal application, corticosteroids induce a pallor of the skin; this reaction has been used to grade the antiinflammatory potency of the compounds tested, but there is no evidence to indicate that corticosteroids cause vasoconstriction of nasal blood vessels.[

162]

Acknowledgment Thanks to Rhian Rhys, consultant radiologist at the Royal Glamorgan Hospital, Llantrisant, Wales, UK, for providing the CT scan for Fig. 47-2 .

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57. Bridger GP: Physiology of the nasal valve, Arch Otolaryngol 92:543, 1970. 58. Cole P: The respiratory role of the upper airways: a selective clinical and pathological review, St Louis, 1993, Mosby–Year Book. 59. Strohl KP, O'Cain CF, Slutsky A: Alae nasi activation and nasal resistance in healthy subjects, J Appl Physiol 52:1432, 1982. 60. Jones AS, Lancer JM: Does submucosal diathermy to the inferior turbinate reduce nasal resistance to airflow in the long term? J Laryngol Otol 101:448, 1987. 61. Dahlstrom A, Fuxe K: The adrenergic innervation of the nasal mucosa of certain mammals, Acta Otolaryngol (Stockh) 59:65, 1964. 62. Malm L: Sympathetic influence on the nasal mucosa, Acta Otolaryngol (Stockh) 83:20, 1977. 63. Cauna N, Cauna D: The fine structure and innervation of the cushion veins of the human nasal respiratory mucosa, Anat Rec 181:1, 1975. 64. Wright JW: A consideration of the vascular mechanism of the nasal mucous membrane and its relations to certain pathological processes, Am J Med Sci 109:516, 1895. 65. Burnham HH: An anatomical investigation of blood vessels of the lateral nasal wall and their relation to turbinates and sinuses, J Laryngol Otol 50:569, 1935. 66. Beickert P: Halbseitenrhythmus der vegetativen innervation, Archiv fur Ohren-Nasen-und Kehlkopifheikonde 157:404, 1951. 67. Stoksted P, Thomsen K: Changes in the nasal cycle under stellate ganglion blockade, Acta Otolaryngol Suppl 109:176, 1953. 68. Eccles R: The domestic pig as an experimental animal for studies on the nasal cycle, Acta Otolaryngol 85:431, 1978. 69. Eccles KS, Eccles R: Nasal vasodilation induced by electrical stimulation of the vagus nerve, Rhinology 20:89, 1982. 70. Schumacher MJ: Rhinomanometry, J Allergy Clin Immunol 83:711, 1989. 71. Cole P, Roithmann R, Roth Y, et al: Measurement of nasal patency: a manual for users of the Toronto systems and others interested in nasal patency measurement, Ann Otol Rhinol Laryngol Suppl 171:1, 1997. 72. Eccles R: Evaluation of the nasal airway and nasal challenge. In Mackay IS, Bull TR, editors: Scott-Brown's otolaryngology, Oxford, 1997, ButterworthHeinemann, p 4/4/1. 73. Grymer LF, Hilberg O, Elbrond O, et al: Acoustic rhinometry: evaluation of the nasal cavity with septal deviations, before and after septoplasty, Laryngoscope 99:1180, 1989. 74. Roithmann R, Cole P, Chapnik J, et al: Acoustic rhinometry in the evaluation of nasal obstruction, Laryngoscope 105:275, 1995. 75. Tomkinson A, Eccles R: Acoustic rhinometry, Curr Opin Otolaryngol Head Neck Surg 4:7, 1996.

76. Syabbalo NC, Bundgaard A, Entholm P, et al: Measurement and regulation of nasal airflow resistance in man, Rhinology 24:87, 1986. 77. Cole P: Nasal airflow resistance. In Mathew OP, Sant' Ambrogio G, editors: Respiratory function of the upper airway, New York, 1988, Marcel Dekker, p 391. 78. Morris S, Jawad SM, Eccles R: Relationships between vital capacity, height and nasal airway resistance in asymptomatic volunteers, Rhinology 30:259, 1992. 79. Vig PS, Zajac DJ: Age and gender effects on nasal respiratory function in normal subjects, Cleft Palate Craniofac J 30:279, 1993. 80. Havas T, Gutierrez MC, Morton J, et al: Nasal airway resistance in patients with cystic fibrosis, Aust J Otolaryngol 1:434, 1994. 81. Polgar G, Kong GP: The nasal resistance of newborn infants, J Pediatr 67:557, 1965. 82. Stocks J, Godfrey S: Nasal resistance during infancy, Respir Physiol 34:233, 1978. 83. Jones AS, Viani L, Phillips D, et al: The objective assessment of nasal patency, Clin Otolaryngol 16:206, 1991. 84. Broms P: Rhinomanometry: III. Procedures and criteria for distinction between skeletal stenosis and mucosal swelling, Acta Otolaryngol 94:361, 1982. 85. Eccles R, Reilly M, Eccles KS: Changes in the amplitude of the nasal cycle associated with symptoms of acute upper respiratory tract infection, Acta Otolaryngol 116:77, 1996. 86. Flanagan P, Eccles R: Spontaneous changes of unilateral nasal airflow in man: a re-examination of the “nasal cycle,” Acta Otolaryngol 117:590, 1997. 87. Flanagan P, Eccles R: The normal range of unilateral nasal air-flow in healthy-human volunteers, J Physiol 491P:53P, 1996. 88. Takeuchi H, Jawad M, Eccles R: Changes in unilateral nasal airflow in patients with seasonal allergic rhinitis measured in and out of season, Auris Nasus Larynx 27:141, 2000. 89. Jawad SM, Eccles R: Effect of pseudoephedrine on nasal airflow in patients with nasal congestion associated with common cold, Rhinology 36:73, 1998. 90. Quine SM, Aitken PM, Eccles R: Effect of submucosal diathermy to the inferior turbinates on unilateral and total nasal airflow in patients with rhinitis, Acta Otolaryngol 119:911, 1999. 91. Stoksted P: Rhinometric measurements for determination of the nasal cycle, Acta Otolaryngol Suppl 109:159, 1953. 92. Hasegawa M, Kern EB: The human nasal cycle, Mayo Clin Proc 52:28, 1977. 93. Eccles R: The central rhythm of the nasal cycle, Acta Otolaryngol 86:464, 1978. 94. Kayser R: Die Exacte Messung der Luftdurchgangigkeit der Nase, Arch Laryngol Rhinol 3:101, 1895. 95. Sen H: Observations on the alternate erectility of the nasal mucous membrane, Lancet 1:564, 1901.

96. Heetderks DL: Observations on the reaction of normal nasal mucous membrane, Am J Med Sci 174:231, 1927. 97. Gilbert AN, Rosenwasser AM: Biological rhythmicity of nasal airway patency: a re-examination of the nasal cycle, Acta Otolaryngol 104:180 1987. 98. Havas TE, Cole P, Gullane PJ, et al: The nasal cycle after laryngectomy, Acta Otolaryngol 103:111, 1987. 99. Fisher EW, Liu M, Lung VJ: The nasal cycle after deprivation of airflow: a study of laryngectomy patients using acoustic rhinometry, Acta Otolaryngol 114:443, 1994. 100. Bhole MV, Karambelkar PV: Significance of nostrils in breathing, Yoga-Mimamsa 10:1, 1968. 101. Mohan SM, Eccles R: Effect of inspiratory and expiratory airflow on congestion and decongestion in the nasal cycle, Indian J Physiol Pharmacol 3:191, 1989. 102. Malcolmson KG: The vasomotor activities of the nasal mucous membrane, J Laryngol Otol 37:3, 1959. 103. Eccles R, Lee RL: Nasal vasomotor oscillations in the cat associated with the respiratory rhythm, Acta Otolaryngol 92:357, 1981. 104. Eccles R: Sympathetic control of nasal erectile tissue, Eur J Respir Dis 64:150, 1983. 105. Geterud A, Rundcrantz H: The effect of a local decongestant in acute rhinitis as related to body position, Rhinology 21:155, 1983. 106. Hasegawa M, Saito Y: Postural variations in nasal resistance and symptomatology in allergic rhinitis, Acta Otolaryngol 88:268, 1979. 107. Hasegawa M: Posture induced nasal obstruction in patients with allergic rhinitis, Clin Otolaryngol 19:135, 1994. 108. Cole P, Haight JS: Posture and the nasal cycle, Ann Otol Rhinol Laryngol 95:233, 1986. 109. Rao S, Potdar A: Nasal airflow with body in various positions, J Appl Physiol 28:162, 1970. 110. Davies AM, Eccles R: Reciprocal changes in nasal resistance to airflow caused by pressure applied to the axilla, Acta Otolaryngol 99:154, 1985.

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111. Haight JS, Cole P: Unilateral nasal resistance and asymmetrical body pressure, J Otolaryngol 15(suppl 16):1, 1986. 112. Takagi K, Kobayasi S: Skin pressure-vegetative reflex, Acta Med Biol 4:31, 1955. 113. Kuno Y: Human perspiration, Springfield, Illinois, 1956, Charles C Thomas. 114. Richerson H, Seebohm P: Nasal airway response to exercise, J Allergy 41:269, 1968.

115. Dallimore NS, Eccles R: Changes in human nasal resistance associated with exercise, hyperventilation and rebreathing, Acta Otolaryngol 84:416, 1977. 116. Forsyth RD, Cole P, Shephard RJ: Exercise and nasal patency, J Appl Physiol 55:860, 1983. 117. Hasegawa M, Kabasawa Y, Ohki M, et al: Exercise-induced change of nasal resistance in asthmatic children, Otolaryngol Head Neck Surg 93:772, 1985. 118. Syabbalo NC, Bundgaard A, Widdicombe JG: Effects of exercise on nasal airflow resistance in healthy subjects and in patients with asthma and rhinitis, Bull Eur Physiopathol Respir 21:507, 1985. 119. Niinimaa V, Cole P, Mintz Z, et al: The switching point from nasal to oronasal breathing, Respir Physiol 42:61, 1980. 120. Tatum AL: The effect of deficient and excessive pulmonary ventilation on nasal volume, Am J Physiol 65:229, 1923. 121. Hasegawa M, Saito Y, Watanabe K, et al: Changes of nasal airway resistance caused by breathing holding: I. Results in normal subjects, Jpn J Otol (Tokyo) 79:30, 1976. 122. McCaffrey TV, Kern EB: Response of nasal airway resistance to hypercapnia and hypoxia in man, Ann Otol Rhinol Laryngol 88:247, 1979. 123. Babatola FD, Eccles R: Nasal vasomotor responses in man to breath holding and hyperventilation recorded by means of intranasal balloons, Rhinology 24:271, 1986. 124. McCaffrey TV, Kern EB: Response of nasal airway resistance to hypercapnia and hypoxia in the dog, Acta Otolaryngol 87:545, 1979. 125. Chavanne L: Secretion nasale et glandes endocrines, Annals d' Otolaryngologic 5:401, 1937. 126. Holmes H, Goodell H, Wolf S, et al: The nose: an experimental study of reactions within the nose in human subjects during varying life experiences, Springfield, Illinois, 1950, Charles C. Thomas. 127. Ellegard E, Karlsson G: Nasal congestion during the menstrual cycle, Clin Otolaryngol 19:400, 1994. 128. Ellegard E, Karlsson G: Nasal congestion during pregnancy, Clin Otolaryngol 24:307, 1999. 129. Iyengar BK: Light on Pranayama, London, 1981, Unwin Paperbacks. 130. Kristof M, Servit Z, Manas K: Activating effect of nasal airflow on epileptic electrographic abnormalities in the human EEG: evidence for the reflex origin of the phenomenon, Physiol Bohemoslov 30:73, 1981. 131. Servit Z, Kristof M, Strejckova A: Activating effect of nasal and oral hyperventilation on epileptic electrographic phenomena: reflex mechanisms of nasal origin, Epilepsia 22:321, 1981. 132. Werntz DA, Bickford RG, Shannahoff-Khalsa D: Selective hemispheric stimulation by unilateral forced nostril breathing, Hum Neurobiol 6:165,1987.

133. Werntz DA, Bickford RG, Bloom FE, et al: Alternating cerebral hemispheric activity and the lateralization of autonomic nervous function, Hum Neurobiol 2:39, 1983. 134. Clarke RW, Jones AS: Nasal airflow sensation (editorial), Clin Otolaryngol 20:97, 1995. 135. Jones AS, Lancer JM, Shone G, et al: The effect of lignocaine on nasal resistance and nasal sensation of airflow, Acta Otolaryngol 101:328, 1986. 136. Jones AS, Crocher R, Wight RG, et al: The effect of local anaesthesia of the nasal vestibule on nasal sensation of airflow and nasal resistance, Clin Otolaryngol 12:461, 1987. 137. Eccles R, Jones AS: The effect of menthol on nasal resistance to air flow, J Laryngol Otol 97:705, 1983. 138. Eccles R, Jawad MS, Morris S: The effects of oral administration of (-)-menthol on nasal resistance to airflow and nasal sensation of airflow in subjects suffering from nasal congestion associated with the common cold, J Pharm Pharmacol 42:652, 1990. 139. Isenberg C, Schafer K, Braun HA: The effect of menthol on discharge pattern of cold receptors, Pflugers Arch 400:R17, 1984. 140. Schafer K, Braun HA, Isenberg C: Effect of menthol on cold receptor activity, J Gen Physiol 88:757–776, 1986. 141. Eccles R: Menthol and related cooling compounds, J Pharm Pharmacol 46:618, 1994. 142. Eccles R, Griffiths DH, Newton CG, et al: The effects of d and l isomers of menthol upon nasal sensation of airflow, J Laryngol Otol 102:506, 1988. 143. Watson HR, Hems R, Rowsell DG, et al: New compounds with the menthol cooling effect, J Soc Cosmetic Chemists 29:185, 1978. 144. Eccles R: Role of cold receptors and menthol in thirst, the drive to breathe and arousal, Appetite 34:29, 2000. 145. Andersson KE, Bende M: Adrenoceptors in the control of human nasal mucosal blood flow, Ann Otol Rhinol Laryngol 93:179, 1984. 146. Eccles R: Nasal airflow and decongestants. In Naclerio RM, Durham SR, Mygind N, editors: Rhinitis mechanisms and management, New York, 1999, Marcel Dekker, p 291. 147. Graf P, Juto JE: Sustained use of xylometazoline nasal spray shortens the decongestive response and induces rebound swelling, Rhinology 33:14, 1995. 148. Morris S, Eccles R, Jawad MS, et al: An evaluation of nasal response following different treatment regimes of oxymetazoline with reference to rebound congestion, Am J Rhinol 1:109, 1997. 149. Blue JA: Rhinitis medicamentosa, Ann Allergy 26:425, 1968. 150. Toohill RJ, Lehman RH, Grossman TW, et al: Rhinitis medicamentosa, Laryngoscope 91:1614, 1981. 151. Flanagan P, Eccles R: Physiological versus pharmacological decongestion of the nose in healthy human subjects, Acta Otolaryngol 118:110, 1998.

152. Howarth PH: Mediators of nasal blockage in allergic rhinitis, Allergy 52:12, 1997. 153. Doyle WJ, Boehm S, Skoner DP: Physiological-responses to intranasal dose-response challenges with histamine, methacholine, bradykinin, and prostaglandin in adult volunteers with and without nasal allergy, J Allergy Clin Immunol 86:924, 1990. 154. Vayonis AG, Seroky JT, Doyle WJ, et al: Intranasal histamine, methacholine, and bradykinin challenge in children with and without allergy, Am J Rhinol 9:1, 1995. 155. Shelton D, Eiser N: Histamine receptors in the human nose, Clin Otolaryngol 19:45, 1994. 156. Secher C, Kirkegaard J, Borum P, et al: Significance of H1 and H2 receptors in the human nose: rationale for topical use of combined antihistamine preparations, J Allergy Clin Immunol 70:211, 1982. 157. Eccles R, Wilson H: A kallikrein-like substance in cat nasal secretion, Br J Pharmacol 49:712, 1973. 158. Doyle WJ, Skoner DP, White M, et al: Pattern of nasal secretions during experimental influenza virus infection, Rhinology 34:2, 1996. 159. Proud D, Reynolds CJ, Lacapra S, et al: Nasal provocation with bradykinin induces symptoms of rhinitis and a sore throat, Am Rev Respir Dis 173: 613, 1988. 160. Rees GL, Eccles R: Sore throat following nasal and oropharyngeal bradykinin challenge, Acta Otolaryngol 114:311, 1994. 161. Pongracic JA, Naclerio RM, Reynolds CJ, et al: A competitive kinin receptor antagonist (DArg0 , Hyp3 , DPhe7 )-bradykinin, does not affect the response to nasal provocation with bradykinin, Br J Clin Pharmacol 31:287, 1991. 162. Mygind N, Naclerio RM: Intranasal corticosteroids. In Naclerio RM, Durham SR, Mygind N, editors: Rhinitis mechanisms and management, New York, 1999, Marcel Dekker, p 221. 163. Schmidt BM, Timmer W, Georgens AC, et al: The new topical steroid ciclesonide is effective in the treatment of allergic rhinitis, J Clin Pharmacol 39:1062, 1999.

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Section D - Pharmacology

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Chapter 48 - Principles of Pharmacotherapeutics

William Kelly Hengameh Heidarian Raissy

This chapter gives the clinician an overview of the pharmacotherapeutic principles that affect the individual patient response to medication. Specific aspects of drugs used in the therapy of asthma and allergy are used to illustrate these principles. These examples are not meant to confer superiority of one product or one form of therapy over another, rather to convey how knowledge of these principles is used to individualize patient therapy to achieve optimal outcomes. More in-depth discussions of the pharmacology, pharmacokinetics, and pharmacodynamics are found in the individual chapters for each drug class. Factors affecting therapeutic response can be categorized broadly as either drug or patient factors. Drug factors include pharmacologic properties, structure-activity relationships, physicochemical properties (i.e., lipophilicity or hydrophilicity), formulation or delivery system, and mechanism of elimination from the body. Patient factors include both normal physiologic and pathophysiologic differences. Physiologic variables include age, genetic, and sex differences, as well as normal diurnal patterns and dietary differences. Pathophysiologic differences include disease state and disease severity, other concomitant diseases, and therapies. Figure 48-1 depicts the factors involved in determining the response to any given pharmacotherapeutic intervention.

GOALS OF THERAPY The general goal of therapy of any disease is to minimize the impact of the disease on the patient's life. This includes measures to relieve symptoms, prevent progression of disease, prevent morbidity and mortality, and minimize the potential for adverse effects of the medication. Ideally, these goals would be achieved with the most convenient and affordable medication; however, the ultimate goal is to provide the most cost-effective therapy (i.e., that therapy that provides the greatest degree of health for cost). Realistically, the ideal goal of normal health (cure of the disease) may not be achievable in either asthma or other allergic disorders; therefore, both the clinician and the patient often must make compromises to derive the best achievable outcome. Although understanding of clinical pharmacologic principles is essential to the rational use of medications, achieving optimal therapeutic outcomes requires attention to a multitude of factors, including numerous psychosocial issues that can

Figure 48-1 Factors that determine the relationship between prescribed dosage and outcome.

This equation assumes reversible binding of the drug-receptor complex and that the maximum effect (Emax ) occurs with occupation of all available receptors. The rate of association and disassociation of the drug-receptor complex are represented by k1 and k2 . The applicable Equation 48-2 or 48-3 describes the hyperbolic relationship pictured in Figure 48-2 , A, in which no drug produces no effect, 50% of the maximum effect occurs when C equals the dissociation constant for the drugreceptor interaction (Kd = k2 /k1 , also referred to as the

Figure 48-2 Representative dose-response curves. A and B represent the same curves on linear and log-linear scales, respectively. Individual curves A and B represent full agonists with different potencies and similar efficacy. Curve C represents a compound with both different potency and efficacy (partial agonist). All three curves could also represent the same agonist against different levels of functional antagonist. ED50A , ED50C , Effective dose that produces 50% of the maximal effect for either drug A or drug C (indicated by subscript letters).

2

This equation is often reparameterized to give the Hill[ ] equation that was originally described to represent the association between oxygen and hemoglobin as follows:

where the EC50 is the effective concentration that produces half of the maximal response. Figure 48-2 , B represents the effect plotted against the logarithm of the concentration. This provides a linear relationship with the log drug concentration and effect between 20% and 80% of maximal effect. A number of drugs have been characterized in this manner, including the β2 agonists and methylxanthine bronchodilators. Understanding specific aspects of the classic dose-response curves allows the clinician to derive a clear perspective of similarities and differences of drugs in similar pharmacologic classes (β2 agonists) or physiologic classes (bronchodilators). If the concentration-response curves depicted in Figure 48-2 represented three different drugs in the same pharmacologic class, drugs A and B would represent two drugs of equal efficacy but different potencies and drug C would represent a drug of both a different potency and a different efficacy or intrinsic activity (partial 3

agonist).[ ] Although drug C represents both decreased potency and only partial agonistic

791

3

efficacy compared with drugs A and B, potency and efficacy are not necessarily linked. [ ] For example, the β2 agonists albuterol and salmeterol are both partial 4

agonists, with 85% and 60% of the efficacy of isoproterenol on bronchial β2 receptors, respectively.[ ] However, salmeterol is more potent in vitro than both albuterol and isoproterenol (EC50 measurements of 53•nM, 2500•nM, and 200•nM, respectively).[

4]

In contrast, the curves in Figure 48-2 could also represent the dose-response curves of differing activities for one drug at differing receptors (β1 vs. β2 receptors) or 5

represent activity of the same drug-receptor at differing physiologic conditions such as desensitization (B vs. C).[ ] Thus, evaluating dose-response curves on different tissues and under differing conditions can provide further insight into clinical phenomena. Direct-Versus Indirect-Acting Agonists Often agonist drugs act by stimulating the production of an intracellular intermediate. The guanine nucleotide regulatory binding proteins (G proteins) comprise a 1

superfamily of these intracellular receptors.[ ] After binding to the membrane-bound portion of the receptor, a conformational change occurs, leading to intracellular binding to a G protein that then alters activity, leading to the production of a substance usually referred to as a second messenger. β2 agonists act in this manner, 1

activating the enzyme adenyl cyclase to enhance cyclic adenosine 3′, 5′-monophosphate (cAMP) production.[ ] Other drugs, such as glucocorticosteroids, first penetrate the plasma membrane then bind to a specific receptor. The activated receptor complex is translocated to the nucleus, where it works by inducing gene

6]

transcription.[

Although both β2 agonists and glucocorticosteroids obviously work indirectly at the subcellular level, indirect-acting drugs are operationally defined by whether the onset of activity occurs rapidly after receptor binding (direct) or whether there is a considerable disassociation (lag time) between receptor binding and pharmacologic response (indirect). For direct-acting agents, the onset of effect is primarily the function of the association constant (k1 , see Equation 48-1 ), assuming diffusion of the drug to the receptor site is not limited. Intensity and duration are a function of the affinity (Kd ), which can also be expressed as the drug7

receptor complex half-life.[ ] For indirect-acting drugs, the onset of effect is more complex and may relate more directly to dose and binding affinity.[

7]

Structure-Activity Relationships Many drugs are chemical modifications of endogenous hormones or mediators. Chemical structural modifications can alter selectivity, efficacy, and potency of drugs in different ways. For example, changing a catecholamine to a resorcinol (isoproterenol to metaproterenol) decreases potency without altering efficacy or selectivity. [8]

On the other hand, changing the N-terminal from dibutyl to a tertiary butyl group (metaproterenol to terbutaline) enhances β2 receptor selectivity, decreases 5 8

efficacy (partial agonist), and increases potency.[ ] [ ] These changes can also significantly alter the pharmacokinetic properties of the drugs, which may have equally profound effects on the clinical efficacy and utility. Because of asymmetric substitution on the b carbon of the side chain, all synthetic β2 agonists exist as racemic mixtures of two optical isomers (i.e., the same chemical structure but mirror images). Levorotatory substitution (R configuration), as with naturally occurring catecholamines (l-epinephrine), confers significantly 9

greater intrinsic activity because receptors are stereoselective. [ ] For example, (R)-albuterol is approximately 100 times more potent than (S)-albuterol, whereas the 9

ratio of activity for the (R) and (S) isomers of terbutaline, formoterol, and salmeterol are 1000:1, 1000:1, and 50:1, respectively.[ ] There is no evidence that the less active isomer inhibits the effect of the more active isomers when the racemic mixture is administered, as would be expected from the marked difference in potency (i. e., binding affinity). In vitro studies suggest that the therapeutically inactive isomers (distomers) of isoproterenol and albuterol may induce increased responsiveness 10

of bronchial smooth muscle, whereas the more active isomers (eutomers) are devoid of this effect (see Chapter 43 ).[ ] Examples of other asthma drugs that occur as racemic mixtures include: zileuton, budesonide, and ipratropium bromide. Finally, because most biologic systems are relatively stereoselective, each isomer may have distinctly different pharmacokinetic patterns.[

10] [11]

Clinical Correlates of Receptor Theory The concept of reversible binding and the law of mass action can be readily appreciated with drugs that act as antagonists, such as antihistamines and 12

anticholinergics. Figure 48-3 shows dose-response curves of two antihistamines against histamine-induced wheal in human skin.[ ] With increasing concentrations of histamine, any given concentration of antihistamine is less effective. This phenomenon can explain why the dosage of any given antihistamine may need to be increased to achieve a similar effect during times of a high pollen count on a purely pharmacodynamic basis, although other factors may also play a role.

The log concentration-response nature of the dose-response curves helps explain why, with drugs like theophylline with its narrow therapeutic index, increasing the dose to achieve a concentration increase from 5 to 10•mg/L results in a significant clinical improvement but that increasing from 15 to 20•mg/L may result in a minimal additional improvement despite the same absolute increase in concentration

Figure 48-3 Dose-response curves of histamine in the human skin. Peripheral inhibition of histamine-induced wheal by loratadine (L; 10, 20, 40•mg) and cetirizine (C; 2.5, 5, 10•mg) 8 hours after drug. (From De Vos C: Clin Exp Allergy 19:503–507, 1989.)

Figure 48-4 Duration of action of increasing doses of two β2 agonists in airway smooth muscle.

Figure 48-5 Schematic representation of the interrelationship of the absorption, distribution, and elimination of a drug and its concentration at its locus of action. (From Benet LZ, Kroetz DL, Sheiner LB: Pharmacokinetics. In Hardman JG, Limbird LE, Molinoff PB et al, editors: Goodman & Gilman's pharmacologic basis of therapeutics, ed 9, New York, 1996, McGraw-Hill.)

Figure 48-6 The relationship between drug concentration and therapeutic effect and toxic effect for any drug. (From Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics.)

After a single dose of a drug, the amount of drug is equivalent to the fraction of the dose that is bioavailable (F), so that the Vd determines the increase in Cp after a single dose of the drug, as follows:

The Vd is also affected by the physicochemical characteristics of the drug (lipophilicity and protein binding) and the physiologic characteristics of the patient (hydration status and pH). The principal clinical utility of the Vd is that it helps determine an appropriate loading dose of a drug. If the Vd is known, the dose to achieve any given increase in plasma concentration of a drug can be estimated. Elimination After entry into the body, the process of elimination of drugs is begun. Drugs that are relatively water-soluble (atropine or cetirizine) may be excreted unchanged, either filtered by the renal glomeruli or actively secreted by organic acid or base transport systems in the renal tubules or bile ducts (e.g., cromolyn and nedocromil). However, because of their lipophilic nature, most drugs are first metabolized to more polar compounds and then excreted. These more polar metabolites may be 39]

inactive (21- and 36-hydroxylated acids of montelukast)[

40]

or active (cetirizine, the major metabolite of hydroxyzine).[

The phase I metabolic biotransformation of drugs, which consists of introducing or exposing a functional group on the parent compound, is generally enzymatic and primarily occurs in the liver. Other organs, such as the lungs, kidneys, and gastrointestinal tract, may also serve as significant sites of metabolism. The majority of drug biotransformations are catalyzed by the cytochrome P-450 family of heme-containing membrane proteins localized in the smooth endoplasmic reticulum of tissues. Oxidative reactions catalyzed by these enzymes require the reduced form of nicotinamide adenine dinucleotide phosphate and molecular oxygen. It is now known that the cytochrome P-450 mixed-function oxidase system consists of a superfamily of enzymes (12 P-450 gene families identified in humans) with varying specificity for xenobiotic substrates.[

41]

Three cytochrome P-450 isozyme families (CYP1, CYP2, and CYP3) encode the enzymes responsible for most drug

41 biotransformations.[ ]

The phase II conjugation reactions (glucuronidation, acetylation, and sulfation) resulting from a covalent linkage generally occur in the cytosolic fraction of the cell, although some glucuronyl transferases occur in the microsomal membrane as well. Conjugation reactions may or may not follow an initial phase I biotransformation. As a result of low substrate specificity among P-450 isozymes, more than one may catalyze a given biotransformation; however, many drugs are primarily metabolized by one subfamily. For example, CYP3A4 is involved in the majority of all drug metabolic conversions, including a number of antihistamines, the 32 39 42

41

leukotriene antagonists, and corticosteroids.[ ] [ ] [ ] The level of specific enzyme activity is genetically determined.[ ] Phenotypic differences in the ability to metabolize drugs through polymorphically controlled pathways not only determines rate of metabolism but also may put some patients at greater risk of toxicity from 9

some drugs. As stated previously, enzymatic biotransformation is also stereoselective, generally favoring more rapid inactivation of the eutomer.[ ] Other factors that 43]

affect enzyme activity include disease state, age, the presence of enzyme inhibitors or inducers, and possibly gender.[ activity is illustrated in Figure 48-7

44 .[ ]

Although there is some P-450

The effect of age on metabolic enzyme

Figure 48-7 Theophylline plasma clearance in relation to age. Reflects maturity of the P-450 mixed-function oxidase system. (From Milsap RL, Hill MR, Szefler SJ: Special pharmacokinetic considerations in children. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics.)

For the vast majority of drugs, the clearance remains constant over the range of concentrations experienced clinically, even though the processes for clearing the drugs (enzymatic metabolism and active secretion) are potentially saturable processes. Thus, the amount of drug eliminated over a unit of time increases as the concentration increases, producing a constant percentage of drug lost per unit time (first-order elimination kinetics). In contrast, if saturation occurred, a constant amount of drug is eliminated per unit time (zero-order kinetics). In this case, clearance changes with serum concentrations. For chronic dosing of drugs undergoing first-order elimination, it is easy to see that, with the clearance unchanging, any change in dosing rate will result in a proportional change in steady-state concentration. Figure 48-8 depicts what occurs with serum concentrations in first-order (linear kinetics) with increasing doses, and Figure 48-9 depicts what occurs with drugs undergoing saturable elimination (Michaelis-Menten or nonlinear kinetics). Theophylline, a drug with a narrow therapeutic index, occasionally exhibits nonlinear kinetics in the usual therapeutic range of serum concentrations, particularly in children.[

18]

Figure 48-8 Representation of the relationship between dose and area under the concentration time curves (or steady-state plasma concentrations) for a drug undergoing first-order elimination (linear kinetics).

where AUC is the total area under the serum concentration time curve. Occasionally the clinician may see the term oral clearance. This refers to clearance calculated from oral dosing when absolute bioavailability is unknown and should not be confused with actual clearance. Drugs with a high first pass effect will have higher oral clearances than actual clearances; however, the oral clearance would be the more appropriate value to use in determining oral dosage regimens for the drug. Drugs undergoing first order elimination kinetics can be characterized by their half-life. The half-life is the time it takes to eliminate one half of the drug from the body or to reduce the serum concentration by one half. The elimination half-life of a drug is dependent on both of the previously discussed physiologically defined pharmacokinetic variables Vd and Cl, as follows:

where T½ is half-life, 0.693 is the natural log constant, and KE, the elimination rate constant, is the slope of the log concentration time curve. Both physiologic variables affect the half-life. The Cl determines how much of the blood is cleared of drug, and the Vd determines how much drug is presented to the clearing organ in that unit of blood. As can be seen, only changes in clearance affect steady-state serum concentrations. Changes in Vd affect the time to reach steady-state concentrations but not the final concentration that is determined by the dose and clearance. For example, a highly lipophilic inhaled corticosteroid will have the same steady-state serum concentration as a more hydrophilic one with a lower Vd if they have the same clearance. The clinical utility of the half-life is threefold. The first is to determine a reasonable dosing interval for chronically administered drug. The half-life, in conjunction with the absorption rate, determines the fluctuation of serum concentrations over the dosing interval. The elimination half-life will generally determine the

Figure 48-9 Representation of the relationship between dose and area under the concentration time curves (or steady-state plasma concentrations) for a drug undergoing saturable elimination (nonlinear kinetics).

Figure 48-10 Fundamental pharmacokinetic profile for repeated administration of a first-order eliminated drug. CL, Clearance, F, bioavailable fraction of dose; Cf, concentration of oral drug.

where Km represents the plasma concentration at which half of the maximal rate of elimination (vm ) is reached. If plasma concentrations are much less than Km , elimination will follow first order kinetics and appear linear. Steady-state can be estimated by the following equation:

As the dosing rate approaches maximal elimination rate, the denominator approaches zero and Cpss increases disproportionately (see Figure 48-9 ).

PHARMACODYNAMIC VARIABILITY With the advent of therapeutic drug monitoring and the individualizing of patient dosing in the 1970s and 1980s, it was widely assumed that the large variability in response was accounted for by pharmacokinetic differences. However, in recent years the study of pharmacokinetics in connection with pharmacodynamics has shown that pharmacodynamic variability plays an important role in interindividual and intraindividual response to drugs. Some of the factors that produce pharmacodynamic differences, such as tolerance or tachyphylaxis, have been well documented and mechanistically explained, whereas others, such as functional antagonism, the effect of inflammation on receptor activity, pharmacogenetics, and chronopharmacology, have been observed but less well investigated. Intact tissue or in vivo pharmacodynamics illustrate factors affecting response other than classic mass action receptor theory. Receptors and tissues out of the milieu of disease may respond quite differently. For instance, although asthmatic patients and intact lung tissue from asthmatic patients demonstrate bronchial hyperresponsiveness, bronchial smooth muscle from asthmatic patients isolated from the inflammatory milieu demonstrates no more responsiveness than that isolated from nonasthmatic patients.[

45]

Pathophysiologic Factors Pathophysiologic factors that alter response to drugs can include changes in both pharmacokinetics and pharmacodynamics. The pharmacokinetic changes are often obvious (e.g., decreased aerosol delivery during severe airways obstruction and decreased metabolic clearance of theophylline secondary to hypoxemia), whereas others are less obvious (e.g., increased penetration of theophylline into the central nervous system with acidosis and low serum albumin). However, it is the alteration of the concentration- or dose-response curves by pathophysiologic changes that has engendered a great deal of interest in recent years.

798

Functional antagonism is a phenomenon that has been used to explain changes in the dose-response characteristics of bronchodilators. Functional antagonism is 32

defined as the reciprocal relationship between two agonists with opposing action.[ ] Potential bronchodilators are often screened in vitro with isolated smooth muscle preparations that are contracted with low concentrations of histamine or an acetylcholine derivative. It has been shown that increasing the contractile stimulus 46 47

concentration increases the amount of the relaxing agonist required, thus shifting the dose-response curve to the right (see Figure 48-2, B ). [ ] [ ] Partial agonists are more easily antagonized than full agonists; therefore, the expression of shifting the dose-response curve is dependent on the contractile agonist used because partial contractile agonists are less likely to induce the effect.[

32]

For example, β2 agonist dose-response curves are readily shifted by methacholine but not

leukotriene D4 or serotonin despite equal degrees of bronchoconstriction.[

47]

In human bronchi, histamine and methacholine produce similar degrees of functional

48] [49]

antagonism and shift the dose-response curves of β2 agonists and theophylline to the right.[ diminished response in patients presenting with increased bronchospasm.[

Clinical studies with both theophylline and β2 agonists have shown

17] [18]

Besides the increased release and production of functional antagonists, the airways inflammation in asthma has been implicated in altering the response to both β2 1] [50]

agonists and corticosteroids through both direct and indirect mechanisms at the postreceptor level.[

It has been shown that the proinflammatory cytokines 1

released in asthma can produce heterologous desensitization to β2 agonists and vasoactive intestinal peptide.[ ] In addition, β2 receptor numbers are decreased and β2 agonist binding is decreased after antigen challenge. Studies have also shown that allergic inflammation, possibly through the production of local cytokines, reduces glucocorticoid receptor-binding affinity or glucocorticoid receptor-DNA binding.[

50] [51]

Patient Factors Given the large number of variables already discussed that may account for altered response to drugs, the potential for true interindividual differences in response may exist that may be genetically determined or as yet undefined. Interest in the effects from β2 agonist receptor polymorphism as a contributor to adverse effects from β2 agonists is an example.[

52] [53]

Thus, even in the face of very similar clinical expression of disease (although somewhat crudely defined as level of baseline

forced expiratory volume in 1 second [FEV1 ]) and consistency of delivered dose or attainment of the same serum concentration, markedly differing responses occur. 1] [43] [54]

These differences may be genetically determined, sex-based, or age-based.[

Lew and colleagues reported greater pharmacodynamic sensitivity to

methylprednisolone in women but also more rapid elimination so that the two effects counteract each other.[

43]

Chronopharmacology 55 56 57

A number of studies have suggested the timing of dosing may have a significant effect on therapeutic outcome.[ ] [ ] [ ] The body undergoes significant circadian rhythms with changes in endogenous hormone secretion. Nonasthmatic normal subjects have a circadian change in pulmonary function, and a number of asthmatic subjects have an exaggerated decrease that generally nadirs at 4 to 5am and peaks in the early afternoon. Patients with nocturnal asthma have demonstrated increased bronchial hyperresponsiveness and increased leukotriene excretion at night. The dosing of montelukast once nightly was designed to produce maximal drug concentration in the late night/early morning hours.[

39]

Studies with both oral and inhaled corticosteroids have demonstrated an improved response when 55 56

administered as a single dose at 3 pm without an increase in adrenal suppression compared with an 8 am dose.[ ] [ ] The mechanism for this diurnal variability in response to corticosteroids is still uncertain. In addition, the dose-response curve to inhaled β2 agonists is shifted to the right in patients awakening from nocturnal asthma.[

56]

Although this may be explained by either functional antagonism or cytokine down-regulation of β2 receptors, the mechanism is still unknown.

Tolerance

Tolerance or desensitization of agonist receptors with continued stimulation is a well-described phenomenon in clinical pharmacology. Desensitization of β2 receptors has been the most extensively studied and is reviewed in more detail in Chapter 49 . It is known that desensitization of β2 receptors may occur by at least three mechanisms: (1) phosphorylation, (2) sequestration, and (3) down-regulation. [

1] [8] [32]

Phosphorylation of the β2 receptors follows short-term exposure

(minutes) and results in a decreased binding affinity between drug and receptor. Sequestration also follows short-term exposure but requires drug occupancy and reverses immediately after dissociation of the drug from receptor. However, if the agonist remains coupled for a sufficient period of time (hours), down-regulation occurs in which receptor numbers diminish. Interestingly, the susceptibility of β2 receptors to down-regulation appears to be genetically determined (see Chapter 49 for more in-depth discussion). Polymorphisms of the β2 -receptor at codon 16 and codon 27 determine the extent of down-regulation of the receptors following the exposure to the β2 agonist.[

52]

The glycine substitution at codon 16 (GLY-16) form of the receptor down-regulates to a much greater extent compared with the

arginine form (ARG-16) of the receptor. [

58]

The heterozygous ARG-16/GLY-16 is intermediately desensitized compared with the homozygous GLY-16. On the 52

other hand, glutamate substitution at 27 (GLU-27) protects against down-regulation compared with the glutamine form GLN-27 of the receptor.[ ] However, the GLY-16 overcomes any protective effect of GLU-27. In addition to the receptor-based mechanisms, a post-receptor mechanism of desensitization is now believed to be likely. Enhanced breakdown of cAMP by an increase in phosphodiesterase (PDE) isozyme activity would account for the particularly rapid development of tolerance to β2 agonists in inflammatory and immunocompetent cells.[

32]

Members of the PDE-IV family of isozymes found in inflammatory cells are either directly

activated by protein kinase A or induced by activation of the cAMP-responsive elements. Increased PDE-IV levels have been found in human mononuclear 32]

leukocytes harvested from atopic individuals,[

associated with β2 -receptor dysfunction.

Clinically important aspects of β2 receptor desensitization include the following[

1] [8]

: (1) shortened duration of bronchodilator effect, (2) greater apparent

desensitization in β2 receptors in other tissues than bronchial smooth muscle

799

(which may be a function of relative receptor numbers), (3) improvement of receptor affinity and reversal of down-regulation by glucocorticoids, (4) apparent ability to easily overcome desensitization with increased dosage, and (5) apparent leveling off of degree of desensitization over time. One aspect of β2 receptor desensitization that is of interest is the greater ability of full agonists (isoproterenol) to induce desensitization than partial agonists (albuterol, salmeterol). The clinical importance of this is unknown. The ability of inhaled corticosteroids to prevent tolerance to β2 agonists has been inconsistent. Lastly, it is important to realize that, because desensitization is a receptor phenomenon, cross-tolerance is the rule; thus changing or alternating β2 agonists would not improve efficacy. Although glucocorticoid resistance occurs, it is thought to be a result of inflammation and not due to agonist-induced tolerance.[

50]

Tolerance or desensitization has

not been reported for drugs that work by inhibiting enzymatic breakdown, and theophylline, a phosphodiesterase inhibitor, has not been reported to induce tolerance. [18]

Although early studies had reported desensitization to H1 antihistamines, this phenomenon has not been confirmed in more recent investigations.[

35]

A proposed

mechanism would be up-regulation of the H1 receptor similar to that seen with β-blocker administration. The early reports may have been due to worsening of the disease or noncompliance with the antihistamine.[

35]

PHARMACOGENETICS Pharmacogenetics involves the genetic determination of pharmacokinetics and pharmacodynamics as well as the possibility of genetic polymorphisms of drug receptors affecting the severity of disease.[

53]

This section focuses on how genetics may affect the pharmacodynamics at the receptor level. The β2 receptor has nine 52]

different polymorphisms, which differ from the accepted wild type sequence by a single base change in the coding sequence of the gene.[ determine the extent of down-regulation of the

52 58 receptors.[ ] [ ]

Some polymorphisms

Recent evidence suggests that the homozygous ARG-16 and not the GLY-16 predisposes to

worsening asthma during regular use of short-acting β2 agonists. [

59]

This suggests a disconnect between the genetic determinants for β2 receptor desensitization and

β2 agonist–induced decrease in lung function. The third polymorphism is at codon 164, which can be either threonine or isoleucine. The isoleucine variant demonstrates markedly diminished agonist binding to the receptor, suggesting a significantly reduced response to any β2 agonist.[ rare and so of little consequence in the overall population. [

53]

However, this variant is very

52] [53]

Other genetic variants that determine drug response include the polymorphism of the transcription factor binding for the 5-lipoxygenase gene that reduces response to 53]

5-lipoxygenase inhibitors.[

A recent preliminary report also suggests that a variant of leukotriene C4 synthase allele is associated with an improved response to 60]

cysteinyl leukotriene antagonists.[

Investigations of polymorphisms of muscarinic, glucocorticoid, and H1 receptors have so far failed to detect functionally

53]

relevant alterations.[

DRUG INTERACTIONS Pharmacodynamic drug interactions may occur at the receptor or cellular level or at the functional level, whereas pharmacokinetic interactions are those occurring as a result of absorption, distribution, metabolism, or elimination changes. Some interactions may involve both pharmacodynamic and pharmacokinetic changes. Drug interactions may produce beneficial results, as well as potentially adverse effects. Numerous drugs with the potential for adverse consequences are frequently administered together without problems. Indeed, it is difficult to determine the risk of adverse consequences of drug interactions because the denominator (total number of patients receiving the combination) is largely unknown. Because adverse drug reactions are increasingly recognized as a significant medical problem leading to up to 300,000 hospitalizations per year in the United States, the clinician needs to be cognizant of the possibility of enhancing adverse drug reactions through drug interactions.

Case reports and evaluation of spontaneous reporting systems, such as Medwatch used by the FDA, tend to exaggerate the significance of interactions because only the more severe reactions are reported. For example, a review of the FDA's spontaneous reporting system for 48 reports of adverse events in patients who received 61

concomitant quinolone antibiotics (ciprofloxacin and norfloxacin) and theophylline reported that 35% of patients experienced seizures.[ ] On the other hand, a retrospective review of a Veteran's Affairs (VA) Medical Center population revealed that 39 patients on theophylline received 59 courses of ciprofloxacin and only 1 62

patient had an adverse outcome.[ ] This dichotomy can contribute to the skepticism of the importance of drug interactions. However, the one patient with the adverse outcome required a 13-day hospitalization at a cost of $5492, which resulted in an overall increased cost of $93 for each prescription of the potential interaction. Drugs associated with severe toxicities and a narrow therapeutic index are more likely to be associated with significant drug interactions. In addition, drug interactions are more likely to be detected with drugs whose serum concentrations are routinely monitored in clinical practice. Adverse consequences from drug interactions can also occur as a result of severe exacerbation of disease resulting from the lost effect from a drug. Many drug interactions go undetected in initial clinical trials unless there is reason to suspect a possible interaction. For instance, how many patients received the combination of erythromycin and theophylline or erythromycin and terfenadine with or without adverse consequences before the publication of the interactions? However, once a mechanism and seriousness of a drug interaction is established, newer drugs are tested for potential of producing drug interactions. Most new asthma drugs are tested against theophylline. Erythromycin can produce life-threatening interactions with theophylline (seizures and death), yet a number of patients have received the combination without any problems. This may be due to the variability of the interaction, the initial dosage of theophylline, the dosage of erythromycin, and the interpatient variability in response to a given concentration of theophylline. Two recent studies have attempted to determine the magnitude of the problem with theophylline drug interactions. The first VA study previously described looked at 913 patients receiving theophylline chronically who also had a prescription for either cimetidine (140 courses), erythromycin (93 courses), or ciprofloxacin (59 courses).[ cimetidine and one ciprofloxacin).

62]

Only 2 of 292 (0.81%) patients with potential interactions were admitted for theophylline toxicity (one

800

63

The other study evaluated the rate of exposure to theophylline drug interactions over a 1-year period in a state Medicaid population,[ ] in which 37% of patients (6619 total) taking theophylline were prescribed an interacting drug. The overall exposure rate to potential theophylline drug interactions was 17.8%. Thus, the apparent exposure to potentially clinically significant adverse drug interactions is high. Although the actual risk of a serious outcome appears low, the seriousness of the adverse effects warrants preventive measures to reduce the risk. Pharmacodynamic Interactions Pharmacodynamic interactions are often used to benefit patients, such as the use of corticosteroids to up-regulate β2 receptor numbers and improve binding affinity, the additive bronchodilation from anticholinergics, and the enhanced efficacy of adding long-acting inhaled β2 agonists to inhaled corticosteroids, which may

partially be due to the β2 agonists priming the glucocorticoid receptor. β2 -receptors that are down-regulated by isoproterenol can be up-regulated by dexamethasone. Isoproterenol causes a decrease in half-life of receptor mRNA from 12 to 5 hours, but this is more than compensated for by the addition of dexamethasone, which increases its transcription rate fourfold, resulting in a net increase in mRNA levels.[

32]

The increase in β2 -receptor mRNA occurs early after adding corticosteroids, 32

maximizing by 1 hour, but 12 or more hours is required for receptor numbers to peak at twofold to threefold greater numbers in tissue.[ ] However, pharmacodynamic interactions may be harmful, such as with the use of nonselective β-blockers in asthmatic subjects or the potential additive hypokalemia produced by diuretics and β2 agonists. On the other hand, the short-acting β-blocker esmolol has been used to treat the tachyarrhythmias seen in theophylline overdose that are at least partially mediated by catecholamine release.[

18]

Although ciprofloxacin inhibits the metabolism of theophylline, the seizures may also be due to a 62]

pharmacodynamic interaction in that quinolones inhibit γ-aminobutyric acid receptor binding, potentially lowering the seizure threshold.[ Pharmacokinetic Interactions

The vast majority of drug interactions fall into the pharmacokinetic category. Drugs that are most likely to undergo significant interactions are those drugs that are primarily eliminated by hepatic metabolism. Understanding which of the P-450 isozymes is involved in the metabolism of a drug enhances the predictive capability for interactions. For example, fluoxetine is a potent inhibitor of CYP2D6 isozyme but produces no inhibition of the metabolism of theophylline (CYP1A2 and CYP3A).[

41]

An understanding of the time course of pharmacokinetic interactions allows the clinician to appropriately monitor patients to avoid potential adverse consequences. 64

Enzyme induction requires synthesis of new enzymes, thus a 3- to 4-day delay occurs for new protein synthesis before any change is seen.[ ] The peak effect is dependent on the half-life of the inducing drug (i.e., the peak effect from rifampin with a 4- to 6-hour half-life occurs sooner than from phenobarbital with a 24- to 72hour half-life). The offset is also delayed by the time it takes to eliminate the inducer and then eliminate the excess enzymes. The maximum effect is dependent on the

Figure 48-11 Time course of change in steady-state theophylline concentrations after introduction and discontinuation of the competitive P-450 enzyme inhibitor cimetidine. Although the onset and offset begin immediately, the maximum effect requires 5 half-lives. (From Vestal RE, Thummel KE, Musser B: Br J Clin Pharmacol 15:411–418, 1983.)

Figure 48-12 Compilation of all the factors that determine the time course for pharmacodynamic response to the indirect-acting glucocorticoids. AUC, Area under the curve. kon and koff designate the drug association (on) and disassociation (off) constants for the rate of drug binding to and unbinding from receptors. (From Jusko WJ: J Clin Pharmacol 30:308–310, 1990.)

REFERENCES Pharmacodynamics

1. Liggett SB, Levi R, Metzger H: G-protein coupled receptors, nitric oxide, and the IgE receptor in asthma, Am J Respir Crit Care Med 152:394, 1995. 2. Lalonde RL: Pharmacodynamics. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics. 3. Ross EM: Pharmacodynamics. In Hardman JG, Limbird LE, Molinoff PB et al, editors: Goodman & Gilman's The pharmacological basis of therapeutics, ed 9, New York, 1996, McGraw-Hill. 4. Johnson M, Butchers PR, Coleman RA: The pharmacology of salmeterol, Life Sci 52:2131, 1993. 5. Hochhaus G, Hendeles L, Harman E, et al: PK/PD analysis of albuterol action: application to a comparative assessment of β2 -adrenergic drugs, Eur J Pharma Sci I:73, 1993. 6. Jusko WJ: Corticosteroid pharmacodynamics: models for a broad array of receptor-mediated pharmacologic effects, J Clin Pharmacol 30:308, 1990. 7. Holford NH, Sheiner LB: Pharmacokinetic and pharmacodynamic modeling in vivo, CRC Crit Rev Bioeng 5:273, 1981. 8. Nelson HS: β-adrenergic bronchodilators, N Engl J Med 333:499, 1995. 9. Waldeck B: Enantiomers of bronchodilating β2 -adrenoceptor agonists: is there a cause for concern? J Allergy Clin Immunol 103:742, 1999. 10. Page CP, Morley J: Contrasting properties of albuterol stereoisomers, J Allergy Clin Immunol 104:S31, 1999. 11. Borgstrom L, Nyberg L, Jonsson S, et al: Pharmacokinetic evaluation in man of terbutaline given as separate enantiomers and as the racemate, Br J Clin Pharmacol 27:49, 1989. 12. De Vos C: New antihistamines, Clin Exp Allergy 19:503, 1989. Pharmaceutics 13. Moren F: Pressurized aerosols for oral inhalation, Int J Pharm 8:1, 1981. 14. Kong AN, Ludwig EA, Slaughter RL, et al: Pharmacokinetics and pharmacodynamic modeling of direct suppression effects of methylprednisolone on serum cortisol and blood histamine in human subjects, Clin Pharmacol Ther 46:616, 1989. 15. Kelly HW, Murphy S: Corticosteroids for acute, severe asthma, DICP Ann Pharmacother 25:72, 1991. 16. Self TH, Ellis RF, Abou-Shala N, Amarshi N: Is theophylline use justified in acute exacerbations of asthma? Pharmacotherapy 9:260, 1989. 17. Kelly HW, Murphy S: Beta-adrenergic agonists for acute severe asthma, Ann Pharmacother 26:81, 1992. 18. Edwards DJ, Zarowitz BJ, Slaughter RL: Theophylline. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug

monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics. 19. Dolovich MA: Influence of inspiratory flow rate, particle size, and airway caliber on aerosolized drug delivery to the lung, Respir Care 45:597, 2000. 20. Fink JB: Metered-dose inhalers, dry-powder inhalers, and transitions, Respir Care 45:623, 2000. 21. Ahrens R, Lux C, Bahl T, et al: Choosing the metered-dose inhaler spacer or holding chamber that matches the patient's need: evidence that the specific drug being delivered is an important consideration, J Allergy Clin Immunol 96:288, 1995. 22. Devadason SG, Everard ML, MacEarlan C, et al: Lung deposition from the Turbuhaler in children with cystic fibrosis, Eur Respir J 10:2023, 1997. 23. Kelly HW: Comparative potency and clinical efficacy of inhaled corticosteroids. Respir Care Clin North Am 5:537, 1999. 24. Thorsson L, Edsbacker S, Conradson TB: Lung deposition of budesonide from Turbuhaler is twice that from a pressurized metered-dose inhaler P-MDI, Eur Respir J 7:1839, 1994. 25. Agertoft L, Pedersen S: Importance of the inhalation device on the effect of budesonide, Arch Dis Child 69:130, 1993. 26. Hess DR: Nebulizers: principles and performance, Respir Care 45:609–622, 2000. 27. Alvine GF, Rodgers P, Fitzsimmons KM, et al: Disposable jet nebulizers: how reliable are they? Chest 101:316, 1992. Pharmacokinetic Principles 28. Evans WE: General principles of applied pharmacokinetics. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics. 29. Food and Drug Administration: Regulations requiring manufacturers to assess the safety and effectiveness of new drugs and biological products in pediatric patients. November 1998 (Docket No. 97N-0165). 30. Benet LZ, Kroetz DL, Sheiner LB: Pharmacokinetics. In Hardman JG, Limbird LE, Molinoff PB, et al, editors: Goodman & Gilman's The pharmacological basis of therapeutics, ed 9, New York, 1996, McGraw-Hill. 31. Adkins JC, Brogden RN: Zafirlukast: a review of its pharmacology and preliminary clinical findings in patients with asthma. Drugs 54:1, 1997. 32. Jenne JW, Kelly HW. β2 agonists. In Murphy S, Kelly HW, editors: Pediatric asthma (Lung Biology in Health and Disease Series/126), New York, 1999, Marcel Dekker. 33. Lipworth BJ: New perspectives on inhaled drug delivery and systemic bioactivity, Thorax 50:105, 1995. 34. Kelly HW, Murphy S: Should anticholinergics be used in acute severe asthma? DICP Ann Pharmacother 24:409, 1990.

35. Simons FER: H1 -receptor antagonists: clinical pharmacology and therapeutics, J Allergy Clin Immunol 84:845, 1989. 36. Johnson M: Pharmacodynamics and pharmacokinetics of inhaled glucocorticoids, J Allergy Clin Immunol 97:169, 1996. 37. Vichyanond P, Irvin CG, Larsen GL, et al: Penetration of corticosteroids into the lung: evidence for a difference between methylprednisolone and prednisolone, J Allergy Clin Immunol 84:867, 1989. 38. Greos LS, Vichyanond P, Bloedow DC, et al: Methylprednisolone achieves greater concentrations in the lung than prednisolone: a pharmacokinetic analysis, Am Rev Respir Dis 144:586, 1991. 39. Seidenberg BC, Reiss TF: Montelukast- an antileukotriene treatment for asthma. In Drazen JM, Dahlen SE, Lee TH, editors: Five-lipoxygenase products in asthma.(Lung Biology in Health and Disease Series/120), New York, 1998, Marcel Dekker. 40. Simons FE, Simons KJ: Pharmacokinetic optimization of histamine H1 -receptor antagonist therapy, Clin Pharmacokinet 21:372, 1991. 41. Slaughter RL, Edwards DJ: Recent advances: the cytochrome P-450 enzymes, Ann Pharmacother 29:619, 1995. 42. Frey BM, Frey FJ: Clinical pharmacokinetics of prednisone and prednisolone, Clin Pharmacokinet 19:126, 1990. 43. Lew KH, Ludwig EA, Milad MA, et al: Pharmacodynamics and drug action: gender-based effects of methylprednisolone pharmacokinetics and pharmacodynamics, Clin Pharmacol Ther 54:402, 1993. 44. Milsap RL, Hill MR, Szefler SJ: Special pharmacokinetic considerations in children. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied Pharmacokinetics: Principles of Therapeutic Drug Monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics. Pharmacodynamic Variability 45. Cerrina J, Ladurie ML, Labet C, et al: Comparison of human bronchial muscle responses to histamine in vivo with histamine and isoproterenol agonists in vitro, Am Rev Respir Dis 134:57, 1986. 46. Karlsson AJ, Persson CG: Influences of tracheal contraction on relaxant effects in vitro of theophylline and isoproterenol, Br J Pharmacol 74:73, 1981. 47. Jenne JE, Shaughnessy TK, Druz WS, et al: In vivo functional antagonism between isoproterenol and bronchoconstrictants in the dog, J Appl Physiol 63:812, 1987. 48. McWilliams BC, Menendez R, Kelly HW, et al: Effects of theophylline on inhaled methacholine and histamine in asthmatic children, Am Rev Respir Dis 130:193, 1984. 49. van Amsterdam RGM, Meurs H, Ten Berge RE, et al: Role of phosphoinositide metabolism in human bronchial smooth muscle contraction and in functional antagonism by beta-adrenoceptor agonists, Am Rev Respir Dis 142:1124, 1990. 50. Adcock IM: Steroid resistance in asthma: molecular mechanisms, Am J Respir Crit Care Med 154:S58, 1996.

51. Spahn JD, Leung DY, Surs W, et al: Reduced glucocorticoid binding affinity in asthma is related to ongoing allergic inflammation, Am J Respir Crit Care Med 151:1709, 1995. 52. Liggett SB. β2 -adrenergic receptor pharmacogenetics, Am J Respir Crit Care Med 161:S197, 2000. 53. Hall IP: Pharmacogenetics of asthma, Eur Respir J 15:449, 2000. 54. Chandler MH, Clifton GD, Burki NK, et al: Pulmonary function in the elderly: response to theophylline bronchodilation, J Clin Pharmacol 30:330, 1990. 55. Pincus DJ, Szefler SJ, Ackerson LM, et al: Chronotherapy of asthma with inhaled steroids: the effects of dosage timing on drug efficacy, J Allergy Clin Immunol 95:1172, 1995.

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56. Beam WR, Weiner DE, Martin RJ: Timing of prednisone and alterations of airways inflammation in nocturnal asthma, Am Rev Respir Dis 146:1524, 1992. 57. Beaty R, Harman E, Molino L, et al: The dose-response of albuterol during acute attacks of nocturnal asthma, Am Rev Respir Dis 145:A66, 1992. 58. Tan S, Hall IP, Dewar J, et al: Association between β2 -adrenoceptor polymorphism and susceptibility to bronchodilator desensitization in moderately severe asthmatics, Lancet 350:995, 1997. Pharmakogenetics 59. Israel E, Drazen JM, Liggett SB, et al: The effect of polymorphisms of the β2 -adrenergic receptor on the response to regular use of albuterol in asthma, Am J Respir Crit Care Med 162:75, 2000. 60. Sampson AP, Siddiqui S, Buchanan D, et al: Variant LTC4 synthase allele modifies cysteinyl leukotriene synthesis in eosinophils and predicts clinical response to zafirlukast, Thorax 55:S28, 2000. Drug Interactions 61. Grasela TH, Dreis MW: An evaluation of the quinolone-theophylline interaction using the Food and Drug Administration spontaneous reporting system, Arch Intern Med 152:617, 1992. 62. Hamilton RA, Gordon T: Incidence and cost of hospital admissions secondary to drug interactions involving theophylline, Ann Pharmacother 26:1507, 1992.

63. Pashko S, Simons WR, Sena MM, et al: Rate of exposure to theophylline-drug interactions, Clin Ther 16:1068, 1994. 64. Brouwer KL, Dukes GE, Powell JR: Influence of liver function on drug disposition. In Evans WE, Schentag JJ, Jusko WJ, editors: Applied pharmacokinetics: principles of therapeutic drug monitoring, ed 3, Vancouver, Wash, 1992, Applied Therapeutics. 65. Vestal RE, Thummel KE, Musser B, Mercer GD: Cimetidine inhibits theophylline clearance in patients with chronic obstructive pulmonary disease: a study using stable isotope methodology during multiple oral dose administration, Br J Clin Pharmacol 15:411, 1983. 66. Lalonde RL, Koob RA, McLean WM, et al: The effects of cimetidine on theophylline pharmacokinetics at steady state, Chest 83:221, 1983. 67. Davis RL, Quenzer RW, Kelly HW, et al: Effect of the addition of ciprofloxacin on theophylline pharmacokinetics in subjects inhibited by cimetidine, Ann Pharmacother 26:11, 1992.

803

Chapter 49 - Beta-Adrenergic Agonists

Harold S. Nelson

1

2

Epinephrine by injection for the treatment of bronchial asthma was introduced in 1903 by Bullowa and Kaplan.[ ] In 1924, Chen and Schmidt[ ] observed that the actions of ephedrine, the active principle of the Chinese drug Ma Huang, which had been used for centuries in the treatment of pulmonary disease, resembled those of epinephrine. This finding led to the use of ephedrine as the first effective oral adrenergic bronchodilator. Introduced in 1941, isoproterenol was not useful by mouth because, as with all catecholamines, it was rapidly inactivated by conjugation in the gut wall as well as through the action of the enzyme catechol Omethyltransferase (COMT). Isoproterenol, however, had almost exclusively beta (β)-adrenergic stimulating properties, lacking the alpha (α)-adrenergic side effects associated with the use of epinephrine. The development of metaproterenol in 1961 offered a significant advance because it was resistant to inactivation by COMT and therefore could be used orally. The duration of action of metaproterenol by any route of administration exceeded that of isoproterenol. After Lands differentiated between beta-1 (β1 )-adrenergic and beta-2 (β2 )-adrenergic receptors, efforts were directed toward the development of drugs that had predominantly β2 -adrenergic or bronchodilator actions.

CATECHOLAMINE STRUCTURE-FUNCTION RELATIONSHIP

3

There are two major pathways of modification of the basic structure of the catecholamines[ ] ( Figure 49-1 ). The first is modification in the 3,4-hydroxyl groups on 4

the benzene ring, which are required for the action of COMT. This modification has been accomplished by repositioning the hydroxyl groups,[ ] as in metaproterenol, terbutaline, and fenoterol, where the groups are located at the 3,5-hydroxyl positions, or by substituting a hydroxymethyl group for the 3-hydroxyl, in 5

6

7

8

the case of albuterol (salbutamol),[ ] pirbuterol,[ ] and salmeterol, [ ] or substituting a formylamino group[ ] in the case of formoterol. All these modifications result in prolonged bronchodilator action. 9

4

6

More selectivity for the β2 -receptor can be achieved by increasing the bulk of the side chain. For albuterol,[ ] terbutaline,[ ] and pirbuterol, [ ] a tertiary butyl group replaces the isopropyl group of isoproterenol and metaproterenol; in the case of fenoterol the substituent (a 4-hydroxybenzyl moiety) is larger.[

10]

However, the β2

specificity of fenoterol appears to be less than for drugs with the tertiary butyl terminal group. In addition to increasing β2 -adrenergic specificity, increasing the size of the terminal-amino group substituent protects the drug against degradation by monoamine oxidase (MAO), further increasing the duration of bronchodilation.[ Two β2 -adrenergic agonists have been developed with remarkable duration of bronchodilation, still detectable after 24 hours.[

11]

4]

Salmeterol xinafoate and

formoterol fumarate closely resemble the noncatecholamine, selective β2 -adrenergic agonists in their phenylethanolamine component; however, they both possess long lipophilic side chains, with the side chain of salmeterol being considerably larger than that of formoterol.[

5] [7]

Because of its lipophilicity, formoterol is thought 12

to enter the plasma membrane, from where it is gradually released into the aqueous phase to react with the β-adrenergic receptor.[ ] Salmeterol displays even greater lipophilicity and probably does not reenter the aqueous phase. Its prolonged action is thought to result from the extended side chain reacting with a specific site within the β-adrenergic receptor, the so-called exo-site, resulting in repeated stimulation of the portion of the receptor connected with adenyl cyclase. [

7]

Modifications made after the introduction of isoproterenol have resulted in four groups of sympathomimetic bronchodilator drugs. The first group is represented by the nonselective catecholamine isoproterenol. Drugs in the second category are relatively nonselective but are noncatecholamine and therefore resistant to inactivation by COMT; metaproterenol and fenoterol are longer acting but have little increase in β2 -adrenergic selectivity over isoproterenol. The third group comprises drugs that are longer acting, are active by mouth, and show selectivity for the β2 -adrenergic receptors; this group includes terbutaline, albuterol, and pirbuterol. The fourth group is the newest category of β-adrenergic bronchodilators, represented by formoterol and salmeterol. These drugs are β-adrenergic selective and, at least when administered by inhalation, produce bronchodilation that may persist for longer than 12 hours.

SPECIFIC β-ADRENERGIC BRONCHODILATORS Catecholamines and Related Compounds Ephedrine.

13

Ephedrine is a relatively weak bronchodilator.[ ] Tablets containing fixed combinations of ephedrine and theophylline were introduced in the late 1930s and were the most popular form of bronchodilator in the United States for three decades. At present, however, no reason exists to use ephedrine or the combination tablets for treatment of asthma.

804

Figure 49-1 Structure of some β-adrenergic agonists.

Figure 49-2 Mean forced expiratory volume in 1 second (FEV1 ) as a percentage of the predicted value on the first and last days of a 12-week comparison of salmeterol, albuterol, and placebo. For the second dose, placebo was given to the salmeterol and placebo groups and albuterol to the albuterol group. (Modified from Pearlman DS, Chervinsky P, LaForce C, et al: N Engl J Med 327:1420, 1992.)

(Modified from Pearlman DS, Chervinsky P, LaForce C, et al: N Engl J Med 327:1420, 1992.) inhalation of 50••g of salmeterol is 30 to 48 minutes; maximum response is generally attained by 180 minutes; and FEV1 remains significantly above baseline for 33]

longer than 12 hours in the majority of patients[ up to 12 hours. salmeterol.[

11]

[35]

34]

( Figure 49-2 ).[

Single doses of salmeterol have effectively reduced exercise-induced bronchoconstriction for

Increased FEV1 and protection against methacholine challenge have been demonstrated at 24 hours after single doses of both formoterol and

Doses of 100••g bid, rather than the U.S.-approved dose of 50••g bid (42••g by Food and Drug Administration [FDA] designation), somewhat more 36

effectively increased morning and evening peak expiratory flow (PEF) rates and reduced daytime symptoms, but at the expense of increased side effects. [ ] Two double-blind parallel comparisons of salmeterol (42••g bid) with albuterol (180••g qid) and placebo demonstrated greater control of both daytime and nighttime symptoms, reduced requirements for rescue bronchodilators, and improvement in both morning and evening PEF rates with salmeterol versus both albuterol and 33] [37]

placebo.[

ROUTES OF ADMINISTRATION Oral Administration

Administration of a β2 -adrenergic agonist by the oral route is not optimal because the dose is limited by the side effects of tremor and palpitations. It is usually 38]

tremor that is dose limiting.[

The intensity of tremor usually declines over 2 weeks with continued administration.

The dose required to produce bronchodilation by the oral route is large compared with that required by inhalation. Terbutaline, although resistant to COMT, is variably absorbed (25% to 80%) and is conjugated in the gut wall and on first passage through the liver, resulting in bioavailability by the oral route of only 7% to 39]

26%.[

Albuterol has a similar bioavailability.

806

Several placebo-controlled studies have indicated that, when employed as a sole bronchodilator, the oral β2 -adrenergic agents do improve pulmonary function and ameliorate asthma symptoms.[

40] [41]

Both oral albuterol and oral terbutaline provide bronchodilation that peaks after 2 hours[

Oral extended-release formulations of albuterol[

44]

45]

and terbutaline[

25]

and persists for 4 to 8 hours.[

42] [43]

have been developed. Compared with sustained-release theophylline, the extended-release β 46]

agonists provide symptom control similar to that obtained with theophylline blood levels of 10 to 20••g/ml.[ Parenteral Administration

When epinephrine or one of the selective β2 -adrenergic agonists is administered by subcutaneous injection, the bronchodilator response is observed within 5 minutes, and some effect may persist for up to 3 or 4 hours, depending on the dose administered.[ subcutaneous dose, the resulting bronchodilation was equal to or somewhat greater with In England, albuterol has been employed by continuous infusions [

49]

47]

When epinephrine and terbutaline were given in the same

48 terbutaline.[ ]

in the treatment of acute asthma. In the United States, intravenous (IV) isoproterenol was

49 asthma.[ ]

occasionally used in children with refractory The similar effectiveness of IV and aerosol treatment even in severe asthma[ abandonment of IV β2 -adrenergic agonists for treatment of asthma.

51]

has generally led to

Aerosol Administration Administration of β2 -adrenergic agonists by inhalation is an appealing approach because onset is rapid and the therapeutic ratio of bronchodilation to side effects is greatly increased. The noncatecholamine β2 -adrenergic agonists generally produce 75% of maximum bronchodilation within 5 minutes, [ attained for 30 to 90 minutes.[

53]

Significant bronchodilation is usually maintained for more than 4 hours in single-dose studies[

53]

52]

but peak effect is not

but may be less with chronic

treatment.[

54] [55]

Formoterol is also rapid in onset,[

studies) may exceed 24

11 hours.[ ]

12]

but with peak bronchodilation after about 4 hours and a duration of bronchodilation that (in single-dose 33]

Salmeterol has a somewhat slower onset of bronchodilation,[

but with a similar time to peak and similar duration to formoterol.

[11]

This delay in onset with salmeterol usually is of little consequence with regular twice-daily administration because the effects from the previous dose 12 hours earlier still persist. The clinically employed aerosol doses usually produce near-maximal bronchodilation. However, when sufficiently larger doses of β2 -adrenergic bronchodilators are administered, a significant bronchodilator response can be demonstrated that is well beyond that produced by smaller doses. The response is log-linear so that to 56]

double the bronchodilation, a tenfold increase in dose is required ( Figure 49-3 ).[ acute asthma.

These higher doses would ordinarily be employed only in the treatment of severe

Systemic versus Aerosol Administration The bronchodilator response to terbutaline has been directly compared when the drug was administered intravenously or by inhalation[ by

57]

and when given orally and

58 inhalation.[ ]

Figure 49-3 Dose-response to inhaled albuterol administered by metered-dose inhaler (MDI) and intermittent positive-pressure nebulization breathing (IPPB). (Modified from Nelson HS, Spector SL, Whittsett TL, et al: J Allergy Clin Immunol 72:371, 1983.)

(Modified from Nelson HS, Spector SL, Whittsett TL, et al: J Allergy Clin Immunol 72:371, 1983.) In both studies the same degree of bronchodilation could be achieved by either route of administration, but the aerosol route resulted in fewer side effects for the

57] [58]

same degree of improvement in airflow.[ parenteral

59 60 administration.[ ] [ ]

Furthermore, it has not been possible to demonstrate any preferential effect on more peripheral airways with

With the development of the long-acting β agonists, there remains no obvious role for oral β agonists in adults.[ 62]

Oral β-adrenergic agonists may still have a limited role in children too young to employ a metered-dose inhaler (MDI) with spacer,[ mask with a

63 spacer[ ]

61]

although the use of a face

or nebulizer makes aerosol therapy generally available to all ages.

PATIENT AND DEVICE VARIABLES IN INHALATION THERAPY Metered-Dose Inhalers versus Jet Nebulizers 56

Equal bronchodilation is produced by a lower stated dose from a fluorocarbon-propelled MDI than from a pressure-driven nebulizer (see Figure 49-3 ).[ ] This greater efficiency of the MDI results from less aerosol being retained in the apparatus than in the typical jet nebulizer, where less than 50% of a 2-ml volume may 64

leave the nebulization chamber.[ ] A greater percentage of the medication is delivered from a nebulizer using a larger (3- to 5-ml) volume, but at the cost of a proportionally longer time for nebulization. Jet nebulizers are less efficient than MDIs also because nebulizers are often operated continuously, so up to two thirds of the medication may be exhausted into the room air during expiration. Commercial nebulizers differ in the percentage of volume of liquid in the chamber, which is eventually nebulized, the rate of nebulization, and the percentage of aerosol generated, which is in the respirable range (1 to 5••g). Because of these differences, the delivery rate of medication in particles the proper size for inhalation 65]

into the lungs has been shown to vary over a sevenfold range among commercially available nebulizers.[

807

Patient Errors in Use of Metered-Dose Inhalers 66 67 68 69 70

Surveys of patients' aerosol use have confirmed that improper technique is common and may reduce or cause complete loss of therapeutic effects. [ ] [ ] [ ] [ ] [ ] In surveys of asthmatic patients who had originally been instructed in the proper use of MDI aerosols, direct observation revealed unsatisfactory technique in 14% to 66 70

48%. [ ] [ ] Common errors were failure to inhale slowly or to hold the breath at the end of inhalation, poor coordination of inhalation and canister actuation, or switching from oral to nasal inhalation when the aerosol cloud enters the oral cavity. Errors of this magnitude would certainly lead to major losses in the benefits of aerosol therapy. Proper Metered-Dose Inhaler Technique

Even with proper patient technique, only a small percentage of the discharged dose is inhaled into the lungs from nebulizers or MDIs ( Box 49-1 ). Studies on the fate 71]

of radiolabeled aerosols have indicated that the major site of deposition is in the mouth and oropharynx.[

Deposition in the lung is increased by exhaling gently and

72 capacity.[ ]

completely and initiating slow inhalation from residual volume rather than functional residual Particles leaving an MDI consist largely of propellant, which must evaporate for the particles to achieve a size suitable for entering the lungs. It would be anticipated that a longer distance from actuator to oropharynx would allow more complete evaporation of propellant as well as slowing of the particles. This expectation is borne out by studies that show a twofold increase in 73]

lung delivery when MDIs are discharged 4•cm in front of the open mouth, rather than with the lips closed about the actuator.[

Impaction in the upper airway is 72 73

favored by turbulence, which increases with the rate of flow; therefore inhaling at a low flow rate over 5 to 6 seconds increases lung deposition. [ ] [ ] Because the aerosol must settle out in the lungs by gravity, a period of breath holding at the end of inspiration increases drug dose; 10 seconds appears to be optimal. With these 74]

techniques a maximum of 12% to 14% of the dose released by an MDI can be deposited in the lungs.[

An extension of the open-mouth approach is use of an extender between the MDI and the mouth. These extenders, also termed spacers or holding chambers, direct the aerosol into the mouth. The added distance results in decreased impaction velocity and increased evaporation of propellant, Box 49-1. Technique for Inhalation of Metered-Dose β-Adrenergic Aerosols 1. Shake the canister thoroughly. 2. Place the mouthpiece 4•cm in front of the open mouth, or use a spacer between actuator and mouth. Placing actuator between lips is a less effective alternative. 3. Breathe out gently and completely. 4. Discharge the inhaler while taking a slow, deep breath (ideally 5 to 6 seconds). 5. Hold the breath at full inspiration for 10 seconds.

both of which increase penetration into the lungs. A variety of designs have been studied, including tube spacers[ and without valves and reservoir-type size of the

75 device.[ ]

80 devices.[ ]

75] [76] [77]

78] [79]

and pear and conical spacers[

with

Although valves may increase efficiency in some patients, the advantage is probably small. More important is the

Greater bronchodilation has been reported with spacers 750•ml or greater in volume than with those of smaller volume. It is likely that the

volume is more important than whether the spacer is of commercial or home design.[

69]

Extensive trials have been conducted with spacer devices, leading to variable conclusions.[

81] [82]

It appears that the advantage of spacers is primarily in patients with 69

faulty inhalation technique, especially those with difficulty synchronizing activation of the inhaler with inspiration.[ ] Considering the frequency with which such problems are reported in patients who regularly employ the inhalers, spacers may benefit a significant percentage of patients.

It has been reported that a greater degree of bronchodilation results when treatments of β-adrenergic agonists from MDIs are separated by several minutes between doses rather than administered one immediately after another. However, the difference in bronchodilation between consecutive or spaced delivery has been modest at 83]

best.[

As a result of the Montreal Agreement signed in 1987, the use of chlorofluorocarbon (CFC) propellants in medical devices is scheduled to be phased out as suitable alternative propellants or devices become available. Development has followed two lines, use of hydrofluoroalkane (HFA) propellants, which do not have the same harmful effects on the ozone layer as the CFC propellants, and dry powder inhalers. β-adrenergic agonists in HFA-propellant MDIs share many of the characteristics of MDIs containing CFCs. The dry powder inhalers avoid the problems associated with coordination of activation and inhalation but do require a greater inspiratory flow rate for efficient drug delivery, although this usually does not present a problem because a common problem with MDIs is too-rapid inhalation. Patients can 84]

generally generate adequate inspiratory flow even when symptomatic. [

NONBRONCHODILATOR ACTIONS OF β2 -ADRENERGIC AGONISTS The beneficial effects of the β2 -adrenergic agonists are thought to result primarily from relaxation of bronchial smooth muscle. Nevertheless, β2 -adrenergic 85

receptors are widely distributed in the lungs and on cells associated with the asthmatic inflammatory response.[ ] In addition to relaxation of bronchial smooth muscle, as would be anticipated, other β2 -adrenergic responses have been documented that could be of additional benefit in the treatment of asthma ( Box 49-2 ). Exposure of preparations of canine trachea to β2 -adrenergic agonists leads to increased movement of chloride ions and water into the bronchial lumen.[ Agonists increase the beating frequency of cilia in human bronchial explants.[

87]

86]

β2

In healthy humans, β2 agonists have been reported to increase mucus flow; whether 88

the increase is secondary to a change in the character of the periciliary fluid, a direct effect on the cilia, or both is unclear.[ ] Patients with bronchial asthma and chronic obstructive pulmonary disease (COPD) usually have a decreased rate of mucociliary clearance. In some studies, β2 agonists have increased mucociliary clearance in patients

808

Box 49-2. Nonbronchodilator Actions of β2 -Adrenergic Agonists 1. Increased mucociliary clearance a. Increased ion and water secretion b. Increased ciliary beat frequency 2. Protection of respiratory epithelium against bacteria 3. Suppression of microvascular permeability 4. Inhibition of cholinergic neurotransmission 5. Inhibition of mediator release a. Basophils b. Mast cells c. Lung tissue 6. Inhibition of function a. Eosinophils b. Macrophages/dendritic cells c. T lymphocytes d. Bronchial epithelial cells 7. Priming the glucocorticoid receptor

with asthma,[

88] [89]

whereas in others they have not increased clearance.[

90] [91]

Overall the clinical importance of this action of the β2 -adrenergic bronchodilators is

not clear. In vitro studies with respiratory epithelium cultures have demonstrated protection by salmeterol against mucosal damage and cilia shedding caused by Pseudomonas aeruginosa[

92]

and Haemophilus influenzae.[

93]

Increased vascular permeability and mucosal edema are thought to contribute importantly to airflow obstruction, and transudated plasma proteins contribute to the tenacious sputum characteristic of asthma. The level of α1-macroglobulin in induced sputum after inhalation of histamine was used to assess vascular permeability. With placebo treatment, levels were increased 30 minutes after histamine, and these levels were reduced by about 50% after formoterol.[

94]

The effect of the β2

agonists was probably through β2 -adrenergic receptors on the endothelial cells. When human bronchial rings are contracted in vitro by electrical stimulation, inhibition of the contraction by β2 -adrenergic agonists is 10 to 100 times greater than when the bronchial rings have been contracted by methacholine.[

95]

It has been suggested that the greater inhibition of the contraction produced by electrical

stimulation by β2 agonists is caused by modulation of cholinergic transmission by prejunctional β2 -adrenergic receptors. In vitro studies have demonstrated that the β2 -adrenergic agonists decrease antigen-induced release of mediators from human basophils[

96]

and mast cells.[

97]

In

chopped human lung preparations, the doses required for inhibition of mediator release are of the same order as required for in vitro relaxation of human bronchial smooth muscle [

98]

and are consistent with levels attained clinically.[

99]

Not only does it block histamine release, but salmeterol also has been shown to block release

of leukotrienes C4 and D4 (LTC4 , LTD4 ) and prostaglandin D2 (PGD2 ) from human lung fragments[ [101]

100]

and tumor necrosis factor alpha (TNF-α) from mast cells.

It should be noted, however, that therapeutic doses of β2 -adrenergic agonists do not ordinarily suppress immediate skin tests.[

102]

Inhibition of mediator release by β2 -adrenergic agonists is thought to contribute to their blocking the bronchoconstrictor response to exercise, cold dry air 103]

hyperventilation, and osmolar challenge.[

Support for an in vivo suppression of mast cell mediator release comes from the demonstration that β2 agonists

suppress the bronchoconstriction induced by adenosine monophosphate (AMP), which acts indirectly by release of mast cell mediators, to a greater degree than that 104]

produced by methacholine, which acts directly through neural pathways.[

Beta-2-adrenergic receptors are present on eosinophils, bronchial epithelial cells, alveolar macrophages, monocytes, and dendritic cells. In vitro studies have not shown suppression of secretory or other activity of eosinophils by β2 agonists.[ increase in serum[

106]

or sputum [

107]

105]

The long-acting inhaled β2 agonists have had no effect on the allergen-induced

eosinophils, but have reduced eosinophil cationic protein (ECP) levels in plasma after allergen challenge. Eight weeks'

treatment with formoterol significantly reduced elevated submucosal eosinophils on bronchial biopsy.[

108]

Bronchoalveolar lavage (BAL) performed before and after 109]

4 to 8 weeks of regular treatment with salmeterol revealed no effect on total cells, mast cells, eosinophils, lymphocytes, or activation markers on lymphocytes.[ [110] [111]

However, BAL fluid levels of ECP and oxidative metabolism of stimulated alveolar macrophages were reduced.[

production of interleukin-12 (IL-12) by human monocytes and dendritic cells.[ inhibit thromboxane B2 (TBX2) release from alveolar macrophages, [

113]

112]

109]

β2 agonists inhibited in vitro the

Other cytokines were not suppressed. The β2 agonists have also been shown to

allergen-induced mononuclear cell proliferation, secretion of granulocyte-macrophage

colony-stimulating factor (GM-CSF), human leukocyte antigen-DR (HLA-DR) surface antigen expression,[ 115 cells,[ ]

114]

interferon gamma (IFN-γ) and interleukin-4 (IL-4) 116

production by human peripheral blood mononuclear and TNF-α production by lipopolysaccharide-activated T helper precursor cells. [ ] Formoterol added to TNF-α–stimulated bronchial epithelial cells reduced GM-CSF production but increased release of IL-8. When formoterol was combined with budesonide, there was additive suppression of GM-CSF, and IL-8 production was suppressed.[

117]

Although these effects on activation of inflammatory cells may be useful, they should not be considered to be antiinflammatory actions that can replace those of inhaled corticosteroids (ICS). The favorable clinical results with the combination of long-acting beta agonists (LABAs) and ICS led to in vitro investigations of their possible interactions. These studies found that the LABAs, when added to suboptimal concentrations of ICS, resulted in enhanced translocation of the glucocorticoid

118

receptor into the nucleus of cells.[ ] The mechanism appears to be through priming of the glucocorticoid receptor by mitogen-activated protein kinases (MAPKs) generated as a result of prolonged stimulation of the β2 -adrenergic receptor.

ADVERSE EFFECTS Pharmacologically Mediated Side Effects The principal side effect of β2 -adrenergic therapy is tremor, which is caused by direct stimulation of β2 -adrenergic receptors in skeletal muscles ( Box 49-3 ). Tremor is inseparable from the bronchodilator action but does decrease significantly over 2 weeks of continuous therapy.[

119]

809

Box 49-3. Adverse Reactions Reported with Use of β-Adrenergic Agonists

Side Effects Consistent with Pharmacologic Activity Tremor Increased heart rate; palpitations Prolonged QTc interval; arrhythmias Myocardial ischemia Transient increase in hypoxia Hyperglycemia Hypokalemia Hypomagnesemia

Central nervous system stimulation and seizures

Adverse Reactions Not Attributable to Pharmacologic Actions Paradoxical bronchoconstriction Bronchodilator subsensitivity

Controversial Associations with β Agonists Epidemics of increased deaths from asthma Worsening of asthma with regular use • Decline in pulmonary function • Decreased asthma control and increased exacerbations - Inhibition of receptor-steroid DNA binding - Increased airway inflammation - Increased IgE synthesis • Loss of bronchoprotection • Increased bronchial hyperresponsiveness - Effect of S-enantiomer

Increased heart rate and palpitations are less common with the selective β2 -adrenergic agonists than with the nonselective β agonists, such as isoproterenol, fenoterol, and metaproterenol. Nevertheless, these effects are to some degree inseparable from bronchodilator activity because increased heart rate and palpitations can result from β2 -adrenergic relaxation of the vasculature supplying the skeletal muscles, resulting in decreased peripheral resistance and secondarily in reflex sympathetic stimulation of the heart.[ 121]

of those in the right atrium[

120]

In addition, the β2 -adrenergic receptors in the myocardium (14% of the β-adrenergic receptors in the left ventricle and 26%

) may contribute directly to the increased heart rate and cardiac output.

122] [123]

All β2 -adrenergic agonists increase the QTc interval,[

with the non-β2 -selective agonist fenoterol increasing the interval to a greater extent than the selective 122]

β2 agonist albuterol at doses producing equal bronchodilation.[

In medical conditions not related to β2 -adrenergic agonist therapy, this delay in depolarization has 124]

been associated with ventricular arrhythmias and sudden death in patients with ischemic heart disease as well as in apparently healthy individuals.[ selective β2 -adrenergic compounds have a potential for inducing arrhythmias

[125] [126]

Even the

in susceptible individuals.

Studies in animals treated with β-adrenergic agonists alone or in combination with theophylline have demonstrated arrhythmias and focal myocardial necrosis.[

127]

128]

Even selective β2 agonists have produced myocardial ischemia in susceptible individuals. [

Administration of β-adrenergic agonists or aminophylline may cause a transient fall in arterial oxygen tension (Pao2 ) of greater than 5•mm Hg in up to 50% of 129

patients with asthma.[ ] Bronchial obstruction in asthma is not uniform, and there is normally compensatory vasoconstriction of the pulmonary arteries that perfuse underventilated segments. Dilation of these vessels due to a direct β2 -adrenergic effect on the vessels plus β2 -induced increases in cardiac output can increase perfusion of underventilated areas, leading to fall in Pao2 . Although usually not profound and of brief duration, the frequency with which this fall in Pao2 is observed suggests precautionary administration of oxygen-enriched air before beginning intensive therapy with β2 -adrenergic agonists in acutely ill patients. When a patient is initially placed on β2 -adrenergic stimulants, there are a number of metabolic responses to which tolerance rapidly develops with continued 130 131

administration.[ ] [ ] Hyperglycemia occurs from glycogenolysis, but the response declines rapidly with chronic stimulation and has not caused problems even in β-adrenergic agonist treatment of diabetic patients. Hypokalemia results from direct stimulation of the Na+ -K+ pump in the cell membrane. Although problems have not been recognized with chronic therapy, administration of therapeutic doses of β2 agonists by nebulizer or injection, or multiple doses from MDIs has produced decreases in serum potassium of 0.4 to 0.9•mmol/L lasting up to 1 hour.[

132] [133]

This degree of fall, particularly in patients who are already relatively hypokalemic

from ICS or diuretics, and particularly in a setting of hypoxemia, could increase the potential for serious arrhythmias.[ urinary excretion of magnesium, [

135]

136]

which may also move intracellularly, as does potassium.[

134]

β2 -adrenergic stimulation increases

Very modest decreases in serum Mg++ have been recorded during

intensive β2 -adrenergic therapy. Patients were studied while receiving chronic oral β agonist therapy and again 2 months after discontinuation of therapy. [

137]

Serum potassium and skeletal muscle magnesium levels were reduced both while the patients were taking and after they had discontinued oral β agonists. Skeletal muscle potassium, on the other hand, was normal on both occasions. These findings suggest that these electrolytes are not likely to be affected by regular use of inhaled β2 -adrenergic agonists. β2 -Adrenergic bronchodilators, except for ephedrine, are not thought to gain entrance to the central nervous system (CNS). The nervousness reported by some patients is thought to arise from stimulation of receptors in the peripheral muscles. Significant CNS symptoms or convulsions were not observed in 40 patients with

acute overdoses of albuterol.[

138]

However, seizures were reported in a 7-year-old girl treated with oral terbutaline. [

139]

β2 Agonists, unlike theophylline, do not stimulate gastric acid secretion and thus do not present a problem in treating patients with a history of peptic ulcer disease. [140]

However, when employed systemically, β-adrenergic agonists do, like theophylline, relax the gastroesophageal sphincter.

There are sporadic reports of the occurrence of sudden severe bronchospasm during or immediately after use of inhaled β-adrenergic agonists. An analysis of reports received by the Center for Drug Evaluation and Research of the FDA revealed that two thirds were associated with use of MDIs and one third with nebulizer treatments.[

141]

The episodes often

810

occurred with the first use of a new canister or bottle of nebulizer solution. The bronchospasm was often accompanied by symptoms suggesting a systemic reaction, such as nasal congestion, flushing, pruritus, warmth, or laryngeal stridor. The episodes usually subsided spontaneously, rarely resulting in respiratory arrest or intubation. One can only speculate about the relation of these symptoms to the drug, to other components of the medication, or to underlying disease. It seems likely that at least some of these bronchospasm episodes represented acute vocal cord adduction triggered by the irritation of the aerosol cloud. 142]

Other adverse reactions have been documented with aerosols, particularly nebulizer solutions.[ hypoosmolarity or hyperosmolarity of the cells and release

145 mediators.[ ]

143 144 solutions[ ] [ ]

Among the documented mechanisms have been marked

and the presence of preservatives in the solutions,[

144] [145]

some of which may act directly on mast

Six patients who reported acute bronchospasm after using salmeterol were tested with inhalations of placebo, albuterol, and 146

salmeterol by both MDIs and dry powder inhalers. [ ] Acute bronchoconstriction was reproduced after use of the placebo and salmeterol MDIs, but not after albuterol (presumably because the more rapid onset of bronchodilation with albuterol was protective) and not after salmeterol from a dry powder inhaler (since the reaction was presumably triggered by the propellant or an excipient). Subsensitivity Subsensitivity, or tolerance, is a diminished responsiveness that develops in many physiologic systems when receptors are repeatedly exposed to increased levels of their agonist. Subsensitivity should be differentiated from the tachyphylaxis occurring with drugs such as ephedrine that have action that partly depends on displacement of norepinephrine from sympathetic nerve endings. Tachyphylaxis is caused by exhaustion of the displaceable norepinephrine and occurs rapidly, as the term implies. Subsensitivity to β2 -adrenergic agonists, on the other hand, is probably a result of down-regulation of the β-adrenergic receptors and develops over 147]

days or weeks. [

148] [149]

Although some investigators have failed to observe bronchodilator subsensitivity in the course of long-term administration of β-adrenergic bronchodilators, [ 53] [54]

other studies have established its occurrence beyond any reasonable doubt.[

Down-regulation and decreased β-adrenergic responsiveness of the bronchial 150

epithelial cells has also been demonstrated after 7 days of inhaled albuterol (180••g qid).[ ] To demonstrate adrenergic subsensitivity, it is necessary to have a period with reduced or no β2 -adrenergic exposure, usually for 2 to 4 weeks. The loss of bronchodilator response affects the duration of action more than the peak effect attained.[

53] [54]

This finding may explain why the cumulative dose-response curve is often not altered by chronic β agonist therapy.[

131] [132]

The clinical significance of this loss of bronchodilator response may be debated. If the duration of time that the FEV1 remains 15% above baseline is used as a measure of continuing significant bronchodilation, this is decreased by approximately 50% for the noncatecholamine β2 -adrenergic bronchodilators. On the other hand, the peak bronchodilator effect is better preserved, and thus the short-acting β2 -adrenergic bronchodilators continue to perform well as rescue medications even after development of subsensitivity. More important for current therapeutic practices is the question of subsensitivity developing to the LABAs. Studies employing the LABA formoterol suggest that the initial bronchodilation is not fully retained with chronic administration.[ 151 Pauwels[ ]

31] [33] [151]

In the studies by FitzGerald[

31]

and

the initial bronchodilation observed with 12••g bid of formoterol was not fully sustained, but after a small initial decline, the subsequent level of

bronchodilation was maintained over 6 and 12 months without further loss ( Figure 49-4 ). In the study by Bensch et al[

32]

the area under the curve of FEV1 over 12

hours was unchanged from the initial value after 12 weeks for the 12-•g dose but was significantly reduced with dosing of 24••g bid. Subsensitivity to the bronchodilator action of salmeterol has not yet been demonstrated. Related to the question of β agonist subsensitivity is the concern that chronic use of the LABAs might blunt the response to short-acting β2 agonists, creating a problem during exacerbations of asthma. This question has been addressed by studying dose-response curves to albuterol during chronic salmeterol administration [152]

( Figure 49-5 ),[

153]

[154]

The first two studies showed clearly that there was no reduction in the absolute values for FEV1 attained after 1 and 12 months of regular use of salmeterol. The

as well as by examining the response to albuterol in the emergency department (ED) in patients chronically using or not using salmeterol.

ED study confirmed that, even in the setting of severe asthma, there was no impairment in the response to nebulized albuterol. Loss of Bronchoprotection Bronchoprotection should be distinguished from bronchodilation. Bronchoprotection refers to the ability of pretreatment with β2 -adrenergic agonists to prevent bronchoconstriction due to a variety of stimuli, including histamine, methacholine, hyperventilation with cold dry air, and exercise. It appears well established that, if there has been a β agonist–free period, the bronchoprotection provided by the first dose of a β agonist will not be duplicated subsequently during the course of regular β agonist therapy[

155] [156] [157] [158] [159]

and to apply to protection against exercise[

157]

as

( Figure 49-6 ). This has been shown to apply to short-acting[

159]

and long-acting[

155] [156] [157] [158]

β2 agonists

Figure 49-4 Changes in mean peak expiratory flow in the morning during the run-in period (1600••g of budesonide [BUD] per day), on days 1 through 14, and at months 1 through 12 of treatment with 200••g (low dose) or 800••g (high dose) per day with or without 12••g of formoterol (FOR). (Modified from Pauwels RA, Lofdahl CG, Postma DS, et al: N Engl J Med 337:1405, 1997.)

(Modified from Pauwels RA, Lofdahl CG, Postma DS, et al: N Engl J Med 337:1405, 1997.)

811

Figure 49-5 Cumulative dose-response to albuterol before and after 1 month of regular treatment with salmeterol or with placebo. Half the patients were receiving regular inhaled corticosteroids (ICS), and half received only as-needed ipratropium. There were no significant differences in the response to albuterol. (Modified from Nelson HS, Berkowitz RB, Tinkelman DA, et al: Am J Respir Crit Care Med 159:1556, 1999.)

(Modified from Nelson HS, Berkowitz RB, Tinkelman DA, et al: Am J Respir Crit Care Med 159:1556, 1999.)

Figure 49-6 Changes in logarithm PC20 for methacholine in patients receiving treatment with formoterol and after cessation of treatment. *p 10 puffs

Budesonide Turbuhaler

200–400••g

400–600••g

>600••g

• 200••g/puff

1–2 puffs

2–3 puffs

>3 puffs

Adults

Flunisolide

500–1000••g

1000–2000••g

>2000••g

• 250••g/dose

2–4 puffs

4–8 puffs

>8 puffs

Fluticasone propionate

88–264••g

264–660••g

>660••g

• MDI: 44, 110, or 220••g/puff

2–6 puffs (44••g)

2–6 puffs (110••g)

>6 puffs (110••g)

2 puffs (110••g) • DPI: 50, 100, or 250••g/dose

2–6 inh (50••g)

>3 puffs (220••g) 3–6 inh (100••g)

>6 inh (100••g) >2 inh (250••g)

Triamcinolone acetonide

400–1000••g

1000–2000••g

>2000••g

• 100••g/puff

4–10 puffs

10–20 puffs

>20 puffs

Beclomethasone dipropionate

84–336••g

336–672••g

>672••g

• 42••g/puff

2–8 puffs

8–16 puffs

>16 puffs

• 84••g/puff

1–4 puffs

4–8 puffs

>8 puffs

Budenoside Turbuhaler

100–200••g

200–400••g

>400••g

1–2 inh

>2 inh

Children

• 200••g/puff Flunisolide

500–750••g

100–1250••g

>1250••g

• 250••g/dose

2–3 puffs

4–5 puffs

>5 puffs

Fluticasone propionate

88–176••g

176–440••g

>440••g

• MDI: 44, 110, or 220••g/dose

2–4 puffs (44••g)

4–10 puffs (44••g)

>4 puffs (110••g)

2–4 puffs (110••g)

>2 puffs (220••g)

• DPI: 50, 100, or 250••g/puff

2–4 inh (50••g)

2–4 inh (100••g)

>4 inh (100••g)

Triamcinolone acetonide

400–800••g

800–1200••g

>1200••g

• 100••g/puff

4–8 puffs

8–12 puffs

>12 puffs

MDI, Metered-dose inhaler; DPI, dry powder inhaler; inh, inhalations.

895

portions of the lung.[

490]

As a result, lower doses of BDP using an HFA-containing MDI provide equivalent to superior efficacy compared with BDP delivered using 491

the traditional CFC propellants. [ ] At present, Qvar is the only available inhaled GC (BDP) in an HFA-containing MDI. Phase 3 studies are currently underway using FN and FP in an HFA-containing MDI. The device itself also significantly contributes to delivery of the drug to the lower airway. For example, budesonide delivered via the DPI (Turbuhaler) results in twice the lower airway deposition of the MDI.[

492]

The use of a spacer device with MDIs can significantly enhance the delivery of the drug into the lower airways

while decreasing the amount of drug deposited on the oropharynx.[ an inhaled GC delivered through an MDI.

493] [494]

In this way, a simple holding chamber can effectively improve the therapeutic index of

Bioavailability.

The systemic bioavailability of an inhaled GC is the sum of the bioavailability from both the oral and pulmonary routes. Oral bioavailability results from absorption of the GC from the GI tract. There is a wide range of oral bioavailability among the available inhaled GCs, with values ranging from less than 2% for FP to 23% for 495 496

TAA.[ ] [ ] Pulmonary bioavailability comes from the drug absorbed from the lung. Because all GC preparations delivered to the lower airway are absorbed, they eventually enter into the systemic circulation and contribute to the total bioavailability. Because 99% of FP undergoes first-pass metabolism, FP's systemic bioavailability comes almost exclusively from the pulmonary route, whereas for drugs such as BDP, TAA, and FN, which have significantly less first-pass metabolism, both oral and pulmonary absorption accounts for their systemic bioavailability. Clearance, Volume of Distribution, and Elimination Half-life.

497]

All the inhaled GCs display rapid systemic clearance.[

As a result, the elimination half-life values of these compounds depend more on the volume of distribution 498]

than clearance. The volume of distribution is a measure of tissue distribution that is related to the lipophilicity of the drug. [

Because FP is among the most

492 496 499 500 distribution.[ ] [ ] [ ] [ ]

lipophilic GCs, it has the greatest volume of The elimination half-life of an inhaled GC depends on both the systemic clearance rate and the volume of distribution. Because of the large volume of distribution noted with FP, this compound also displays the longest elimination half-life at 7.8 hours. [499]

493 501 502

The other inhaled GCs have elimination half-life values ranging from 0.1 to 0.2 hour for BDP to 2.8 hours for BUD.[ ] [ ] [ ] Because the elimination halflife of FP is much longer than for the other drugs, it takes longer for FP to reach steady-state levels. It should be noted that for equal amounts of any drug absorbed, the resulting steady-state concentrations will be similar.[

497]

503 504 505

FP's larger volume of distribution and a longer terminal half-life have been used to explain its greater ability to suppress the HPA axis [ ] [ ] [ ] compared with BUD, especially with doses exceeding 1000••g/day. Although the greater volume of distribution and longer terminal half-life of FP may contribute to its greater propensity to suppress the HPA axis, a large volume of distribution does not necessarily imply a greater potential for systemic effects, because GCs circulate primarily in an inactive protein-bound form. The active unbound form is independent of the volume of distribution, with clearance and extent of protein binding the most important variables. FP's ability to suppress cortisol production, especially at higher doses, may also be explained by the observation that FP binds to the GR

with greater affinity than the other inhaled GCs. Pulmonary Retention.

The longer the drug is available within the airway, the greater its ability to inhibit inflammation. Thus drugs with longer pulmonary retention times are likely to 26

display increased efficacy. Retention appears to be related to lipophilicity; the more lipophilic the GC, the greater the retention time. Esmailpour et al[ ] investigated the retention time and distribution of FP between central and peripheral lung tissue and serum in vivo by having 17 subjects undergoing lobectomy for bronchial carcinomas inhale FP (1•mg) from 2.8 to 21.6 hours before surgery. The investigators found FP in the peripheral lung tissue for up to 16 hours after a single administration. In addition, the concentration of FP in the central lung tissue was three to four times that in the peripheral lung tissue, which in turn was approximately 100 times greater than that found in the serum. Although BUD is less lipophilic than FP, recent in vitro studies have suggested that BUD's retention time is as long or longer than FP. Budesonide forms long-chain fatty acid conjugates within the airway epithelial cells. These BUD conjugates result in budesonide remaining in the lung for longer periods.[

506] [507]

Glucocorticoid Receptor Binding Affinity.

The affinity with which a GC binds to its receptor (GR) also contributes to its effectiveness. In general, GCs that bind the GR with the greatest affinity have the greatest antiinflammatory actions. FP and mometasone furoate bind the GR with greatest affinity, followed by BMP (the active metabolite of BDP), BUD, TAA, and FN.[

497]

Efficacy in Children and Adults Multiple studies have demonstrated inhaled GC therapy to be effective in children with all levels of asthma severity. This discussion highlights three placebocontrolled studies evaluating the efficacy of inhaled GCs in children with persistent asthma. In the Dutch Chronic Non-Specific Lung Disease (DCNSLD) Study in 508

1992,[ ] 116 children with moderately severe asthma were randomized to receive either BUD (600••g/day) or placebo in addition to regularly administered salbutamol (0.2•mg) three times daily over a 22-month period. The children receiving BUD had a 1.4 doubling-dose decrease in BHR at 4 months of therapy that continued to decrease throughout the study. BUD use was also associated with an 11% improvement in prebronchodilator FEV1 after 2 months of therapy, with a difference of 14% by 12 and 22 months compared with placebo. In addition, 40% of the children randomized to placebo required withdrawal from the study secondary to poor asthma control, and almost 50% required at least one oral GC burst, whereas only 14% of BUD patients required prednisone for poor asthma control. This study has been criticized in that all patients received regularly administered inhaled β2 agonists, which may have interfered with the reported results. In addition, more than 50% of the subjects had been receiving inhaled GCs before randomization into the study.

896

Figure 52-13 Geometric mean provocation concentration (PC20 ) values for methacholine, measured at 12 hours after inhalation of the study medication in children with asthma randomly assigned to treatment with beclomethasone, salmeterol, or placebo. Primary outcome measure, protection against methacholine-induced bronchoconstriction, was assessed at baseline and follow-up (2 weeks after end of treatment) and at 3, 6, 9, and 12 months. When PC20 for methacholine was measured 12 hours after inhalation of the study medication, protection afforded by beclomethasone (expressed as change from baseline value) was significantly greater than that afforded by placebo at 3 months (P = 0.01), 6 months (P 50%; 15% BA

Height: BDP 157.1•cm vs theophylline 158.6•cm Mean change in height: BDP 4.4•cm/yr vs theophylline 6.0• cm/yr; male predominance

reversibility Mean FEV1 74.1% Prior IGC use: 70%; 10% BA reversibility Mean FEV1 89% No prior IGC use: 55%

510

CAMP group[ ] (2000) 60 months: multicenter, DB, PC, RZ

BUD: DPI (Turbuhaler) 200• •g, necrodomil 8••g, or placebo bid

1041 subjects 8.9•yr/5–12•yr

Mild/moderate asthma PC20 ≤12.5•mg/ml Mean FEV1 94% No prior IGC use: 64%

Growth significantly slower in BDP (−0.28 SDS) vs salmeterol group (− 0.03 SDS; P = 0.001) Mean increase 6.1 vs 4.7•cm in salmeterol and BDP groups Linear growth: BDP 3.96•cm vs salmeterol 5.40•cm (P = 0.004) vs placebo 5.04•cm (P = 0.02)•cm Growth suppression most prominent in first 3•mo Mean height increase: placebo 6.15•cm; FP (50••g) 5.94•cm; FP (100••g) 5.73•cm (P = 0.308) Growth velocity: placebo 6.10• cm/yr; FP (50••g) 5.91•cm/yr; FP (100••g) 5.67•cm/yr Mean height increase at 48• mo: BUD 22.7•cm; nedocromil 23.7•cm; placebo 23.8•cm Growth velocity most affected in first year; no difference by end of study

DB Double blind; RZ, randomized; PC, placebo controlled; BDP, beclomethasone dipropionate; BUD, budesonide; FP, fluticasone propionate; qid, four times daily; tid, three times daily; bid, twice daily; MDI, metered-dose inhaler; DPI, dry powder inhaler; FEV1′ , forced expiratory volume in 1 second; PC20′ , provocation concentration to lower baseline FEV1 by 20%; BA, beta (β-) agonist; SDS, standard deviation score; GC glucocorticoid; mo, months. † With 80 matched nonasthmatic controls. * Growth was primary outcome variable.

901

Figure 52-15 Mean height at baseline and after 3, 6, 9, and 12 months of glucocorticoid treatment. Overall, during months 1 through 12, mean increase in height was 3.96•cm in beclomethasone dipropionate (BDP) group (square), 5.04•cm in salmeterol group (triangle), and 5.04 in placebo group (circle). Effect of BDP on height appeared to be greatest during months 1 through 3. After this period, slopes of the lines were parallel for three treatment groups. Effect of BDP on growth differed significantly from the effect of placebo at 6 months (P = 0.002), 9 months (P Th1 profile

DTH(?) (1°)

LT/PHA, LT/Ag, IL-2

Thymic involution

T cell subset alteration

••Immunization

*

DTH, Delayed skin test hypersensitivity; LT/PHA, decreased mitogen-induced lymphocyte proliferation; LT/Ag, decreased antigen-induced lymphocyte proliferation; IL-12, decreased interleukin-12 production; NK, decreased natural killer cell activity; CH, contact hypersensitivity; MLR, decreased mixedlymphocyte responses; IL-2, decreased interleukin-2 production; recall, decreased responses to anergy panel skin tests; 1°, decreased sensitization to dinitrochlorobenzene (DNCB); Th1, Th2, T helper cell types 1 and 2. * Decreased CMI response after immunization with certain antigens. † May be associated with inhibitory serum factors.

986

with a measles virus–induced decrease in monocyte production of the IL-12 required for Th1 responses.[ also likely occurs. NK function is also altered, with reduced cytotoxic reactivity.

150]

A direct inhibitory effect of the virus on T lymphocytes

Decreased DTH skin test reactivity has been found during the first several weeks of infectious mononucleosis, accompanied by decreased lymphocyte responses to 152

PHA, antigen, and allogeneic cells, with evidence for decreased IL-2 production after stimulation through the CD3 pathway.[ ] There is also increased relative frequency of CD8+ T cells and suppressor T cell activity. These CD8+ cells account for the characteristic increased numbers of atypical lymphocytes in the blood and in the paracortical areas of lymph nodes and spleen. These cells are also likely responsible for the impaired pokeweed mitogen (PWM)-stimulated B cell responses seen in these patients. After primary infection, Epstein-Barr virus (EBV), the cause of most cases of infectious mononucleosis, persists throughout life in resting memory B cells of asymptomatic individuals. This dormant EBV virus may be reactivated on breakdown of cellular immunity, possibly from impaired 152

153

cytotoxic T cell control of the EBV-infected B cells.[ ] This reactivation may result in uncontrolled infection and rarely lymphoma.[ ] By contrast, development of an excessive T cell effector response to EBV may dismantle normal B cell function, rendering the individual hypogammaglobulinemic. Prolonged Depression of Cell-Medicated Immunity Hodgkin's Disease

Prolonged anergy secondary to systemic disease has probably been studied most extensively in Hodgkin's disease (HD) ( Table 57-6 ). Partial depression of CMI 154

frequently occurs despite apparent clinical remission.[ ] In one study, 66% of HD patients reacted to at least one and 23% reacted to two of six recall DTH skin test antigens, compared with such reactivity in 100% and 66% of age-matched normals, respectively. Although findings are somewhat contradictory, DTH responses are generally depressed more often in those with more advanced disease, particularly when there is the lymphocyte-depleted (histiocytic) pattern in lymphoid tissues and associated blood lymphopenia. Lymphopenia is apparently caused mainly by decreases in CD4+ cells, with decreased CD8+ cell levels seen in more advanced disease. There is also decreased NK cell activity during active disease, but not in remission. Also, decreased capacity for primary CMI sensitization is seen when using suboptimal doses of agents such as DNCB.

TABLE 57-6 -- Prolonged Depression of Cell-Mediated Immunity (CMI) Condition

In Vivo Defect

In Vitro Blood Cell Defect

Hodgkin's disease

DTH (recall, 1°)

LT/PHA, LT/Ag, MLR, NK

Allograft rejection, CH

CD4, IL-2, suppressor cells

*

Defective T cells Sarcoidosis

DTH (recall, 1°); CH

LT/PHA, LT/Ag, CD4, IL-10 Spontaneously activated T cells and monocytes in BAL

Cancer

DTH (variable), CH, Inflammation

LT/Ag, possible suppressor cells

Systemic lupus erythematosus

DTH (variable)

LT/PHA, LT/Ag (variable)

*

CD4, decreased CD8 suppressor activity IL-2/-12 immunoregulatory alterations Rheumatoid arthritis

DTH

(?)

Disseminated TB

DTH

CD4; LT/PHA; LT /Ag (PPD) Possible suppressor cells

Lepromatous leprosy

DTH (lepromin)

LT/PHA, LT/Ag (lepromin, others), CH Allograft Th2 profile with decreased IL-12 receptors Suppressor T cells (?)

Disseminated CMC

†

DTH (coccidioidin)

LT/Ag (coccidioidin), coccidiodomycosis

DTH

LT/PHA, LT/Ag

‡

‡

Monocyte chemotaxis AIDS

DTH, CH

CD, LT/PHA, LT/Ag Monocyte function

Primary biliary cirrhosis

DTH, CH

LT/PHA

Uremia (chronic)

DTH, CH

LT/PHA, MLR

Allograft rejection

Suppressor cells

DTH, Delayed skin test hypersensitivity; recall, decreased responses to anergy panel skin tests; 1°, decreased sensitization to DNCB; CH, contact hyper-sensitivity; LT/PHA, decreased mitogen-induced lymphocyte proliferation; LT/Ag, decreased antigen-induced lymphocyte proliferation; MLR, decreased mixed-lymphocyte responses; NK, decreased natural killer cell activity; IL-2, IL-10, IL-12, decreased interleukin-2, -10, or -12 production; BAL, bronchoalveolar lavage; PPD, purified protein derivative; CMC, chronic mucocutaneous candidiasis. * May be associated with inhibitory serum factors. † Mannan from Candida may inhibit CMI. ‡ Selective defect to Candida organisms in some patients, global defect in others.

987

154

The mechanisms underlying depressed CMI in HD are not defined and likely are complex.[ ] The lesional T cell in HD is most often an atypical CD4+ cell with a surface marker pattern not typical of Th0, Th1, or Th2. In addition to the lymphopenia already noted, most studies show some decreased in vitro proliferation and IL2 production in blood lymphocytes induced by antigens or mitogens. The depressed in vitro responses may result partly from increased suppressive activity of adherent blood cells that elaborate excessive amounts of prostaglandin E, as well as the decreased IL-2 production and (in advanced disease) intrinsic T cell deficits, which may be responsible for disease spread.[

155]

Other immunoregulatory disturbances likely occur, particularly involving CD8+ cells and immunoregulatory

factors such as antilymphocyte and anti–MHC-II antibodies and inhibitory molecules secreted by the HD tumor cells.[

154]

Unexplained discrepant findings and inconsistent correlations between immunologic and clinicopathologic patterns make biologic and prognostic interpretation difficult in HD. At present it is reasonable to conclude that CMI in HD is frequently affected by the stage of disease and immunomodulatory effects of treatment. Impairment of CMI by HD or its treatment at least predisposes patients to certain opportunistic infections, which play major roles in the morbidity and mortality of HD. Sarcoidosis 156 157

Sarcoidosis is characterized by formation of noncaseating granulomas, most often involving the lung, and frequently depressed CMI.[ ] [ ] Depressed DTH reactivity, particularly in quantitative skin testing, has been seen in more than 50% of those with systemic involvement, with an associated decrease in the intensity of local lymphocyte accumulation and decreased CD4 reactivity. Primary sensitization with agents such as DNCB induces positive DTH responses in only 10% to 15% of sarcoidosis patients (vs. 90+% of normal subjects). CD4+ lymphopenia and decreased in vitro responses to mitogens and PPD also characterize sarcoidosis. These alterations are seen mainly during active disease, particularly in more severe and extensive involvement, with intermediate responses during clinical remission. The etiology of this peripheral anergy is unclear, with some evidence suggesting contributions by increased levels of IL-10 and vitamin D3 . [

156]

Of note, DTH tests are

less frequently depressed in presentations of sarcoidosis characterized by erythema nodosum and joint symptoms as well as adenopathy and an overall excellent

prognosis (Löfgren's syndrome). 157

At the same time, however, evidence suggests increased regional T cell activities in sarcoid.[ ] There may be an increased frequency of spontaneously “activated” T cells with increased production of cytokines function in the recruitment and retention of monocytes for granuloma development. The phenotypic and functional properties of cells recovered by bronchoalveolar lavage (BAL) fluid and present in tissues obtained by transbronchial biopsy may differ significantly from that in the 158

blood.[ ] The BAL population contains macrophages and increased numbers of lymphocytes, predominantly CD4+ T cells, some of which are activated, secreting amore of a Th1 profile than autologous blood T cells. IL-1 production by the BAL macrophages may be increased in sarcoidosis. The frequently increased serum levels of angiotensin-converting enzyme (ACE) produced by stimulated macrophages suggest increased macrophage numbers and secretory activity in sarcoidosis. Some recent studies suggest an expansion of a lung-restricted T cell population in the airways in sarcoid.[

159]

In summary, alterations in blood lymphocyte patterns and DTH skin test reactivity in sarcoidosis seem to parallel the extent and severity of the disease but may also reflect trafficking of immunoreactive cells to pulmonary tissue and other involved sites. Although this anergy pattern is associated with increased predisposition to tuberculosis and certain fungal infections, host defenses do not appear to be as compromised as in advanced HD. Nonlymphomatous Malignancies

Although some patients exhibit CMI responses to apparent tumor antigens,[ tumor-bearing metastatic

161 162 patients.[ ] [ ]

162 disease.[ ]

74] [160]

the paradoxical situation of depressed overall CMI responses is also seen in some

In fact, some observers find depressed DTH skin test reactivity at the initial tumor diagnosis to be prognostic of likely recurrent or

Infectious complications also appear more often in those with depressed CMI, mainly with CMV, herpes, and opportunistic intracellular

163

microbes.[ ] The mechanisms underlying these depressed DTH responses are not clarified. Evidence from different studies, however, indicates depressed nonspecific inflammatory reactivity, decreased antigen-induced lymphocyte responses, and immunoregulatory factors produced by tumors, including transforming growth factor beta (TGF-β) and prostaglandins of tumor cell origin, with associated suppressor activities of lymphocytes and monocytes. In summary, depression of CMI is seen in some nonlymphomatous malignancies and correlates to some degree with extent of tumor involvement, ultimate prognosis, and immunoregulatory alterations. Rheumatic Disorders

Interest in possible CMI alterations in systemic lupus erythematosus (SLE) was stimulated by the findings of B cell hyperactivity and defects in certain T cell 164

functions.[ ] Some findings suggesting altered CMI include (1) modestly decreased DTH skin test reactivity in some reports; (2) frequent blood lymphopenia with variable CD4/CD8 ratios; (3) variable depression in lymphocyte proliferative responses to mitogens, antigens, and autologous cells (in a mixed-leukocyte reaction), possibly related to altered antigen presentation by dendritic cells;[ IL-12

165 secretion;[ ]

164]

(4) possibly decreased CD8+ suppressor cell activity, associated with an imbalance of IL-6 and

and (5) decreased IL-2 production and responsiveness.

The mechanisms underlying these altered T cell responses in SLE are still not defined, although roles are postulated for lymphocytotoxic antibodies, antiidiotypic 166 167 168

antibodies, and altered intracellular signaling after antigen binding. [ ] [ ] [ ] The in vivo clinical significance of these depressed in vitro T cell responses in SLE remain unclear. There may be defective CMI protective responses against herpes zoster in SLE, but no impressively increased incidence of infection with other obligate intracellular microorganisms independent of treatment side effects. In summary, SLE is characterized by a state of immune imbalance reflected by exaggerated and abnormal antibody formation, with T cell function that is frequently defective.

988

The role of T cell function in the pathogenesis of SLE is still uncertain; however, defective CMI responses to agents such as the herpes zoster virus resulting from SLE or its treatment may play a part in the overall morbidity of SLE patients. Abnormal CMI and T cell immunoregulatory responses may also be seen in rheumatoid arthritis (RA). Several groups have described depressed DTH skin test 169

reactivity in 20% to 50% of untreated RA patients, apparently unrelated to disease activity.[ ] In juvenile rheumatoid arthritis (JRA), or juvenile chronic arthritis, several lines of indirect evidence suggest altered T cell function, including (1) activation markers expressed on synovial T cells suggesting previous activation in vivo, (2) persistent oligoclonally expanded T cell populations accumulating preferentially in the synovial compartment, (3) some TCR complementarity-determining region 3 sequence similarities between different clones in an individual patient, and (4) T cell–derived cytokines of predominantly Th1 type, with decreased IL-4 secretion.[

170]

It is unclear whether these alterations are pathogenic in RA.

Chronic Infections

Certain chronic infections, particularly in predominantly intracellular loci, may be associated with depressed CMI. In some cases the defective CMI response is selective for the antigens composing the infecting organism. In other patients the defect becomes broad-based anergy with progression of the disease. Irrespective of 171

etiology, the degree of fever, fatigue, and leukocytosis correlates well with likelihood of anergy.[ ] For example, depressed overall DTH skin test reactivity frequently occurs in those with extensive or miliary tuberculosis, possibly related in part to poor nutrition or lymphopenia with suppressor cell effects. However, some patients with localized tuberculosis may be tuberculin negative, particularly older individuals with less symptoms.[ particularly susceptible to infection with other acid-fast microbial pathogens, such as Nocardia.

142] [172]

Patients with impaired CMI may be

[163]

173

Leprosy presents the fascinating immunologic and clinical picture of a spectrum of host responses to the same microorganism, Mycobacterium leprae.[ ] At one extreme, some patients develop the relatively benign clinical pattern of tuberculoid leprosy. Their cutaneous lesions feature prominent granuloma formation with high concentrations of Th1-type cytokines (IL-12) and a paucity of organisms in lesional tissue. Associated with this are uniformly positive late-onset (weeks) DTH skin test reactions to lepromin (Mitsuda), generally preserved capacity for expressing induced contact sensitivity, and positive in vitro lymphocyte responses to

lepromin and tuberculin. At the other extreme, individuals with lepromatous leprosy have a much more devastating disease, with many organisms in the lesions, high concentrations of IL-4 and IL-10, negative lepromin skin test reactions often progressing to broad-based anergy, and absent in vitro lymphocyte responses that persist even after prolonged chemotherapy. Impaired responsiveness to lepromin usually persists, whereas in vitro T cell responsiveness to mitogens and other antigens may recover after prolonged chemotherapy. Intermediate clinical forms of the disease may be associated with variable degrees of CMI deficiency. The pathogenesis of impaired CMI in the lepromatous form is complex, likely involving decreased expression of IL-12 receptors on T cells after interaction with M. leprae.[

173]

Fungal Infections

Human tissues may support the growth of numerous species of fungi. However, the normal function of natural and acquired defense mechanisms, especially CMI, 174]

makes the occurrence of progressive systemic infection an unusual event.[

Chemokines may also play a major role in attracting immune cells to the site of fungal

175 invasion.[ ]

However, the frequency of disseminated fungal infections has increased considerably in recent years because of decreased host defenses due to certain underlying diseases or their treatment. The disseminated fungal infections themselves can be associated with anergy. For example, a small subset of patients with coccidioidomycosis develop severe disseminated disease with negative coccidioidin DTH skin tests in the face of normal DTH reactivity to other antigens.[

176]

177

Genetic factors also play a role in susceptibility to disseminated disease.[ ] A similar picture has been reported in extensive or disseminated histoplasmosis. In vitro T cell proliferative responses to mitogens are frequently depressed in a broad array of disseminated fungal disorders. As in the case of tuberculosis, this deficit may reflect modulation of CMI by a number of immunologic and nonimmunologic host factors. Of interest, CMI directed against different antigens of Cryptococcus may be protective or nonprotective against spreading infection.[

178]

Candidiasis presents a particular spectrum of disorders. Local infections of skin and mucosal surfaces are common, and a majority of normal adults exhibit positive 179

DTH skin test to Candida antigens.[ ] Impaired CMI predisposes to extensive esophageal and possibly disseminated infection. Indeed, Candida likely causes the majority of opportunistic fungal infections at this time, possibly due to a switch from Th1-type to Th2-type host responses. Chronic mucocutaneous candidiasis 180

(CMC) is a spectrum of disorders in which patients have persistent and recurrent candidiasis of the skin, nails, and mucous membranes.[ ] Some patients have a genetic predisposition. There is a variable incidence of associated autoimmune diseases, polyglandular endocrine dysfunction, alopecia, vitiligo, malabsorption, and neoplasms, particularly thymoma. Depressed CMI responses to Candida occur in most CMC patients, and some have a more broad-based anergy with associated diminished T cell responses to all antigens A common immunologic abnormality is failure of the patient's T lymphocytes to produce cytokines that are essential for expression of CMI to Candida. Mannan, a component of Candida, may play a role in inhibiting CMI. The pathogenic significance of these findings is still uncertain, although clinical improvement persists after antifungal treatment only if the defects in CMI are corrected.[

180]

Acquired Immunodeficiency Syndrome

Perhaps the most striking broad-based depression of CMI seen in developed societies occurs in acquired immunodeficiency syndrome (AIDS).[

18] [181] [182]

find that a cumulative “skin test score” or DTH responses to a panel of three antigens (Candida, mumps, and tetanus) is a sensitive correlate of HIV

Some

183 status.[ ]

181 183 disease.[ ] [ ]

Some

studies have found an impressive correlation between the presence of skin test anergy and decreased CD4 levels and worsening clinical Decreased DTH skin test reactivity is seen in a minority of asymptomatic human immunodeficiency virus (HIV) antibody–positive individuals, with less anergy seen in 182]

adolescents than in adults.[

Depressed CMI likely plays a major role in the increased frequency of

989

tuberculosis, candidiasis, and other opportunistic infections seen in patients with advanced AIDS. However, an analysis by a CDC committee has found factors limiting the usefulness of anergy skin testing, including problems with standardization and reproducibility of the tests, the low risk for tuberculosis associated with a diagnosis of anergy, and the lack of apparent benefit of preventive therapy for groups of anergic HIV-infected individuals. Therefore the committee no longer recommends routine use of anergy testing in conjunction with PPD testing in screening programs for M. tuberculosis infection conducted in HIV-infected persons in 140] [184]

the United States.[

Other viral infections may be associated with depressed CMI reactivity. The possible protective or pathogenic roles of CMI in systemic CMV infection is still 185]

debated.[

The occurrence of depressed CMI during chronic HBV infection is discussed earlier.

Miscellaneous Conditions 186

Depressed DTH skin test reactivity with preserved in vitro lymphocyte responses has been observed in alcoholic cirrhosis. [ ] Impaired expression of CMI in chronic renal insufficiency manifests as prolonged graft survival, blunted expression of DTH, impaired immunosurveillance, and increased susceptibility to infection 187

by intracellular organisms, particularly M. tuberculosis. The pathogenesis of this depressed CMI is complex, with apparently no correction during hemodialysis.[ ] Depressed CMI often occurs in intestinal lymphangiectasia associated with an excessive loss of CD4+/CD45RA+ lymphocytes into the lumen of the gastrointestinal tract and an imbalance toward Th2 responses.[ prognosis in the anergic

188]

Anergy has been reported in 45% of patients with heart failure associated with cardiomyopathy, with a poorer

189 individuals.[ ]

In summary, these examples of altered CMI in disease indicate that partial depression of DTH test responses is much more common than complete suppression. Such depression may occur transiently with any acute illness, particularly certain viral infections. However, more prolonged suppression is generally associated with more extensive disease, frequently with lymphopenia, and often with worse prognosis. Intensive study in recent years has uncovered several potentially important underlying mechanisms, including (1) active suppression by T cells, macrophages, and soluble factors; (2) impaired in vitro lymphocyte responses seen often but not uniformly; and (3) altered production of cytokines, particularly decreases in IL-12, and possibly a skewing toward a Th2-type response.

MODULATION OF CELL-MEDIATED IMMUNITY Therapeutic agents can affect the in vivo expression of CMI reactivity either by modulating the development or persistence of the state of sensitization or by affecting the expression of CMI. It is important to remember that information is sometimes available only from animal models or during multidrug treatment of humans. In designing strategies for treatment, one should also remember that, to date, most approaches used clinically involve a broad suppression of all CMI responses, not only those thought to be pathogenic. Therefore the defenses against invasion by infectious pathogens and mutant cells may be compromised as well. However, this

field is changing rapidly, with hopes for more antigen-specific clinical immunosuppression. Also, measures designed to increase rather than decrease CMI responses (where appropriate) are under intensive investigation and are discussed here briefly. Corticosteroids Corticosteroids (CS) have long been considered potent suppressors of the DTH response in several species, including humans. Pharmacologic doses of CS can 190

variably depress DTH skin test responses, generally after days to weeks of therapy.[ ] Skin test reactivity returns to pretreatment levels within several weeks after cessation of CS treatment. Long-term CS therapy may result in breakdown of quiescent infectious foci containing intracellular parasites such as M. tuberculosis and certain fungi. This finding is associated with increased likelihood of dissemination of the infection. The mechanisms underlying the suppressive effects of CS on human CMI responses are still not completely understood. CS administration is followed within hours 191

by transient increases in levels of blood neutrophils and decreases in monocytes, CD4 cells, and total lymphocytes, whereas CD8 levels are usually unchanged. [ ] A similar temporal pattern of transient effects is observed regardless of the duration of the CS therapy. Animal and limited human studies suggest that CS administration leads to altered trafficking of lymphocytes from blood to bone marrow and possibly decreased recirculation from extravascular sites due to alterations 190

in cell membrane characteristics. However, no alterations in CAM expression in DTH sites have been reported.[ ] Previous evidence suggested that these effects occur through genomic alterations in which binding of CS to specific cytosolic receptors is followed by interaction with selective regulatory sites within DNA. This 192]

leads to altered transcription of genes for several proinflammatory components.[

As a result, there is suppression of the synthesis of selected cytokines,

particularly the Th1 type, due in part to induction of TGF-β synthesis. Of interest, some cytokines can modulate CS secretion.[ macrocortin, which can suppress formation of proinflammatory

193]

CS also induce synthesis of

194 prostaglandins.[ ] 194

CS also affect the function of monocytes and macrophages, which is potentially important in CMI reactions.[ ] Also, the in vitro chemotactic responses of monocytes are depressed. IL-1, IL-2, and IL-4 production can be inhibited, but responses to exogenous IL-2 are not affected. In vivo studies suggest that a steroidinduced decreased Fc receptor and C3 receptor function in the reticuloendothelial system may be important in the clearance of immune complexes and in modulating NK cell activity. In higher concentrations, steroids may exert rapid-onset nongenomic effects, suppressing a number of other intracellular events, such as synthesis of immunoglobulin and certain complement components, release of lysosomal enzymes and leukotriene B4 (LTB4 ), and even apoptosis.[

195]

These effects could contribute to the

hypogammaglobulinemia, decreased wound healing, and generally poor inflammatory responsiveness seen in some patients receiving sizable steroid doses for a prolonged period. This may lead to an increased incidence of infections in such individuals, particularly with facultative intracellular pathogens. Prolonged use of more moderate CS doses (e.g., up to 20•mg of prednisone daily in adults) results in modest impairment of granulocyte entry into inflammatory sites. B lymphocyte function appears to be least affected by CS therapy. The depression of immune/inflammatory

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196]

responses and metabolic alterations in those treated daily with sizable doses of prednisone is less prominent in those who receive alternate-day steroid treatment. [ Purine Antimetabolites Mercaptopurines such as 6-mercaptopurine (6-MP) and azathioprine, 6-MP's imidazole derivative, act at multiple loci in the suppression of purine synthesis by 197

actively replicating cells.[ ] Because of this cell cycle action, already developed immune responses are less readily suppressed than when a primary sensitization can be anticipated (e.g., organ transplantation). DTH expression appears to be suppressed more than humoral immunity by these drugs. This suppression is seen mainly in the response to new antigens but can even occur to some degree in well-established DTH responses, primarily because of these agents' antiinflammatory effects. Blood levels of large lymphocytes with NK cell activity and monocytes are reduced more than those of small lymphocytes, probably because of suppressed proliferation of cells in the bone marrow. There is a decrease in the mononuclear cell inflammatory exudate in skin windows of humans receiving these drugs, but only modest and inconsistent suppression of antigen-induced proliferation and cytokine production by blood lymphocytes obtained from patients treated with 6-MP and azathioprine. Alkylating Agents 197

Cyclophosphamide has emerged as the most widely used alkylating agent.[ ] It offers at least a theoretic advantage when compared to purine antimetabolites in that cyclophosphamide acts against both replicating and nonreplicating T and B cells involved in immune responses by cross-linking effects leading to denaturation of DNA and RNA. Therefore the likelihood exists of a suppressive effect of cyclophosphamide on immunocompetent cells sensitized by prior exposure to the antigen and in a present resting or slowly dividing state. The effects of cyclophosphamide in human immune responses are complex and somewhat variable, depending on 197

the dose, timing, and duration of therapy.[ ] Cyclophosphamide in larger doses appears to be effective in suppressing secondary and ongoing immune responses. However, lower cyclophosphamide doses may preferentially inhibit suppressor T cell responses, leading to enhanced CMI or humoral immune responses. Methotrexate (MTX) is a potent antimetabolite interfering with folate metabolism, with resultant inhibition of several key enzyme systems, and leading to 197

accumulation of adenosine, a pronounced immunosuppressant.[ ] Use of very large doses of MTX in cancer chemotherapy frequently led to serious adverse side effects. In recent years, MTX has been employed in psoriasis and RA in much lower doses (about 15•mg once weekly) with fairly impressive beneficial effects and less toxicity.[

198]

It is not clear whether CMI is consistently depressed by MTX at these lower dosages. Concomitant use of folic acid may reduce toxicity without 199]

altering beneficial clinical effects.[

Other Immunosuppressive Agents Cyclosporin A (CsA) is a noncytotoxic, cyclic peptide of fungal origin that reduces T cell activation and IL-2 secretion, primarily by inhibiting Ca++ -dependent signal transduction. The latter occurs following CsA binding to cytosolic cyclophilins and subsequent inhibition of the phosphatase activity of calcineurin and 197] [200]

dephosphorylation of nuclear factors of activated T cells (NFATs).[ responsible for the improved survival of cadaveric

200 allografts.[ ]

CsA has become a mainstay in controlling allograft rejection and likely is primarily

DTH skin test reactivity is frequently suppressed in CsA-treated patients.

Tacrolimus, a macrolide, has similar immunologic, clinical, neurotoxic and nephrotoxic effects as CsA but is absorbed more consistently and is more potent. Tacrolimus has been used with some success to control transplant rejection when CsA has not been successful.[ inflamed skin has led to its use as a topical therapy in atopic dermatitis (see Chapter 86 ).

201]

The capacity of tacrolimus to be absorbed into

Mycophenolate is a recently developed immunosuppressive agent that acts to inhibit T and B cell proliferation by blocking the production of guanosine nucleotides 202

required for DNA synthesis.[ ] Mycophenolate also inhibits adherence of lymphocytes to vascular endothelium. Mycophenolate has been used to enhance rejection control regimens and to allow dose reduction of agents such as CsA. Antibody Therapy Intravenous (IV) injection of polyclonal immunoglobulin has multiple immunomodulatory effects, including effects on presentation of antigens to T cells and 203

proliferation of T cells.[ ] IV immunoglobulin may decrease allograft rejection and inhibit in vitro mixed-lymphocyte reactions, but it is unclear whether in vivo expression of DTH is affected. Monoclonal anti–T cell antibodies, particularly against epitopes in the CD3 complex, have been used to control allograft rejection, GVHR, and some connective 204 205

tissue diseases, with associated decreases in CMI.[ ] [ ] Frequent, rapid elimination of murine monoclonal antibodies (mAbs) resulted from formation of human antimurine antibodies by patients treated repeatedly. Subsequently, “humanized” mAbs, in which the Fc portion of the murine antibody molecules is replaced with the Fc fragment of human IgG, are being used preferentially.[

206]

The use of carefully humanized anti-CD3 mAbs has been associated with longer half-life of the 207]

donated antibodies, prominent T cell depletion, and less adverse effects due to cytokine release.[

Conjugation of the anti-CD3 mAb to a toxin (immunotoxin) has

208 trials.[ ]

lead to even more striking T cell depletion, but side effects have limited clinical Because of the postulated particular role of CD4+ lymphocytes in mediating pathogenic CMI, trials of anti-CD4 mAbs have been tried in RA and other disorders with evidence of immunosuppression even when the humanized anti209 210

CD4 mAb does not deplete the CD4+ cells.[ ] [ ] Treatments with mAbs against other epitopes on the T cell surface or endothelial cells, including CD28, CD18, leukocyte function–associated antigen (LFA), very late antigen-4 (VLA-4), and intercellular adhesion molecule (ICAM), are under current investigation, with experimental evidence that at least some of these suppress DTH. [

211] [212]

Cytokines

The diverse biologic effects of this group of naturally occurring compounds are discussed in Chapters 10 and 11 . With particular regard to CMI, experimental animal evidence

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suggests a complex interaction of cytokines in immune inflammatory reactions, with Th1-type cytokines (IFN-γ, IL-12) promoting DTH and down-modulating IgEmediated inflammation.[

81] [82]

A converse pattern is seen after administration of IL-4 and IL-10. The situation in humans appears less clear-cut.[

81]

However, 213 214

information concerning the effect of cytokines on human CMI is emerging from ongoing studies of IL-12 and IL-4 effects in several human diseases.[ ] [ ] Adjuvant cytokine therapy is also being tried in immunocompromised hosts, infectious diseases, and particularly in malignancy and bone marrow transplantation. [214] [215]

215

In preliminary studies, use of IL-12 as an adjuvant has enhanced CMI and protective responses against HIV infection.[ ] TNF-α stimulates migration of dendritic cells to lymphoid tissue, and GM-CSF enhances the antigen-presenting capacity of such cells important in CMI development. Studies in larger patient groups will be required to determine whether any observed effect on CMI is consistent and relevant to clinical effects.

Microbial Products

It has long been known that injection of mycobacterial preparations (e.g., BCG), heat-killed mycobacteria in complete Freund's adjuvant, and the peptidoglycan 216 217

component muramyl dipeptide (MDP) enhances the development of CMI and resistance to repeated infection.[ ] [ ] Recent studies have shown that MDP and its analogs enhance the maturation and functional capacity of monocyte-derived dendritic cells. New adjuvant formulations with attenuated vaccinia virus and “naked 218

DNA” are of potential promise alone or in combination with cytokines such as IL-12 and IFN-γ.[ ] Plasmid DNA vaccine encoding microbial or tumor components has been shown to induce both CMI and humoral immunity. As mentioned earlier, injections of immunostimulatory nucleotide sequences obtained from certain 83

bacteria can skew immune responses to accompanying protein antigens from Th2 to Th1.[ ] It is not yet clear whether such alterations lead to increase in protective CMI responses against certain infections or tumors. As a result of these findings, clinical trials have been initiated to determine whether such agents enhance 74

resistance to tumors. [ ] Some successful local effects of BCG injection into melanoma have been reported, possibly because of the prominent local DTH reaction induced. However, variable systemic effects have been obtained, possibly because of varying effects of BCG on T lymphocytes, NK cells, and macrophages. Thymic Factors

Because of increasing evidence of the role of the thymus in normal T cell development, the in vivo and in vitro biologic effects of thymic extracts are being investigated. In clinical trials, fractions such as thymosin fraction 5 (a collection of polypeptides), thymosin-α1 , thymulin, and thymostimulin have been reported to correct some spontaneous or induced immunodeficiency states and augment T cell responses in certain cancers and other diseases.[ immune effects have been observed, depending on the particular thymic agent employed.

219]

However, different patterns of

Miscellaneous Agents

Levamisole, an antihelminthic drug, has been reported to restore DTH to varying degrees of skin reactivity, increase T cell and NK cell levels, and enhance in vitro T cell responses in anergic patients with tumors, in whom levamisole is often used therapeutically. However, its effects on in vivo CMI have been inconsistent in other reports.[

220] [221]

Sensitization of HIV-infected patients with DNCB has been reported to enhance general DTH skin test reactivity.[

222]

Oral zinc administration may enhance

depressed CMI, possibly through stimulation of thymic activity.[ infection may result from suppressed CMI

223]

In contrast, in persons exposed repeatedly to opioids and marijuana, increased susceptibility to

224 responses.[ ]

Summary A variety of agents may modulate human CMI responses. Most of the clinical trials have involved suppression of CMI, largely because of altered trafficking of reactive cells and immunologically nonspecific antiinflammatory effects. It is frequently unclear how these biologic effects relate to any clinical efficacy observed. Because of the nonspecific nature of the inhibition, protective CMI responses may also be suppressed. More recently, attempts to enhance CMI responses with a variety of approaches have led to a fascinating array of effects, the clinical significance of which is still uncertain. Injection of a combination of agents may be 225

required to induce CMI strongly through Th1-type responses.[ ] Approaches that result in antigen-specific suppression or enhancement of CMI responses would be potentially of greater clinical utility. However, concerns about side effects and cost-effectiveness of such treatment have been expressed.

CONCLUSIONS Current concepts on the induction and expression of CMI involve not only the traditional DTH reaction but also the broad context of cell-mediated responses. Experimental and clinical data are increasing regarding the possible roles of CMI in health and disease. DTH reactivity probably plays at least a partial role in the containment of some microbial infections, but sometimes at a significant cost to the patient in the form of adverse inflammatory effects. Certain systemic diseases with associated depressed CMI responses are characterized by increased prevalence of some of these same bacterial, fungal, and viral infections. These acquired states of depressed CMI cannot be explained by any unifying hypothesis. Depending on the disease, one may find in vivo depletion of immunocompetent lymphocytes, decreases in antigen-induced proliferation needed for strong and persistent CMI, impairment of effector mediator release, deficient response of inflammatory cells such as macrophages, augmented suppressor cell activity, or combinations of these mechanisms. Complicating the situation may be the presence of serum factors that modulate CMI in vivo. On the other hand, there is currently at least indirect evidence that CMI may be pathogenic in some putative autoimmune diseases, although it is still unclear whether CMI initiates the reaction or only acts secondarily after tissue damage from other causes. The vast array of in vitro cell responses may seem confusing and contradictory at present. Such data have generally not provided unequivocal proof of a particular role for CMI in clinical disorders. However, the rapid recent growth in the knowledge about cellular immunoregulatory mechanisms and

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the technologic advances for separating immunocompetent cell populations obtained from lesional areas provide encouragement that the pathogenic significance of CMI responses in health and disease will be clarified in the future.

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127. Molberg O, McAdam S, Lundin KE, et al: T cells from celiac disease lesions recognize gliadin epitopes deamidated in situ by endogenous tissue transglutaminas, Eur J Immunol 31:1317, 2001. 128. Stafford EA, Rose NR: Newer insights into the pathogenesis of experimental autoimmune thyroiditis, Int Rev Immunol 19:501, 2000. 129. Stassi G, Zeuner A, Di Liberto D, et al: Fas-FasL in Hashimoto's thyroiditis, Clin Immunol 21:19, 2000. 130. Drugarin D, Negru S, Koreck A, et al: The pattern of a Th1 cytokine in autoimmune thyroiditis, Immunol Lett 71:73, 2000. 131. Koh LD, Napolitano G, Singer DS, et al: Graves' disease: a host defense mechanism gone awry, Int Rev Immunol 19:633, 2000. 132. Lee RS, Schlumberger M, Caillou B et al: Phenotypic and functional characterisation of tumour-infiltrating lymphocytes derived from thyroid tumours, Eur J Cancer 32A:1233, 1996. 133. Betterle C, Dalpra C, Greggio N, et al: Autoimmunity in isolated Addison's disease and in polyglandular autoimmune diseases, Ann Endocrinol 62:193, 2001. 134. Peterson P, Uibo R, Krohn KJ: Adrenal autoimmunity: results and developments, Trends Endocrinol Metab 11:285, 2000. 135. Maclaren N, Chen QY, Kukreja A, et al: Autoimmune hypogonadism as part of an autoimmune polyglandular syndrome, J Soc Gynecol Invest 8:S52, 2001. 136. Kligman I, Jeremias J, Rosenwaks Z, et al: Cell-mediated immunity to human and Escherichia coli 60-kDa heat shock protein in women: association with a history of spontaneous abortion and endometriosis, Am J Reprod Immunol 40:32, 1998. 137. Hayashi T, Faustman D: Defective function of the proteasome in autoimmunity: involvement of impaired NF-kB activation, Diabetes Technol Ther 2:415, 2000. Clinical States Affecting Cell-Mediated Immunity 138. Kay AB: T cells in allergy and anergy, Allergy 54:29, 1999. 139. Klein RS, Flanigan T, Schuman P, et al: Criteria for assessing cutaneous anergy in women with or at risk for HIV infection. HIV Epidemiologic Research Study Group, J Allergy Clin Immunol 103:26, 1999. 140. Slovis BS, Plitman JD, Haas DW: The case against anergy testing as a routine adjunct to tuberculin skin testing, JAMA 283:2003, 2000. 141. Taams LS, Wauben MH: Anergic T cells as active regulators of the immune response, Hum Immunol 61:633, 2000 (review). 142. Ginaldi L, De Martinis M, D'Ostilio A, et al: The immune system in the elderly. II. Specific cellular immunity, Immunol Res 20:109, 1999. 143. Lesourd BM: Nutrition and immunity in the elderly: modification of immune responses with nutritional treatments, Am J Clin Nutr 66:478S, 1997. 144. French AL, McCullough ME, Rice KT, et al: The use of tetanus toxoid to elucidate the delayed-type hypersensitivity response in an older, immunized population, Gerontology 44:56, 1998.

145. Jackson TD, Murtha AP: Anergy during pregnancy, Am J Obstet Gynecol 184:1090, 2001 (review). 146. Robertson SA: Control of the immunological environment of the uterus, Rev Reprod 5:164, 2000.

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147. Chandra RK: Nutrition and immunology: from the clinic to cellular biology and back again, Proc Nutr Soc 58:681, 1999. 148. Sammon AM: Dietary linoleic acid, immune inhibition and disease, Postgrad Med J 75:129, 1999. 149. Beier-Holgersen R, Brandstrup B: Influence of early postoperative enteral nutrition versus placebo on cell-mediated immunity, as measured with the Multitest CMI, Scand J Gastroenterol 34:98, 1999. 150. Karp CL, Wysocka M, Wahl LM: Mechanism of suppression of cell-mediated immunity by measles virus, Science 273:228, 1996. 151. Zweiman B, Pappagianis D, Maibach H, et al: Effect of measles immunization on tuberculin hypersensitivity and in vitro lymphocyte reactivity, Int Arch Allergy Appl Immunol 40:834, 1971. 152. Perez-Blas M, Reguiero JR, Ruiz-Contreras JA: T lymphocyte anergy during acute infectious mononucleosis, Clin Exp Immunol 89:83, 1992. 153. Knecht H, Berger C, Rothenberger S, et al: The role of Epstein-Barr virus in neoplastic transformation, Oncology 60:289, 2001. 154. Poppema S: Immunology of Hodgkin's disease, Baillieres Clin Haematol 9:447, 1996 (review). 155. Schultze JL: Why do B cell lymphoma fail to elicit clinically sufficient T cell immune responses? Leuk Lymphoma 32:223, 1999 (review). 156. Bansal AS, Bruce J, Hogan PG, et al: An assessment of peripheral immunity in patients with sarcoidosis using measurements of serum vitamin D3 , cytokines and soluble CD23, Clin Exp Immunol 110:92, 1997 (review). 157. Kataria YP, Holter JF: Immunology of sarcoidosis, Clin Chest Med 18:719, 1997 (review). 158. Mollers M, Aries SP, Dromann D, et al: Intracellular cytokine repertoire in different T cell subsets from patients with sarcoidosis, Thorax 56:487, 2001. 159. Katchar K, Wahlstrom J, Eklund A, et al: Highly activated T-cell receptor AV2S3(+) CD4(+) lung T-cell expansions in pulmonary sarcoidosis, Am J Respir Crit Care Med 63:1540, 2001. 160. Zinkernagel RM: Immunity against solid tumors? Int J Cancer 93:1, 2001.

161. Czarnecki D, Zalcberg J, Kulinskaya E et al: Impaired cell-mediated immunity of apparently normal patients who had multiple skin cancers, Cancer 76:228, 1995. 162. Triantafillidis JK, Papatheodorou K, Kogevinas M, et al: Prognostic factors affecting the survival of operated patients with colorectal cancer: significance of delayed hypersensitivity skin reactions and nutritional status, Ital J Gastroenterol 27: 419, 1995. 163. Rolston KV: The spectrum of pulmonary infections in cancer patients, Curr Opin Oncol 3:218, 2001. 164. Scheinecker C, Zwolfer B, Koller M, et al: Alterations of dendritic cells in systemic lupus erythematosus: phenotypic and functional deficiencies, Arthritis Rheum 44:856, 2001. 165. Filaci G, Bacilieri S, Fravega M, et al: Impairment of CD8+ T suppressor cell function in patients with active systemic lupus erythematosus, J Immunol 166: 6452, 2001. 166. Yamada A, Minota S, Nojima Y, et al: Changes in subset specificity of anti-T cell autoantibodies in systemic lupus erythematosus, Autoimmunity 14:269, 1993. 167. Williams WM, Isenberg DA: Naturally occurring anti-idiotypic antibodies reactive with anti-DNA antibodies in systemic lupus erythematosus, Lupus 7:164,1998. 168. Tsokos GC, Wong HK, Enyedy EJ, et al: Immune cell signaling in lupus, Curr Opin Rheumatol 12:355, 2000. 169. Coaccioli S, Di Cato L, Marioli D, et al: Impaired cutaneous cell-mediated immunity in newly diagnosed rheumatoid arthritis, Panminerva Med 42:263, 2000 (review). 170. Grom AA, Hirsch R: T-cell and T-cell receptor abnormalities in the immunopathogenesis of juvenile rheumatoid arthritis, Curr Opin Rheumatol 12:420, 2000. 171. Bennett BK, Hickie IB, Vollmer-Conna US, et al: The relationship between fatigue, psychological and immunological variables in acute infectious illness, Aust NZ J Psychiatry 32:180, 1998. 172. Maher J, Kelly P, Hughes P, et al: Skin anergy and tuberculosis, Respir Med 86:481, 1992. 173. Kim J, Uyemura K, Van Dyke MK, et al: A role for IL-12 receptor expression and signal transduction in host defense in leprosy, J Immunol 167:779, 2001. 174. Mencacci A, Cenci E, Bacci A, et al: Cytokines in candidiasis and aspergillosis, Curr Pharm Biotechnol 1:235, 2000 (review). 175. Traynor TR, Huffnagle GB: Role of chemokines in fungal infections, Med Mycol 39:41, 2001. 176. Cox RA, Magee DM: Protective immunity in coccidioidomycosis, Res Immunol 149:417, 1998. 177. Rosenstein NE, Emery KW, Werner SB, et al: Risk factors for severe pulmonary and disseminated coccidioidomycosis: Kern County, California, 1995–1996, Clin Infect Dis 32:708, 2001. 178. McGaha T, Murphy JW: CTLA-4 down-regulates the protective anticryptococcal cell-mediated immune response, Infect Immun 68:4624, 2000.

179. Fidel PL Jr, Sobel JD: The role of cell-mediated immunity in candidiasis, Trends Microbiol 2:202, 1994. 180. Kirkpatrick CH: Chronic mucocutaneous candidiasis, Pediatr Infect Dis J 20: 197, 2001. 181. Klein RS, Flanigan T, Schuman P, et al: Criteria for assessing cutaneous anergy in women with or at risk for HIV infection. HIV Epidemiologic Research Study Group, J Allergy Clin Immunol 103:26, 1999. 182. Smith Rogers A, Ellenberg JH, Douglas SD, et al: The prevalence of anergy in human immunodeficiency virus-infected adolescents and the association of delayed-type hypersensitivity with subject characteristics, J Adolesc Health 27: 384, 2000. 183. Metersky ML, Yang P, Nielsen HS, et al: Identification of human immunodeficiency virus-infected individuals by delayed type hypersensitivity skin testing, Ann Clin Lab Sci 28:272, 1998. 184. Centers for Disease Control and Prevention: Anergy skin testing and tuberculosis prevention and therapy for HIV-infected persons: revised recommendations, MMWR 46(RR-15):1, 1997. 185. Walsh JE, Abinum M, Peiris JS, et al: Cytomegalovirus infection in severe combined immunodeficiency: eradication with Foscarnet, Pediatr Infect Dis J 14:911, 1995. 186. Schirren CA, Jung MC, Zachoval R, et al: Analysis of T cell activation pathways in patients with liver cirrhosis, impaired delayed hypersensitivity and other T cell-dependent functions, Clin Exp Immunol 108:144, 1997. 187. Smirnoff M, Patt C, Seckler B, et al: Tuberculin and anergy skin testing of patients receiving long-term hemodialysis, Chest 113:25, 1998. 188. Fuss IJ, Strober W, Cuccherini BA, et al: Intestinal lymphangiectasia, a disease characterized by selective loss of naive CD45RA+ lymphocytes into the gastrointestinal tract, Eur J Immunol 28:4275, 1998. 189. Vredevoe DL, Woo MA, Doering LV, et al: Skin test anergy in advanced heart failure secondary to either ischemic or idiopathic dilated cardiomyopathy, Am J Cardiol 82:323, 1998. Modulation of Cell-Mediated Immunity 190. Sousa AR, Lane SJ, Atkinson BA, et al: The effects of prednisolone on the cutaneous tuberculin response in patients with corticosteroid-resistant bronchial asthma, J Allergy Clin Immunol 97:698, 1996. 191. Zweiman B, Atkins PC, Bedard PM, et al: Corticosteroid effects on circulating lymphocyte subset levels in normal humans, J Clin Immunol 4:151, 1984. 192. Almawi WY, Hess DA, Rieder MJ: Multiplicity of glucocorticoid action in inhibiting allograft rejection, Cell Transplant 7:511, 1998. 193. Angeli A, Masera RG, Sartori ML, et al: Modulation by cytokines of glucocorticoid action, Ann N Y Acad Sci 876:210, 1999. 194. Rugstad HE: Antiinflammatory and immunoregulatory effects of glucocorticoids mode of action, Scand J Rheumatol Suppl 76:257, 1988.

195. Gold R, Buttgereit F, Toyka KV: Mechanism of action of glucocorticosteroid hormones: possible implications for therapy of neuroimmunological disorders, J Neuroimmunol 117:1, 2001. 196. Sivaraman P, Nussbaumer G, Landsberg D: Lack of long-term benefits of steroid withdrawal in renal transplant recipients, Am J Kidney Dis 37:1162, 2001. 197. Allison AC: Immunosuppressive drugs: the first 50 years and a glance forward, Immunopharmacology 47:63, 2000. 198. Alarcon GS: Methotrexate use in rheumatoid arthritis: a clinician's perspective, Immunopharmacology 47:259, 2000. 199. Endresen GK, Husby G: Folate supplementation during methotrexate treatment of patients with rheumatoid arthritis: an update and proposals for guidelines, Scand J Rheumatol 30:129, 2001. 200. Rovira P, Mascarell L, Truffa-Bachi P: The impact of immunosuppressive drugs on the analysis of T cell activation, Curr Med Chem 7:673, 2000. 201. De Bonis M, Reynolds L, Barros J, et al: Tacrolimus as a rescue immunosuppressant after heart transplantation, Eur J Cardiothorac Surg 19:690, 2001. 202. Mele TS, Halloran PF: The use of mycophenolate mofetil in transplant recipients, Immunopharmacology 47:215, 2000. 203. Ballow M: Mechanisms of action of intravenous immune serum globulin in autoimmune and inflammatory diseases, J Allergy Clin Immunol 100:151, 1997 (review). 204. Flechner SM, Goldfarb DA, Fairchild R, et al: A randomized prospective trial of low-dose OKT3 induction therapy to prevent rejection and minimize side effects in recipients of kidney transplants, Transplantation 69:2374, 2000. 205. Yu XZ, Bidwell SJ, Martin PJ, et al: Anti-CD3 epsilon F(ab′)2 prevents graft-versus-host disease by selectively depleting donor T cells activated by recipient alloantigens, J Immunol 166:5835, 2001. 206. Haraoui B, Strand V, Keystone E: Biologic agents in the treatment of rheumatoid arthritis, Curr Pharm Biotechnol 1:217, 2000. 207. Norman DJ, Vincenti F, de Mattos AM, et al: Phase I trial of HuM291, a humanized anti-CD3 antibody, in patients receiving renal allografts from living donors, Transplantation 70:1707, 2000. 208. Knechtle SJ: Treatment with immunotoxin, Philos Trans R Soc Lond B Biol Sci 356:681, 2001. 209. Isaacs JD, Burrows N, Wing M, et al: Humanized anti-CD4 monoclonal antibody therapy of autoimmune and inflammatory disease, Clin Exp Immunol 110:158, 1997. 210. Schulze-Koops H, Davis LS, Haverty TP, et al: Reduction of Th1 cell activity in the peripheral circulation of patients with rheumatoid arthritis after treatment with a non-depleting humanized monoclonal antibody to CD4, J Rheumatol 25:2065, 1998. 211. Lockwood CM, Elliott JD, Brettman L, et al: Anti-adhesion molecule therapy as an interventional strategy for autoimmune inflammation, Clin Immunol 93:93, 1999.

212. Nicolls MR, Coulombe M, Yang H, et al: Anti-LFA-1 therapy induces long-term islet allograft acceptance in the absence of IFN-γ or IL-4, J Immunol 164: 3627, 2000. 213. Borish LC, Nelson HS, Corren J, et al: Efficacy of soluble IL-4 receptor for the treatment of adults with asthma, J Allergy Clin Immunol 107:963, 2001. 214. Lee P, Wang F, Kuniyoshi J, Rubio V, et al: Effects of interleukin-12 on the immune response to a multipeptide vaccine for resected metastatic melanoma, J Clin Oncol 19:3836, 2001. 215. Gherardi MM, Ramirez JC, Esteban M: Towards a new generation of vaccines: the cytokine IL-12 as an adjuvant to enhance cellular immune responses to pathogens during prime-booster vaccination regimens, Histol Histopathol 16:655, 2001.

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216. Allison AC: Immunological adjuvants and their modes of action, Arch Immunol Ther Exp 45:141, 1997. 217. Vidal V, Dewulf J, Bahr GM: Enhanced maturation and functional capacity of monocyte-derived immature dendritic cells by the synthetic immunomodulator Murabutide, Immunology 103:479, 2001. 218. Pachuk CJ, McCallus DE, Weiner DB, et al: DNA vaccines: challenges in delivery, Curr Opin Mol Ther 2:188, 2000. 219. Munno I, Marinaro M, Gesario A: Immunomodulatory effects of alpha interferon and thymostimulin in patients with neoplasia, Clin Diagn Lab Immunol 2:503, 1995. 220. Holcombe RF, Milovanovic T, Stewart RM, et al: Investigating the role of immunomodulation for colon cancer prevention: results of an in vivo dose escalation trial of levamisole with immunologic endpoints, Cancer Detect Prev 25: 183, 2001. 221. Hajnzic TF, Kastelan M, Lukac J, et al: Immunocompetent cells and lymphocyte reactivity to mitogens in levamisole-treated brain tumor children, Pediatr Hematol Oncol 16:335,1999. 222. Strecker AB, Goldberg B, Mills LB, et al: Improved delayed hypersensitivity skin testing in HIV-infected patients treated with topical DNCB, J Am Acad Dermatol 33:608, 1995. 223. Licastro F, Chiricolo M, Mocchegiani E, et al: Oral zinc supplementation in Down's syndrome subjects decreased infections and normalized some humoral and cellular immune parameters, J Intellect Disabil Res 38, 1994. 224. Friedman H, Klein TW, Newton C, et al: Marijuana, receptors and immunomodulation, Adv Exp Med Biol 373:103, 1995. 225. Moingeon P, Haensler J, Lindberg A: Towards the rational design of Th1 adjuvants, Vaccine 19:4363, 2001.

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Chapter 58 - Immune Complexes and Allergic Disease

Michael M. Frank C. Garren Hester

Much of our understanding of the role that immune complexes play in human diseases is derived from a series of observations made almost 100 years ago by von 1 2

Pirquet and Schick.[ ] [ ] These investigators studied patients treated with horse antidiphtheria antitoxin to decrease the morbidity and mortality of diphtheria. They noted a reproducible syndrome that they termed serum sickness that developed in many of their patients (fever, malaise, cutaneous eruptions, lymphadenopathy, leukopenia, arthralgias, albuminuria, edema). This syndrome was noted 8 to 13 days after the initial injection of the horse antiserum. The first signs were an area of erythema and pruritus at the site of injection, accompanied by fever and malaise. The most common skin eruption was urticaria, although scarlatiniform, morbilliform, and erythema multiforme–like eruptions also occurred. All eruptions were symmetrically distributed, with urticaria being generally the most widespread rash. Lymphadenopathy occurred in most patients during serum sickness and was most pronounced in the region of the injection. The lymph nodes were almost always tender. The authors also noted that enlargement of the lymph nodes often preceded the onset of serum sickness and receded before its termination, thus indicating that the behavior of the lymph nodes may have a predictive value. Leukopenia caused by a reduction in the number of polymorphonuclear neutrophils was noted. Arthralgias occurred much less frequently than the previously mentioned signs and symptoms, with metacarpo-phalangeal and knee joints most often involved. In individuals with joint pain the discomfort was intense despite complete absence of objective findings. Edema, which was an extremely frequent occurrence, was mainly confined to the face. Decrease in edema was said to portend resolution of serum sickness. Albuminuria, in contrast, was much less common and of only modest degree. Von Pirquet and Schick reported that the incidence of serum sickness was directly related to the amount of horse serum injected. Injections of 5 to 15•ml were associated with a 5% to 10% incidence of serum sickness, whereas injections of 100 to 200•ml resulted in serum sickness in more than 85% of patients. If a second injection was administered 6 weeks to 6 months after the first injection, an immediate reaction occurred at the injection site, manifesting as erythema and edema. This reaction was followed by an accelerated systemic reaction, manifesting as the development of serum sickness within 8 days. These accelerated reactions tended

to be severe and required smaller doses of serum for their initiation. Von Pirquet and Schick recognized that the initial latent period was the time required for the development of antibody in the recipient. They suggested that the immediate reaction on reinjection of antigen was attributable to the presence of circulating antibody and that the accelerated reaction was the result of an anamnestic response on secondary exposure to antigen. They surmised that the union of antigen and antibody was in some way directly toxic. The recognition that the products of the immune response could have pathologic effects as well as beneficial results was a landmark in modern understanding of the role of immunity in disease.

IMMUNOCHEMICAL FACTORS IN IMMUNE COMPLEX BIOACTIVITY 3

The sophisticated nature of the structure and function of antigens in model systems is now better understood.[ ] Genetic predisposition is clearly important in these models, but it is still difficult to translate the lessons of genetic endowment to humans. Chemical characteristics of the antigen will affect the type of immunologic response the host makes against the antigen and thus will influence complexes that are formed. In general, a maximal immunologic response is made to highmolecular-weight foreign proteins or glycoproteins, although certain polysaccharides will elicit a strong, often T lymphocyte–independent, antigenic response as well. These antigens provide powerful signals for polyclonal antibody responses. Small, low-molecular-weight antigens, frequently termed haptens, are not potent antigens unless linked to large carrier proteins. Antigens vary in potency; the molecular basis for this variation is unknown but relates in part to the number of lymphocytes in the population that recognize the antigen. Both the chemical groupings on the protein surface and the degree of hydrophilicity are important. Exposure of the host to a large amount of antigen, particularly during a primary immunization, tends to be more immunogenic than exposure to small amounts of antigen. Duration of exposure is also important. The longer an antigen is exposed to the host's immune system, the more potent the immunologic stimulus becomes. Thus, immunization given intradermally or subcutaneously often incites better antibody production than a brief intravenous exposure. Traditionally, depot injections of emulsions in oil have been used to promote long duration of antigen exposure and maximal response.

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The isotype of antibody formed after exposure to antigen affects the biologic activity of the immune complex.[ ] [ ] For example, immunoglobulin G (IgG) subclasses 1, 2, and 3 and IgM may activate complement by the classic pathway, whereas IgA-containing immune complexes activate complement by the alternative pathway. IgD and IgE do not activate complement efficiently. The valence of antibody also varies with the isotype. The valence of IgG, IgD, IgE, and monomeric IgA is 2. Dimeric IgA has a valence of 4, and IgM has an effective valence of 5. Antigen valence also affects immune complex size and composition. By definition, monovalent antigens com-bine with only one antibody binding site and are unable to cross-link antibody molecules. On the other hand, multivalent antigens may bind multiple antibodies with different specificities and form large immune complex lattices. 6

The relative concentration of antigen and antibody is another factor that influences the immunochemical characteristics of immune complexes[ ] ( Figure 58-1 ). During the early stages of immunization, the concentration of antigen is in excess over that of antibody. As the antibody production increases, the number of antibody and antigen molecules reach equivalence. Finally, as the antigen is removed from the circulation, antibody excess is achieved. In antibody excess, antigen valences are saturated, and the molar ratio of antigen and antibody approximates the valence of the antigen. In general, immune complexes formed in antibody excess are smaller than those formed at equivalence. At equivalence, the number of available antigenic sites is about equal to the number of antibody-combining sites, and

immune complexes tend to be large because chances for cross-linking have been optimized. Under conditions of extreme antigen excess, immune complexes are generally small because all antibody-combining sites are saturated and chances for lattice formation are limited. Immune complexes formed at moderate antigen excess are believed to be most pathogenic because they remain in the circulation for relatively long intervals and are large enough to activate complement efficiently. The charge of antigen and antibody is also important in determining tissue localization and pathophysiologic effects of

Figure 58-1 Graph of precipitin curve. Fixed amount of immune serum is placed in series of test tubes, and increasing amounts of antigen are added to each. At first, small immune complexes are formed in antibody excess, but as equivalence is reached, large-lattice immune complexes are formed and result in visible precipitates. Eventually, antigen excess is reached, and only small immune complexes are formed and do not precipitate. (From Yancey KB, Lawley TJ: J Am Acad Dermatol 10:711, 1984.)

(From Yancey KB, Lawley TJ: J Am Acad Dermatol 10:711, 1984.) 7

immune complexes.[ ] Positively charged immune complexes tend to localize to the basement membrane zone of skin and renal glomeruli, whereas neutral or 8

negatively charged complexes do not.[ ] Because these basement membranes are negatively charged, the complexes appear to bind through ionic interactions. In a similar fashion, cationic antigens may also bind to the basement membrane zone of glomeruli and only after tissue localization bind antibody. In renal glomeruli the negatively charged lamina rara interna and the lamina rara externa appear to be major sites of these interactions.[

7]

The ability of immune complexes to activate the various mediator pathways may play a vital role in their damage-producing effects and in their metabolism. For example, this ability of such complexes to activate complement may be important to their ability to induce inflammatory effects. Such interactions also may act to solubilize relatively insoluble immune complexes, affecting their ability to precipitate and presumably their rate and site of clearance.

CLEARANCE OF IMMUNE COMPLEXES Circulating immune complexes occur frequently in all normal individuals exposed to antigens, but only under exceptional circumstances do immune complexes lodge in tissue and cause disease. In most cases, circulating immune complexes are efficiently removed by cells of the reticuloendothelial system (RES), which is 9 10 11

composed of circulating and tissue-fixed phagocytic cells that typically possess both IgG Fc and C3 receptors.[ ] [ ] [ ] For this reason, some investigators use the term mononuclear phagocyte system for the RES to emphasize the role of these tissue-fixed phagocytes. Circulating immune complexes that have IgG or C3 in their lattices are bound by these receptors and then phagocytosed. Many studies have attempted to address the factors that affect the metabolism of circulating immune complexes, allowing them to be deposited in various organs. If injected intravenously into sensitized animals, antigen is rapidly removed from the circulation and is found mainly in the liver, spleen, and lung, all elements of the RES. Studies using injections of pre-formed immune complexes have shown that the rate of removal of the immune complexes from the circulation is related to the 12

size of the complexes.[ ] Large immune complexes that have sedimentation coefficients greater than 19S are rapidly removed from the circulation and lodge predominantly in the liver. Smaller immune complexes tend to circulate longer, but neither the large nor the small immune complexes are prone to deposit in renal glomeruli or vascular walls. Modifications of antibody that affect the Fc portion of the molecule, such as reduction and alkylation or destruction of the Fc portion, may result in prolonged circulation of immune complexes, with a larger percentage of the complexes being deposited in tissue. Clearly, IgG antibody without a functioning Fc fragment is unable to activate the classic complement pathway and tends not to bind to RES cells with Fc and complement receptors. The role of complement in the clearance of immune complexes is being explored. For example, if cobra venom factor is used to deplete serum complement, then 12

large immune complexes are injected that would normally efficiently activate complement, the clearance of these complexes is not markedly impaired.[ ] However, recent work in primates and human volunteers indicates that large immune complexes that efficiently activate complement are rapidly bound to the surface of

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red blood cells (RBCs) by means of C3b receptors on the RBC membranes and then are transported to the liver, where these immune complexes are deposited and 11 13 14 15

phagocytosed by Kupffer's cells.[ ] [ ] [ ] [ ] Immune complexes bound in this way to erythrocytes do not become deposited in the kidney or arterial walls when the complexes traverse these tissues. In contrast, in the absence of complement activation and binding, a larger percentage of the complexes apparently escape efficient clearance and are deposited in tissue sites other than RES organs. One set of studies of RES clearance in humans examined the clearance of chromium 51 (51 Cr)–radiolabeled IgG-coated autologous erythrocytes. Many patients with autoimmune diseases, circulating complexes, and tissue damage from these complexes demonstrated clearance abnormalities with RBC probes, suggesting an 10

impairment in the function of the Fc receptors of their RES macrophages.[ ] The clearance of such antibody-coated cells, mediated primarily by the Fc receptors of the spleen, is extremely abnormal in patients with systemic lupus erythematosus (SLE) who have circulating immune complexes. These abnormalities may be associated with a decreased rate of removal of immune complexes from the circulation. Thus, experimental observations in humans support the hypothesis that abnormalities of RES function may exist in some clinical situations in which immune complexes are deposited in tissues and cause damage. However, the situation in

humans is complex. [

9] [10] [11]

11]

Clearance of soluble immune complexes also has been studied in humans.[

In the first set of studies, radiolabeled tetanus toxoid/anti–tetanus toxoid immune 14

complexes were injected into volunteers and patients with immune complex–related diseases such as SLE.[ ] The immune complexes bound to erythrocyte complement receptor 1 (CR1) in a complement-dependent manner. The number of CR1 receptors per erythrocyte correlated with uptake. A fraction of RBCs with a high CR1/erythrocyte ratio bound a disproportionate number of the complexes, presumably because there are clusters of CR1 on the erythrocyte that increase their 15

efficiency in immune complex binding. [ ] Patients with a low CR1/erythrocyte ratio, or hypocomplementemia, had a lower degree of erythrocyte binding and a rapid initial clearance of the immune complexes from the circulation because of rapid hepatic uptake. Thereafter the rate of clearance was slower and conformed to a monoexponential function as the RBC-bound immune complexes were cleared. If the complexes were preopsonized (i.e., allowed to bind complement before injection), immune complexes bound more efficiently to erythrocytes, and the initial rapid clearance was not seen. 16

In a later set of experiments the clearance of hepatitis B surface antigen (HBsAg)/anti-HBsAg complexes was examined in patients with SLE.[ ] Again, the initial hepatic phase of clearance was more rapid in patients with SLE, and the splenic phase was slower. Interestingly, 12% of the complexes initially sequestered in the liver were subsequently released from the liver. Although the reasons are unclear, it has been suggested that efficient processing of immune complexes by complement prolongs their circulatory half-life in such a way as to ensure specific elimination by Fc and complement receptors of the RES and to prevent nonspecific, systemic deposition in vulnerable tissue sites.[

17]

Although most interest in RES function and immune complex clearance focuses on autoimmune disease and its pathogenesis, clearance of IgG-sensitized RBCs as a 18 19

measure of clearance was found to be strikingly abnormal first in rats given quantities of ethanol and then in patients with alcoholism.[ ] [ ] This modern experimental approach to the study of immune complexes in humans may provide the first clear explanation of the propensity for such patients to develop overwhelming infection.

IMMUNE COMPLEX INTERACTION WITH Fcγ-RECEPTOR–BEARING CELLS Most analyses of immune complex effects consider factors that influence the formation of immune complexes, how the complexes interact with mediators, and the effect of the various mediator pathways and their products on cells in the region where the mediators are generated. This section discusses the factors that control the deposition of immune complexes in tissues, their ability to activate mediators such as complement, and the effect of complement inflammatory peptides on immigration of cells. In some models, such as Heymann nephritis in the rat, antibodies form to renal tubular antigens, termed fraction 1A (Fx1A), and immune complexes form within the kidney, but the pathophysiologic consequences of such complex formation are considered to be similar to those that occur when pre20

formed immune complexes are deposited in the kidney.[ ] Detailed studies have shown that this formulation is too simple. Studies have suggested that inflammatory cells triggered by the interaction of immune products with their receptors act much earlier in the train of events. One receptor set that has been examined in greater detail in recent years is the family of Fc receptors. Fc receptors belong to the immunoglobulin (Ig) supergene 21

family.[ ] General features of the family are that they are all glycoproteins and all have a ligand-binding alpha (α) subunit consisting of Ig domains. The extracellular domains of the α subunits are highly conserved. For example, there is 70% to 98% homology in the Fc gamma receptor (FcγR) group and even 40%

homology between FcγR and FcepsilonRI, but considerable heterogeneity exists in the transmembrane and cytoplasmic domains. On phagocytic cells there are three subfamilies of FcγR. The FcγRI subfamily has high affinity for IgG and will bind monomeric Ig. FcγRII and FcγRIII subfamilies bind with low affinity (Ka 106 ). Therefore these receptors, assayed on B lymphocytes or phagocytic cells, will bind immune complexes because they can form multiple sites of attachment, but they will not bind Ig monomer well. A total of eight genes are present for Fc receptors: three for high-affinity receptors FcγRIA, B, and C and five genes for low-affinity receptors FcγRIIA, B, and C (only FcγRIIB is present in the mouse) and FcγRIIIA and B. The low-affinity genes are on chromosome 1 region q22 of humans. The high-affinity genes are also on chromosome 1 but are more scattered. In general, these receptors trigger cells when they are crossligated with immune complexes. There is relatively little control over their function at the level of the immune complex; most immune complexes bind to most or all of the receptors. The differences in function of the various receptors in general reside in different cytoplasmic segments and transmembrane domains. For example, human neutrophils have surface FcγRIIA, which contains an immunoreceptor tyrosine-based activation motif (ITAM), a sequence motif characteristic of activation FcγRs and B cell and T cell receptors. When crossligated, these receptors release proinflammatory mediators.

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B lymphocytes have FcγRIIB. The extracellular domains of these two receptors are 96% identical and bind immune complexes in the same way. However, their cytoplasmic tails are quite different, and a 13–amino acid sequence in the cytoplasmic domain of FcγRIIB, called immunoreceptor tyrosine-based inhibition motif (ITIM), delivers strong inhibitory signals to the activated B cell. On ligation with the B cell receptor (BCR), the ITIM sequence becomes tyrosine-phosphorylated, creating a binding domain for an inhibitory signaling molecule that hydrolyzes activated membrane inositol phosphate, called SH2-containing inositol phosphatase (SHIP), thereby disrupting ITAM activation and BCR-mediated calcium influx. BCR-induced cell proliferation is also blocked because phosphorylation interferes 21]

with the activation and recruitment of mitogen-activated protein (MAP) and other protein kinases.[ BCR-mediated antigen processing and

22 23 internalization.[ ] [ ]

and is associated with the overproduction of autoantibodies.

In the mouse, FcγRIIB1 has also been reported to interfere with

Decreased FcγRIIB expression on germinal center B cells has been demonstrated in lupus-prone mice

[24]

Fc epsilon receptor I (FcepsilonRI), FcγRI, and FcγRIII require at least one additional chain for receptor assembly at the cell surface and signaling: the dimeric γ subunit or the γ subunit and zeta (ζ) subunit. In most phagocytes there appears to be a two-phase activation process. In the first phase, ligation of the extracellular domain of the receptor activates the src family of protein kinases, which are responsible for tyrosine phosphorylation of cytoplasmic ITAM. In the second phase, other kinase families such as the syk family are activated and bind the phosphorylated ITAM. Ultimately this leads to elevation of intracellular calcium ions, release of inflammatory mediators (e.g., leukotrienes, prostaglandins, hydrolases), and transcription of cytokine genes. A polymorphism in FcRγIIIA has been identified in SLE patients, especially patients with associated nephritis, and is correlated with decreased binding to natural killer (NK) cells and monocytes that may interfere with natural host defense and immune complex processing. [

25]

Similarly, polymorphisms in FcγRIIIB have been linked to a patient's susceptibility to rheumatoid arthritis.

[26]

It is becoming possible to determine what each of these genes is doing by developing knockout mice with gene deletions. The particular cytoplasmic amino acid motifs that trigger phagocytosis have been determined, and how altering these genes affects specific immunopathologic disease models has been explored.

IMMUNE COMPLEXES AND INDUCTION OF IMMUNE RESPONSE Immune complexes between antigen and antibody may form at the onset and during the course of an immune response. Depending on the nature of the antigen (i.e., particulate or soluble protein), the size of the immune complex, the antigen/antibody ratio, the concentration, and the ability to activate complement, immune complexes can interact with virtually all types of immune cells possessing Fc or complement receptors and can initiate or augment the response. In general, immune complexes containing low amounts of antibody (antigen excess), as might be found early in the immune response, tend to elicit or further support a response to antigen, whereas complexes containing excessive amounts of antibody (antibody excess), as might be found late, tend to suppress the response. In vitro studies have shown that pre-formed immune complexes bind B lymphocytes and stimulate B cell proliferation under a variety of experimental conditions. The mechanisms of action on the antibody response are complex, however, and variably depend on the presence of T lymphocytes, macrophages, and complement as well as low amounts of specific antibody within the complexes.[

27] [28] [29] [30] [31] [32] [33]

Normal serum contains small amounts of natural anti-bodies of the IgM, IgG, and IgA isotypes that can react with foreign or self antigens, and these complexes may have the potential to elicit an immune response. Specific IgM and very low amounts of IgG antibodies administered passively will enhance the immune response to 34 35

low concentrations of T cell–dependent particulate antigens (erythrocytes, dextran) that otherwise do not induce a response alone.[ ] [ ] Interestingly, IgM exhibits its effects over a wide range of concentrations to induce the production of IgM antibodies of the same specificity, but low concentrations are required to promote antigen deposition in the spleen and the generation of specific IgG antibodies.[

36] [37]

Furthermore, this process requires an intact complement system and the 38 39

interaction of complement-opsonized IgM complexes with complement receptors CR1 and CR2.[ ] [ ] Solubilized immune complexes containing the C3 degradation fragment C3d can bind CR2 (CD21), a 140-kD protein on the membrane of mature B lymphocytes. This C3d bound to antigen increases the affinity of binding to the B cell. CR2 is physically associated with B cell–signaling proteins CD19, CD81 (TAPA-1), and Leu-13, and this complex cross-linked with membrane 40

Ig contributes greatly to B cell activation and proliferation.[ ] C3d bound to antigen or to an immune complex functions as an adjuvant, enhancing the immunogenicity of an antigen and dramatically lowering the threshold for B cell activation and ultimately the threshold for specific antibody production. In the mouse, specific IgG antibodies can also enhance IgM and IgG titers to particulate antigens and soluble proteins, but in contrast to the mechanism of action of IgM, IgG enhancement appears to depend primarily on antigen processing and presentation to antigen-specific T cells through macrophage FcγRI and FcγRIIB2 receptors, with little assistance from lymphocyte CR1/CR2 or FcγRIIB1 receptors. [

39] [41] [42]

However, studies with human materials have shown otherwise, and 43 44 45

both complement receptors (CR1, CR2) as well as FcγRII on B lymphocytes may participate in IgG-induced antigen presentation.[ ] [ ] [ ] There also appears to be a synergistic effect of IgG and IgM natural antibodies on B cell presentation of soluble antigen to antigen-specific T cells after complement binding, and CR2 has 45]

been shown to enhance the interaction between FcγRII and suboptimal concentrations of IgG within the immune complex.[

During the course of a strong immune response, antigens form complexes with natural antibodies or antibodies generated during the primary response. Immune complex formation leads to their entrapment in secondary lymphoid organs, the formation of germinal centers, and generation of memory B cells. Immune complexes that activate complement and interact with erythrocyte CR1 have an increased capacity to bind resting B lymphocytes through CR2; these interactions in

46 47 48

turn may lead to increased immune complex deposition in the spleen.[ ] [ ] [ ] Early studies demonstrated that germinal center formation in the spleen requires both antibody and complement, and that immune complexes formed were associated

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49 50

with marginal zone antibody-forming cells and follicular dendritic cells (FDCs) during germinal center development.[ ] [ ] Serving as antigen-presenting cells (APCs) for somatically hypermutated B cells, FDCs bind but do not endocytose immune complexes and are able to harbor complexes on their surface for weeks to 51

52

months.[ ] Immune complex retention is first observed outside the primary follicles of the spleen and is a highly radioresistant mechanism.[ ] However, the transport of immune complexes into the follicles, presumably either by a sessile population of B lymphocytes in the marginal zone or by FDCs, is a radiosensitive process, indicating that trapping and transport mechanisms are indeed different and that immune complex retention does not necessarily induce a germinal center 53 54 55

response. [ ] [ ] [ ] The development of germinal centers is a T cell–dependent event driven by massive proliferation of B lymphocytes. Immune complex–bound FDCs are thought to be crucial for the growth and selection of high-affinity, memory B cells in the germinal center and the subsequent production of high-affinity 56 57 58 59

antibody. Also, CR1/CR2 and Fcγ receptors on FDCs have been shown to play important roles in the generation of a strong memory response.[ ] [ ] [ ] [ ] Splenic localization of immune complexes with generation of a memory response is not an antigen-specific process, and the retention of antigen within the lymphoid follicles and resultant germinal center formation are highly dependent on immune complex enhancement of the immune response. The prophylaxis against Rh-hemolytic disease in the newborn entails the therapeutic use of IgG anti-Rh (Rhesus) D antibodies to suppress an immune response. To prevent immunization against fetal RhD+ erythrocytes via transplacental passage, women are routinely administered human IgG anti-Rh D antibodies; this therapy in 60

turn dramatically reduces the occurrence of hemolytic disease of the newborn.[ ] This was a major advance in clinical immunology, but the mechanism by which the administered antibody inhibits the immune response remains unclear. IgG antibodies have been shown to suppress both IgM and IgG antibody responses to particulate antigens, soluble antigens, and antigens administered in adjuvants, although the primary response to antigen appears more affected than the secondary response.[

61]

High-affinity antibodies are more active, and no one IgG subclass appears superior in activity. T cell priming remains unaffected, and suppression still

occurs in complement-deficient states.[

62]

Immune complex ligation to the inhibitory FcRγIIB1 has been implicated as one possible mechanism of suppression in the 21]

mouse, and this ligation may interrupt the processes of B cell activation, proliferation, and antigen presentation.[ 42

Mice deficient in this receptor develop enhanced 63

antibody responses to soluble immune complexes.[ ] However, IgG is capable of suppressing antibody responses to erythrocytes even in deficient mice, [ ] suggesting that FcγRIIB1 predominantly serves to prevent normal antibody responses after antigen presentation from reaching abnormal levels. Another mechanism for the Rh effect is that enhanced immune adherence of immune complexes to phagocytes by Fc receptors may increase erythrocyte clearance and decrease adherence to activated B cells, thus inhibiting the immune response. It has been demonstrated that larger complexes, or complexes formed in antibody excess, have a high affinity for phagocytes, as opposed to smaller immune complexes, with suboptimal concentrations of antibody, which tend to have a high affinity for antigenpresenting monocytes and lymphocytes.[

64] [65]

A third explanation proposes that IgG has the ability to mask all antigenic epitopes, thereby interfering with the

ability of activated B cells to recognize the antigen within the immune complex.[

61]

The ability of F(ab′)2 fragments of IgG to suppress a response also supports this

mechanism. The binding of immune complexes by APCs, however, is not affected, which may reflect the weak inhibition of the secondary and memory responses to antigen.

ENDOTHELIAL CELLS, CELL ADHESION MOLECULES, AND CYTOKINES It is reasonable to ask how immune complexes induce vasculitis and other forms of tissue injury. The ability of immune complexes to interact with and activate various mediator pathways is well known. Therefore, for example, complement proteins may be activated to generate a host of inflammatory complement peptides (see Chapter 6 ). Similarly, direct damage to vessels plays an integral part in the development of these diseases. Increasing evidence also clearly indicates that endothelial cells are not passive bystanders in the evolution of immuno-logically mediated inflammation and tissue 66 67 68 69 70

damage, as occurs in immune complex–mediated diseases.[ ] [ ] [ ] [ ] [ ] The ability of these cells to synthesize proinflammatory mediators such as interleukin1 (IL-1), IL-6, IL-8, as well as proteolytic enzymes and tissue factor, indicates their potential for an active role in the inflammatory response. In addition, endothelial cells are known to synthesize and express a series of important cell-surface glycoproteins known as cell adhesion molecules (CAMs). CAMs are cell surface receptors that mediate cell-cell and cell-matrix interactions and play critical roles in diverse biologic processes, including inflammation, wound healing, coagulation, tumor metastasis, cellular growth, and cellular differentiation. At least five different CAM families exist: the integrins, immunoglobulin supergene family, selectins, adherins, and cartilage-link proteins. Endothelial cells express or can be induced to express CAMs from most of these families. Some CAMs on endothelial cells mediate the attachment of leukocytes by binding to CAMs on the leukocytes. This “lock and key” phenomenon demonstrates specificity and is a highly regulated process. Thus, intercellular adhesion molecule-1 (ICAM-1) is present on endothelial cells and binds to the CAM leukocyte function–associated antigen-1 (LFA-1) on leukocytes. Furthermore, ICAM-1 expression on endothelial cells can be substantially increased by proinflammatory mediators, such as IL-1, tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and lipopolysaccharides (LPS), and has been shown to be complement 71

dependent during acute inflammatory lung injury.[ ] These factors in turn lead to enhanced leukocyte binding and subsequent migration into tissue. In addition, three CAMs—E-selectin, also termed endothelial cell leukocyte adhesion molecule-1 (ELAM-1); vascular cell adhesion molecule-1 (VCAM-1); and P-selectin, also termed granular membrane protein-140 (GMP-140)—are not normally expressed by endothelial cells, but their expression can be induced rapidly by proinflammatory mediators (e.g., IL-1, TNF-α), which up-regulate E-selectin (ELAM-1) and VCAM-1, or by histamine, which up-regulates GMP-140. Because ELAM-1 and GMP-140 preferentially bind polymorphonuclear neutrophil leukocytes (PMNs), these CAMs are critical in the generation of acute tissue inflammation.

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It has also become clear that the time course of CAM induction or up-regulation is an important aspect of the evolution of the character of the inflammatory cell infiltrate. GMP-140 and ELAM-1, which are rapidly but transiently expressed on the surface of endothelial cells, preferentially bind PMNs, whereas the somewhat later appearance of VCAM-1 or up-regulation of ICAM-1 may be more responsible for monocyte and lymphocyte binding. Immune Complexes and Cytokine Release

Immune complex–mediated injury is associated with a number of inflammatory cytokines. Dermal vascular injury is highly dependent on IL-1β expression, and both 72

IL-1β and TNF-α are required for an optimal immune complex–induced injury in the lung.[ ] Immune complexes also induce the release of IFN-γ and IL-2, and consequently, release of IFN-γ is highly correlated with increased FcγRIIA expression on human monocytes and enhanced antigen presentation, as well as with increased synthesis of essential complement components during the inflammatory response.[

73] [74] [75] [76]

Complement opsonization of immune complexes in turn 77

affects cytokine profiles, and inefficient opsonization is associated with increased IL-1β and IL-8 secretion from leukocytes.[ ] Complement C1q-bound immune complexes have also been shown to induce secretion of interleukin-8 (IL-8) from human umbilical vein endothelial cells (HUVECs) and from synoviocytes of patients with juvenile rheumatoid arthritis (JRA).[

78] [79]

The chemotactic complement fragment C5a has been widely studied and is associated with the production

of IL-1β, TNF-α, and IL-8, as well as the sometimes anti-inflammatory IL-6 in human leukocytes.[

80] [81]

Cytokine patterns characteristic of a proinflammatory T helper cell 1 (Th1) type of immune responses depend on the immune complex antigen/antibody ratio. As the 82

ratio approaches equivalence and antibody excess, a cytokine pattern shift from a Th1 to T helper cell 2 (Th2) type of response has been reported.[ ] During this shift, IL-6 and IL-10 are released and have been shown to down-regulate the secretion of TNF-α and IL-1β, thus augmenting the stimulatory effects of 83] [84]

proinflammatory cytokines on vascular permeability and neutrophil accumulation. [

In addition, interleukin-4 (IL-4) release has been found to down-regulate 73

the IFN-γ–induced expression of FcγRIIA and up-regulate the expression of the inhibitory receptor, FcγRIIB2.[ ] An immune complex–induced shift from proinflammatory to antiinflammatory cytokine release and the consequential modulatory effects on cellular expression of FcγRs may prove essential to the attenuation of immune complex–induced inflammation.

ANIMALS MODELS OF ANTIBODY-MEDIATED AND IMMUNE COMPLEX–MEDIATED TISSUE DAMAGE Arthus Reaction The Arthus reaction is named after Maurice Arthus, who in 1903 noted that daily injections of horse serum into the skin of rabbits resulted in a localized cutaneous 85

reaction characterized by erythema, edema, hemorrhage, and necrosis. [ ] This reactivity was found to be transferable with the serum of the immunized rabbit, and it was associated with the development of antibody to the horse serum. The Arthus reaction has been studied in great detail, and it is known that the cutaneous 86

inflammation is caused by a local vasculitis of the small blood vessels of the skin.[ ] The vasculitis is caused by the formation of antigen-antibody complexes in the region of the vessel walls, their binding to cells with Fc receptors, and the subsequent activation of a variety of mediators of inflammation. The type of reaction just described, in which an animal is actively immunized and the same antigen is then injected intradermally, is called the active Arthus reaction. Several variations exist: (1) the direct passive Arthus reaction, in which immune serum is administered intravenously to an unimmunized animal, followed by the immunizing antigen intradermally; (2) the reverse passive Arthus reaction, in which antibody is given locally and antigen intravenously; and (3) the local passive Arthus reaction, in which both antibody and antigen are administered locally. Active Arthus reactions reach peak intensity between 4 and 10 hours. If sufficient antigen and antibody are injected, there is pronounced hemorrhage and edema in the site of the reaction, followed in some cases by necrosis 8 to 24 hours after injection. Histopathologic studies of the Arthus reaction have shown that PMNs rapidly marginate in the small blood vessels, penetrate the walls, and accumulate in the surrounding tissue ( Figure 58-2 ). Intravascular clumping of platelets also occurs. The PMNs undergo leukocytoclasis, the walls of the blood vessels become swollen, and the endothelial cells appear damaged. As the reaction progresses,

hemorrhage from the damaged cutaneous vessels may become prominent. Immunofluorescent studies have shown that the earliest event is the deposition of antigenantibody complexes and complement in and around blood vessel walls. Platelets are not required in the Arthus reaction, but PMNs are required for the full expression of the reaction. Even in the absence of PMNs, edema may be noted in some patients, although it tends to be less striking than in the presence of neutrophils. Until recently, it seemed clear that phagocytes played a role in the second phase of the Arthus reaction, during which neutrophil immigration was induced by chemotactic complement fragments. As noted in the section on Fc receptors, the situation is much more complex now. First, it is clear that histamine release plays a 87

role in the deposition of immune complexes in tissue, as suggested by earlier work of Kniker and Cochrane[ ] and reinforced by the later finding that histamine release was required for an Arthus reaction using guinea pig antibodies, in which the histamine releasing γ1 can be separated from the non–histamine-releasing γ2 . [88]

Both were required for an Arthus reaction. Immune complex–mediated serum sickness in humans is also preceded by a wave of histamine release (see later

section). The Arthus reaction is attenuated in strains of mice that have been developed with depleted mast cells,[

89]

and as mentioned earlier, the cutaneous Arthus 10

reaction is quite depressed in animals that have a knockout of the FcRγ chain gene and that thereby lack FcγRI, FcγRIII, and FcepsilonRI.[ ] In the latter case, mice studied using the reverse passive Arthus technique, in which antigen is given intravenously and antibody is injected in the skin, had minimal Arthus reactivity and minimal infiltration with neutrophils. Other necessary elements of the Arthus reaction have been further dissected. It is known that precipitating antibody is required and that nonprecipitating antibody is less effective,

1003

Figure 58-2 Histopathologic features of guinea pig Arthus reaction after 4 hours, with massive infiltration of polymorphonuclear neutrophils in wall of small cutaneous blood vessel, leukocytoclasis, endothelial cell swelling, and extravasation of erythrocytes. (From Lawley TJ, Frank MM. In Parker C, editor: Clinical immunology, Philadelphia, 1980, WB Saunders.)

(From Lawley TJ, Frank MM. In Parker C, editor: Clinical immunology, Philadelphia, 1980, WB Saunders.) even if it is capable of activating the complement system. An intact complement pathway is required for optimal reactions, although the classical pathway is not needed. [

90]

The requirement of the Fcγ receptors and complement in immune complex–mediated inflammation has become increasingly evident, but their 91 92 93 94

independent and synergistic effects can be highly variable depending on the organ affected and the animal model used.[ ] [ ] [ ] [ ] For example, in murine models of immune complex deposition in the lung and the skin, FcγRIII and complement C5a receptor (C5aR) on mast cells play interdependent roles in inducing 80] [81]

inflammation.[

In the lung, complement C3 has also been shown to be influential in recruitment of neutrophils and increased proinflammatory cytokine 95

production; however, C3 is not required for a cutaneous reaction.[ ] C5aR triggering has proven to be a C3-independent process in the skin of the mouse, providing an explanation for a normal Arthus in C3-deficient mice. In murine models of immune complex deposition in the peritoneum, complement and mast cells are involved in initial neutrophil infiltration and TNF-α production, and depending on the strain of mouse, FcγRI on macrophages and complement mediate the 92] [96]

remaining course of the inflammatory response.[

Clearly the details of the model are of critical importance in determining the pathologic consequences.

Forssman Shock 97 98

Forssman shock is a reaction noted in guinea pigs that resembles anaphylaxis.[ ] [ ] It is mediated by antibody directed against a lipopolysaccharide structural tissue antigen termed the Forssman antigen. The Forssman antigen is composed of five sugar residues attached to a ceramide moiety in which the terminal sugar N 99]

acetylgalactosamine (GalNAc) is linked by an α, 1 → 3 linkage to the penultimate GalNAc.[

Animals are either Forssman positive or Forssman negative,

depending on whether they have the enzymes necessary to form this rigid sugar side chain. A Forssman-negative animal, such as the rabbit, responds to injections of Forssman antigen vigorously with the formation of large amounts of antibody. Intravenous injection of IgG anti-Forssman antibody into a Forssman-positive animal, such as the guinea pig, results in a striking clinical reaction. The guinea pig develops respiratory distress within minutes of the injection, caused by exudation of fluid into alveoli and bronchi. This is rapidly followed by convulsions, hypotension, hemorrhage into the lung, and death. Although this reaction resembles anaphylaxis, it is believed to have a different mechanism. Forssman shock does not require homocytotropic antibody, cannot be blocked by antihistamines or epinephrine, and does 100 101

not appear to be dependent on mast cell degranulation.[ ] [ ] Unlike anaphylaxis, complement is required for Forssman shock, and more specifically, an intact classic complement pathway is necessary. Presumably, mediators released on complement activation cause endothelial cell and alveolar damage with edema formation, hemorrhage, and death. Anaphylaxis 102

The anaphylaxis reaction was first described in 1902 by Portier and Richet,[ ] who were studying sea anemone toxicity and had injected extracts of tentacles into dogs. They found that dogs, given a second injection several weeks after the first, developed rapid onset of respiratory distress, diarrhea, and hematemesis followed by death. Analysis of this reaction led to the realization that immunoglobulin E (IgE) antibody bound to mast cells plays a central role in this reaction pattern. IgE is not an absolute requirement, however, because anaphylaxis can be induced in IgE-deficient mice.[ Chapter 83 ).

103]

Presumably, IgG substitutes for IgE in these animals (see

Acute Serum Sickness In the 1950s, with the advent of radiolabeled isotopes, it became possible to study the pathophysiologic basis of serum sickness in animals. Investigators developed a rabbit model of

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Figure 58-3 Graph of time course of “one-shot” serum sickness. Radio-labeled bovine serum albumin is injected intravenously into normal rabbits at day 0, and sequence of immunologic events is followed. At about day 8, immune complexes are formed, complement levels fall, and the animals become ill. By day 15, foreign antigen and immune complexes are no longer detectable, complement levels are rising, and animals are recovering. (From Lawley TJ, Frank MM In Parker C, editor: Clinical immunology, Philadelphia, 1980, WB Saunders.)

(From Lawley TJ, Frank MM In Parker C, editor: Clinical immunology, Philadelphia, 1980, WB Saunders.) 104 105 106 107

the disease.[ ] [ ] [ ] [ ] Rabbits were injected intravenously with radiolabeled bovine serum albumin ( Figure 58-3 ). After an initial period of intravascular equilibration lasting several minutes and intravascular-extravascular equilibration lasting several days, the concentration of the antigen in the circulation declined slowly because of physiologic degradation of the injected serum protein. Between days 6 and 8, the rate of decline in the concentration of the radiolabeled protein suddenly accelerated. This coincided with the appearance in the circulation of rabbit antibodies directed against bovine serum albumin. The antibody bound to the antigen-forming immune complexes, and these were cleared from the circulation by cells of the RES. During the period of immune complex formation, usually days 8 to 13, many pathophysiologic events took place. First, immune complex levels rose, reached a peak, and then declined. At the same time, serum complement levels declined. The animals developed clinical illness, including arthritis, glomerulonephritis, and vasculitis. The glomerulonephritis was characterized by swelling of endothelial cells and proteinuria, but little hematuria and few granulocytes in the renal glomeruli. In this regard it is interesting that, if one treats the rabbits with drugs that block the PMN response or decrease the level of the late complement components, one finds little change in the renal pathologic condition of this acute glomerulonephritis. Immunofluorescence stud-ies demonstrated antigen, host immunoglobulin, and C3 deposited along the glomerular basement membrane in a typical granular pattern. On electron microscopic examination of kidney biopsy specimens, only swelling of endothelial cells was seen. As the reaction progressed, subepithelial electron-dense deposits were found in some rabbits, but by this time, immunofluorescence studies were negative. The arteritis associated with the acute serum sickness model in rabbits was also studied in detail. There is a high incidence of arteritis in the coronary artery outflow tract and at branching points of the aorta. PMNs enter the site of intimal proliferation, and their presence is followed by the appearance of degraded internal elastic

media and adventitia, with resulting fibrinoid necrosis of the vessel. On immunofluorescence, host immunoglobulin, antigen, and C3 were found roughly in the region of the internal elastic lamina, but these immunoreactive materials were rapidly removed and were gone in several days. It was suggested that the PMNs present in the lesions phagocytose these complexes. In contrast to the findings in glomerulonephritis, materials that decrease complement activity or inhibit the PMN response diminished or blocked the development of arteritis. At the time of the development of serum sickness in this animal model, there were large immune complexes in the circulation of the animal, and animals that become sick regularly showed the presence of complexes that were greater than 19S in their sedimentation characteristics. It was believed that these complexes are deposited in tissues, thereby causing the disease. Nevertheless, it proved difficult to duplicate this sort of disease by the injection of pre-formed complexes into animals. 108

A rabbit model of chronic serum sickness also has been developed.[ ] The animals are given frequent repeated injections of a foreign protein over a period of weeks. In animals that develop an antibody response, renal disease occurs after 4 to 10 weeks of injections. This is manifested initially by proteinuria but progresses to renal failure if the injections are maintained. The dose of antigen administered is critical and must be specifically tailored so that antigen excess is achieved. In addition, circulating immune complexes must be formed and circulate in the animal. These animals do not develop the arteritis characteristic of acute serum sickness. The renal disease is manifested mainly by a proteinuria that can become so severe that it causes nephrotic syndrome. The pathologic changes found in the kidneys depend in part on the vigor of the antibody response to the injections of antigen. Animals that have a moderate antibody response show thickening of the glomerular basement membrane without considerable cellular proliferation or PMN infiltrates. Animals that respond by making high-titer antibody have glomerular lesions that are more inflammatory in nature, with infiltrates of PMNs. In addition, these animals also have mesangial proliferation and crescent formation. Antigen, antibody, and complement deposits have been identified in these lesions. Arthritis Several animal models have been developed to mimic the pathology of human rheumatoid arthritis (RA). The most widely studied model is type II collagen–induced arthritis (CIA). In susceptible strains of rodents and primates, immunization with heterologous collagen type II in adjuvant induces polyarthritis with characteristic inflammatory synovitis, pannus formation, cartilage destruction, and bone erosion. CIA can also be transferred to recipients using antibodies to collagen type II, whether as a hyperimmune serum concentrate, polyclonal IgG anti-bodies, or monoclonal antibodies specific for different collagen domains. The role of T cells in disease progression has been demonstrated and shown to be genetically linked to the major histocompatibility complex class II (MHC II) molecules in

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susceptible mice of several H2 haplotypes.[

109]

In addition, germline deletions in Vβ T cell antigen receptor (TCR) genes render mice resistant to CIA, and antibodies

to T cell CD4 can block disease onset. High levels of specific anticollagen antibodies are also found, suggesting a major role for B cells as well. B cell–deficient mice show resistance to CIA. Other studies have suggested the participation of Fc receptors and complement in disease pathogenesis.[

110] [111]

112] [113]

A recent model of arthritis used KRN TCR transgenic mice on a C57Bl/6 (wild-type)x NOD (nonobese diabetic) background.[

At 3 to 5 weeks of age, these

mice (abbreviated K/BXN) spontaneously develop joint disorders. Characteristic symptoms of human RA rapidly follow with elevated levels of proinflammatory cytokines and antibody production, although rheumatoid factor is not elevated. Arthritis in K/BXN mice is initiated by the cross-reactivity of KRN TCR transgenic cells and a peptide bound to NOD-derived Ag7 molecules of MHC II–positive cells, and this reaction in turn stimulates specific B cells to produce antibody. This peptide, or autoantigen, was identified as glucose-6-phosphate isomerase (GPI), a glycolytic enzyme, and high levels of anti-GPI antibodies have been recently found 114 115

in the serum and synovial fluid of RA patients.[ ] [ ] Transfer of immunoglobulins from K/BXN mice induces arthritis in healthy inbred strains of mice, and genetic mapping of susceptible strains of mice reveal two chromosomal regions of importance: (1) the proximal region on chr2 containing the complement gene C5 locus and (2) the distal region on chr1 containing the Sle1 locus that controls SLE-like disease in mice.[ depleted

116]

This mode of transfer does not occur in neutrophil-

117 recipients.[ ]

METHODS OF DETECTING CIRCULATING IMMUNE COMPLEXES 4 118

Many assays are available that detect circulating immune complexes. [ ] [ ] These range from relatively insensitive but easily performed assays that rely on physical characteristics of immune complexes, such as cryoprecipitation, to more complex assays that rely on other characteristics of the immune complexes, such as complement binding or binding of complexes to immunologically specific cell surface receptors. Sensitive radioimmunoassays can now detect circulating immune complexes containing IgG, IgM, IgA, or IgE as the immunoglobulin, as well as assays that specifically detect bound fragments of C3. It should be noted, however, that most immune complex assays are not antigen specific. In most cases in which circulating immune complexes are detected, the antigen is unknown. Additionally, both false-positive and false-negative results can occur in immune complex assays, although the precise substances responsible will vary with each assay. For example, aggregated immunoglobulin, anticoagulants, endotoxin, and free deoxyribonucleic acid (DNA) are some of the substances that may cause false-positive results. In studies of immune complex levels in humans, usually for research purposes, it is thus best to use at least two different assays based on different immunologic principles to minimize these effects. Several of the most sensitive and widely used assays are discussed next. 125

I-C1q Binding Assay

The hexamer C1q is a subcomponent of the first component of complement and will bind by its multiple binding sites to immune complexes containing IgG 119 120

subclasses 1, 2, or 3 or IgM by noncovalent attachment to a specific site on the Fc portion of the immunoglobulin.[ ] [ ] C1q will also bind to polyanionic substances in serum, such as bacterial endotoxin or DNA, resulting in spurious assay results unless reaction conditions are controlled. The 125 I-C1q binding assay is performed by the addition of a known amount of 125 I-C1q to test sera that have been pretreated with disodium edetate (ethylenediaminetetraacetic acid [EDTA]). [121] [122]

The EDTA dissociates the C1 in the test sera and frees C1q. The radio-labeled C1q binds to immune complexes containing IgG subclasses 1, 2, or 3 or IgM, and the complex is then detected. The sensitivity range of this assay is broad; test sera containing DNA or endotoxin do not give false-positive results with this method because they are soluble in the low-percentage polyethylene glycol (PEG) used in this assay. However, aggregated immunoglobulin or heparin in test samples will give false-positive results in the 125 I-C1q binding assay. 123

A variation of this assay is the C1q solid-phase assay in which the C1q is bound to the sides of plastic test tubes.[ ] Immune complexes in serum samples will bind to the solid-phase C1q and be detected by radiolabeled antibody against human IgG or IgM. This assay has the advantage of detecting specific classes of antibody in the immune complexes, whereas the fluid-phase125 I-C1q binding assay will not. It must be recognized that in some cases autoantibodies to C1q may be responsible

for false-positives in the C1q binding assay. These autoantibodies were found to co-sediment with normal monomeric IgG and bind to the collagen-like region of 124]

C1q.[

Solid-Phase Anti-C3 Assay 125

The solid-phase anti-C3 assay employs anti–human C3 bound to a solid-phase matrix.[ ] The anti-C3 captures C3 in the test serum. If bound to immune complexes, the C3 is captured as well and detected with a radiolabeled anti-Ig. This assay is sensitive and is not affected by non–immune complex substances such as polyanions, endotoxin, and rheumatoid factor. It is also not inhibited by native C3 in the test sera because the solid-phase anti-C3 antibody is present in vast excess. [126]

Raji Cell Radioimmunoassay Raji cells are a human lymphoblastoid cell line originally derived from a patient with Burkitt's lymphoma. These cells have cell surface receptors for complement components C1q, C3b, C3bi, and C3d; Raji cells lack surface immunoglobulin; and their cell surface Fc IgG receptors are either of low affinity or low in number and 127 128 129 130

do not interfere with the assay.[ ] [ ] [ ] [ ] Immune complexes containing complement components bind to the surface of Raji cells and thus can be isolated for detection. After incubating with radiolabeled antiimmunoglobulin, the amount of radioactivity adhering to the Raji cells is used as a measure of bound immune complexes. IgG-containing circulating immune complexes are detected in this system by using radiolabeled anti–human IgG, IgA-containing circulating immune complexes that bind with anti–human IgA, and IgM-containing circulating immune complexes that bind with radiolabeled anti–human IgM. Although the Raji cell assay is sensitive and reproducible, warm reactive antilymphocyte antibodies found in sera of patients with certain diseases (e.g., SLE) may cause false-positive results.

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Conglutinin Assay Conglutinin is a 75,000-D nonimmunoglobulin protein found in certain bovine sera that will bind to a cleavage fragment of human C3 known as iC3b. Immune 131 132

complexes containing iC3b will bind to conglutinin attached to a solid-phase substrate and can then be detected.[ ] [ ] Although this assay is sensitive and easy to perform, iC3b may be converted to another C3 fragment, C3dg, within 1 to 8 hours of its generation; this mechanism may allow some circulating immune complexes in test sera to escape detection. Monoclonal Rheumatoid Factor Assay Rheumatoid factors (RFs) are immunoglobulins that bind to IgG. Because RFs have a higher binding affinity for aggregated IgG or immune complexes containing

133 134

IgG than that for monomeric IgG, they can be used to detect circulating immune complexes.[ ] [ ] The most common RF assay uses RFs bound to a solid-phase matrix in a competitive inhibition assay. IgG-containing circulating immune complexes in test sera compete with radiolabeled, aggregated human immunoglobulin for binding to monoclonal RFs. The percentage inhibition of radiolabeled immunoglobulin binding to the solid-phase RFs gives an estimate of the amount of circulating immune complexes present in the test area. Monoclonal RF assays detect IgG-containing circulating immune complexes without dependence on their complement-fixing ability. However, RFs in the test sera may give false-negative results.

DISEASES MEDIATED BY CIRCULATING IMMUNE COMPLEXES For years it has been clear that a selected group of diseases is characterized by either the deposition of immune complexes in damaged organs or the presence of circulating immune complexes. Previous editions of this book considered vasculitis, SLE, and glomerulonephritis in this group. However, it has never been clear how the distinctive features of disease ranging from temporal arteritis to glomerulonephritis could be explained simply by the formation of circulating immune complexes. Now, we have much more information about several of these diseases, and the earlier formulations have proved to be oversimplified. Most experts in the field of 135

vasculitis do not believe that circulating immune complexes provide a sufficient explanation for the range of diseases.[ ] It is now recognized that many of the diseases with leukocytoclastic vasculitis as a common feature are characterized by the presence of circulating antibody to distinctive cellular antigens. 136

The most prominent of these antigens is the ANCA (antineutrophil cytoplasmic antibody) family.[ ] For example, patients with Wegener's granulomatosis have a very high incidence of ANCA with specificity for a neutrophil granular protein, proteinase 3. Patients with crescentic and necrotizing glomerulonephritis have a higher than normal incidence of an ANCA that recognizes myeloperoxidase. These proteins may be expressed on the surface of activated neutrophils and in some cases on endothelial cells as well. They may well play an essential role in the development of vasculitis. As with CAMs and their role in cellular infiltration, T cell immunity similarly has been demonstrated in these diseases, and in many cases T cells have been demonstrated in lesions. Thus the set of diseases clearly mediated by circulating immune complexes is becoming ever more circumscribed. We emphasize serum sickness in this discussion because all the pathologic features of the disease appear with the presence of circulating immune complexes and disappear when they are cleared from the circulation. The relevance of this disease to SLE is emphasized. Glomerulonephritis is discussed briefly. Once again, the focus of modern research has deemphasized the importance of circulating immune complexes in the disease and focused attention on two additional 137

mechanisms to explain many cases.[ ] The first mechanism of disease induction follows the generation of an immune response to an antigen of the glomerular matrix. In situ immune complexes form, and the inflammatory cascade is initiated. A model for the mechanism is Heymann's nephritis in the rat (see earlier discussion).[

20]

A second mechanism is the induction of an antigen not usually present on glomerular epithelial cells by the release of cytokines and other mediators, 137

such as prostaglandins, in the region of the glomerulus, with a subsequent immune response.[ ] The glomerular lesions induced may be inflammatory in type or noninflammatory. For example, it has been suggested that membranous nephropathy follows the deposition of antibody, complement activation, and deposition of C5b-9 on the glomerular epithelial cell. Although complement is activated, there is little or no cellular response. This is an example of a noninflammatory lesion. Inflammatory lesions follow the generation of the appropriate cytokines and chemotactic factors, with important contributions to the lesions by cellular elements, including neutrophils, macrophages, mesangial cells, and platelets. Serum Sickness

The most common cause of serum sickness today is a hypersensitivity reaction to drugs. This reaction is usually manifested by fever, malaise, and urticarial or morbilliform cutaneous eruptions. Some patients may develop arthritis-arthralgias, nephritis, neuropathy, or vasculitis. Generally, serum sickness occurs 1 to 3 weeks after the start of administration of the medication, although it can occur within 12 to 36 hours in individuals who have been sensitized during a previous exposure. Medications frequently implicated in drug-induced serum sickness include penicillin, sulfonamides, hydantoins, phenylbutazone, and thiazides, as well as cefaclor in children. Foreign antisera and blood products are also capable of producing serum sickness. Drug-induced serum sickness usually abates within days after discontinuation of the agent, although with long-acting or repository drugs, the reaction may persist longer. Unfortunately, a thorough analysis of the pathophysiology of this type of serum sickness that evaluates the individual roles of immunoglobulin type, platelets, PMNs, and complement components in disease development is not yet available. An example of current analysis and manifestations of immune complex–related diseases is serum sickness induced by injection of horse globulin. In one study, patients with bone marrow failure were undergoing treatment with 1•mg/kg/ day of horse antithymocyte globulin (ATG) administered intravenously for 14 or 28 138

days.[ ] The patients also received 1 to 1.5•mg/kg/day of methylprednisolone. Of the 35 patients treated in this protocol, 30 developed typical signs and symptoms of serum sickness 8 to 13 days after beginning therapy, including fever and malaise (100%), skin rashes (93%),

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Figure 58-4 Hand of patient with bone marrow failure who developed serum sickness during therapy with horse antithymocyte globulin. Seventy-five percent of patients who developed cutaneous signs of serum sickness had this thin band of erythema or purpura at junction of skin on sides of hands, feet, fingers, or toes with that of dorsolateral surface. (From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

(From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.) arthralgias (67%), myalgias (67%), gastrointestinal disturbances (67%), and lymphadenopathy (20%). Of patients who had cutaneous signs of serum sickness, 75% developed a previously undescribed cutaneous eruption, which may be a specific sign of serum sickness. This eruption consisted of a band of erythema along the sides of the hands and feet and fingers and toes at the junction of skin on the palmar or plantar surface with that of the dorsolateral surface ( Figure 58-4 ). Often, this was not only the first cutaneous sign of serum sickness but also the first sign of serum sickness itself, occurring on the average at day 6 ± 1 after the initiation of ATG therapy and disappearing a few days later. In addition, patients were followed sequentially with immune complex and complement level determinations. Patients who developed serum sickness developed high levels of circulating immune complexes, as measured by the 125 I-C1q binding assay, that reached a peak at day 10 ± 1, coincident with peak clinical disease activity ( Figure 58-5 ). Patients with serum sickness also had pronounced decreases in serum C3 and C4 levels coincident with disease onset ( Figure 58-6 ). The lowest levels of C3 and C4 were closely correlated with the peak immune complex values. Furthermore, when lesional skin biopsy specimens were obtained during clinical serum sickness and examined with direct immunofluorescence techniques, deposits of IgM and C3 were found in the blood vessel walls of six of nine and seven of nine patients, respectively ( Figure 58-7 ). IgE and IgA deposits were also frequently present. These data clearly support the concept that serum sickness in humans, as in the rabbit model, is an immune complex–mediated disease. Vasculitis Vasculitis is usually defined as inflammation of blood vessels resulting in local or remote tissue damage. The involved blood vessels may be small, such as the postcapillary venules of skin, or large vessels, such as the temporal artery or the arch of the aorta. The types of inflammation vary from necrosis from infiltration with PMNs to granulomatous changes in the vessel wall and perivascular tissue, to intimal proliferation with fibrosis and scarring of the media. As mentioned earlier, it is not clear at present that vasculitis is simply a manifestation of the presence of circulating immune complexes. Nevertheless, it is suggested that immune complexes are present in the disease. For example, in necrotizing vasculitis of small and medium-sized vessels, histologic studies of sites of cutaneous vasculitis reveal endothelial cell swelling and necrosis, hemorrhage, fibrin deposition, a neutrophil-rich infiltrate, and leukocytoclasis. Immunopathologic study of early skin lesions (less than 24 hours old) reveals immunoglobulin, complement, and fibrin in blood vessel walls. Bacterial, mycobacterial, and viral antigens have also been detected in affected vessels of some individuals with cutaneous necrotizing

Figure 58-5 Time course of immune complex formation in patient treated with antithymocyte globulin who developed serum sickness. The 125 I-C1q binding assay and Raji cell IgG radioassay were employed. In the Raji cell assay, results are expressed as Ti /Ui . T i , Mean of duplicate values of test serum; U i , upper 95% confidence limit of normal serum in assay. (From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

(From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

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Figure 58-6 Serum C3 and C4 levels fall precipitously during serum sickness. Same patient as in Figure 58-5 . (From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

(From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.) vasculitis. The presence of immune complexes at lesional sites is also suggested by electron-dense deposits at the endothelial basement membrane zone in areas where immunoreactants are deposited in vessels.[ sensitive radioimmunoassays ( Figure 58-8

139]

Evidence indicates the presence of circulating immune complexes in a high percentage of patients using

140 ).[ ]

Systemic Lupus Erythematosus SLE is a multisystem disease characterized by a myriad of immunologic abnormalities, including the production of autoantibodies, hypergammaglobulinemia, suppressor T cell abnormalities, low levels of serum complement (especially during intervals of active disease), and the presence of circulating immune complexes. Because SLE clinically resembles serum sickness, it was suspected that circulating immune complexes might play a role in its pathogenesis. Before measurement of circulating immune complexes was possible, evidence indicated that immune complexes were important

Figure 58-7 Direct immunofluorescence of lesional skin biopsy during serum sickness. Small blood vessels in papillary dermis reveal deposits of IgM. (From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

(From Lawley TJ, Bielory L, Gascon P, et al: N Engl J Med 311:1407, 1984.)

Figure 58-8 Graph showing high frequency of circulating immune complexes in patients with leukocytoclastic vasculitis and their relative absence in patients with lymphocytic vasculitis. (From Mackel SE, Tappeiner G, Brumfield H, et al: J Clin Invest 64:1652, 1979.)

(From Mackel SE, Tappeiner G, Brumfield H, et al: J Clin Invest 64:1652, 1979.) in SLE. Direct immunofluorescence microscopy of kidney biopsy specimens from SLE patients with nephritis revealed glomerular deposits of immunoglobulin, 141

complement, and DNA, a known antigen in SLE patients.[ ] Similarly, immuno-globulin and complement were found in the blood vessel walls of SLE patients with active necrotizing vasculitis and at the basement membrane zone of skin of patients with cutaneous lesions of SLE. When assays for the detection of circulating

immune complexes became available, a high percentage of SLE patients were found to have these reactants in their serum.[

142] [143]

More importantly, there has generally been a correlation between the levels of circulating immune complexes and the disease activity in SLE patients. Fc-mediated 144

RES clearance studies of SLE patients, using autologous erythrocytes coated with IgG, have shown defective FcR-specific clearance.[ ] The prolonged RES clearance in SLE patients is correlated with both high levels of circulating immune complexes and clinical disease activity. Studies in the same patients after their disease improved with treatment revealed significant correlation among clinical improvement, improvement of Fc-mediated clearance, and decreased levels of 145

circulating immune complexes.[ ] For these reasons, circulating immune complexes and FcR-specific clearance defects are believed to be important elements in the pathogenesis of SLE. Studies have also shown that patients with SLE have decreased numbers of C3 receptors (specifically, CR1 receptors) on their erythrocytes. [146] [147]

Part of this deficiency may be genetic and inherited, but the major portion of the defect is acquired. Erythrocytes with normal numbers of C3b receptors 148

lose a portion of those receptors when infused into patients with SLE.[ ] It is believed that the complement-dependent immune complex clearance mechanisms discussed earlier are associated with removal of the complement receptor as well as the erythrocyte-bound immune complexes as the RBCs pass through the sinusoids of the RES. Glomerulonephritis 1

As previously discussed, the first suggestions that immune mechanisms were operative in some forms of renal disease came from Von Pirquet and Schick,[ ] who noted the development of transient renal impairment (edema and albuminuria) in patients with serum sickness. Schick[ development of poststreptococcal

149]

also noted that the time course for the

1009

glomerulonephritis was similar to that of some hypersensitivity reactions. Subsequent animal studies have confirmed this hypothesis and demonstrated that there are 105 137 144 145 146 147 148 149 150 151 152

at least two general types of immunologically mediated glomerular disease.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] The first type is produced by immunization of animals with homologous or heterologous basement membrane zone antigens. Antibodies are then deposited in the glomerular basement membrane zone of the immunized animal and activate complement, resulting in the recruitment of inflammatory cells and tissue damage. The human counterpart of this disease is Goodpasture's syndrome. Anti–glomerular basement membrane zone disease is believed to account for less than 5% of cases of glomerulonephritis in humans. The second type of immunologically mediated glomerular disease in humans may be immune complex mediated. Studies of animal models in which circulating immune complexes are induced reveal deposits of immunoglobulin and complement in a “lumpy-bumpy” pattern in the glomeruli of animals that develop renal 105

disease.[ ] The human counterparts of these animal models are believed to include, for example, poststrepto-coccal glomerulonephritis, lupus nephritis, and membrano-proliferative glomerulonephritis. This is surmised because the immunofluorescent pattern of the immunoglobulin and complement deposits in these diseases closely resembles that seen in the animal models ( Figure 58-9 ). However, studies using sensitive immune complex assays have yielded variable results. In one study of 100 patients with various types of renal disease, only 18 were found to have circulating immune complexes.[

153]

In contrast, other studies have found

154 155

that substantial numbers of patients with renal disease, especially lupus nephritis, have evidence of circulating immune complexes. [ ] [ ] There is a general agreement that circulating immune complexes cannot be detected as often as one would predict on the basis of animal models. As discussed earlier, it is clear that immune complex deposits in glomeruli can develop in situ.[

137]

Antigen can be deposited in the glomerulus, fix antibody, and activate

Figure 58-9 Direct immunofluorescence of renal biopsy of patient with lupus nephritis showing heavy deposits of IgG in mesangium as well as in peripheral capillary loops. (Courtesy James Balow, U.S. Public Health Service, Bethesda, Md. Data from Davies KA et al: J Clin Invest 90:2075, 1992; Frank MM: J Lab Clin Med 122:487, 1993; Frank MM et al: N Engl J Med 300:518, 1979; Gómez F et al: N Engl J Med 331:1122, 1994; Lambert PH et al: J Clin Lab Immunol 1:1, 1978; Lawley TJ: In Fauci A, editor: Clinics in immunology and allergy, Philadelphia, 1981, WB Saunders.)

(Courtesy James Balow, U.S. Public Health Service, Bethesda, Md. Data from Davies KA et al: J Clin Invest 90:2075, 1992; Frank MM: J Lab Clin Med 122:487, 1993; Frank MM et al: N Engl J Med 300:518, 1979; Gómez F et al: N Engl J Med 331:1122, 1994; Lambert PH et al: J Clin Lab Immunol 1:1, 1978; Lawley TJ: In Fauci A, editor: Clinics in immunology and allergy, Philadelphia, 1981, WB Saunders.) complement, resulting in tissue damage. Mechanisms by which circulating antigens localize to glomeruli include charge-dependent mechanisms in which cationic antigens bind to anionic areas of the glomerular basement membrane.[

156] [157] [158]

Alternatively, cationized IgG molecules may bind to glomeruli and then bind

150 151 152 153 154 155 156 157 158 159 160 reaction.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]

their specific antigen and initiate an inflammatory Anionic antigens may localize to glomeruli in another way. This first involves the interaction of cationic proteins generated by infiltrating leukocytes or platelets with anionic sites in the glomeruli, followed by interaction of the circulating anionic antigens with the cationic proteins bound to the glomeruli. This action then serves to initiate in situ immune complex formation.

LABORATORY FINDINGS

11 161

In most clinical settings the diagnosis of immune complex-related tissue injury is made using a combination of clinical and laboratory findings.[ ] [ ] Most hospitals do not have assays for immune complexes available. Moreover, even when such assays are available, their usefulness is limited. Circulating immune complexes are found in many clinical settings, and in many cases their presence does not indicate the presence of immune complex–induced disease. The formation of immune complexes and their removal from the circulation are believed to be common events. For example, many infectious diseases are associated with the presence of apparently nonpathogenic circulating immune complexes. Moreover, the biochemical characteristics of immune complexes that predispose or preclude their deposition in tissue are not fully known. Some diseases, such as SLE, are regularly associated with the presence of immune complexes in the circulation. Others, such as membranoproliferative glomerulonephritis, are less frequently associated with the finding of circulating immune complexes, as assessed by current methods, even though many believe that immune complexes in the glomeruli are in part responsible for the disease. In some cases this finding indicates that immune complexes are required for initiation of disease but are not necessary once inflammation, scarring, and repair mechanisms of tissue are underway. In other cases this finding represents a lack of proper specificity of the immune complex assay. Finally, as mentioned, immune complexes also may form locally in tissues and never be present in the circulation. Tests used to suggest the presence of immune complexes in the circulation or tissues include tests of complement activity and complement component levels. Reduced levels of complement are taken to suggest immunologic activation. A fall in titer of C4 and C3 is suggestive of classic pathway activation. A fall in titer of C3, factor B, or properdin suggests alternative pathway activation. The former pattern of complement component utilization is often seen in SLE; the latter pattern is often noted in membranoproliferative glomerulonephritis. Many difficulties arise with the use of complement component levels to determine the presence of immune complexes, however, even when the complexes are of the type that activates one of the complement pathways. A large amount of immune complexes must be present before sufficient complement activation occurs to cause a detectable fall in titer. However, only small amounts of complexes of the right type may be sufficient to produce tissue injury. Because complement is highly efficient in inducing inflammation, and

1010

because the circulating numbers of complement component molecules are high (e.g., an average person has 3.5 × 1015 molecules of C3 per milliliter of plasma), a small amount of activation may be sufficient to induce injury but may not be detected. Moreover, complement acts as an acute-phase reactant, and the levels of most components rise during inflammation, thereby masking any concomitant fall in titer. For these reasons, complement component levels are useful parameters to 162

monitor in diseases such as SLE, in which experience has shown that the levels are indicative of disease activity.[ ] However, complement component levels are much less useful in other diseases, such as vasculitis, in which complement may participate in tissue injury, but a fall in plasma complement levels is regularly unappreciated. In an effort to develop more reliable complement activation tests, investigators have begun to use tests that examine (1) the level of complement component cleavage fragments, (2) the presence of neoantigens formed only when complement is activated, and (3) the presence of complexes between components such as C3b and properdin or C1 and C1 inhibitor, the presence of which suggests complement activation in vivo. C3d is a small fragment formed after sequential C3 degradation steps. This protein is not found in the circulation or in body fluids under normal circumstances. The finding of the cleavage fragment indicates probable ongoing in vivo complement activation, as found in active SLE and in joint fluid from patients with active RA.[

163] [164]

The second new method examines blood or body fluid for the presence of neoantigens that form when complement is activated. The complement activation sequence is associated with the formation of many enzyme complexes, such as the complex between C4b and C2a and the complex that occurs between C3b and Bb in the early steps of the classic and alternative pathways. Similarly, formation of the membrane attack complex (MAC) C5b, 6, 7, 8, and 9 occurs by interaction of the late components during activation of classic and alternative pathways. These complexes are associated with the formation of neoantigens, antigens not present on the individual complement proteins but found after the conformational changes associated with these protein-protein interactions. One of the most well-studied situations 165 166

is the series of neoantigens found on activation of the late components C5b, 6, 7, 8, and 9.[ ] [ ] These neoantigens, recognized by antibody, are not present on any of the individual components C5b, C6, C7, C8, or C9. One of the important neoantigenic determinants can be formed in vitro when C9 is allowed to polymerize into a multimeric structure, poly-C9. Antibody to poly-C9 does react with the C5b, 6, 7, 8, 9 complex but does not react with native C9. The neoantigen can be found 165 167

in glomeruli of patients with SLE glomerulonephritis and in the lesional skin of patients with active SLE.[ ] [ ] It is present in the blood in some patients with ongoing immunologic injury, as in the plasma from patients with Guillain-Barré syndrome and even in the spinal fluid of patients with immunologically induced central nervous system disease.[

168]

The third type of test examines the formation of complexes in a different way. Instead of attempting to detect neoantigens, the test looks directly for the linkage of individual proteins that are not physically linked under usual circumstances. For example, activation of the classic pathway is associated with activation of C1. This activation is down-regulated by the formation of a complex between C1 inhibitor and the activated C1. Such a complex does not normally exist in the circulation but can be detected during activation of the classic complement pathway.[

169]

Other even more indirect tests have been used to detect the presence of immune complexes. The finding of IgG/IgM-containing cryoprecipitates demonstrates the presence of one type of immune complex. Anti-DNA autoantibody suggests the presence of autoimmunity with circulating immune complexes, just as does the presence of many tissue-specific antibodies. Similarly, the presence of specific antigens, such as HBsAg in the appropriate clinical setting, suggests immune complex– mediated disease. In most immune complex–related diseases, the erythrocyte sedimentation rate is elevated, although this is not always a reliable criterion of disease activity. [

170]

As mentioned earlier, an important element in diagnosis of many types of immune complex disease is the finding of tissue deposition of immune complexes or products of complement activation on tissue biopsy. Often this finding provides essential diagnostic information in a difficult case. Again, however, such deposition may be evanescent, and such immunoreactive products may be gone within hours of their deposition.

TREATMENT Immune complex–related diseases range in clinical expression from SLE and serum sickness to vasculitis and glomerulonephritis. The approach to therapy is different in each of these diseases, and these approaches are not reviewed here. In theory, therapy consists in removal of the offending antigen, decreasing inflammation that produces tissue damage, and decreasing antibody production. In some patients the direct removal of antibody or immune complexes by extensive plasmapheresis has been attempted, and in theory it might also be possible to facilitate the removal of complexes by the RES. Removal of the antigen, when the antigen is known, is an excellent approach to therapy. Serum sickness is usually self-limited and is rarely life threatening when the drug that induced the disease is discontinued or the administration of foreign protein is stopped. In these patients, supportive care is often sufficient. Decreasing

tissue inflammation is usually possible through the use of glucocorticoids or nonsteroidal antiinflammatory drugs (NSAIDs). However, glucocorticoids are associated with serious side effects, and it may not be practical to administer these agents in doses sufficient to ameliorate the disease being treated. CR1, the cell membrane C4b/C3b fragment receptor, has been cloned and expressed in a water-soluble form using the techniques of genetic engineering. The material promotes the decay dissociation of the C3 convertases of the classic and alternative pathway and the degradation of C3b. It has been shown to be useful in decreasing inflammation in 171

models of myocardial infarct and in delaying acute graft rejection and may well prove useful in the treatment of human disease. [ ] Other cell-bound complementregulatory proteins have been cloned and expressed in various cell systems. Use of these proteins is being tested, and it is certain that they will be used clinically in the next few years. C5a plays an important role in inducing immigration of PMNs to inflammatory sites, and monoclonal anti-C5 antibodies have been prepared that 172] [173] [174]

inhibit C5a function and appear relatively nontoxic. These antibodies are currently being studied in a variety of diseases.[

Intravenous immunoglobulin (IVIG) preparations have become available for the treatment of a variety of autoimmune diseases. IVIG exerts its effects on many immune functions, and several mechanisms of action have been postulated.[

175]

IVIG interferes with various steps of the complement activation

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cascade and down-regulates complement-induced inflammation. Monomeric IgG in IVIG is also able to compete for activation FcγR receptors (in particular FcγRIII) and attenuate FcγR-mediated immune complex phagocytosis and clearance. In patients with idiopathic thrombocytopenic purpura (ITP), IVIG therapy is believed to 176]

prevent immune complex–induced platelet destruction indirectly through an FcγR blockade.[

In support of this mechanism, it was shown that the administration 177]

of IgG Fc fragments dramatically reduces glomerular damage in a rat model of immune complex–induced nephritis.[

Interestingly, in a murine model of ITP,

178 mice.[ ]

IVIG does not inhibit platelet clearance and destruction in FcγRIIB-deficient In normal mice, IVIG administration was found to increase FcγRIIB expression, suggesting that this inhibitory receptor is essential in modulating an FcγRIII-induced inflammatory response. This finding may help explain the ability of IVIG to interfere with proinflammatory cytokine release and augment B cell and T cell proliferative responses to inflammatory agents. Antiidiotypic antibodies in 179

IVIG preparations that may bind and neutralize potentially cytotoxic autoantibodies and that suppress autoimmune-like activity have been implicated as well.[ ] Despite the uncertainty surrounding the mechanism of action in the clinical situation in which it is used, IVIG is a safe therapeutic agent that may prove useful for a variety of autoimmune diseases.[

180]

Unfortunately, IVIG is extremely expensive at this time.

Decreasing antibody production by the use of cytotoxic agents has an important place in therapy of many immune complex–related diseases, but current agents often lack sufficient specificity, often do poorly in inhibiting the B cells responsible for an ongoing immune response, and are attended by many serious side effects. The role of plasmapheresis is considerably more controversial. This approach to therapy is believed to be beneficial in many self-limited diseases when large amounts of antibody directed at a tissue-specific antigen are made and cause disease, as is believed to be the case in Goodpasture's syndrome and perhaps in Guillain-Barré syndrome. Earlier studies suggested that this approach is considerably less useful for diseases in which ongoing production of immune complexes leads to local tissue inflammation and damage. Plasmapheresis may be followed immediately by increased antibody production, re-formation of complexes, and renewed tissue damage.

Currently the use of plasmapheresis with concomitant cytotoxic therapy is being used. This approach may prove useful in some immune complex–mediated diseases. In theory, it may also be possible to augment the ability of the RES to remove immune complexes from the circulation. Some immunomodulatory agents, such as levamisol, may have this ability. However, both experimental and clinical data to support this contention are still lacking.

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Exp Med 155:460, 1982. 158. Ward HJ, Cohen AH, Border WA: In situ formation of subepithelial immune complexes in the rabbit glomerulus: requirement of a cationic antigen, Nephron 36:257, 1984. 159. Gallo GR, Caulin-Glaser T, Lamm ME: Charge of circulating immune complexes as a factor in glomerular basement membrane localization in mice, J Clin Invest 67:1305, 1981. 160. Agodoa LYC, Gauthier VJ, Mannik M: Antibody localization in the glomerular basement membrane may precede in situ immune deposit formation in rat glomeruli, J Immunol 134:880, 1985. Laboratory Findings 161. Wagner E, Haixiang J, Frank MM: Complement and kinins: mediators of inflammation. In Henry JB, editor: Clinical diagnosis and management by laboratory methods, Philadelphia, 2001, Saunders. 162. Atkinson JP: Complement activation and complement receptors in systemic lupus erythematosus, Springer Semin Immunopathol 9:179, 1986. 163. Sturfelt G, Sjöholm AG: Complement components, complement activation, and acute phase response in systemic lupus erythematosus, Int Arch Appl Immunol 75:75, 1984. 164. Mollnes TE, Lea T, Mellbye OJ, et al: Complement activation in rheumatoid arthritis evaluated by C3dg and the terminal complex, Arthritis Rheum 29:715, 1986. 165. Falk RJ, Dalmasso AP, Kim Y, et al: Neoantigen of the polymerized ninth component of complement: characterization of a monoclonal antibody and immunohistochemical localization in renal tissue, J Clin Invest 72:560, 1983. 166. Sanders ME, Schmetz MA, Hammer CH, et al: Quantitation of activation of the human terminal complement pathway by ELISA, J Immunol Methods 85:245, 1985. 167. Biesecker G, Lavin L, Ziskind M, et al: Cutaneous localization of the membrane attack complex in discoid and systemic lupus erythematosus, N Engl J Med 306:264, 1982. 168. Sanders ME, Koski CL, Robbins D, et al: Activated terminal complement in cerebrospinal fluid in Guillain-Barré syndrome and multiple sclerosis, J Immunol 136:4456, 1986.

1014

169. Oleesky DA, Daniels RH, Williams BD, et al: Terminal complement complexes and C1/C1inhibitor complexes in rheumatoid arthritis and other arthritic

conditions, Clin Exp Immunol 84:250, 1991. 170. Cox HC, Liang MH: The erythrocyte sedimentation rate, Ann Intern Med 104: 515, 1986. Treatment 171. Kalli KR, Hsu P, Fearon DT: Therapeutic uses of recombinant complement protein inhibitors, Springer Semin Immunopathol 15:417, 1994. 172. Wang Y, Rollins SA, Madri JA, et al: Anti-C5 monoclonal antibody therapy prevents collagen-induced arthritis and ameliorates established disease, Proc Natl Acad Sci U S A 92:8955, 1995. 173. Vakeva AP, Agah A, Rollins SA, et al: Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy, Circulation 97:2259, 1998. 174. Huber-Lang MS, Sarma JV, McGuire SR, et al: Protective effects of anti-C5a peptide antibodies in experimental sepsis, FASEB J 15:568, 2001. 175. Hester CG, Frank MM: The use of intravenous immunoglobulin. In Austin KF, Frank MM, Atkinson JP, Cantor H, editors: Samter's Immunologic diseases, Philadelphia, 2001, Lippincott, Williams & Wilkins. 176. Fehr J, Hofmann V, Kappeler V: Transient reversal of thrombocytopenia in idiopathic thrombocytopenic purpura by high-dose intravenous immunoglobulin, N Engl J Med 306:1254, 1982. 177. Gomez-Guerrero C, Duque N, Casado MT, et al: Administration of IgG Fc fragments prevents glomerular injury in experimental nephritis, J Immunol 164: 2092, 2000. 178. Samuelsson A, Towers TL, Ravetch JV: Anti-inflammatory activity of IVIG mediated through the inhibitory Fc receptor, Science 291:484, 2001. 179. Dietrich G, Kaveri SV, Kazatchkine MD: Modulation of autoimmunity by intravenous immune globulin through interaction with the function of the immune/ idiotypic network, Clin Immunol Immunopathol 62:S73, 1992. 180. Basta M, Kirshbom P, Frank MM, et al: Mechanism of therapeutic effect of high-dose intravenous immunoglobulins: attenuation of acute, complementdependent immune damage in a guinea pig model, J Clin Invest 84:1974, 1989.

1015

Chapter 59 - Primary Immunodeficiency Diseases

Rebecca Hatcher Buckley

Physicians caring for infants, children, and adults with presumed allergic disorders are impressed by the frequency with which patients have a complaint of repeated infections. Most often the complaint is without foundation because the symptoms are from an allergic rather than an infectious cause. Within this group of patients, however, several will have a primary immunodeficiency, and many more such diseases likely will be found in the allergy-immunology care setting than in a primary care setting. The approaches to proper diagnosis and treatment of these complex disorders change as new information emerges. It is therefore important that the allergist-immunologist be current on the rapidly expanding knowledge about these genetically determined diseases. 1

2

Since Bruton discovered agammaglobulinemia in 1952,[ ] more than 100 other primary immunodeficiency syndromes have been described.[ ] These disorders may involve one or more components of the immune system, including T, B, and natural killer (NK) lymphocytes; phagocytic cells; and complement proteins. Most are recessive traits, some of which are caused by mutations in genes on the X chromosome, others in genes on autosomal chromosomes (see Table 59-1 ). The conditions discussed in this chapter do not include all the known primary immunodeficiency syndromes; the ones selected were chosen because of new information about them, their relative importance, or their connections to allergic diseases. Immunodeficiency diseases are characterized by unusual susceptibility to infection, but they may also have associated autoimmune or allergic manifestations. Knowledge of the particular infectious agents involved and the anatomic sites most often affected in a given patient can provide clues as to the most likely type of defect. Patients with B cell, phagocytic cell, or complement defects have recurrent infections with encapsulated bacterial pathogens. By contrast, patients with T cell defects have problems with opportunistic infections involving viral and fungal agents. These patients also begin to fail to thrive shortly after these problems develop. Excessive routine use of antibiotics has altered the textbook presentation of many of these conditions, however, so their detection requires not only a high index of suspicion but also extensive knowledge on the part of the allergist-immunologist about their fundamental causes and various modes of presentation. Paradoxically, some immunodeficiency diseases are accompanied by excessive production of immunoglobulin E (IgE) antibodies or of autoantibodies. Because antibiotics may mask infection susceptibility, allergic or autoimmune problems may be the presenting illnesses of these patients. Patients with primary 3 4 5

immunodeficiency also have an increased incidence of malignancy, particularly those with defects that involve deficiencies of B or T cell function.[ ] [ ] [ ] Whether this is attributable to defective immune elimination of tumor cells or to increased susceptibility to infection with agents associated with malignancy is unknown. 2

Compared with acquired immunodeficiency disorders, genetically determined immunodeficiency is seemingly rare.[ ] B cell defects greatly outnumber those affecting T cells, phagocytic cells, or complement proteins. Selective immunoglobulin A deficiency (A Def) is the most common, with reported incidences ranging 6

from 1 in 333 to 1 in 700. [ ] Primary immunodeficiency is diagnosed more often in childhood, when it occurs predominantly in males, than in adult life, when it occurs slightly more often in females than in males. Until the past decade there was little insight into the fundamental problems underlying a majority of immunodeficiencies. Many disorders have now been mapped to specific chromosomal locations, however, and the fundamental biologic errors have been identified in an impressive number (see Table 59-1 ).[

7] [8] [9] [10]

Within the past 9 years the molecular bases of six X-linked immunodeficiency disorders have been discovered: X-linked agammaglobulinemia,[ immunodeficiency with hyper–immunoglobulin lymphoproliferative

17 18 disease,[ ] [ ]

13 M,[ ]

Wiskott-Aldrich

8 14 syndrome,[ ] [ ]

X-linked severe combined

and nuclear factor kappa B essential modulator (NEMO)

granulomatous disease (CGD) had been identified several years

21 earlier,[ ]

19 20 deficiency.[ ] [ ]

15 16 immunodeficiency,[ ] [ ]

11] [12]

X-linked

X-linked

The abnormal gene in X-linked chronic

and the gene encoding properdin (mutated in properdin deficiency) has also been cloned.

[22]

Autosomal recessive immunodeficiencies for which the molecular bases have been discovered include leukocyte adhesion deficiency type 1 (LAD-1), [ adenosine deaminase (ADA) deficiency,[

25]

purine nucleoside phosphorylase (PNP) deficiency,[

histocompatibility complex (MHC) antigen

31 32 deficiency,[ ] [ ]

interleukin-7 receptor alpha-chain (IL-7Rα)

35 deficiency,[ ]

26]

ataxia-telangiectasia,[

zeta-chain-associated protein 70 (ZAP-70)

recombinase activating genes 1 and 2

33 deficiency,[ ]

36 deficiencies,[ ]

27]

23] [24]

28] [29] [30]

DiGeorge syndrome,[

Janus kinase 3 (Jak-3)

Artemis gene deficiency,[

37]

1016

TABLE 59-1 -- Locations of Faulty Genes in Primary Immunodeficiency Diseases Chromosome

Disease

1q21

MHC class II antigen deficiency caused by RFX5 mutation

1q25

Chronic granulomatous disease (CGD) caused by gp67phox deficiency

1q42–43

Chédiak-Higashi syndrome

2p11

Kappa-chain deficiency

2q12

CD8 lymphocytopenia caused by ZAP-70 deficiency

*

5p13

SCID caused by IL-7 receptor alpha-chain deficiency

*

6p21.3

MHC class I antigen defect caused by mutations in TAP-1 or TAP-2

6p21.3

(?)Common variable immunodeficiency and selective IgA deficiency

6q22-q23

Interferon-γR1 mutations

*

*

*

*

major

34 deficiency,[ ]

*

*

*

7q11.23

CGD caused by gp47phox deficiency

8q21

Nijmegen breakage syndrome caused by mutations in Nibrin

9p13

Cartilage-hair hypoplasia caused by mutations in endoribonuclease RMRP

10p13

SCID (Athabascan, radiation sensitive) caused by mutations in Artemis gene

10p13

DiGeorge syndrome/velocardiofacial syndrome

11p13

IL-2 receptor alpha-chain deficiency

11p13

SCID caused by RAG-1 or RAG-2 deficiencies

11q22.3

AT, attributable to AT mutation, causing DNA-dependent kinase deficiency

11q23

CD3 gamma-chain or epsilon-chain deficiency

12p13

Autosomal recessive hyper-IgM caused by mutations in AID gene

13q

MHC class II antigen deficiency caused by RFXAP mutation

14q13.1

Purine nucleoside phosphorylase (PNP) deficiency

14q32.3

Immunoglobulin heavy-chain deletion

16p13

MHC class II antigen deficiency caused by CIITA mutation

16q24

CGD caused by gp22phox deficiency

17

Human nude defect

19p13.1

SCID caused by Janus kinase 3 (Jak-3) deficiency

19p13.2

Agammaglobulinemia caused by mutations in Igα gene

20q13.11

SCID caused by adenosine deaminase (ADA) deficiency

21q22.3

Leukocyte adhesion deficiency type 1 (LAD-1) caused by CD18 deficiency

*

22q11.2

Agammaglobulinemia caused by mutations in λ5 surrogate light-chain gene

*

* * *

* * *

* *

*

*

* *

*

* * * *

22q11.2

DiGeorge syndrome

Xp21.1

CGD caused by gp91phox deficiency

Xp11.23

Wiskott-Aldrich syndrome (WAS) caused by WASP deficiency

Xp11.3-p21.1

Properdin deficiency

Xq13.1

X-linked SCID caused by common gamma-chain (γc ) deficiency

Xq22

X-linked agammaglobulinemia caused by Btk deficiency

Xq24–26

X-linked lymphoproliferative syndrome caused by mutations in SH2D1A gene

Xq26

* *

* *

* *

Immunodeficiency with hyper-IgM caused by CD154 (CD40 ligand) deficiency *

Xq28

Anhidrotic ectodermal dysplasia with immunodeficiency caused by mutations in NEMO

*

IL, Interleukin; IgA, immunoglobulin A; AT, ataxia-telangiectasia; see Table 59-2 for other abbreviations. * Gene cloned and sequenced; gene product known.

38] [39]

activation-induced cytidine deaminase (AID) deficiency,[

the genes for these diseases have obvious implications for gene

and interferon gamma receptor (IFN-γR) 1 and 2 deficiencies.[

10 therapy.[ ]

40] [41]

The discovery and cloning of

The rapidity of these advances indicates that many more discoveries will be made soon.

A committee of the World Health Organization (WHO) has published several versions of a classification of primary immunodeficiency diseases over the past three 42

decades, most recently in 1999.[ ] Table 59-1 lists numerous conditions for which the molecular bases are already known.[ the more common immunodeficiency diseases and related factors.

10]

Table 59-2 provides abbreviations for

X-LINKED DEFECTS X-Linked Agammaglobulinemia 1

X-linked agammaglobulinemia (XLA) was the first recognized human host defect, discovered by Colonel Ogden Bruton in 1952.[ ] Most patients with this or other

43

antibody deficiency syndromes are identified because they have recurrent infections with encapsulated bacterial pathogens.[ ] Because of passive immunity from maternally transmitted immunoglobulin G (IgG) antibodies, boys with XLA usually remain well during the first few months of life, except for occasional problems with mucous membrane infections (e.g., conjunctivitis, otitis), because of the lack of secretory IgA.

1017

TABLE 59-2 -- Abbreviations for Common Immunodeficiency Disorders and Associated Factors Abbreviation

Disorder/Factor

ADA

Adenosine deaminase

A Def

Selective IgA deficiency

AID

Activation-induced cytidine deaminase

ATM

Ataxia-telangiectasia mutation

Btk

Bruton tyrosine kinase

CII TA

MHC class II transactivator

CD154

CD40 ligand

CGD

Chronic granulomatous disease

CID

Combined immunodeficiency

CVID

Common variable immunodeficiency

EBV

Epstein-Barr virus

γc

Common cytokine receptor gamma chain

GVHD

Graft-versus-host disease

Hyper-IgE

Hyperimmunoglobulinemia E syndrome

Hyper-IgM

Hyperimmunoglobulinemia M syndrome

IVIG

Intravenous immunoglobulin

Jak-3

Janus kinase 3

LAD

Leukocyte adhesion deficiency

MHC

Major histocompatibility complex

NEMO

Nuclear factor kappa B essential modulator

NK

Natural killer (lymphocytes)

Phox

Phagocyte oxidase

PK

Protein kinase

PMA

Phorbol myristate acetate

PNP

Purine nucleoside phosphorylase

RAG

Recombinase activating gene

SCID

Severe combined immunodeficiency

TAP-1 and -2

Transporter of antigen peptides 1 and 2

TNF

Tumor necrosis factor

WASP

Wiskott-Aldrich syndrome protein

XLA

X-linked agammaglobulinemia

ZAP-70

Zeta-chain-associated protein 70 44

Thereafter they are highly susceptible to infections with organisms such as pneumococci, streptococci, and Haemophilus influenzae.[ ] They may also experience infections with other high-grade pathogens, such as meningococci, staphylococci, Pseudomonas organisms, and various Mycoplasma species. Because XLA patients have a profound deficiency of antibodies of all isotypes, the infections may be systemic (e.g., meningitis, septicemia) or may involve mucous membrane surfaces (sinusitis, pneumonia, otitis, conjunctivitis, gastrointestinal and urinary tract infections), joints (septic arthritis), or skin (cellulitis, abscesses). Growth and development are usually normal despite chronic or recurrent bacterial infections, unless bronchiectasis or persistent enteroviral infections develop.[ [46]

45]

Chronic fungal infections are not usually a problem, and Pneumocystis carinii pneumonia is rare. Except for the hepatitis and enteroviruses, viral infections are

45

usually handled normally.[ ] Polioviruses (in the form of live poliovirus vaccine) are examples of such enteroviruses, causing viremia because of a lack of secretory IgA antibodies, then central nervous system (CNS) infection because of a lack of circulating IgG antibody. Paralysis occurs from reversion of the vaccine virus to a more neurotropic form. Various echoviruses and coxsackieviruses also have caused chronic, progressive, and eventually fatal CNS infections in many XLA patients. [45] [46]

In addition to septic arthritis, patients with XLA may have joint inflammation similar to that seen in rheumatoid arthritis.[ [47]

44]

Infections with Ureaplasma urealyticum

and viral agents such as echoviruses, coxsackieviruses, and adenoviruses have been identified from joint fluid cultures of patients, even from those receiving

intravenous immunoglobulin (IVIG) replacement therapy.[

44]

Immunologic Defects

Concentrations of all serum immunoglobulins are extremely low. Tests for antibodies to blood group substances and for antibodies to vaccine antigens (e.g., diphtheria, tetanus, H. influenzae, pneumococci) help distinguish XLA from transient hypogammaglobulinemia of infancy or from protein-losing states, because antibody formation is profoundly impaired in XLA but not in these conditions. Polymorphonuclear functions are usually normal if IgG antibodies are provided, but some patients with XLA have had transient neutropenia early in the course of a serious infection or even without an obvious cause. Few if any circulating B cells can be detected by flow cytometry in XLA patients. Some bone marrow precursor B cells can be found, but these cells are strongly 48

biased to a fetal-like repertoire of V-D-J gene usage.[ ] The tonsils, adenoids, and peripheral lymph nodes are very small because of the absence of germinal centers. Circulating T cells and NK cells are relatively increased in Bruton's disease (infantile XLA), and the percentages of the two T cell subsets are normal in most patients. Both T and NK cell functions are normal in patients with Bruton's disease, as are the structure and content of the thymus and thymus-dependent areas of peripheral lymphoid tissues. Molecular Basis

In 1993, two groups of investigators independently and almost simultaneously discovered the mutated gene in XLA. Because XLA had been precisely mapped to position Xq22, one group successfully used the technique of positional cloning to identify an abnormal gene in patients with this defect.[

12]

For other reasons, the

11 signaling[ ]

second group had sought and found a B cell–specific tyrosine kinase important in murine B lymphocyte ; the kinase was found to be encoded by a gene on the mouse X chromosome. When cloned, the human gene counterpart was found to reside at Xq22, and the gene product was identical to that found by the first group. This intracellular signaling tyrosine kinase has been named Bruton tyrosine kinase (Btk) in honor of Dr. Bruton. Btk is a member of the tec family of cytoplasmic tyrosine kinases (e.g., Lck, Fyn, Lyn), which are found in many types of hematopoietic cells.[ expressed at high levels in all B-lineage cells, including pre-B cells.[ 11 series.[ ]

49]

11] [12]

Btk is

Btk has not been detected in any cells of T lineage but has been found in cells of the myeloid

Thus far, all males with known XLA (by family history) have had low or undetectable Btk messenger ribonucleic acid (mRNA) and kinase activity ( Figure 59-1 ). A mutation at position 28 of the murine Btk

1018

Figure 59-1 Locations of mutant proteins in B cells identified in primary immunodeficiency diseases. Each mutant protein is identified by a white x. Ig, Immunoglobulin; HLA, human lymphocyte antigen; SLAM, signaling lymphocyte activation molecule; BLNK, B cell linker adapter protein; β2 -M, beta-2 microglobulin; Btk, Bruton tyrosine kinase; TAP1, TAP2, transporters of processed antigen; RFX, RFXAP, CIITA, RFXANK, transcription factors. From Buckley RH: N Engl J Med 343:1317, 2000.

From Buckley RH: N Engl J Med 343:1317, 2000. gene has been shown to be the basis for the B cell defect in CBA/N mice. Because such mice have a much milder antibody deficiency than boys with XLA, it was speculated that humans with mutations in the nonkinase encoding part of the Btk gene might have a less severe immunodeficiency. However, many different

50

mutations of the human Btk gene (greater than 300) have already been described.[ ] These defects have encompassed most parts of the coding portions of the gene, and no clear correlation has been made between the location of the mutation and the clinical phenotype. Female carriers of XLA can be identified by the finding of nonrandom X chromosome inactivation in their B cells or by the detection of the mutated gene (if known 51

in the family).[ ] Prenatal diagnosis of affected or nonaffected male fetuses has also been accomplished by detection of the mutated gene in chorionic villus or amniocentesis samples. Studies of Btk protein, enzymatic activity, or mRNA have also permitted identification of X-linked inheritance in some agammaglobulinemic boys with no family history.[

52]

Interestingly, Btk is also expressed in cells of myeloid lineage, considering the well-known occurrence of intermittent neutropenia in 53 54

boys with XLA, particularly at the onset of acute infection.[ ] [ ] Conceivably, Btk is only one of several signaling molecules participating in myeloid maturation, and neutropenia might be observed in XLA only when rapid production of such cells is needed. XLA has been reported in association with growth hormone deficiency in nine cases.[

55]

Treatment and Prognosis

Except in those who develop polio, other persistent enteroviral infections, rheumatoid arthritis, or lymphoreticular malignancy (with an incidence as high as 6% having been reported), the overall prognosis for patients with XLA is reasonably good if IVIG replacement therapy is instituted early. However, there is considerable clinical heterogeneity even within the same pedigree.[

56] [57]

Systemic infection can be prevented by administration of IVIG at a dose of 400•mg/kg every 3 to 4

58 weeks.[ ]

Despite this therapy, however, many patients go on to develop persistent enteroviral infections or crippling sinopulmonary disease because no effective means exists for replacing secretory IgA at the mucosal surface. Chronic antibiotic therapy is usually necessary in addition to IVIG infusions for effective management of XLA patients with pansinusitis or bronchiectasis. X-Linked Immunodeficiency with Hyper–Immunoglobulin M Because they lack IgG, IgA, and IgE antibodies, patients with the hyper-IgM syndrome (hyperimmunoglobulinemia M, hyper-M) resemble those with XLA in their 59 60

susceptibility to encapsulated bacterial infections.[ ] [ ] They also have diminished lymphoid tissue. Coexistent neutropenia and the fundamental T cell immunodeficiency are considered possible explanations for the occurrence of P. carinii pneumonia in many such patients.

1019

Immunologic Defects

Concentrations of serum IgG, IgA, and IgE are very low, whereas the serum IgM concentration is either normal or elevated and polyclonal. Low-molecular-weight IgM molecules are present in some patients and may give falsely high IgM values in radial immunodiffusion assays. There is an increased frequency of autoantibody formation. [

59] [60]

Hemolytic anemia and thrombocytopenia may occur, and transient, persistent, or cyclic neutropenia is common.

Until the past decade the X-linked hyper-M syndrome was classified as a B cell defect because only IgM is produced. Moreover, crude tests of T cell functions were usually normal in such patients. However, normal numbers of B lymphocytes are usually present in the circulation of these patients. Also, more than a decade ago, B cells from some hyper-M patients were shown to have the capacity to synthesize IgM, IgA, and IgG normally when co-cultured with a “switch” T cell line, indicating that in these patients the defect may lie in T-lineage cells.[

61]

Molecular Basis

The abnormal gene in X-linked hyper-IgM was localized to Xq26 [

62]

and identified by six groups almost simultaneously in 1993.[ 65 cells,[ ]

13] [14] [63] [64]

The gene product is

a surface molecule now known as CD154, or CD40 ligand, on the surfaces of activated helper T (Th) which interacts with CD40 molecules on B cells[ ( Figure 59-2 ). CD40 is a type I integral membrane glycoprotein belonging to the tumor necrosis factor (TNF) and nerve growth factor receptor family; it is expressed on B cells, monocytes, some carcinomas, and a few

13]

Figure 59-2 Locations of mutant proteins in activated CD4+ T cells identified in primary immunodeficiency diseases. ATM, Ataxia-telangiectasia mutation; NFAT, nuclear factor of activated T cells; Jak-3, Janus kinase 3; WASP, Wiskott-Aldrich syndrome protein; ZAP-70, zeta-chain-associated protein 70; SH2DIA, SLAMassociated protein. From Buckley RH: N Engl J Med 343:1317, 2000.

From Buckley RH: N Engl J Med 343:1317, 2000. other types of cells. Cross-linking of CD40 on either normal or X-linked hyper-M B cells with a monoclonal antibody to CD40 or with soluble CD154 in the presence of cytokines (IL-2, IL-4, or IL-10) causes the B cells to undergo proliferation and isotype switching and to secrete various types of immunoglobulins. CD154 is a type II integral membrane glycoprotein with significant sequence homology to TNF; it is found only on activated T cells, primarily of the CD4 phenotype. [65]

Mutations in the gene encoding CD154 in X-linked hyper-M patients result in a lack of signaling of their B cells by their activated T cells. Therefore, hyper-M B cells fail to undergo isotype switching and produce only IgM. Of further importance to effective immune responses, the lack of stimulation of CD40 also results in failure of these patients' B cells to up-regulate CD80 and CD86. The latter are important co-stimulatory molecules that interact with CD28/CTLA4 on T cells. [ Failure of the molecules in these pathways to interact results in a propensity for tolerogenic T cell signaling.

66]

Lymph node histologic features are abnormal, showing only abortive germinal center formation and a severe depletion and phenotypic abnormalities of follicular

dendritic cells.[

67]

Many distinct point mutations or deletions in the gene encoding CD154 have been identified, giving rise to frame shifts, premature stop codons, 68 69

and single amino acid substitutions, most of which were clustered in the TNF homology domain located in the carboxy-terminal region.[ ] [ ] A highly polymorphic microsatellite dinucleotide (CA) repeat region in the 3′ untranslated end of the gene for CD154 is useful for detecting carriers of X-linked hyper-IgM and for making a prenatal diagnosis of this condition.[

70]

1020

Treatment and Prognosis

Because the prognosis is not good for many patients with X-linked hyper-M, the treatment of choice is a human leukocyte antigen (HLA) identical bone marrow transplant.[

71]

Otherwise, the treatment for this condition is the same as that for agammaglobulinemia, monthly IVIG infusions.[

58]

In addition to opportunistic 60

infections such as P. carinii pneumonia, there is an increased incidence of malignancy in hyper-M syndrome, particularly hepatocellular carcinoma.[ ] Mutations in the gene encoding CD154 are believed to account for both defective negative selection of autoreactive thymocytes and defective recognition of tumor cells because of tolerogenic signaling of peripheral T cells. Nuclear Factor Kappa B Essential Modulator Deficiency The NEMO deficiency is a newly recognized syndrome characterized most often clinically as anhidrotic ectodermal dysplasia with associated immunodeficiency 19 20

(EDA-ID).[ ] [ ] The condition results from mutations in the IKBKG gene at position 28q on the X chromosome that encodes nuclear factor of kappa B (NFκB) essential modulator. Germline loss-of-function mutations cause the X-linked dominant condition incontinentia pigmenti and are lethal in male fetuses. Mutations in the coding region of IKBKG are associated with EDA-ID, and stop codon mutations in IKBKG are associated with osteopetrosis, lymphedema, and EDA-ID (OLEDA-ID). Neither type of mutation abolishes NF-κB signaling entirely. The NEMO immunodeficiency has been variable, with some EDA-ID patients showing impaired antibody responses to polysaccharide antigens.[ have presented with hyper-IgM. seriousness of their infections.

[20]

19]

Some patients

OL-EDA-ID patients' immune cells respond poorly to lipopolysaccharide, IL-1, IL-18, TNF-α, and CD154, accounting for the

Immunodeficiency with Thrombocytopenia and Eczema: Wiskott-Aldrich Syndrome WAS is an X-linked recessive syndrome characterized by eczema, thrombocytopenic purpura with normal-appearing megakaryocytes but small defective platelets, and undue susceptibility to infection.[ bruising.

9] [42]

Patients usually present during infancy with prolonged bleeding from the circumcision site, bloody diarrhea, or excessive

Atopic dermatitis and recurrent infections usually develop during the first year of life. Asthma and other atopic diseases may also be seen. In younger patients, infections are usually those produced by pneumococci and other encapsulated bacteria, resulting in otitis media, pneumonia, meningitis, or sepsis. Later, infections with opportunistic agents such as P. carinii and the herpesviruses become more problematic. Autoimmune cytopenias and vasculitis are common in those who live beyond infancy. There may be considerable variability in the degree of infection-susceptibility or in the extent to which atopic manifestations are expressed. However, survival beyond the teenage years is rare; infections and bleeding are the major causes of death, although a 12% incidence of fatal malignancy also occurs in these patients.[

72]

Immunologic Defects

Patients with WAS have an impaired humoral immune response to polysaccharide antigens, as evidenced by absent or greatly diminished isohemagglutinins and poor 42 73 74

or absent antibody responses to polysaccharide antigens.[ ] [ ] [ ] In addition, antibody titers to protein antigens fall with time, and anamnestic responses are often poor or absent. There is an accelerated rate of synthesis as well as hypercatabolism of albumin, IgG, IgA, and IgM, resulting in highly variable immunoglobulin 74]

concentrations. [

Most often these patients have a low serum IgM, elevated IgA and IgE, and a normal or slightly low IgG concentration. Flow cytometry of blood

lymphocytes has shown moderately reduced percentages of CD3, CD4, and CD8 T cells, and lymphocyte responses to mitogens are moderately depressed. [

72]

Molecular Basis 75

8

The mutated gene responsible for WAS was mapped to Xp11.22–11.23 [ ] and isolated in 1994 by Derry and colleagues.[ ] They found that the gene was limited in expression to lymphocytic and megakaryocytic lineages. The gene product, a 501-amino-acid proline-rich protein that lacks a hydrophobic transmembrane domain, was designated WASP (WAS protein). WASP has been shown to bind CDC42H2 and rac, members of the Rho family of guanosine triphosphatases (GTPases) that 76] [77] [78]

are important in actin polymerization.[

79]

In addition, WASP was found to associate physically with Nck through Src homology 3 domains.[

varied number of mutations in the WASP gene have been identified in WAS patients, of infection susceptibility or other problems in one mutations in the WASP

86 gene.[ ]

83 series[ ]

but not in

84 85 others[ ] [ ]

[80] [81] [82]

A large and

with some correlation between the site of the mutation and severity

(see Figure 59-1 ). Isolated X-linked thrombocytopenia is also caused by

Carriers can be detected by the finding of nonrandom X-chromosome inactivation in several hematopoietic cell lineages or by

detection of the mutated gene (if known in the family).[

87] [88] [89]

Prenatal diagnosis of WAS can also be made by chorionic villus sampling or amniocentesis if the 90] [91]

mutation is known in that family. Two families with apparent autosomal inheritance of a clinical phenotype similar to WAS have been reported,[ case a girl was shown to have this mutation as an X-linked

and in one

92 defect.[ ]

Treatment and Prognosis

Numerous patients with WAS have had complete correction of both the platelet and the immunologic abnormalities by HLA-identical sibling bone marrow 71

transplants after being conditioned with irradiation or busulfan and cyclophosphamide. [ ] Success has been minimal with T cell–depleted haploidentical stem cell transplants in WAS, primarily because of the requirement for pretransplant immunosuppression to permit engraftment, the long time course to immunoreconstitution

when T cells are depleted, resistance to engraftment, and a high mortality from preexisting opportunistic infections in this setting.[ been achieved in the treatment of WAS with matched unrelated donor (MUD) transplants when done in those under 5 years of cord blood transplants will be similarly successful because T cells can be left in the donor cell suspension in both cases.

71] [93]

Recently, some success has

93 94 age.[ ] [ ]

It is likely that matched

1021

Unless immunoreconstitution can be achieved, patients with the clinically more severe forms of WAS usually die in childhood. Vasculitis is a major problem in many older children with WAS. Several patients who required splenectomy for uncontrollable bleeding had impressive rises in their 95 96

platelet counts and have done well clinically while being administered prophylactic antibiotics and IVIG.[ ] [ ] The higher platelet counts also permitted the use of high-dose aspirin or other nonsteroidal antiinflammatory drugs (NSAIDs) in the control of the vasculitis. Unless the patient is under 5 years of age and an appropriate unrelated matched donor exists, splenectomy is likely to remain the treatment of choice for WAS patients lacking an HLA-identical donor until gene therapy can be perfected. The most common cause of death in WAS patients currently is Epstein-Barr virus (EBV)–induced lymphoreticular malignancy. [

83]

X-Linked Lymphoproliferative Disease X-linked lymphoproliferative disease (XLP), or Duncan's disease, is a recessive trait characterized by an abnormal immune response to infection with EBV, resulting in (usually) fatal malignant or nonmalignant immune cell proliferation or in immunodeficiency syndromes.[ experience infectious mononucleosis.

[97]

97] [98]

Affected boys are apparently healthy until they

XLP has three major phenotypes: fulminant infectious mononucleosis (50%), B cell lymphomas (20%), and

98 (30%).[ ]

hypogammaglobulinemia The mononucleosis is fatal primarily because of extensive liver necrosis caused by polyclonally activated CD8+ cytotoxic T cells that recognize EBV-infected autologous B cells. The mean age of presentation is less than 5 years. Most patients surviving the primary infection developed lymphomas or hypogammaglobulinemia. NK function is also greatly depressed. 99

17 18 100

The defective gene in XLP was localized to the Xq26-q27 region, [ ] cloned, and identified as SH2D1A.[ ] [ ] [ ] SH2D1A is expressed in thymocytes, T cells, and NK cells. The gene encodes a novel protein composed of a single SH2 domain. It functions as an inhibitory adapter protein for a high-affinity self-ligand called signaling lymphocyte activation molecule (SLAM) present on the surfaces of T and B cells. The adapter protein, SLAM-associated protein (SAP), or SH2D1A, normally serves to inhibit signal transduction by SLAM so that T cell proliferation does not continue unchecked in response to EBV and possibly other infections. 101

Boys who have mutations in SH2D1A fail to inhibit this signaling, which is mediated by SHP-2. The SH2D1A protein also associates with 2B4 on NK cells.[ ] Ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2- or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.[

102]

Recently it has been discovered that patients with mutations in SH2D1A may present with common variable immunodeficiency (CVID), even without antecedent clinical EBV infection. Male siblings in one arm of two separate pedigrees had only CVID, whereas in another arm of each pedigree male siblings presented with

103 104

fulminant infectious mononucleosis.[ ] [ ] Because the SH2D1A gene was found altered in both arms of each family, these findings indicate that XLP must be considered when more than one male patient with CVID is encountered in the same family. Approximately half the limited number of patients with XLP given HLAidentical related or unrelated, unfractionated bone marrow transplants are currently surviving without sign of the disease.[

71] [104]

X-Linked Chronic Granulomatous Disease 105]

X-linked CGD is characterized by defective intracellular killing of bacterial and fungal organisms.[

The true incidence is unknown, but X-linked CGD is

106 births.[ ]

estimated to occur at a frequency of 1 in 500,000 During infancy, patients with CGD begin to have infections with catalase-positive organisms, such as Staphylococcus aureus, Serratia marcescens, Pseudomonas species, and Salmonella species, but not with catalase-negative organisms, such as Streptococcus 107

pneumoniae or Haemophilus influenzae. [ ] Abscess formation is characteristic, occurring in the skin, lymph nodes, liver, lungs, or other viscera. When present, abscesses often require surgical drainage. Osteomyelitis caused by bacteria (particularly S. marcescens) and later by fungi (especially Aspergillus species) is more frequent in CGD than in any other form of immunodeficiency. Although most cases present in infancy or childhood, late presentations from 13 to 43 years of age have been documented.[

108]

Immunologic Defects

Neutrophil counts are normal or elevated, and the chemotactic, adherence, and phagocytic functions of these cells are normal. In CGD the immunologic defect lies 105

solely in the inability of the phagocyte to kill ingested organisms. [ ] During normal phagocytosis a “respiratory burst” occurs in which nicotine adenine dinucleate phosphate + (NADP+) is converted to nicotinamide adenine dinucleotide phosphate (NADPH), which is then moved from the cytosol to an electron transport chain in the cell membrane driven by NADPH oxidase. A 66-kD flavoprotein appears to be the receptor for NADPH in the membrane, and it is linked to cytochrome b245, the terminal component of the electron transport chain. Cytochrome b245 is capable of directly reducing molecular oxygen to superoxide. Superoxide then dismutates to hydrogen peroxide, which is directly toxic to microorganisms or interacts with a chloride ion to produce hypochlorite. Defects exist in this electron transport chain in CGD. The diagnosis of CGD is made when one can demonstrate an inability of neutrophils from the patient to undergo a respiratory burst after phagocytosis or phorbol myristate acetate (PMA) stimulation, that is, to generate superoxide ions. The generation of superoxide ions can be demonstrated by measurement of 109 110

chemiluminescence, by nitroblue tetrazolium (NBT) dye reduction, or (preferably) by a respiratory burst assay that involves flow cytometry.[ ] [ ] As a result of ineffective destruction of phagocytized organisms in these conditions and compensatory responses by lymphoid cells, granulomatous lesions are formed, especially 105]

in the liver and gastrointestinal and urinary tracts.[ Molecular Basis

105]

Approximately 65% of patients with CGD have an X-linked (X-CGD) mode of inheritance, and the remainder have autosomal recessive (AR-CGD) inheritance.[ The

1022

111 112

defect in X-CGD is attributable to mutations in the gene that encodes the heavy chain of the cytochrome b245 heterodimer.[ ] [ ] This heavy chain is a glycoprotein of 91 kD, referred to as gp91phox (for phagocyte oxidase), and does not contain the cytochrome activity. The gene encoding gp91phox is at position Xp21.1 on the short arm of the X chromosome.[

21]

Treatment and Prognosis

When first discovered, CGD was found to be usually fatal in early childhood. Aggressive treatment with antibiotics, especially trimethoprim-sulfamethoxazole, 107

antistaphylococcal antibiotics, and aminoglycosides, has reduced the frequency and severity of bacterial infections.[ ] Fungal infections, especially with Aspergillus species, have been a more important cause of death. Amphotericin B, with alternate-day (nonirradiated) normal granulocyte transfusions, is the initial treatment of choice. Once Aspergillus infections are controlled with the latter therapy, chronic therapy with itraconazole or voriconazole appears to be very effective in preventing recurrences.[

113]

A double-blind placebo-controlled study on the efficacy and safety of subcutaneous injections of IFN-γ in preventing serious infections was conducted in 128 patients with CGD. The investigators reported a significant reduction in the number of serious infections (as defined by need for hospitalization) in CGD patients 114] [115]

who received IFN-γ subcutaneously three times a week, compared with the number in placebo-treated CGD patients.[ phagocytic cell oxidative or microbicidal functions.

However, no improvement was seen in

Bone marrow transplantation has not been as successful in correcting the underlying defects in CGD as it has in correcting defects in lymphocyte function because CGD patients often have chronic indolent bacterial or fungal infections that are very difficult to control when the necessary chemotherapeutic ablation is given to gain graft acceptance.[

71]

If an HLA-identical sibling donor is available and the transplant can be performed early in life, however, an unfractionated marrow

transplant can be curative with either standard pretransplant conditioning regimens[

116]

117]

or nonmyeloablative conditioning.[

X-Linked Recessive Severe Combined Immunodeficiency Disease 9] [42] [118] [119]

Severe combined immunodeficiency (SCID) is a fatal syndrome characterized by profound deficiencies of T and B cell function.[ description of SCID in

120 1950,[ ]

it has become evident that the genetic origins of this condition are quite diverse.

common form, accounting for approximately 46% of U.S.

[9] [119] [121]

Since the initial

X-linked SCID (SCID-X1) is the most

119 122 cases.[ ] [ ]

Figure 59-3 Relative frequencies of the different genetic types of severe combined immunodeficiency disease (SCID) in 165 patients seen consecutively over 4

decades. ADA, Adenosine deaminase; Jak-3, Janus kinase 3; AutoRec, autosomal recessive; CHH, cartilage-hair hypoplasia; RD, reticular dysgenesis; RAG, recombinase activating gene; IL-7Rα Def, interleukin-7 receptor alpha-chain deficiency; γc Def, X-linked recessive.

Figure 59-4 Interleukin-2 receptor gamma chain gene (IL2RG)–complementary deoxyribonucleic acid (cDNA) map showing exons, with cDNA numbers corresponding to first coding nucleotide of each exon; protein domains; and sites of mutations found in 87 unrelated families with X-linked severe combined immunodeficiency disease (SCID). Shaded boxes, Identical mutations found in unrelated patients. From Puck JM, Pepper AE, Henthorn PS, et al: Blood 89:1970, 1997.

From Puck JM, Pepper AE, Henthorn PS, et al: Blood 89:1970, 1997. cases likely will be found, such as one patient who had an apparent reversion of a documented mutation in the gene encoding γc .[

138]

Treatment and Prognosis 118 122

58

SCID is a pediatric emergency.[ ] [ ] Replacement therapy with IVIG fails to halt the progressively downhill course.[ ] Unless bone marrow transplantation from HLA-identical or haploidentical donors can be performed, death usually occurs before the patient's first birthday and almost invariably before the second. On the other hand, transplantation in the first 3.5 months of life offers a greater than 97% chance of survival.[

122] [139]

Therefore, early diagnosis is essential. Bone marrow 122] [139]

transplantation in SCID does not require pretransplant chemotherapy, because such infants are unable to reject grafts.[

Recent studies have shown that the immune reconstitution effected by stem cell transplants results from thymic education of the transplanted allogeneic stem cells. [126]

139

The thymic output appears to occur sooner and to a greater degree in infants transplanted in the neonatal period than in those transplanted later.[ ] Unfortunately, white blood cell counts and manual differentials are not routinely done on the cord blood. Because infants affected with SCID exhibit lymphopenia in the cord blood, this would be an effective way to screen for this defect.[

122] [140]

This should alert the physician to perform studies of T cell phenotype and function

and to examine the blood mononuclear cells for expression of γc . [

141]

Currently, there are more than 400 SCID patients surviving worldwide as a result of successful

71]

bone marrow transplantation.[

Currently, optimism is high that primary immunodeficiency diseases with identified molecular defects will be correctable by gene therapy. Within the past 2 years, a normal

1024

γc complementary deoxyribonucleic acid (cDNA) was successfully transduced into autologous marrow cells of five infants with SCID-X1 by retroviral gene transfer, 142 143

with subsequent full correction of their T and NK cell defects.[ ] [ ] This success offers hope that gene therapy will eventually be the treatment of choice for all patients with SCID or other genetically determined immunodeficiency diseases with a known molecular basis.

AUTOSOMAL DEFECTS WITH KNOWN MOLECULAR BASIS Autosomal Recessive Severe Combined Immunodeficiency Disease 120]

Autosomal recessive SCID was the first of the SCID syndromes to be described, reported initially by Swiss workers in 1958.[

This pattern of inheritance is less

118 119 122 123 Europe.[ ] [ ] [ ] [ ]

common in the United States than in Mutated genes on autosomal chromosomes have been identified in six genetic types of SCID: ADA deficiency, Jak-3 deficiency, IL-7Rα deficiency, recombinase activating gene (RAG-1 or RAG-2) deficiency, Artemis gene product deficiency, and CD45 119] [144]

deficiency. Other causes will likely be discovered.[ Adenosine Deaminase Deficiency

25 118 119 122

An absence of the enzyme ADA has been observed in approximately 15% of patients with SCID.[ ] [ ] [ ] [ ] Patients with ADA deficiency have the same clinical problems of susceptibility to opportunistic bacterial, viral, and parasitic diseases as described for SCID-X1 and the same susceptibility to GVHD from allogeneic T cells in blood products or bone marrow. However, certain features distinguish ADA deficiency, including the presence of rib cage abnormalities similar to a rachitic rosary and multiple skeletal abnormalities of chondroosseous dysplasia on radiographic examination; these occur predominantly at the costochondral junctions, at the apophyses of the iliac bones, and in the vertebral bodies, where a “bone-in-bone” effect can be seen. Immunologic Defects.

Patients with ADA deficiency usually have a much more profound lymphopenia than infants with other types of SCID, with mean absolute lymphocyte counts of less

118 122

118 122 145

than 500/mm3 ; they rarely have elevated percentages of B or NK cells.[ ] [ ] ADA-deficient patients do have normal NK function,[ ] [ ] [ ] and after T cell function is effected by bone marrow transplantation without pretransplantation chemotherapy, they generally have excellent B cell function. The reason is that ADA deficiency affects primarily T cell function, which is absent, just as in all the other forms of SCID. In further contrast to infants with other types of SCID, however, a few ADA-deficient patients have been found to have rare Hassall's corpuscles in their thymuses and changes suggestive of early thymic differentiation.[ milder forms of this condition have been reported, leading to delayed diagnosis of immunodeficiency even into

25]

Moreover,

146 adulthood.[ ]

Molecular Basis.

25]

The gene encoding ADA was mapped to chromosome 20q13-ter and was cloned and sequenced.[

The ADA deficiency caused by mutations in this gene results in

[25]

pronounced accumulations of adenosine, 2′-deoxyadenosine, and 2′-O-methyladenosine. These latter two metabolites directly or indirectly lead to apoptosis of thymocytes and circulating lymphocytes, which causes the immunodeficiency. Adenosine and deoxyadenosine are also apparent suicide inactivators of the enzyme Sadenosylhomocysteine (SAH) hydrolase, resulting in the accumulation of SAH. SAH is a potent inhibitor of virtually all cellular methylation reactions.[

147]

Treatment and Prognosis.

As with other types of SCID, ADA deficiency can be cured by HLA-identical or haploidentical T cell–depleted bone marrow transplantation without pretransplant 122]

chemotherapy,[

which remains the treatment of choice.[

71] [122]

Enzyme replacement therapy with polyethylene glycol–modified bovine ADA (PEG-ADA) 148] [149] [150]

administered subcutaneously once weekly has resulted in both clinical and immunologic improvement in more than 100 ADA-deficient patients.[ 122

However, the immunocompetence achieved is not as effective as with bone marrow transplantation. [ ] Therefore, PEG-ADA therapy should not be initiated if bone marrow transplantation is contemplated because it will confer graft rejection capability on the infant. ADA deficiency is the first genetic defect in which gene 151] [152] [153] [154] [155]

therapy was attempted; those particular efforts were not very successful until recently.[ in the ADA gene has been reported.

Spontaneous in vivo reversion to normal of a mutation

[156] [157]

Janus Kinase 3 Deficiency

SCID patients with autosomal recessive SCID caused by Jak-3 resemble all other types in their susceptibility to infection and to GVHD from allogeneic T cells. However, they have lymphocyte characteristics that resemble those of patients with X-linked SCID, including an elevated percentage of B cells and very low percentages of T and NK cells.[

118] [122]

Because Jak-3 is the only signaling molecule known to be associated with γc , it was a candidate gene for mutations leading

to autosomal recessive SCID not attributable to ADA deficiency[ [34] [122] [161] [162]

158] [159] [160]

( Figure 59-5 ). Thus far, more than 20 patients who lack Jak-3 have been identified.

As with SCID-X1 patients, they have very low or no NK activity. Even after successful T cell reconstitution by transplantation of haploidentical 71

stem cells, they fail to develop NK cells.[ ] Moreover, as with SCID-X1 patients, they often fail to develop normal B cell function after transplantation despite their high numbers of B cells. Their failure to develop NK cells or B cell function is believed to be caused by the defective function of the multiple cytokine receptors that share γc .

Interleukin-7 Receptor Alpha-Chain Deficiency

Because mice whose genes for either the alpha chain of the IL-7 receptor or of IL-7 itself have been mutated are profoundly deficient in T and B cell function but 161

have NK cell function,[ ] naturally occurring mutations in these genes were sought in some of the author's patients who had previously been shown not to have either γc or Jak-3 deficiency and who had T− B+ NK+ SCID. Mutations in the gene for IL-7Rα on chromosome 5p13 have thus far been found in 16 of the author's 35 122

patients[ ] [ ] (see Table 59-1 and Figure 59-3 ). These findings imply that the T cell but not the NK cell defect in SCID-X1 and Jak-3–deficient SCID results from an inability to signal through IL-7R (see Figure 59-5 ). These patients developed normal B cell function after nonablative haploidentical bone marrow stem cell transplantation despite lacking donor B cells, which also suggests that the B cell defect in SCID-X1 is not caused by failure of IL-7 signaling.

1025

Figure 59-5 Diagram showing that Janus kinase 3 (JAK-3) is the major signal transducer for the common gamma chain (γc ) shared by multiple cytokine receptors. Mutations in the gene encoding Jak-3 result in a form of autosomal recessive severe combined immunodeficiency disease (SCID) that mimics X-linked SCID type 1 (SCID-X1) phenotypically. IL, Interleukin; R, receptor; XCID, X-linked combined immunodeficiency disease. Courtesy Sarah Russell, MD, and Warren Leonard, MD.

Courtesy Sarah Russell, MD, and Warren Leonard, MD. Recombinase Activating Gene Deficiency

Infants with autosomal recessive SCID caused by RAG-1 or RAG-2 deficiency resemble all others in their infection susceptibility and complete absence of T or B cell function. However, their lymphocyte phenotype differs from that of patients with SCID caused by γc , Jak-3, IL-7Rα, or ADA deficiencies in that RAG-deficient infants lack both B and T lymphocytes and have primarily NK cells in their circulation (T− B− NK+ SCID). This finding indicated a possible problem with their antigen receptors, and indeed many patients with this phenotype have been found to have mutations in the genes that encode RAG-1 or RAG-2.[ mutations result in a functional inability to form antigen receptors through genetic recombination.

36] [164] [165]

Such

165]

In addition, patients with Omenn's syndrome also have mutations in RAG-1 or RAG-2 genes, resulting in partial and impaired V(D)J recombinational activity.[ [166]

Omenn's syndrome is characterized by the development soon after birth of a generalized erythroderma and desquamation, diarrhea, hepatosplenomegaly, hypereosinophilia, and greatly elevated serum IgE levels. These problems are caused by circulating activated oligoclonal T lymphocytes that do not respond normally 167] [168]

to mitogens or antigens in vitro.[

169]

Circulating B cells are not found, and lymph node architecture is abnormal due to a lack of germinal centers.[ 71 transplantation.[ ]

syndrome is fatal unless corrected by bone marrow Omenn's syndrome, but not for RAG-1– or RAG-2–deficient SCID.

Omenn's

Pretransplant chemotherapy is required for successful bone marrow transplantation in

Artemis Gene Product Deficiency

The most recently discovered cause of human SCID is a deficiency of a novel V(D)J recombination/deoxyribonucleic acid (DNA) repair factor that belongs to the metallo-β-lactamase superfamily. This factor is encoded by a gene on chromosome 10p called Artemis. A deficiency of this factor results in an inability to repair DNA after double-stranded cuts have been made by RAG-1 or RAG-2 gene products in rearranging antigen receptor genes from their germline configuration. Similar to RAG-1/RAG-2–deficient SCID, this defect results in another form of T− B− NK+ SCID, also called Athabascan SCID. In addition, patients with this type of SCID have increased radiation sensitivity of both skin fibroblasts and bone marrow cells. CD45 Deficiency 170 171

Another recently discovered molecular defect causing SCID is a mutation in the gene encoding the common leukocyte surface protein CD45.[ ] [ ] This hematopoietic cell–specific transmembrane protein tyrosine phosphatase functions to regulate Src kinases required for T and B cell antigen receptor signal transduction. A 2-month-old male infant presented with a clinical picture of SCID and was found to have a very low number of T cells but a normal number of B 170

cells. [ ] The T cells failed to respond to mitogens, and serum immunoglobulins diminished over time. The patient was found to have a large deletion at one CD45 allele and a point mutation causing an alteration of the intervening sequence 13 donor splice site at the other. A second case of SCID caused by CD45 deficiency has 171]

been reported.[

T Cell Activation Defects Defects in T cell activation are characterized by the presence of normal or elevated numbers of blood T cells that appear phenotypically normal but fail to proliferate or produce cytokines in response to stimulation with mitogens, antigens, or other signals delivered to the T cell antigen receptor (TCR) because of defective signal transduction from the TCR to intracellular metabolic pathways ( Figure 59-6 ). This condition may be caused by mutations in genes for a variety of cell surface molecules or signal transduction molecules; several examples are described next. These patients have problems similar to those of other T cell–deficient individuals, and some with severe T cell activation defects may resemble SCID patients clinically. Defective Expression of T Cell Receptor–CD3 Complex 172

The first type of defective expression in the TCR-CD3 complex (Ti-CD3) was found in two male siblings in a Spanish family.[ ] The proband presented with severe infections and died at 31 months of age with autoimmune hemolytic anemia and viral pneumonia. His lymphocytes had responded poorly to mitogens and to anti-CD3 in vitro and could not be stimulated to develop cytotoxic T cells. However, his antibody responses to protein antigens had been normal, indicating normal T helper cell function. His 12-year-old brother was healthy, but he had almost no CD3-bearing T cells and had IgG2 deficiency similar to his sibling. The defect in this family was shown to be due to mutations in the CD3γ chain.[

172]

The second form of Ti-CD3 disorder was found in a 4-year-old French boy who had recurrent Haemophilus influenzae pneumonia and otitis media in early life but later

1026

Figure 59-6 T cell signal transduction pathway. T cell antigen receptor (TCR) spans plasma membrane in association with CD3 and ζ, CD4 or CD8, CD28, and CD45. Cytoplasmic protein tyrosine kinases (PTKs) associated with the TCR are activated on antigen binding to TCR. These PTKs include Lck, Fyn, zeta-chainassociated protein 70 (ZAP-70), and Syk. PTK activation results in the phosphorylation of phospholipase Cγ1 and the activation of other signaling molecules. Distal signaling events, including protein kinase C (PKC) activation and Ca++ mobilization, result in the transcription of genes encoding interleukin-2 (IL-2) and other proteins, culminating in T cell activation, differentiation, and proliferation. Ionomycin and phorbol myristate acetate (PMA) can be used to mimic distal signaling events. Mutations in the gene encoding ZAP-70 result in greatly impaired T cell activation, in addition to abnormal thymic selection resulting in CD8 deficiency. PIP2 , Phosphatidylinositol bisphosphate; DG, diacylglycerol; IP3 , inositol triphosphate; MAP, mitogen-activated protein; NFAT, nuclear factor of activated T cells; AP-1, transcription factor. Modified from Elder ME: Pediatr Res 39:744, 1996.

Modified from Elder ME: Pediatr Res 39:744, 1996. was healthy. He had a partial defect in expression of Ti-CD3, resulting in about a half-normal percentage of CD3+ cells, all with very low CD3 staining on flow cytometry. His T cells did not proliferate in response to anti-CD3 or anti-CD2 and did not express the IL-2 receptor or have normal calcium influx after these 173

treatments. However, the T cells did respond normally to stimulation with anti-CD28 or antigens such as tetanus.[ ] The defect was shown to be due to two independent CD3epsilon gene mutations, leading to defective CD3epsilon-chain synthesis and preventing normal association and membrane expression of the TiCD3 complex.[

174]

Defective Expression of Major Histocompatibility Complex Antigens MHC Class I Antigen Deficiency.

An isolated deficiency of MHC class I antigens is rare, and the resulting immunodeficiency is milder than that in SCID, contributing to a later age of presentation. Sera from affected patients contain normal quantities of class I MHC antigens and β2 -microglobulin, but class I MHC antigens are not detected on any cells in the body. There is a deficiency of CD8+ but not of CD4+ T cells. Mutations have been found in two genes within the MHC locus on chromosome 6 that encode the 175 176 177 178 179

peptide transporter proteins, TAP-1 and TAP-2[ ] [ ] [ ] [ ] [ ] (see Table 59-1 and Figure 59-1 ). TAP proteins function to transport peptide antigens from the cytoplasm across the Golgi apparatus membrane to join the alpha chain of MHC class I molecules and β2 -microglobulin. The complex can then move to the cell surface; if the assembly of the complex cannot be completed because there is no peptide antigen, the MHC class I complex is destroyed in the cytoplasm.[

180]

MHC Class II Antigen Deficiency.

181

Many patients with this autosomal recessive syndrome are of North African descent.[ ] More than 70 patients have been identified. They present in infancy with persistent diarrhea, often associated with cryptosporidiosis, bacterial pneumonia, Pneumocystis infection, septicemia, and viral or candidal infections. However, their immunodeficiency still is not as severe as in SCID, as evidenced by their failure to develop BCG-osis or GVHD from nonirradiated blood transfusions.[

181]

MHC class II–deficient patients have a very low number of CD4+ T cells but normal or elevated numbers of CD8+ T cells. Lymphopenia is only moderate. The MHC class II antigens, HLA-DP, DQ, and DR, are undetectable on blood

1027

B cells and monocytes. These patients have impaired antigen-specific responses caused by the absence of these antigen-presenting molecules. In addition, MHC antigen–deficient B cells fail to stimulate allogeneic cells in mixed leukocyte culture. Lymphocytes respond normally to mitogens but not to antigens. The thymus and other lymphoid organs are severely hypoplastic. The lack of class II molecules results in abnormal thymic selection, because recognition of HLA molecules by 182

thymocytes is central to both positive and negative selection. The abnormal selection results in circulating CD4+ T cells that have altered CDR3 profiles.[ ] The associated defects of both B and T cell immunity and of HLA expression emphasize the important biologic role for HLA determinants in effective immune cell cooperation. Molecular Basis.

31

Four different molecular defects resulting in impaired expression of MHC class II antigens have been identified[ ] (see Table 59-1 and Figure 59-1 ). In one defect there is a mutation in the gene on chromosome 1q that encodes a protein called RFX5, a subunit of RFX, a multiprotein complex that binds the X box motif of MHC II promoters.[

183]

A second form is caused by mutations in a gene on chromosome 13q that encodes a second 36-kD subunit of the RFX complex, called RFX-

associated protein (RFXAP).[

184]

The most recently discovered and most common cause of MHC class II defects are mutations in RFXANK, the gene encoding a

third subunit of RFX.[

31]

In a fourth type there is a mutation in the gene on chromosome 16p13 that encodes a novel MHC class II transactivator, CIITA, a non-DNA185]

binding coactivator that controls the cell-type specificity and inducibility of MHC II expression. [ of MHC class II molecules on the surfaces of B cells and macrophages.

All these defects cause impairment in the coordinate expression

p56 Lck Deficiency 186

A 2-month-old male infant who presented with bacterial, viral, and fungal infections was found to be lymphopenic and hypogammaglobulinemic.[ ] B and NK cells were present, but there was a low number of CD4+ T cells. Mitogen responses were variable. The T cells failed to express the activation marker CD69 when stimulated through the T cell receptor but did express it when stimulated with PMA and a calcium ionophore, suggesting a proximal signaling defect. Molecular studies revealed an alternatively spliced transcript for p56 lck that lacked the kinase domain (see Figure 59-2 ). Zeta-Chain-Associated Protein 70 Deficiency

Patients with CD8 lymphocytopenia caused by ZAP-70 deficiency present during infancy with severe, recurrent, sometimes fatal infections similar to those in SCID patients; however, they often live longer and present later than SCID patients. More than eight cases have been reported, and a majority were Mennonites. [

33] [187]

[188]

They have normal, low, or elevated serum immunoglobulin concentrations and normal or elevated numbers of circulating CD4+ T lymphocytes but essentially no CD8+ cells. These CD4+ T cells fail to respond to mitogens or to allogeneic cells in vitro or to generate cytotoxic T lymphocytes. By contrast, NK activity is normal. The thymus of one patient exhibited normal architecture; there were normal numbers of CD4:CD8 double positive thymocytes but an absence of CD8+ thymocytes. 187 188

This condition has been attributed to mutations in the gene encoding ZAP-70, a non-src family protein tyrosine kinase important in T cell signaling[ ] [ ] (see Figure 59-6 ). The gene is on chromosome 2 at position q12. ZAP-70 has been shown to have an essential role in both positive and negative selection in the thymus [189]

(see Figure 59-5 ). The hypothesis as to why there are normal numbers of CD4+ T cells is that thymocytes can use the other member of the same tyrosine kinase family, Syk, to facilitate positive selection of CD4+ cells. In addition, there is a stronger association of Lck with CD4+ than with CD8+ cells. Syk is present at fourfold higher levels in thymocytes than in peripheral T cells, possibly accounting for the lack of normal responses by the CD4+ blood T cells. CD8 Deficiency Caused by CD8a Gene Mutation

Another cause of CD8 deficiency (in addition to ZAP-70 deficiency and MHC class I antigen deficiency) was discovered in a 25-year-old Spanish man with a history of recurrent respiratory infections since childhood. Immunoglobulins and antibodies were normal, as were T cell proliferation studies and NK cell function. However, he was found to have a complete absence of CD8+ T cells. Molecular studies revealed a missense mutation in both alleles of the immunoglobulin domain of the CD8a gene in the patient and in two of his sisters.[ Combined Immunodeficiency

190]

The term combined immunodeficiency (CID) is used to distinguish patients with low but not absent T cell function from those with SCID. Three examples are given next. Purine Nucleoside Phosphorylase Deficiency 26

More than 40 patients with CID have been found to have purine nucleoside phosphorylase (PNP) deficiency.[ ] Deaths have occurred from generalized vaccinia, varicella, lymphosarcoma, and GVHD mediated by T cells from nonirradiated allogeneic blood or bone marrow. Two thirds of patients have had neurologic abnormalities ranging from spasticity to mental retardation. One third of patients developed autoimmune diseases, the most common being autoimmune hemolytic anemia. Immunologic Defects.

Most patients have normal or elevated concentrations of all serum immunoglobulins. PNP-deficient patients are as profoundly lymphopenic as those with ADA deficiency, with absolute lymphocyte counts usually less than 500/mm.3 Analyses of lymphocyte subpopulations with monoclonal antibodies have demonstrated a profound deficiency of T cells and of T cell subsets but increased numbers of cells with NK phenotype and function. T cell function is low but not absent and is variable over time. Molecular Basis.

The gene encoding PNP is on chromosome 14q13.1 and has been cloned and sequenced. A variety of mutations have been found in the PNP gene in patients with 191

PNP deficiency.[ ] Unlike ADA deficiency, serum and urinary uric acid are deficient because PNP is needed to form the urate precursors hypoxanthine and xanthine. Prenatal diagnosis is possible. Treatment and Prognosis.

PNP deficiency is invariably fatal in childhood unless immunologic reconstitution can be achieved. Bone marrow transplantation is the treatment of choice but thus far has been successful in only three patients.[

71] [192]

1028

Ataxia-Telangiectasia

Ataxia-telangiectasia (AT) is a complex CID syndrome with associated neurologic, endocrinologic, hepatic, and cutaneous abnormalities.[

42] [193]

The most

194

prominent features are progressive cerebellar ataxia, oculocutaneous telangiectasias, chronic sinopulmonary disease, a high incidence of malignancy,[ ] and variable humoral and cellular immunodeficiency. The ataxia typically becomes evident shortly after the child begins to walk and progresses until he or she is confined to a wheelchair, usually by 10 to 12 years of age. The telangiectasias develop at 3 to 6 years of age. Recurrent, usually bacterial, sinopulmonary infections occur in about 80% of these patients. Fatal varicella occurred in one patient, and transfusion-associated GVHD has also been reported.[

195]

Immunologic Defects.

196

Selective IgA deficiency is found in from 50% to 80% of patients with AT, attributable in part to hypercatabolism of IgA in some patients. [ ] IgE concentrations are usually low, and the IgM may be of the low-molecular-weight variety. IgG2 or total IgG may be decreased. Specific antibody titers may be decreased or normal. There is impaired (but not absent) cell-mediated immunity in vivo, as evidenced by delayed skin test anergy and prolonged allograft survival. In vitro tests of lymphocyte function have generally shown moderately depressed proliferative responses to T and B cell mitogens. The percentages of CD3+ and CD4+ T cells are only modestly low, and there are usually normal or increased percentages of CD8+ T cells and (sometimes) elevated numbers of γ/δ T cell receptor–positive cells. Studies of immunoglobulin synthesis have shown both T helper cell and intrinsic B cell defects. The thymus is hypoplastic, exhibits poor organization, and is lacking in Hassall's corpuscles. Cells from AT patients as well as cells of heterozygous carriers have increased sensitivity to ionizing radiation, defective DNA repair, and frequent chromosomal 196 197

abnormalities.[ ] [ ] The sites of chromosomal breakage in more than 50% of patients involve the genes that encode the TCR on chromosome 7 and the immunoglobulin heavy chains on chromosome 14, most likely accounting for the combined T and B cell abnormalities seen. These rearrangements may be clonal and may either be stable or undergo malignant transformation. The malignancies reported in AT usually have been of the lymphoreticular type, but adenocarcinoma and other forms also have been seen; the incidence of malignancy in unaffected relatives is also increased. Molecular Basis.

Inheritance of AT follows an autosomal recessive pattern. The mutated gene (ATM) responsible for this defect was mapped by RFLP analysis to the long arm of 27 196 198

chromosome 11 (11q22–23) and was cloned.[ ] [ ] [ ] The gene product is a phosphatidyl-inositol 3 kinase-like protein that also has similarities to the catalytic subunit of DNA-dependent protein kinase, localized predominantly to the nucleus and believed to be involved in mitogenic signal transduction, meiotic recombination, and cell cycle control.[

199] [200] [201]

Of the mutations identified up to now, a majority were expected to completely inactivate the ATM protein by

truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments.[

193] [202] [203]

Treatment and Prognosis.

196

No satisfactory definitive treatment for AT has been found.[ ] The prognosis is exceedingly poor for these patients, although some have reached adulthood. The most common causes of death are lymphoreticular malignancy and progressive neurologic disease. Cartilage-Hair Hypoplasia

In 1965 an unusual form of short-limbed dwarfism associated with frequent and severe infections was reported among the Pennsylvania Amish; non-Amish cases 42 204

have since been described.[ ] [ ] These patients have short and pudgy hands with redundant skin, metaphyseal chondrodysplasia, hyperextensible joints of hands and feet but an inability to extend the elbows completely, and fine, sparse, light hair and eyebrows. These features led to the term cartilage-hair hypoplasia (CHH). Radiographically the bones show scalloping and sclerotic or cystic changes in the metaphyses. In contrast to ADA deficiency, in which the predominant changes are in the apophyses of the iliac bones, the ribs, and vertebral bodies, the chondrodysplasia in CHH principally affects the limbs. Severe and often fatal varicella infections, progressive vaccinia, and vaccine-associated poliomyelitis have been observed. Associated conditions include deficient erythrogenesis, Hirschsprung disease, and an increased risk of malignancies. Immunologic Defects.

Three patterns of immune dysfunction in CHH have emerged: defective antibody-mediated immunity, defective cellular immunity (most common form), and SCID. NK cells, however, are increased in number and function. Pathogenesis.

205

CHH is an autosomal recessive condition, and the defective gene has recently been mapped to chromosome 9p21-p13 in Amish and Finnish families.[ ] The endoribonuclease RNase MRP consists of an RNA molecule bound to several proteins. It has at least two functions: cleavage of RNA in mitochondrial DNA synthesis and nucleolar cleaving of pre–ribosomal RNA (rRNA.) Recently, numerous mutations were found in the untranslated RMRP gene that cosegregate with the 206]

CHH phenotype.[

The authors conclude that mutations in RMRP cause CHH by disrupting a function of RNase MRP RNA that affects multiple organ systems.

[206]

Treatment and Prognosis.

Bone marrow transplantation has resulted in immunologic reconstitution in some CHH patients with the SCID phenotype.[ immunodeficiency have lived to adulthood, some even to old age.

122]

Those with milder types of

ANTIBODY DEFICIENCY SYNDROMES Autosomal Recessive Agammaglobulinemia Autosomal recessive conditions that resemble XLA phenotypically are caused by mutations in the genes that encode immunoglobulin heavy or light chains or their associated signaling molecules, leading to agammaglobulinemia or hypogammaglobulinemia. In • chain,[ receptor signaling molecule),

[209]

and B cell linker (BLNK) gene

Autosomal Recessive Hyper–Immunoglobulin M

210 mutations,[ ]

207]

λ5/14.1 (surrogate light chain),[

circulating B cells are absent.

208]

Igα (B cell antigen

211

212

Not all males with hyper-M have a mutation in the gene encoding CD154,[ ] and there are several examples in females,[ ] indicating that this condition has more than one genetic cause. In such patients, the B cells are unable to switch from IgM-secreting to IgG-, IgA-, or IgE-secreting cells, even when co-cultured with monoclonal antibodies to CD40 and a variety of cytokines.[

211]

Thus, in these patients,

1029

the condition truly is a B cell defect. CD40 has been present on the surfaces of the B cells of most patients, although recently three patients with this syndrome were 213]

found to have mutations in the CD40 gene.[

The defect in many patients has recently been identified as caused by mutations in a gene on chromosome 12p13 that encodes an activation-induced cytidine 38 39

deaminase (AID), an RNA-editing enzyme specifically expressed in germinal center B cells.[ ] [ ] A deficiency of AID results in impaired terminal differentiation of B cells and a failure of isotype switching. There is a lack of immunoglobulin gene somatic hypermutation. Unlike X-linked hyper-IgM, patients with autosomal recessive hyper-M resulting from AID mutations have lymphoid hyperplasia because they do have germinal centers, although defective. As with the X-linked form, treatment is monthly infusions of IVIG. Thymic Hypoplasia: DiGeorge Syndrome Thymic hypoplasia results from dysmorphogenesis of the third and fourth pharyngeal pouches during early embryogenesis, leading to hypoplasia or aplasia of the 214

thymus and parathyroid glands.[ ] Other structures forming at the same age are also frequently affected, resulting in anomalies of the great vessels (right-sided aortic arch), esophageal atresia, bifid uvula, upper limb malformations, congenital heart disease (conotruncal, atrioventricular septal defects), a short philtrum of the 215]

upper lip, hypertelorism, an antimongoloid slant to the eyes, mandibular hypoplasia, and low-set, often notched ears.[ of hypocalcemic seizures during the neonatal period.

The diagnosis is usually suspected because

Since the original description of this syndrome, it has become apparent that a variable degree of hypoplasia is more frequent than total aplasia of the thymus and parathyroid glands.[

216] [217]

Some children have minimal difficulty with life-threatening infections and grow normally; such patients are often referred to as having

216 syndrome.[ ]

partial DiGeorge Those with complete DiGeorge syndrome may resemble patients with SCID in their susceptibility to infections with opportunistic pathogens (e.g., fungi, viruses, Pneumocystis carinii) and to GVHD from nonirradiated blood transfusions. Many clinical similarities exist among DiGeorge syndrome, the velocardiofacial syndrome (VCFS), fetal alcohol syndrome, and retinoic acid toxicity.[ Immunologic Defects

214]

216 217

Patients with DiGeorge syndrome are usually only mildly lymphopenic. [ ] [ ] The percentage of T cells is variably decreased, resulting in a relative increase in the percentage of B cells. B cell function is impaired only to the extent of needing T helper cells. Immunoglobulin concentrations are usually normal, although IgE 216 217

may be elevated and IgA may be low.[ ] [ ] Monoclonal antibody analyses of blood lymphocytes have demonstrated that, despite a decreased number of CD3+ T cells, proportions of CD4+ and CD8+ cells are usually normal. Responses of blood lymphocytes after mitogen stimulation have been absent, reduced, or normal, depending on the degree of thymic deficiency, and such variability suggests that most T lymphocytes present are intrinsically normal. Thymic tissue, when found, does contain Hassall's corpuscles and a normal density of thymocytes; corticomedullary distinction is present. Lymphoid follicles are usually present, but lymph node paracortical areas and thymus-dependent regions of the spleen show variable degrees of depletion. Molecular Basis

DiGeorge syndrome has occurred in both males and females. It is rarely familial, but three cases of apparent autosomal dominant inheritance have been reported. 30] [218] [219]

Microdeletions of specific DNA sequences from chromosome 22q11.2 (the DiGeorge chromosomal region) have been shown in a majority of patients,[ [220] [221]

and several candidate genes have been identified in this region.[

28] [30] [222] [223]

There appears to be an excess of 22q11.2 deletions of maternal origin.[

224]

29] [225] [226]

Another deletion associated with DiGeorge syndrome and VCFS has been identified on chromosome 10p13.[ Treatment and Prognosis

No immunologic treatment is needed for patients with the partial form. If patients with DiGeorge syndrome do not have a severe cardiac lesion, they have few clinical problems except possible seizures and developmental delay. Because of variability in the severity of the immunodeficiency, it is difficult to evaluate claimed benefits of fetal thymus transplantation.[

227]

Transplantation of HLA-DR-matched, cultured, mature, thymic epithelial explants has successfully reconstituted the

immune function of some infants with the complete DiGeorge syndrome.[

228]

Three patients with complete DiGeorge syndrome have experienced immunologic 229]

reconstitution after unfractionated HLA-identical bone marrow transplantation.[

LEUKOCYTE DEFECTS Interferon Gamma Receptor Mutations Disseminated BCG infections occur in infants with SCID or with other severe T cell defects. In approximately half of patients, however, no specific host defect has 40]

been found. One explanation for this predilection was found in a 2.5-month-old Tunisian female infant who had fatal idiopathic disseminated BCG infection [ 41 immunodeficiency.[ ]

and

in four children from Malta who had disseminated atypical mycobacterial infection in the absence of a recognized In all five children there was consanguinity in their pedigrees. All were found to have a functional defect in the up-regulation of tumor necrosis factor alpha (TNF-α) production by their blood macrophages in response to stimulation with IFN-γ. Further, all lacked expression of IFN-γ receptors on their blood monocytes or lymphocytes, and each was found to have a mutation in the gene on chromosome 6q22-q23 that encodes IFN-γR1. Defects in the IFN-γ receptor 2 (IFN-γR2) have also been identified in patients susceptible to facultative intracellular infectious agents. Interestingly, these children did not appear to be susceptible to infection with agents other than mycobacteria.

T helper type 1 (Th1) cell responses appeared to be normal in these patients. The susceptibility of these children to mycobacterial infections thus apparently results from an intrinsic impairment of the IFN-γ pathway response to these particular intracellular pathogens, showing that IFN-γ is obligatory for efficient macrophage antimyco-bacterial activity.[

40] [41]

1030

Interleukin-12 Receptor β1 Mutation IL-12 is produced by activated antigen presenting cells (APCs) (dendritic cells, macrophages). It promotes the development of Th1 responses and is a powerful inducer of IFN-γ production by T and NK cells. T and NK cells from seven unrelated patients who had severe idiopathic mycobacterial and Salmonella infections failed to produce IFN-γ when stimulated with IL-12. The patients were otherwise healthy. They were found to have mutations in the IL-12 receptor β1 chain, 230] [231]

resulting in premature stop codons in the extracellular domain and, in turn, unresponsiveness to this cytokine, demonstrating IL-12's role in host defense. [ Germline Mutation in Signal Transducer and Activator of Transcription-I

Interferons induce the formation of two transcriptional activators: gamma-activating factor (GAF) and interferon-stimulated gamma factor 3 (ISGF-3). A natural heterozygous germline signal transducer and activator of transcription (STAT-1) mutation associated with susceptibility to mycobacterial but not viral disease was 232

found in two unrelated patients with unexplained mycobacterial disease.[ ] This mutation caused a loss of GAF and ISGF-3 activation but was dominant for one cellular phenotype and recessive for the other. It impaired the nuclear accumulation of GAF but not of ISGF-3 in cells stimulated by interferons, implying that the antimycobacterial but not the antiviral effects of human interferons are mediated by GAF. Leukocyte Adhesion Deficiencies Leukocyte Adhesion Deficiency Type 1.

LAD-1 is attributable to mutations in the gene on chromosome 21 at position q22.3 encoding CD18, a 95-kD β subunit shared by three adhesive heterodimers: (1) LFA-1 on B, T, and NK lymphocytes; (2) complement receptor type 3 (CR-3) on neutrophils, monocytes, macrophages, eosinophils, and NK cells; and (3) p150,95 23] [24] [233]

(another complement receptor) (see Chapter 9 ). [ of the abnormal β chain.

The α chains of these three molecules (encoded by genes on chromosome 16) are not expressed because

Patients with LAD-1 have histories of delayed separation of the umbilical cord, omphalitis, gingivitis, recurrent skin infections, repeated otitis media, pneumonia, 234 235

septicemia, ileocolitis,[ ] [ ] peritonitis, perianal abscesses, and impaired wound healing. Life-threatening bacterial and fungal infections account for the high mortality. Those affected do not have increased susceptibility to viral infections or malignancy. Blood neutrophil counts are usually significantly elevated even when

no infection is present because of an inability of the cells to adhere to vascular endothelium and migrate out of the intravascular compartment. All cytotoxic lymphocyte functions are considerably impaired because of a lack of the adhesion protein LFA-1. Deficiency of LFA-1 also interferes with immune cell interaction and immune recognition. CR-3 binds fixed iC3b fragments of C3 and β-glucans; absence of CR-3 causes abnormal phagocytic cell adherence and chemotaxis and a reduced respiratory burst with phagocytosis. Deficiencies of these glycoproteins can be screened for by cytofluorography of blood leukocytes with monoclonal antibodies to CD18 or to CD11a, b, or c. Because the CD18 gene has been cloned and sequenced, the patient with LAD-1 is another potential candidate for gene therapy.[

236]

Leukocyte Adhesion Deficiency Type 2

LAD-2 is attributable to the absence of neutrophil sialyl-LewisX , a ligand of E-selectin on vascular endothelium.[

237]

LAD-2 was discovered in two unrelated Israeli

238 parents.[ ]

boys, 3 and 5 years of age, each the offspring of consanguineous Both had severe mental retardation, short stature, a distinctive facial appearance, and the Bombay (hh) blood phenotype, and both were secretor negative and Lewis negative. Both patients had had recurrent severe bacterial infections similar to those seen in patients with LAD-1, including pneumonia, peridontitis, otitis media, and localized cellulitis. Also similar to patients with LAD-1, their infections were accompanied by pronounced leukocytosis (30,000 to 150,000/mm3 ) but an absence of pus formation at sites of recurrent cellulitis. In vitro studies revealed a pronounced defect in neutrophil motility. Because the genes for the red blood cell H antigen and for the secretor status encode for distinct α1,2-fucosyltransferases, 238]

and because the synthesis of sialyl-Lewisx requires an α1,3-fucosyltransferase, Etzioni and colleagues[

postulated a general defect in fucose metabolism as the

basis for this disorder. They recently found that GDP-L-fucose transport into Golgi vesicles was specifically impaired[

239]

and then discovered missense mutations in

the GDP-fucose transporter cDNA of three patients with LAD-2. Thus GDP-fucose transporter deficiency is a cause of LAD-2.[

240]

Autosomal Recessive Chronic Granulomatous Disease Patients with AR-CGD have infections similar to those in patients with X-CGD, that is, suppurative adenopathy, pne monia, cutaneous and visceral abscesses, and osteomyelitis caused by infections with catalase-positive bacterial organisms or fungi. Immunologic Defects 105 111

As in X-CGD, the molecular defects in the three forms of AR-CGD lie solely in an ability of the phagocyte to kill ingested organisms.[ ] [ ] In the autosomal forms, defects exist in different components of the electron transport chain. However, as in X-CGD, a diagnosis of AR-CGD is made by demonstration of an inability of neutrophils from the patient to undergo a respiratory burst after phagocytosis or PMA stimulation, as measured by chemiluminescence, NBT dye reduction, or (preferably) by a flow cytometry dye reduction test.[

109] [110]

Unlike those with X-CGD, however, carriers of AR-CGD cannot be detected by the latter methods.

Molecular Bases

Approximately 5% of patients with CGD have a defect in the 22-kD subunit (p22phox ), or light chain, of the cytochrome b245 heterodimer, encoded by a gene on

105

105

chromosome 16q24.[ ] However, in most patients with AR-CGD, cytochrome b245 is intact.[ ] Evaluation of cytochrome-positive AR-CGD patients led to the identification of cytosolic proteins that participate in the respiratory burst. A majority of AR-CGD patients (and 25% of all patients with CGD) lack a 47-kD phosphoprotein (p47phox ) encoded by a gene on

1031

chromosome 7 at position q11.23,[ missing.[

105] [107]

241]

but in approximately 5% of CGD patients a 67-kD protein (p67phox ) encoded by a gene on chromosome 1 at position q25 is

Delineation of the specific molecular abnormality in any given patient with AR-CGD requires molecular techniques.

Treatment and Prognosis

The treatment and prognosis for AR-CGD are the same as for X-CGD (see earlier discussion). Chédiak-Higashi Syndrome 242

The rare Chédiak-Higashi syndrome is characterized by oculocutaneous albinism and susceptibility to recurrent respiratory tract and other types of infections. [ ] The characteristic feature of the disease is giant lysosomal granules, not only in neutrophils, but also in most of the other cells of the body, including melanocytes, [243]

244

neural Schwann cells, renal tubular cells, gastric mucosa, pneumatocytes, hepatocytes, Langerhans' cells of the skin, and adrenal cells.[ ] The granules in neutrophils are positive for peroxidase, acid phosphatase, and esterase. The abnormal lysosomes are unable to fuse with phagosomes, and thus ingested bacteria 245]

cannot be lysed normally. In addition, cytotoxic T lymphocyte and NK cell activity is almost completely absent because of abnormal lysosomal granule function.[ [246]

The fundamental defect in this autosomal recessive disorder was found to be caused by mutations in a gene on human chromosome 1 at position q42–43. [ [249]

This gene is similar to the one mutated in the murine beige defect. [

250]

247] [248]

The protein is postulated to function with other proteins as components of a vesicle 247

membrane–associated signal transduction complex that regulates intracellular protein trafficking.[ ] Approximately 85% of patients with Chédiak-Higashi syndrome develop an “accelerated phase” of the disease, with fever, jaundice, hepatosplenomegaly, lymphadenopathy, pancytopenia, bleeding diathesis, and 251]

neurologic changes.[

Once the accelerated phase occurs, the disease is usually fatal within 30 months unless successful treatment with an unfractionated HLA-

identical bone marrow transplant after cytoreductive conditioning can be accomplished.[

71] [252]

AUTOSOMAL DEFECTS WITH UNIDENTIFIED MOLECULAR BASES

Common Variable Immunodeficiency CVID, also known as “acquired” hypogammaglobulinemia or idiopathic late-onset immunoglobulin deficiency, may appear similar clinically in many respects to 253

46

XLA.[ ] The types of infections experienced and bacterial etiologic agents involved are generally the same for the two defects.[ ] However, patients with CVID generally have a later age of onset of infections than in those with XLA, and the infections may be less severe. Males and females are almost equally affected with CVID. 254

255

CVID has been variably associated with a spruelike syndrome, nodular follicular lymphoid hyperplasia of the intestine, colitis,[ ] small bowel lymphoma,[ ] gastric atrophy, achlorhydria, thymoma, alopecia areata, hemolytic anemia, and pernicious anemia. In addition, such patients have frequent thyroid abnormalities, vitiligo, keratoconjunctivitis sicca, and arthritis. Frequent complications include giardiasis (seen more often here than in XLA), bronchiectasis, gastric carcinoma, lymphoreticular malignancy, and cholelithiasis. Lymphoid interstitial pneumonia, pseudolymphoma, amyloidosis, and noncaseating granulomas of the lungs, spleen, 256]

skin, and liver have also been seen.[

There is a 438-fold increase in lymphomas in affected women in the fifth and sixth decades of life.[

257]

Immunologic Defects

The serum immunoglobulin and antibody deficiencies in CVID may be as profound as those in XLA. Despite normal numbers of circulating immunoglobulinbearing B lymphocytes and the presence of lymphoid cortical follicles, blood B lymphocytes from CVID patients do not differentiate into immunoglobulin-producing 253]

cells when stimulated with pokeweed mitogen (PWM) in vitro, even when co-cultured with normal T cells,[

and they are L-selectin negative, possibly because of

258 activation.[ ]

253

aberrant lymphocyte From these observations, it was believed that the defect or defects in this syndrome are intrinsic to the B cell.[ ] In keeping with this are studies showing a lack of protein kinase C (PKC) activation and translocation to the plasma membrane when CVID B cells are stimulated with phorbol ester or anti-•.[ 4 or

259]

However, CVID B cells can be stimulated to both isotype-switch and synthesize/secrete immunoglobulin when stimulated with anti-CD40 plus IL-

260 261 IL-10.[ ] [ ]

T cells are usually present in normal percentages, although an abnormality of CD4+ T cell differentiation has been reported,[

well as depressed T cell

265 266 267 function.[ ] [ ] [ ]

262] [263] [264]

as

Mitogen-activated T cells from some CVID patients were also found to be deficient in expression of genes for 263

several lymphokines while retaining a normal capacity to proliferate.[ ] In addition, a subset of patients with CVID were reported to have significantly depressed (but not absent) expression of CD40 ligand (CD154) mRNA and surface protein on their activated T lymphocytes, suggesting that inefficient signaling by poorly 268]

expressed CD154 on their T cells could account for failure of their B cells to differentiate.[

Tonsils and lymph nodes are either normal sized or enlarged, and splenomegaly occurs in approximately 25% of patients with CVID. In addition, there is a tendency to autoantibody formation, and lupus erythematosus converting to CVID has been reported.[ such patients acquired human immunodeficiency virus (HIV)

269]

Rarely, CVID has resolved transiently or permanently when some

270 infection.[ ]

Pathogenesis

Because CVID occurs in first-degree relatives of patients with selective IgA deficiency (A Def) and some patients with A Def later become

271 272

panhypogammaglobulinemic, it has long been suspected that these diseases have a common genetic basis.[ ] [ ] The high incidences of abnormal immunoglobulin concentrations, autoantibodies, autoimmune disease, and malignancy in families of both types of patients also suggested a shared hereditary influence.[

273]

This concept is supported by the finding of a high incidence of C4-A gene deletions and C2 rare gene alleles in the MHC class III region in individuals 273 274

275 276

with either A Def[ ] [ ] or CVID,[ ] [ ] suggesting that a susceptibility gene may exist in this region on chromosome 6. These studies also have shown that a small number of HLA haplotypes are shared by individuals affected with CVID and A Def, with at least one of two particular

1032

276

271

haplotypes being present in 77% of those affected.[ ] In one large family with 13 members, two had A Def and three had CVID.[ ] All the immunodeficient patients in the family had at least one copy of an MHC haplotype shown to be abnormally frequent in A Def and CVID: HLA-DQB1 *0201, HLA-DR3, C4B-Sf, C4A-deleted, G11–15, Bf-0.4, C2a, HSP70–7.5, TNF-α-5, HLA-B8, and HLA-A1.[

277]

However, four immunologically normal members of the pedigree also 271

possessed this haplotype, indicating that its presence alone is not sufficient for expression of the defects.[ ] Environmental factors, particularly drugs such as phenytoin, have been suspected of providing the triggers for disease expression in individuals with the permissive genetic background. Clearly, however, CVID is still best regarded as a syndrome that includes many different genetic defects. As mentioned, because of the high rate of new mutations occurring in human X-linked immunodeficiencies, the SH2D1A gene should be studied in male CVID patients. In addition, male patients should be studied for mutations in the BTK and CD154 genes, and both males and females should be evaluated for mutations in the AID and CD40 genes. Treatment and Prognosis

The treatment of patients with CVID is essentially the same as that for XLA.[

58]

Anaphylactic reactions caused by IgE antibodies to IgA are always a possibility in

patients with CVID; thus caution should be used when therapy is initiated with IVIG preparations containing IgA.[

278]

Nonanaphylactic adverse reactions to IVIG 279]

infusions in patients with CVID (usually those with chronic lung or sinus disease) have been associated with elevated plasma TNF-α levels. [ patients with CVID is reasonably good unless severe autoimmune disease or malignancy develops.

The prognosis for

[280]

Selective Immunoglobulin A Deficiency Isolated absence or near-absence (less than 10•mg/dl) of serum and secretory IgA is believed to be the most common primary immunodeficiency disorder. A Def was 6

6

reported with a frequency of 1 in 333 among some blood donors. [ ] Although A Def has been observed in apparently healthy individuals,[ ] it is typically associated 281]

with poor health. When present, infections, as expected, are predominantly in the respiratory, gastrointestinal, and urogenital tracts.[ are essentially the same as in other types of antibody deficiency

282 syndromes.[ ]

Bacterial agents responsible

No clear evidence indicates that patients with A Def have an undue susceptibility to

viral agents. Similar to CVID, A Def is frequently associated with collagen vascular and autoimmune diseases.[

273]

A spruelike syndrome has occurred in adults with

A Def, who may or may not respond to a gluten-free diet. In further similarity to CVID, patients with A Def have an increased incidence of malignancy.[

3]

Immunologic Defects 282 283 284

Serum concentrations of other immunoglobulins are usually normal in patients with A Def, although IgG2 subclass deficiency may be present,[ ] [ ] [ ] and IgM (often monomeric) may be elevated. In vitro cultures of B cells from IgA-deficient patients could be stimulated to produce IgA by the combination of anti-CD40 285

and IL-10. [ ] Children with A Def vaccinated intranasally with killed poliovirus produced local IgM and IgG antibodies. Several of them later contracted rubella, and IgM and IgG antirubella antibodies were found in their secretions during convalescence. 6]

Of possible etiologic and great clinical significance is the presence of antibodies to IgA in the sera of as many as 44% of patients with selective IgA deficiency.[ [286]

Several A Def patients have had severe or fatal anaphylactic reactions after intravenous administration of blood products containing IgA,[

287]

and anti-IgA

278

antibodies (particularly IgE anti-IgA antibodies) have been implicated. [ ] For this reason, only normal donor erythrocytes, washed five times (in 200-ml volumes), or blood products from other A Def individuals should be administered to these patients. Patients with A Def also frequently have IgG antibodies against cow's milk and ruminant serum proteins. These antiruminant antibodies often falsely detect “IgA” in immunoassays that employ goat (but not rabbit) antisera.[ incidence of autoantibodies has also been

288]

A high

273 noted.[ ]

Pathogenesis

The basic defect leading to A Def is unknown. The occurrence of A Def in both males and females and in families is consistent with autosomal inheritance; in many families this appears to be dominant inheritance with variable expressivity. Treatments with phenytoin (Dilantin), d-penicillamine, sulfasalazine, or gold compounds have been suspected as being facilitators of expression of this defect. As already noted, A Def occurs in pedigrees with CVID patients, and molecular genetic studies suggest that the susceptibility genes for these two defects may reside in the MHC class III region as an allelic condition on chromosome 6.[

271] [272] [276] [277]

Treatment and Prognosis

Currently, no treatment exists for A Def beyond the vigorous treatment of specific infections with appropriate antimicrobial agents. IVIG (99% IgG) is not indicated 58]

because most A Def patients make IgG antibodies normally.[ with IgE antibodies to

58 278 IgA.[ ] [ ]

Moreover, many IVIG preparations contain sufficient IgA to cause anaphylactic reactions in those

In some A Def children the defect may spontaneously disappear with time,[

some A Def patients the defect has evolved into Immunoglobulin G Subclass Deficiency

290 CVID.[ ]

281]

289]

whereas in adults it usually is persistent.[

In

283

Many patients have been reported to have deficiencies of one or more subclasses of IgG, despite normal total IgG serum concentrations.[ ] Most of those with absent or very low concentrations of IgG2 have been patients with A Def. IgG2 deficiency should be suspected if patients have had repeated problems with 291

encapsulated bacterial pathogens, because a majority of the antipolysaccharide antibody molecules are of the IgG2 isotype.[ ] However, it is difficult to know the biologic significance of reported IgG subclass protein deficiencies in the absence of documented broad antibody deficiencies, particularly when completely asymptomatic individuals who totally lacked IgG1, IgG2, IgG4, or IgA1 because of heavy-chain gene deletions have been described who produced antibodies normally.[

292]

Similarly, numerous healthy children have been described who had low levels of IgG2 but normal responses to polysaccharide antigens

1033

when immunized.[

293]

On the other hand, patients with the Wiskott-Aldrich syndrome who have a profound antipolysaccharide antibody deficiency have normal

levels of IgG2 protein,[

294]

and even in non-Wiskott patients with recurrent infections, pronounced deficiencies of antipolysaccharide antibodies have been noted in 295

the presence of normal concentrations of IgG2.[ ] When children with low IgG2 subclass levels who had histories of frequent infections were studied in depth, they were found to have broader and more varied patterns of immunologic dysfunction than healthy children with low IgG2 levels, and such a finding suggests that they may have been in the process of evolving into frank CVID.[

296]

From these observations, it can be concluded that IgG subclass measurement is not very helpful in the general assessment of immune function. Such assays provide no information about the patient's capacity to produce specific antibodies to protein, polysaccharide, or viral antigens. Moreover, considerable variability exists in values reported for the different subclasses when aliquots of the same serum sample are tested in different commercial laboratories. Thus, when “abnormalities” are detected, one is still left with the more relevant question: what is the capacity of the patient to make specific antibodies to protein and polysaccharide antigens? IVIG 58]

should not be given to “IgG subclass–deficient” patients unless they are known to have a deficiency of antibodies to a broad array of antigens.[ Transient Hypogammaglobulinemia of Infancy

Unlike patients with XLA or CVID, patients with transient hypogammaglobulinemia of infancy (THI) can synthesize antibodies to human type A and B erythrocytes 297

and to diphtheria and tetanus toxoids.[ ] Often these antibodies are found in normal titers by the time the infant is 6 to 11 months of age, long before their immunoglobulin concentrations become normal. THI has been found in pedigrees of patients with other immune defects, including CVID and SCID. The finding of only 11 cases of THI among more than 10,000 patients whose sera were sent to the author for immunoglobulin studies over a 12-year period indicates that this may not be a common entity.[

297]

58

IVIG therapy is not indicated in THI.[ ] In addition to the known risk of inducing antiallotype antibodies, passively administered IgG antibodies could suppress endogenous antibody formation to infectious agents in the same manner that RhoGAM suppresses anti-D antibodies in Rh-negative mothers delivering Rh-positive infants.

Immunodeficiency with Thymoma Patients with immunodeficiency with thymoma are adults who almost simultaneously develop recurrent infections, panhypogammaglobulinemia, deficits in cell42

mediated immunity, and benign thymoma.[ ] They may also have eosinophilia or eosinopenia, aregenerative or hemolytic anemia, agranulocytosis, thrombocytopenia, or pancytopenia. Antibody formation is poor, and progressive lymphopenia develops, although percentages of Ig-bearing B lymphocytes are usually normal. The thymomas are predominantly of the spindle cell variety, although other types of benign and malignant thymic tumors have also been seen. Severe Combined Immunodeficiency of Unknown Molecular Cause In addition to the patients with SCID for whom the molecular bases are known, 15% to 20% have SCID for which the underlying cause has not yet been identified. [119]

Many other potential candidate genes exist for these unknown types of SCID.[

144]

Severe Combined Immunodeficiency with Leukopenia: Reticular Dysgenesis In 1959, identical-twin male infants who exhibited a total lack of both lymphocytes and granulocytes in their peripheral blood and bone marrow were described. Seven of eight infants reported with this defect died between 3 and 119 days of age from overwhelming infections; the eighth underwent complete immunologic 42]

reconstitution from a bone marrow transplant.[

Recombinant granulocyte colony-stimulating factor (G-CSF) was not successful in the treatment of an infant with

298 condition.[ ]

this The presence of mature normal-appearing granulocytes (although considerably reduced in number) noted in three patients and a normal percentage of T cells in the cord blood of a fourth patient argued against a total failure of stem cell differentiation in this defect. Despite the normal percentage of T cells in the latter patient's cord blood, however, the cells failed to give an in vitro proliferative response to mitogens. The thymus glands of all have weighed less than 1•g, no Hassall's corpuscles have been present, and few or no thymocytes have been seen. An autosomal recessive mode of inheritance seems most likely, but the molecular basis of reticular dysgenesis is not yet known. Combined Immunodeficiency of Unknown Molecular Cause A number of patients with CID have molecular defects as yet undefined. They also present during infancy with recurrent or chronic pulmonary infections, failure to 42]

thrive, oral or cutaneous candidiasis, chronic diarrhea, recurrent skin infections, gram-negative sepsis, urinary tract infections, or severe varicella. [ Immunologic Defects

Serum immunoglobulins may be normal or elevated for all classes, but A Def, pronounced elevation of IgE, and elevated IgD levels have been found in some of these CID patients. Although impaired in most patients, antibody-forming capacity has not been absent. Moreover, plasma cells are usually abundant in the lamina propria and lymph nodes. Other findings include neutropenia and eosinophilia. Studies of cellular immune function have shown skin test anergy to ubiquitous antigens; lymphopenia; and extremely low but not absent lymphocyte proliferative responses to mitogens, antigens, and allogeneic cells in vitro. Peripheral lymphoid tissues demonstrate paracortical lymphocyte depletion. The thymuses are very small and have a paucity of thymocytes and usually no Hassall's corpuscles; however,

in contrast to patients with acquired immunodeficiency syndrome (AIDS), thymic epithelium is intact in these CID patients.

1034

Pathogenesis

An autosomal recessive pattern of inheritance is usually seen in patients with these forms of CID. The molecular basis is unknown. Treatment and Prognosis

Patients with CID of unknown molecular cause usually survive longer than infants with SCID do, but they fail to thrive and die early in life. Patients with CID have been successfully reconstituted by unfractionated matched sibling or unrelated bone marrow or cord blood transplants after chemoablation, but T cell–depleted haploidentical marrow stem cell transplants have not been very successful because of resistance to engraftment. Defective Cytokine (Interleukin) Production Two main types of defects in cytokine production are known. The first is a selective inability to produce IL-2. In the two reported cases, patients had severe recurrent infections in infancy.[

299] [300]

The IL-2 gene was present in both, but no IL-2 message or protein were produced. Other T cell cytokines were produced normally. 301

The second type was seen in a single patient who also presented during infancy with severe recurrent infections and failure to thrive.[ ] She had defective transcription of several lymphokine genes, including IL-2, IL-3, IL-4, and IL-5, possibly attributable to abnormal binding of nuclear factor of activated T cells (NFAT) to response elements in IL-2 and IL-4 enhancers.[

301]

The molecular defects have not been identified in either type of cytokine deficiency.

Recently, two male infants born to consanguineous parents had SCID-like infection susceptibility despite phenotypically normal blood lymphocytes. However, their T cells were unable to produce IL-2, IFN-γ, IL-4, and TNF-α. Electrophoretic mobility shift assays were used to examine the DNA binding of NFAT, and the 302

binding of NFAT to its IL-2 promoter response element was barely detectable before and after T cell stimulation.[ ] The findings indicate that the NFAT/DNA binding defect is responsible for the multiple cytokine deficiency in these two boys. (See Chapter 10 for more information about cytokines.) Hyperimmunoglobulinemia E Syndrome The hyperimmunoglobulinemia E (hyper-IgE) syndrome is a relatively rare primary immunodeficiency syndrome characterized by recurrent severe staphylococcal abscesses of the skin, lungs, and viscera and greatly elevated levels of serum IgE [ 303

303] [304]

( Figure 59-7 ). The disorder was first reported by us in two young boys in 304

1972[ ] ; since then I have evaluated more than 40 patients with the condition, and many other examples have been reported.[ ] These patients all have histories of staphylococcal abscesses involving the skin, lungs, joints, and other sites from infancy; persistent pneumatoceles develop as a result of their recurrent pneumonias

( Figure 59-8 ). The pruritic dermatitis that occurs is not typical atopic eczema and does not always persist; respiratory allergic symptoms are usually absent. I noted coarse facial features in the first two patients,[ very different

303]

which has been a consistent feature of all the patients whom I evaluated. Hyper-IgE syndrome patients look

Figure 59-7 Patient with hyperimmunoglobulinemia E (hyper-IgE) syndrome showing multiple abscesses of face and neck and coarse facial features. From Buckley RH, Wray BB, Belmaker EZ: Pediatrics 49:59, 1972.

From Buckley RH, Wray BB, Belmaker EZ: Pediatrics 49:59, 1972.

Figure 59-8 Chest radiograph of 12-year-old boy with hyper-IgE syndrome. Giant pneumatoceles were present for more than 1 year. A putrid abscess caused by Enterobacter cloacae led to chest tube insertion on the right. Left cyst required emergency excision because of massive hemoptysis and was found to contain an aspergilloma.

Figure 59-9 Kaplan-Meier survival curve for 35 consecutive infants with severe combined immunodeficiency disease (SCID) who received marrow transplants at Duke University Medical Center from HLA-identical (3) or haploidentical (32) donors before they were 3.5 months of age, without pretransplantation chemoablation and without posttransplantation graft-versus-host disease prophylaxis. Thirty-four (97%) survived for periods of 1 month up to 20.2 years after transplantation. The one death occurred from a cytomegalovirus infection.

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233. Kishimoto TK, Springer TA: Human leukocyte adhesion deficiency: molecular basis for a defective immune response to infections of the skin, Curr Probl Dermatol 18:106, 1989. 234. D'Agata ID, Paradis K, Chad Z, et al: Leukocyte adhesion deficiency presenting as a chronic ileocolitis, Gut 39:605, 1996. 235. Rivera-Matos IR, Rakita RM, Mariscalco MM, et al: Leukocyte adhesion deficiency mimicking Hirschprung disease, J Pediatr 127:755, 1995. 236. Fischer A: Gene therapy of lymphoid primary immunodeficiencies, Curr Opin Pediatr 12:557, 2000. 237. Etzioni A, Tonetti M: Leukocyte adhesion deficiency II: from A to almost Z, Immunol Rev 178:138, 2000. 238. Etzioni A, Frydman M, Pollack S, et al: Brief report: recurrent severe infections caused by a novel leukocyte adhesion deficiency, N Engl J Med 327:1789, 1992. 239. Sturla L, Puglielli L, Tonetti M, et al: Impairment of the Golgi GDP-L-fucose transport and unresponsiveness to fucose replacement therapy in LAD-II patients, Pediatr Res 49:537, 2001. 240. Lubke T, Marquardt T, Etzioni A, et al: Complementation cloning identifies CDG-IIc, a new type of congenital disorders of glycosylation, as a GDP-fucose transporter deficiency, Nat Genet 28:73, 2001. 241. Vazquez N, Lehrnbecher T, Chen R, et al: Mutational analysis of patients with p47-phox-deficient chronic granulomatous disease: the significance of recombination events between the p47-phox gene (NCF1) and its highly homologous pseudogenes, Exp Hematol 29:234, 2001. 242. Stolz W, Graubner U, Gerstmeier J, et al: Chédiak syndrome: approaches to diagnosis and treatment, Curr Probl Dermatol 18:93, 1989. 243. Zhao H, Boissy YL, Abdel-Malek Z, et al: On the analysis of the pathophysiology of Chédiak-Higashi syndrome: defects expressed by cultured melanocytes, Lab Invest 71:25, 1994. 244. Holcombe RF, Jones KL, Stewart RM: Lysosomal enzyme activities in Chédiak-Higashi syndrome: evaluation of lymphoblastoid cell lines and review of the literature, Immunodeficiency 5:131, 1994. 245. Merino F, Esparza B, Sabino E: Chédiak-Higashi syndrome natural killer cells: a protein kinase C defective activation/regulation defect? Eur J Pediatr 155:254, 1996. 246. Baetz K, Isaaz S, Griffiths GM: Loss of cytotoxic T lymphocyte function in Chédiak-Higashi syndrome arises from a secretory defect that prevents lytic granule exocytosis, J Immunol 154:6122, 1995. 247. Nagle DL, Karim MA, Woolf EA, et al: Identification and mutation analysis of the complete gene for Chédiak-Higashi syndrome, Nat Genet 14:307, 1996. 248. Barrat FJ, Auloge L, Pastural E, et al: Genetic and physical mapping of the Chédiak-Higashi syndrome on chromosome 1q42–43, Am J Hum Genet 59:625, 1996. 249. Certain S, Barrat F, Pastural E, et al: Protein truncation test of LYST reveals heterogenous mutations in patients with Chédiak-Higashi syndrome, Blood 95:979,

2000. 250. Perou CM, Moore KJ, Nagle DL, et al: Identification of the murine beige gene by YAC complementation and positional cloning, Nat Genet 13:303, 1996.

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251. Aslan Y, Erduran E, Gedik Y, et al: The role of high dose methylprednisolone and splenectomy in the accelerated phase of Chédiak-Higashi syndrome, Acta Haematol 96:105, 1996. 252. Haddad E, LeDeist F, Blanche S, et al: Treatment of Chédiak-Higashi syndrome by allogenic bone marrow transplantation: report of 10 cases, Blood 85:3328, 1995. Autosomal Defects with Unidentified Molecular Basis 253. Cunningham-Rundles C: Clinical and immunologic analyses of 103 patients with common variable immunodeficiency, J Clin Immunol 9:22, 1989. 254. John HA, Sullivan KE, Smith C, et al: Enterocolitis in infantile common variable immunodeficiency: a case report and review of the literature, Dig Dis Sci 41:621, 1996. 255. Washington K, Stenzel TT, Buckley RH, et al. Gastrointestinal pathology in patients with common variable immunodeficiency and X-linked agammaglobulinemia, Am J Surg Pathol 20:1240, 1996. 256. Spickett GP, Zhang JG, Green T, et al: Granulomatous disease in common variable immunodeficiency: effect on immunoglobulin replacement therapy and response to steroids and splenectomy, J Clin Pathol 49:431, 1996. 257. Cunningham-Rundles C, Siegal FP, Cunningham-Rundles S, et al: Incidence of cancer in 98 patients with common varied immunodeficiency, J Clin Immunol 7:294, 1987. 258. Zhang JG, Morgan L, Spickett GP: L-selectin in patients with common variable immunodeficiency (CVID): a comparative study with normal individuals, Clin Exp Immunol 104:275, 1996. 259. Kaneko H, Katagiri-Kawade M, Motoyoshi F, et al: Abnormal B cell response of protein kinase C in some common variable immunodeficiency, Exp Clin Immunogenet 13:36, 1996. 260. Nonoyama S, Farrington M, Ochs HM: Activated B cells from patients with common variable immunodeficiency proliferate and synthesize immunoglobulin, J Clin Invest 92:1281, 1993. 261. Punnonen J, Kainulainen L, Ruuskanen O, et al: IL-4 synergizes with IL-10 and anti-CD40 MoAbs to induce B cell differentiation in patients with common variable immunodeficiency, Scand J Immunol 45:203, 1997.

262. Farrant J, Spickett G, Matamoros N, et al: Study of B and T cell phenotypes in blood from patients with common variable immunodeficiency (CVID), Immunodeficiency 5:159, 1994. 263. Eisenstein EM, Jaffe JS, Strober W: Reduced interleukin 2 (IL 2) production in common variable immunodeficiency is due to a primary abnormality of CD4+ T cell differentiation, J Clin Immunol 13:247, 1993. 264. Funauchi M, Farrant J, Moreno C, et al: Defects in antigen-driven lymphocyte responses in common variable immunodeficiency (CVID) are due to a reduction in the number of antigen-specific CD4+ T cells, Clin Exp Immunol 101:82, 1995. 265. Spickett GP, Webster ADB, Farrant J: Cellular abnormalities in common variable immunodeficiency, Immunodef Rev 2:199, 1990. 266. Jaffe JS, Strober W, Sneller MC: Functional abnormalities of CD8+ T cells define a unique subset of patients with common variable immunodeficiency, Blood 82:192, 1993. 267. Fischer MB, Wolf HM, Hauber I, et al: Activation via the antigen receptor is impaired in T cells, but not in B cells from patients with common variable immunodeficiency, Eur J Immunol 26:231, 1996. 268. Farrington M, Grosmaire LS, Nonoyama S, et al: CD40 ligand expression is defective in a subset of patients with common variable immunodeficiency, Proc Natl Acad Sci USA 91:1099, 1994. 269. Baum CG, Chiorazzi N, Frankel S, et al: Conversion of systemic lupus erythematosus to common variable hypogammaglobulinemia, Am J Med 87:449, 1989. 270. Wright JJ, Birx DL, Wagner DK, et al: Normalization of antibody responsiveness in a patient with common variable hypogammaglobulinemia and HIV infection, N Engl J Med 317:1516, 1987. 271. Ashman RF, Schaffer FM, Kemp JD, et al: Genetic and immunologic analysis of a family containing five patients with common variable immune deficiency or selective IgA deficiency, J Clin Immunol 12:406, 1992. 272. Truedsson L, Baskin B, Pan Q, et al: Genetics of IgA deficiency, APMIS 103:833, 1995. 273. French MA, Dawkins RL: Central MHC genes, IgA deficiency and autoimmune diseases, Immunol Today 11:271, 1990. 274. Schaffer FM, Monteiro RC, Volanakis JE, et al: IgA deficiency, Immunodef Rev 3:15, 1991. 275. Howe HS, So AKL, Farrant J, et al: Common variable immunodeficiency is associated with polymorphic markers in the human major histocompatibility complex, Clin Exp Immunol 84:387, 1991. 276. Schroeder HW, Zhu Z, March RE, et al: Susceptibility locus for IgA deficiency and common variable immunodeficiency in the HLA-DR3, -B8, -A1 haplotypes, Mol Med 4:72, 1998. 277. Fiore M, Pera C, Delfino L, et al: DNA typing of DQ and DR alleles in IgA-deficient subjects, Eur J Immunogenet 22:403, 1995.

278. Burks AW, Sampson HA, Buckley RH: Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia, N Engl J Med 314:560, 1986. 279. Farber CM, Crusiaux A, Schandene L, et al: Tumor necrosis factor and intravenous gammaglobulins in common variable immunodeficiency, Clin Immunol Immunopathol 72:233, 1994. 280. Cunningham-Rundles C: Clinical and immunologic studies of common variable immunodeficiency, Curr Opin Pediatr 6:676, 1994. 281. Buckley RH: Clinical and immunologic features of selective IgA deficiency. In Bergsma D, Good RA, Finstad J, Paul NW, editors: Immunodeficiency in man and animals, Stamford, Conn, 1975, Sinauer Associates. 282. French MAH, Denis KA, Dawkins R, et al: Severity of infections in IgA deficiency: correlation with decreased serum antibodies to pneumococcal polysaccharides and decreased serum IgG2 and/or IgG4, Clin Exp Immunol 100:47, 1995. 283. Preud'Homme JL, Hanson LA: IgG subclass deficiency, Immunodef Rev 2:129, 1990. 284. Sandler SG, Trimble J, Mallory DM: Coexistent IgG2 and IgA deficiencies in blood donors, Transplantation 36:256, 1996. 285. Briere F, Bridon JM, Chevet D, et al: Interleukin 10 induces B lymphocytes from IgA-deficient patients to secrete IgA, J Clin Invest 94:97, 1994. 286. Koskinen S, Tolo H, Hirvonen M, et al: Long-term follow-up of anti-IgA antibodies in healthy IgA-deficient adults, J Clin Immunol 15:194, 1995. 287. Sandler SG, Mallory D, Malamut D, et al: IgA anaphylactic transfusion reactions, Transfus Med Rev 9:1, 1995. 288. Huntley CC, Robbins JB, Lyerly AD, et al: Characterization of precipitating antibodies to ruminant serum and milk proteins in humans with selective IgA deficiency, N Engl J Med 284:7, 1971. 289. Koskinen S: Long-term follow-up of health in blood donors with primary selective IgA deficiency, J Clin Immunol 16:165, 1996. 290. Espanol T, Catala M, Hernandez M, et al: Development of a common variable immunodeficiency in IgA-deficient patients, Clin Immunol Immunopathol 80:333, 1996. 291. Scott MG, Shackelford PG, Briles DE, et al: Human IgG subclasses and their relation to carbohydrate antigen immunocompetence, Diagn Clin Immunol 5:241, 1988. 292. Lefranc MP, Hammarstrom L, Smith CIE, et al: Gene deletions in the human immunoglobulin heavy chain constant region locus: molecular and immunological analysis, Immunol Rev 2:265, 1991. 293. Shackelford PG, Granoff DM, Madassery JV, et al: Clinical and immunologic characteristics of healthy children with subnormal serum concentrations of IgG2, Pediatr Res 27:16, 1990. 294. Nahm MH, Blaese RM, Crain MJ, et al: Patients with Wiskott-Aldrich syndrome have normal IgG2 levels, J Immunol 137:3484, 1986.

295. Ambrosino DM, Umetsu DT, Siber GR, et al: Selective defect in the antibody response to Haemophilus influenzae type b in children with recurrent infections and normal IgG subclass levels, J Allergy Clin Immunol 81:1175, 1988. 296. Shackelford PG, Granoff DM, Polmar SH, et al: Subnormal serum concentrations of IgG2 in children with frequent infections associated with varied patterns of immunologic dysfunction, J Pediatr 116:529, 1990. 297. Tiller TL, Buckley RH: Transient hypogammaglobulinemia of infancy: review of the literature, clinical and immunologic features of 11 new cases, and longterm follow-up, J Pediatr 92:347, 1978. 298. Bujan W, Ferster A, Azzi N, et al: Use of recombinant human granulocyte colony-stimulating factor in reticular dysgenesis, Br J Hematol 81:128, 1992. 299. Weinberg K, Parkman R: Severe combined immunodeficiency due to a specific defect in the production of interleukin-2, N Engl J Med 322:1718, 1990. 300. Disanto JP, Keever CA, Small TN, et al: Absence of interleukin-2 production in a severe combined immunodeficiency disease syndrome with T cells, J Exp Med 171:1697, 1990. 301. Castigli E, Pahwa R, Good RA, et al: Molecular basis of a multiple lymphokine deficiency in a patient with severe combined immunodeficiency, Proc Natl Acad Sci USA 90:4728, 1993. 302. Feske S, Muller JM, Graf D, et al: Severe combined immunodeficiency due to defective binding of the nuclear factor of activated T cells in T lymphocytes of two male siblings, Eur J Immunol 26:2119, 1996. 303. Buckley RH, Wray BB, Belmaker EZ: Extreme hyperimmunoglobulinemia E and undue susceptibility to infection, Pediatrics 49:59, 1972. 304. Buckley RH: The hyper-IgE syndrome, Clin Rev Allergy Immunol 20:139, 2001. 305. Grimbacher B, Holland SM, Gallin JI, et al: Hyper-IgE syndrome with recurrent infections: an autosomal dominant multisystem disorder, N Engl J Med 340:692, 1999. 306. Kirchner SG, Sivit CJ, Wright PF: Hyperimmunoglobulinemia E syndrome: association with osteoporosis and recurrent fractures, Radiology 156:362, 1985. 307. Claassen JL, Levine AD, Schiff SE, et al: Mononuclear cells from patients with the hyper-IgE syndrome produce little IgE when stimulated with recombinant interleukin-4 in vitro, J Allergy Clin Immunol 88:713, 1991. 308. Grimbacher B, Schaffer AA, Holland SM, et al: Genetic linkage of hyper-IgE syndrome to chromosome 4, Am J Hum Genet 65:735, 1999. 309. King CL, Gallin JI, Malech HL, et al: Regulation of immunoglobulin production in hyperimmunoglobulin E recurrent infection syndrome by interferon, Proc Natl Acad Sci USA 86:10085, 1989. Complement Component Deficiencies 310. Figueroa JE, Densen P: Infectious diseases associated with complement deficiencies, Clin Microbiol Rev 4:359, 1991.

311. Winkelstein JA, Sullivan KE, Colten HR: Genetically determined deficiencies of complement. In Scriver CR, Beaudet AL, Sly WS, Valle DL, editors: Metabolic basis of inherited disease, New York, 1995, McGraw-Hill. 312. Hartmann D, Fremeaux-Bacchi V, Weiss L, et al: Combined heterozygous deficiency of the classical complement pathway proteins C2 and C4, J Clin Immunol 17:176, 1997. 313. Frank MM: Complement in disease: inherited and acquired complement deficiencies. In Frank MM, Austen KF, Claman HN, Unanue ER, editors: Samter's immunologic diseases, Boston, 1994, Little, Brown. 314. Puck JM: Prenatal diagnosis and genetic analysis of X-linked immunodeficiency disorders, Pediatr Res 33:S29, 1993. 315. Puck JM, Willard HF: X inactivation in females with X-linked disease, N Engl J Med 338:325, 1998.

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Chapter 60 - Approach to the Patient with Recurrent Infections

Mark Ballow Kathleen M. O'Neil

Immune deficiency diseases are relatively uncommon in the general population, but an appreciation by physicians of the importance of host defenses has increased over the past several decades because of the human immunodeficiency virus (HIV) epidemic. The evaluation of a patient with frequent infections requires a careful history and physical examination directed toward clues that could help categorize the nature of the patient's underlying defect. This discussion initially focuses on the basic principles of function and dysfunction of the human immune system as related to signs and symptoms in immunodeficiency diseases.

MEDICAL HISTORY The approach to the patient with recurrent infection begins with the medical history, which comprises several critical elements ( Table 60-1 ). Age of Onset

In general, the earlier the age of onset of infections, the more severe is the underlying immunodeficiency. For example, patients with severe combined immunodeficiency disease (SCID) who lack function of both cell-mediated and humoral immunity, typically have onset of infection by 4 to 5 months of age. In contrast, patients with B cell deficiencies, even those with the total inability to form antibody, as in Bruton's agammaglobulinemia, are usually well until 7 to 9 months of age. It is at this time that the placentally derived maternal immunoglobulin G (IgG) decreases to below protective levels in the infant, resulting in increased susceptibility to infection.[

1]

Sites of Infection The sites of infection are important in determining the significance of the patient's recurrent infections. Otitis media, sinusitis, pneumonia, gingivitis, meningitis, septicemia, skin infections, and abscesses are all sites of infection often associated with immunodeficiency. On the other hand, recurrent pharyngitis is not typically a serious or significant site of infection. The sites of infection in a patient may also provide insights into the pathophysiology of the immunologic defect. For example, patients with persistent or recurrent gingivitis may have a phagocytic defect or neutropenia. Skin infections may also occur in patients with phagocytic abnormalities and those with B cell deficiency. Recurrent septicemia suggests an opsonic defect: either an inability to generate specific IgG antibody or a lack of the nonspecific opsonins in the late classic or alternative pathway complement components. Delayed separation of the umbilical cord beyond 6 to 8 weeks of age in neonates can suggest a leukocyte adhesion deficiency. The physician must remember that urachal anomalies can also present as delayed umbilical separation.[

2]

Microbiology of the Infections The type of organism responsible for the patient's infections often yields important information about the nature of the defect in host defense. The microorganisms responsible for infection in patients with neutrophil defects, T cell abnormalities, and antibody deficiency disorders are frequently quite distinct. For example, recurrent viral, fungal, mycobacterial, or protozoal infections suggest T cell defects. The resulting immunodeficiency is clearly seen in patients with acquired immunodeficiency syndrome (AIDS) who have recurrent or persistent candidiasis and frequently die of pneumonia as a result of cytomegalovirus or Pneumocystis carinii. In contrast, repeated infections with encapsulated invasive bacteria, such as Streptococcus pneumoniae and Haemophilus influenzae type B, suggest an antibody deficiency disorder. Patients with combined T cell and B cell deficiencies may have problems with all types of microbial agents, including bacteria, fungi, protozoa, and viral agents. Opportunistic infections can often be seen in many of the primary immunodeficiencies. Patients with immunoglobulin A (IgA) deficiency or common variable hypogammaglobulinemia frequently have protracted gastrointestinal (GI) symptoms as a result of Giardia lamblia. Infections with P. carinii, Mycobacterium aviumintracellulare, and other opportunistic organisms suggest a T cell deficiency. Patients with X-linked hypogammaglobulinemia (Bruton's disease) have an increased susceptibility to infection with enteroviruses (echovirus, coxsackievirus), which can lead to meningoencephalitis. Arthritis of the large joints can be caused by Ureaplasma urealyticum. In contrast, recurrent infection with bacteria of low virulence suggests a phagocytic abnormality. For example, the patient with recurrent Staphylococcus aureus skin infections may have an abnormality in neutrophil function. Lymphadenitis or abscesses from which such unusual

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TABLE 60-1 -- Medical History in Patients with Recurrent Infection Factor

Association with Infection

Age at Onset 4–5 months

Combined T/B cell deficiency

7–9 months

B cell deficiency

Site of Infection Otitis media

B cell deficiency

Sinusitis

B cell deficiency

Pneumonia

B cell deficiency

Meningitis

B cell deficiency

Gingivitis

Neutrophil/phagocyte defects

Skin infections

Neutrophil/phagocyte defects

Organ abscesses

Neutrophil/phagocyte defects

Microbiology of Infection Viruses (herpes, varicella, CMV)

T cell deficiency

Fungi Candida

T cell deficiency

Aspergillus

T cell deficiency or phagocyte defects

Parasites Giardia lamblia

B cell deficiency

Pneumocystis carinii

T cell deficiency

Toxoplasma gondii

T cell deficiency

Bacteria

Mycobacteria

T cell deficiency

Encapsulated organisms

B cell or complement deficiency

Low-virulence organisms

Neutrophil/phagocyte defects

Gastrointestinal Disturbances Malabsorption, diarrhea

T cell or B cell deficiency

Lactose intolerance

B cell deficiency

Celiac disease

B cell deficiency

Family History X linked

T cell or B cell deficiency

Autosomal recessive

T cell or B cell deficiency

Adverse Vaccine or Transfusion Reaction Paralytic polio from OPV

B cell or combined T/B cell deficiency

Transfusion reaction

B cell (IgA) or phagocyte (CGD) deficiency

CMV, Cytomegalovirus; OPV, oral poliovirus vaccine; IgA, immunoglobulin A; CGD, chronic granulomatous disease. pathogens as Escherichia coli, Serratia, or Klebsiella are isolated may also suggest a neutrophil or phagocytic abnormality. Aspergillus or other fungal organisms can also cause infection in patients with phagocytic abnormalities. Recurrent Neisseria infection is a hallmark presentation for the congenital complement deficiencies affecting the late complement components (C5, C6, C7, C8). Atypical mycobacterial infection or infection with other intracellular pathogens may suggest a defect in 3] [4] [5]

T helper type 1 (Th1) cytokine signaling, in the form of either cytokine production defects or receptor defects.[ Gastrointestinal Disturbances

Many patients with primary immunodeficiency disease have symptoms and clinical findings referable to the GI tract. In a survey of 248 patients with common 6

variable immunodeficiency disease (CVID), 21% had significant GI disease. [ ] Liver disease occurred in an additional 12%. Bacterial overgrowth of the small bowel, including infections with Yersinia and Campylobacter, parasitic infestations with such organisms as G. lamblia, and chronic viral enteritis caused by enteroviruses and cytomegalovirus (CMV) are relatively common in patients with B cell or T cell defects. The incidence of lactose intolerance is higher in patients 7

with immunodeficiency than in the normal population. [ ] Patients with the X-linked syndrome of immune dysregulation, polyendocrinopathy, and enteropathy 8] [9]

(IPEX) have protracted diarrhea.[

Family History Many of the immunodeficiency diseases are inherited either as an autosomal recessive or an X-linked disorder. Therefore a careful family history is important. For example, chronic granulomatous disease (CGD), a neutrophil immune defect, is inherited as an X-linked disorder in approximately two thirds of patients. WiskottAldrich syndrome, infantile X-linked agammaglobulinemia or Bruton's disease, and hypogammaglobulinemia with hyper-immunoglobulin M syndrome (hyper-IgM) are other examples of X-linked disorders. Consanguinity raises the possibility of an autosomal recessive disorder. CVID and IgA deficiency are familial and are often seen in a setting of other family members with autoimmune disorders, such as pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus (SLE), or autoimmune hematologic diseases. In a family in whom the mother's brother died of recurrent infection in early childhood, the first serious bacterial infection in her son should cause the astute clinician to suspect the possibility of an X-linked immunodeficiency in the boy. Adverse Reactions to Vaccines and Transfusions An adverse vaccine or transfusion reaction may indicate an underlying immunodeficiency. For example, paralytic polio occurs in patients with B cell deficiency and SCID who received live attenuated oral poliovirus vaccine.[

10]

Paralytic disease has also been reported in immunodeficient persons exposed to children who have just

received the oral immunization and who are still actively shedding the poliovirus in their stools.[

10]

Disseminated mycobacterial disease after bacille Calmette-Guérin

(BCG) immunization can be seen in interferon-gamma (IFN-γ)–associated and interleukin-12 (IL-12)–related immune deficiencies.[ reactions can occur in patients with IgA deficiency because of the presence of IgE antibodies to IgA.

4] [11]

Anaphylactic transfusion

[12]

PHYSICAL EXAMINATION As with the medical history, the physical examination may give the clinician important clues to the cause of the patient's

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defect in host defense that underlies the recurrent infections. Repeated pyogenic infections may leave permanent scars. In addition, physical findings may be seen that represent consequences of serious insults to internal organs. Digital clubbing or a loud pulmonic heart sound with a right ventricular heave indicates pulmonary hypertension, which implies that serious pulmonary damage has occurred. Examination reveals physical signs that reflect not only the patient's previous infectious history but also a number of immunodeficiency syndromes associated with certain physical abnormalities and dysmorphisms. The physical examination of the patient with suspected immunodeficiency is discussed in a system-by-system manner ( Table 60-2 ). Growth Parameters

An early onset of recurrent infections, that is, during the first 6 months of life, is frequently accompanied by growth failure and delayed maturation. As in other disorders occurring early in life, such as congenital cardiac or renal disease, severe immunodeficiency will affect growth and development. Normal, or near-normal, height and weight, however, do not rule out the presence of a significant defect in host defense. Dysmorphisms Developmental embryologic abnormalities of the thymus result in atresia or dysplasia and consequently a T cell defect. This immunodeficiency often gives rise to defects in other structures formed from the third and fourth branchial pouches, including the parathyroid glands, the mandible and related structures, and the great blood vessels and the heart, resulting in the syndrome known as DiGeorge anomaly. Neonatal tetany, congenital heart disease, and facial abnormalities, including hypoplastic mandible, small mouth, and low-set posteriorly rotated ears, are frequently recognized before the immunodeficiency manifests. The disease is variable in its expression, however, and patients may have profound T cell deficiency without significant hypoparathyroidism or cardiac anomaly, usually presenting with severe, persistent candidiasis and growth failure.[

13]

Similarly, abnormalities of the bone marrow, where lymphocytes are produced, may result from defects of the connective tissue. Such an abnormality is likely 14

responsible for the defects in bone formation and the immune abnormalities in the syndrome of cartilage-hair hypoplasia (or syndrome, CHH).[ ] Because of the physical findings of short-limbed dwarfism and abnormal hair, children with CHH can be identified early in infancy on the basis of their appearance, before the clinical manifestation of their immune defects. Skin and Mucous Membranes The skin and oral mucosa can give valuable clues to the diagnosis of the underlying disease process. For example, patients with Wiskott-Aldrich syndrome present with recurrent infections, intractable eczema, and petechiae. Candidiasis of the skin or mucus membranes, as mentioned previously, may be an important indication of T cell deficiency. Patients with ataxia-telangiectasia have recurrent infections, cerebellar ataxia, and telangiectasia of the skin, which are easy to identify on physical examination. These TABLE 60-2 -- Physical Examination in Patients with Recurrent Infection Physical Finding/Diagnosis

Association with Infection

Growth failure

Combined T/B cell deficiency

Skin and Oral Mucosa Eczema

Wiskott-Aldrich syndrome, hyper-IgE

Petechiae

Wiskott-Aldrich syndrome

Pyoderma, abscesses

Neutrophil or B cell defects

Candidiasis

T cell or combined T/B cell deficiency

Telangiectasia

Ataxia-telangiectasia

Delayed umbilical separation

Neutrophil adhesion defect

Ear, Nose, and Throat Chronic otitis media: dull tympanic membranes; poor light reflex, scarring, tympanic perforations

B cell deficiency Mannose-binding lectin deficiency

Sinusitis: purulent nasal discharge, purulent postpharyngeal exudate, pharyngeal “cobblestoning”

B cell deficiency

Respiratory Tract Digital clubbing, rales

Defect in any immune component

Wheezing

B cell (IgA) deficiency

Lymphatic System Absent tonsils/nodes

Bruton's disease, combined T/B cell deficiency

Diffuse lymphoid hyperplasia

CVID, CGD, HIV infection

Musculoskeletal System Arthralgia

B cell deficiency

Dermatomyositis

B cell or complement deficiency

Lupuslike syndrome

Complement or IgA deficiency

Neurologic System Ataxia

Ataxia-telangiectasia

Enterovirus encephalitis

B cell deficiency (Bruxton's disease, XLA)

Dysmorphisms Micrognathia, short philtrum, ear anomalies

T cell deficiency DiGeorge anomaly

Short-limbed dwarfism

T cell deficiency Cartilage-hair hypoplasia

IgE, Immunoglobulin E; IgA, immunoglobulin A; CVID, common variable immunodeficiency; CGD, chronic granulomatous disease; HIV, human immunodeficiency virus; XLA, X-linked agammaglobulinemia.

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small vessel abnormalities over the bulbar conjunctivae, bridge of the nose, the ears, and antecubital fossa tend to occur in late childhood, usually several years after the onset of ataxia and infectious problems.[

15]

Intractable seborrhea is often seen in children with immunodeficiency, and a form of skin disease resembling severe

atopic dermatitis is common in the hyperimmunoglobulinemia E syndrome.[

16]

Boys with X-linked agammaglobulinemia (XLA) can develop a dermatomyositis-like rash with livedo reticularis, muscle weakness, neurologic symptoms, developmental failure or regression, and hepatitis resulting from chronic infection with an enterovirus. [

17]

A lupuslike rash with negative or low antinuclear antibody

[18]

(ANA) titers may occur in deficiencies of the early components of the classic complement pathway. Discoid lupus erythematosus, or less often SLE, has been seen in mothers of boys with CGD. Erythroderma in a patient with failure to thrive, eosinophilia, hepatosplenomegaly, and recurrent infections may suggest Omenn's syndrome.[

19]

Silvery hair, pale skin, and photophobia are seen in children with Chédiak-Higashi syndrome.

Candidiasis of the skin or mucous membranes, as noted earlier, may be an important indication of T cell deficiency. Some patients appear to have a selective susceptibility to Candida and present with chronic mucocutaneous candidasis. The major morbidity in these patients is the autoimmune endocrinopathies. A subgroup of these patients also have ectodermal dyplasia, including alopecia, vitiligo, dental and nail dystrophy, and keratopathy in a syndrome called autoimmune polyendocrinopathy and candidasis (APEC).[

20]

Ear, Nose, Mouth, and Throat Recurrent otitis media is a common problem in pediatrics. This infection is also common in persons with antibody deficiency. In studies of children with recurrent otitis media, however, this complaint does not prove to be a reliable indicator of immune deficiency. Increased exposure to viral and bacterial pathogens because the child is in a day care facility or lives in a home with other school-age children or with parents who smoke may contribute to the risk of recurrent otitis media. Examination of the nose and pharynx for the clinical signs of sinusitis is important. Children and adults with antibody deficiency frequently suffer from recurrent infection of the paranasal sinuses. In children the clinical diagnosis of sinusitis is frequently overlooked. As already noted, however, pharyngitis is not a marker for immune defects. The absence of tonsillar tissue is a significant physical finding, particularly in the child with recurrent upper respiratory infection, and should raise the suspicion of Bruton's XLA.[

17]

Chronic periodontitis is seen in patients with neutrophil chemotactic abnormalities. Severe gingivostomatitis, often with dental

erosions, occurs in patients with leukocyte adhesion defects, such as leukocyte adhesion deficiency type 1 (LAD-1). Pulmonary System The examination of the chest is important in the evaluation of the patient with recurrent infections and should include careful auscultation for rales or rhonchi. Not all patients who wheeze have simple asthma. Other conditions that cause wheezing include cystic fibrosis and chronic bronchitis or bronchiectasis related to underlying immunodeficiency. The presence of rales may indicate bronchiectasis or acute pneumonia, suggesting a diagnosis of immune deficiency. Digital clubbing is an important indicator of significant lung disease necessitating a careful workup because it is rarely, if ever, seen in uncomplicated asthma. Cardiovascular System The cardiovascular examination may provide an indication of DiGeorge anomaly. Conotruncal cardiac defects such as tetralogy of Fallot, micrognathia, and ear anomalies may be associated with congenital absence of the thymus and hypoparathyroidism. Physical findings of pulmonary hypertension may be noted in patients with chronic lung disease resulting from repeated infections in the immunodeficient host. Lymphoreticular System The examination of the lymphatic system for hepatosplenomegaly and for the presence or absence of lymphoid tissue is an important aspect of the physical examination in a patient with suspected immunodeficiency. Patients with SCID or infantile XLA do not have palpable lymphoid tissue or visible tonsils. The presence of lymphoid tissue can be misleading, however; adult patients with common variable hypogammaglobulinemia may actually have enlarged lymphoid tissue 21

and even hepatosplenomegaly,[ ] because the reticuloendothelial system undergoes hyperplasia in the absence of opsonic antibody. Draining abscesses of the lymph nodes suggest a phagocyte defect. Two other interesting groups of patients present with perturbations of lymphoid proliferation. Autoimmune lymphoproliferative syndrome (ALPS, Canale-Smith syndrome) is associated with inherited mutations in the tumor necrosis factor TNFRSF6 gene, which encodes for CD95/Fas, and results in a defect in apoptosis. These patients usually present before age 5 with a chronic, nonmalignant lymphadenopathy and massive splenomegaly. ALPS patients also develop hematologic autoimmune diseases. X-linked lymphoproliferative disease is a disease that manifests as an unusual susceptibility to Epstein-Barr virus (EBV) infections. Clinical manifestations include a fulminant and often fatal infectious mononucleosis, a lymphoproliferative disease resulting in lymphoma, and the development of a dysgammaglobulinemia. Neurologic System As mentioned, abnormalities of the neuromuscular system may be the first indicator of ataxia-telangiectasia (AT). Affected patients usually present with a broad22

based gait and stumbling in the first or second year of life, and the cerebellar ataxia progresses with age.[ ] The serum alpha fetoprotein (AFP) is elevated in these children. The onset of overt immunodeficiency is variable in AT, usually occurring after the onset of neurologic disease but occasionally preceding it. Flaccid 10]

paralysis after poliomyelitis vaccination suggests combined immunodeficiency or antibody defects.[

Adults with CVID occasionally develop pernicious anemia,

which, if not diagnosed and treated promptly, may lead to neurologic defects (combined systems disease). Physical examination

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may reveal loss of vibratory sense in the lower extremities, poor finger coordination, a positive Babinski response, and other signs suggesting posterior and lateral column disease. Peripheral nerve findings and changes in mentation may occur in patients with more severe AT. Musculoskeletal System Patients with immune deficiency may have arthritis, either because of joint infection, or apparently aseptic inflammation. Children with antibody deficiency and occasionally those with deficiencies of the complement system are subject to an increased incidence of septic arthritis with pyogenic bacteria. Children and adults with deficiencies of the early classical complement pathway often present with arthritis, frequently in conjunction with dermal vasculitis, resulting in a lupuslike syndrome in a number of cases. Children with antibody deficiency diseases often have arthralgia until placed on adequate immunoglobulin therapy. This presentation is not typically a frank arthritis, however, and is not usually accompanied by physical findings of overt joint inflammation. As noted earlier, however, patients with XLA have an increased incidence of arthritis (25% to 35%) from infection with a mycoplasma organism (U. urealyticum). Patients with infantile XLA and other B 17

cell abnormalities may also present with dermatomyositis, arthralgia, or overt arthritis.[ ] Patients with CHH (short-limbed dwarfism with metaphyseal or spondyloepiphyseal dysplasia) and adenosine deaminase deficiency (rib end cupping and flaring) also have skeletal abnormalities.

DIAGNOSTIC AND EPIDEMIOLOGIC OVERVIEW As just detailed, elements of the history and physical examination will lead the physician evaluating a patient with recurrent infection to an impression that a defect may exist in opsonic function (antibody or complement), mucosal immunity (antibody), T cell function, phagocytic activity, or a broader defect in host defense (combined immunodeficiency). The task then is to identify which of the recognized syndromes the patient best fits and to confirm this with pertinent laboratory 23

studies. Conley et al,[ ] representing the Pan-American Group for Immunodeficiency (PAGID) and the European Society for Immunodeficiencies (ESID), developed diagnostic criteria for many of the primary immunodeficiency disorders. These criteria were established to help physicians formulate a diagnosis using simple, objective guidelines and definitions. Studies of the relative frequencies of the primary immunodeficiency disorders show that antibody deficiency disorders make up about 50% of the total, cellular defects about 10%, combined T and B cell deficiencies 20%, phagocytic defects about 18%, and complement deficiencies 2%.

INNATE IMMUNE DISORDERS: COMPLEMENT AND PHAGOCYTE DEFICIENCIES Complement Protein Deficiencies

This section briefly discusses the presentation of patients with deficiencies of complement proteins from the perspective of the evaluation of the patient with recurrent infection. Chapter 6 discusses the biology of the complement system and recognized complement deficiency states. In general, complement deficiencies present as (1) severe, recurrent or invasive infection with encapsulated bacteria (due to loss of the opsonic function of C3 fragments), (2) immune complex disease (because complement is important in the solubilization of immune complexes and their removal from the circulation), or (3) severe, recurrent or invasive infection due to Neisseria. Congenital, or primary, complement deficiencies are usually inherited as co-dominant traits, which means that the parents of the patients will have half-normal levels of the involved complement component, whereas the patient has a complete absence of the component in question. The complement deficiencies can be broadly divided into those of the early complement components—C1, C2, and C4 of the classical pathway; factors B, D, and P of the alternative pathway; and 24]

mannan-binding protein and its associated proteases (MASP-1, MASP-2) of the lectin pathway—and the late complement components—C5, C6, C7, C8, and C9.[ Persons deficient in C3 share characteristics of both early and late component deficiencies ( Table 60-3 ). 25

Patients lacking one of the early complement components often present with a lupuslike illness.[ ] They may have the typical features of SLE. Occasionally, however, complement deficient individuals may be serology negative; that is, anti-deoxyribonucleic acid (DNA) antibodies are absent and the ANA is present in low titer, if at all. Individuals with C2 deficiency may have true SLE or discoid lupus without significant systemic involvement.[

26]

However, significant renal disease

and systemic vasculitis can occur in patients lacking an early complement component, without other evidence of SLE or autoimmunity.[ deficiency may present as hemolytic-uremic

26]

Homozygous factor H

27 syndrome.[ ]

Deficiencies in the late complement components usually present with recurrent infections caused by Neisseria species, with invasive meningococcal or gonococcal 28

infection, such as recurrent meningococcal meningitis, gonococcal arthritis, or gonococcal septicemia.[ ] It is important to note, however, that patients with deficiencies of early complement components may present with recurrent or invasive neisserial infections as well. Likewise, deficiencies of late complement components may occasionally be associated with vasculitis or other lupuslike illnesses.[

26]

Less frequently, patients with late component

TABLE 60-3 -- Clinical Characteristics of Complement Deficiencies Deficient Component

Disease Association

Early classical pathway components: C1, C4, C2

Lupuslike illness Vasculitis

Late complement components: C5-C8

Recurrent or disseminated Neisseria infection

C3

Recurrent septicemia Vasculitis Glomerulonephritis

Mannose-binding lectin

Recurrent respiratory tract infections and otitis media in early infancy

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deficiencies have developed Raynaud syndrome, scleroderma, or dermatomyositis. Deficiencies of the third component of complement (C3) are associated with both recurrent infections and an increased incidence of lupuslike illness, including 29

glomerulonephritis.[ ] C3 is both an important opsonin and an enzyme crucial in activation of the membrane attack complex (MAC). Assembly of the MAC is required for complement-mediated bacteriolysis in both the classical and the alternative complement pathways. In addition, C3 plays an important role in the 30]

solubilization and clearance of immune complexes from the circulation.[ both recurrent bacterial infection and vasculitic complications.

Thus it is not surprising to find that absence of this pivotal complement protein leads to

The individual with severe or recurrent blood-borne infection caused by encapsulated bacteria (e.g., S. pneumoniae, H. influenzae), invasive meningococcal or gonococcal disease, or immune complex disease should be screened for complement deficiency by testing the total hemolytic complement activity (CH50 ), which tests for classic pathway activity, and if these are normal, alternative pathway function should be tested with the AH50 . Hemolytic activity is very sensitive to heat degradation, so samples must be handled in the cold and serum separated and frozen at −70° C as soon as possible. Abnormalities in CH50 or AH50 should be pursued by analysis of specific component concentrations in serum. Phagocytic Defects Patients with defects in phagocyte function experience repeated infections at locations where the body interfaces with the environment. A history of recurrent gingivitis, skin infections with furunculosis, abscesses of the viscera or perirectal tissues, and lymphadenitis should prompt evaluation of the phagocyte host defenses ( Box 60-1 ). Because of defective phagocyte function, the manifestations of infections may be blunted; erythema, swelling, and pus formation may be limited or absent. Phagocyte defects frequently present as infections caused by bacteria of relatively low virulence, such as S. aureus, fungi, and gram-negative enteric bacteria, including Klebsiella, E. coli, Burkholderia aeruginosa, and Serratia species. Infections often fail to respond optimally to usual courses of antimicrobial agents, and 31]

protracted courses are often required. Many patients also have a history of poor wound healing, reflecting the critical role of phagocytes in tissue repair.[ The evaluation of the patient with suspected phagocytic dysfunction requires an understanding of the steps involved in

Box 60-1. Clinical Presentation of Phagocytic Dysfunction Disease Infections with minimal or no pus Infections with formation of abscesses and granuloma Recurrent infections with bacteria of low virulence Skin infections, furunculosis, organ abscesses, lymphadenitis Poor wound healing Delayed separation of umbilical cord

normal antimicrobial activity of these cells. Normal function of the phagocyte compartment of host defense requires adequate numbers of neutrophils and monocytes, 31

as well as the normal performance of a number of closely integrated functions, to result in the effective killing of a pathogen by the leukocyte.[ ] Defects in neutrophil number (e.g., neutropenia, cyclic neutropenia, Kostmann's syndrome, Schwachman-Diamond syndrome), adherence, deformability, locomotion, chemotaxis, recognition of foreign particles and attachment, phagocytosis, oxidative respiratory metabolism, and intracellular microbial killing have all been reported ( Box 60-2 ). Data from the history and physical examination will help the clinician focus attention on which function is most likely to be defective. Absence of pus at the sites of infection, for example, suggests that the patient either has a decreased number of granulocytes, or that these cells have impaired ability to concentrate at the site of bacterial invasion, that is, defective chemotaxis or adhesion. A critical aspect of host defense is the accumulation of neutrophils at the site of infection. This

Box 60-2. Spectrum of Phagocyte Defects Defective Neutrophil Localization Disorders of neutrophil number Congenital neutropenias • Kostmann syndrome • Cyclic neutropenia • Schwachman-Diamond syndrome • Aplastic anemia Acquired neutropenias • Decreased phagocyte production: drug induced, infection, marrow infiltration • Increased phagocyte destruction: drug induced, autoimmune, neonatal neutropenia

Disorders of leukocyte movement C5 complement deficiency: inability to generate anaphylotoxins Circulating inhibitors of chemotaxis • Immune complex disease • Malignancy Abnormal chemotaxis • Chédiak-Higashi syndrome • Hyper-IgE syndrome • Down syndrome Abnormal leukocyte adhesion • CD18 deficiency • Fucosylation defects • Specific granule deficiency Defective Bactericidal Activity

Disorders of oxidative metabolism Chronic granulomatous disease Glucose-6-phosphate dehydrogenase deficiency Myeloperoxidase deficiency

Disorders of leukocyte granules Chédiak-Higashi syndrome Myeloperoxidase deficiency Specific granule deficiency

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process requires both the elaboration of chemotactic substances and a normal response by leukocytes to chemoattractants. Chemotactic substances important in vivo include complement cleavage fragments and the chemotactic peptides and lipids released by a variety of inflammatory cells, including mast cells in the allergic response. A history of persistent abscesses with exudates, on the other hand, suggests that the phagocytes can migrate to the appropriate site but are defective in bacterial killing. Leukocyte Movement Disorders Chédiak-Higashi Syndrome.

Chédiak-Higashi syndrome is an autosomal recessive disease characterized by partial oculocutaneous albinism and recurrent pyogenic infections, usually caused by 32

S. aureus and beta-hemolytic streptococci. [ ] Many patients develop a lymphoproliferative disorder that results in death at an early age. Mild neutropenia and variable platelet dysfunction with easy bruising may be seen, but immunoglobulin and antibody production is usually normal. The leukocytes of these patients contain giant lysosomal granules in the cytoplasm that do not degranulate normally during phagocytosis.[ of primary and secondary granules. [

34]

33]

These giant granules are the result of an abnormal fusion

Some patients have a decreased number of centriole-associated microtubules.[

35]

The granules do not orient themselves

normally during chemotaxis,[

36]

resulting in abnormal directed motility.[

37]

The diagnosis is made clinically and confirmed by the finding of the characteristic giant cytoplasmic granules on peripheral blood smear examination. The genetic 38]

defect in Chédiak-Higashi syndrome is mutation of the gene LYST, which codes for a cytoplasmic protein involved in vacuole formation and function. [ patients have benefited from therapy with 500 mg of ascorbic acid causes terminal illness.

39 daily.[ ]

Some

In many patients, tissue infiltration by nonmalignant CD8+ T cells and macrophages

[40]

Hyperimmunoglobulinemia E Syndrome.

Known as Job's syndrome, this syndrome was initially reported in two girls with elevated serum IgE, red hair, and fair skin having “cold” abscesses (see Chapter 59 ). In addition to exceedingly high serum levels of IgE, immunologic studies in these patients have revealed a variable defect in neutrophil chemotaxis.[ chemotactic defect may vary within an individual patient over time. Hyper-IgE has been mapped to a region on chromosome 4q in several autosomal dominant, with variable expressivity.

[44]

43 families.[ ]

42]

The

Inheritance is

A variety of treatment modalities have been tried in small numbers of patients, none with conclusive efficacy.[

45]

Leukocyte Adhesion Disorders

Advances in the biology of intercellular adhesion molecules (ICAMs) have led to a greater understanding of the complex processes involved in the homing of leukocytes to sites of inflammation and in their adhesion to and migration through endothelium at inflammatory foci. Leukocyte Adhesion Deficiency Type 1.

A major defect in the phagocyte response to infection that leads to impaired localization of phagocytes at sites of microbial invasion and impaired ingestion of opsonized bacteria is seen in LAD-1.[

46] [47] [48]

These patients present with severe bacterial infections, including pneumonia, otitis media, sinusitis, periodontitis, 49] [50]

and chronic skin infections, often with ulceration. [

Some patients also have delayed separation of the umbilical cord, occurring after 6 weeks of age. In 51

several, periumbilical abscesses have formed. Most patients with LAD-1 have myeloid hyperplasia with granulocytosis and splenomegaly[ ] and a history of poor wound healing. The most common pathogens identified are S. aureus and Pseudomonas aeruginosa. The molecular basis for this immunodeficiency disease is a deficiency in the leukocyte adherence glycoproteins CD11/CD18, also known as leukocyte function-associated antigen-1 (LFA-1), the receptor for intercellular 52]

adhesion molecule-1 (ICAM-1).[

Diagnosis of LAD-1 can be made by flow cytometry with the appropriate monoclonal antibody in most cases, although some patients have detectable but nonfunctional CD11/CD18 heterodimers on their phagocytes.[

53]

Chemotactic and adherence assays demonstrate functional defects in many of these patients.

Neutrophil phagocytosis and respiratory burst response to opsonized zymosan is greatly diminished.[ of deficient LFA-1.

[55]

54]

Natural killer (NK) cell function is reduced in vitro because

Treatment of LAD-1 patients includes antibacterial prophylaxis and aggressive treatment of identified infections. Marrow transplantation has been successful in 56

correcting the immunodeficiency in some patients.[ ] Morbidity and mortality related to transplant procedures, immune suppression of the chronically infected host, and graft-versus-host disease (GVHD) limit the success of this approach, particularly in older patients with significant infections. Successful retroviral-mediated CD18 gene transfer to peripheral blood CD34+ cells from a patient with LAD-1 deficiency suggests that gene therapy might be possible.[

57]

Leukocyte Adhesion Deficiency Type 2.

A new variant of LAD that was inherited as an autosomal recessive trait was described in which affected individuals had severe mental retardation, short stature and 58

recurrent periodontitis, cellulitis without pus, pneumonia, and otitis media.[ ] These children had the Bombay (hh) blood phenotype (absence of sialyl-Lewisx , a component of the core carbohydrate of ABO blood group antigens and the ligand for E-selectin). Sialyl-Lewisx mediates initial adhesion and rolling of neutrophils on activated endothelium (expressing E-selectin) and triggers expression of the high-affinity adhesion molecules, LFA-1 and macrophage antigen-1 (Mac-1). Thus neutrophils of patients with LAD-2 cannot adhere or roll on activated endothelium and lack a major mechanism for the up-regulation of LFA-1 and Mac-1. The defect in LAD-2 appears to be decreased availability of guanosine diphosphate (GDP)-fucose in the lumen of the Golgi apparatus due to a mutation in the GDPfucose transporter gene.[

59]

Distinct mutations in the transporter gene have been identified in Turkish and Arab children with LAD-2. Patients with the Turkish

variant responded to oral fucose therapy, with clinical improvement in infections and increased expression of fucosylated membrane proteins,[

60]

whereas the Arab

61 response.[ ]

children had no The mutated transporter in the Arab children had normal affinity for GDP-fucose but diminished transporter activity, explaining the lack of response to supplemental fucose. Intracellular Killing Disorders

After chemotaxis results in the localization of leukocytes at an inflammatory focus and the phagocytes have adhered to the offending microorganism and ingested it, neutrophils

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must then destroy the infectious agent within its phagosomes. This energy-dependent process involves fusion of phagosomes with lysosomes, proteolytic destruction within the phagolysosome, and death of the infectious agent, a process that depends on reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase. The result is the generation of superoxide anion and the formation of toxic halides that kill the bacteria. Several independent defects have been reported in the complicated process of intracellular bacterial killing. Chronic Granulomatous Disease.

The classic example of this group of diseases is CGD. Patients usually present in early childhood with pneumonia or lung abscesses, lymphadenitis, dermatitis, hepatosplenomegaly, sinusitis, perirectal abscesses, or deeper visceral abscesses. The spectrum of pathogens involved in the infectious complications is distinctive: Staphylococcus species, Enterobacteriaceae, Nocardia species, and fungi such as Candida albicans are responsible for the majority of infections.[ 63 cepacia[ ]

64 species,[ ]

and phytopathogenic Pseudomonas propensity to cause disease in patients with CGD.

62]

Burkholderia

which are rare pathogens in immunocompetent persons without cystic fibrosis, have a particular

In contrast, the common invasive bacterial pathogens of childhood, such as Streptococcus pneumoniae and Haemophilus influenzae, cause no greater morbidity in patients with CGD than in the normal population. These latter organisms generate their own peroxides and are catalaze negative. Consequently, S. pneumoniae and H. influenzae are unable to destroy their self-produced peroxide. The peroxide of bacterial origin then is able to assist in bacterial killing by the defective leukocytes. The microbes causing infection in CGD either are catalase positive or do not produce significant quantities of peroxide. This observation led to the recognition of the defect in CGD: an inability to generate superoxides and other toxic oxygen species, resulting in greatly impaired killing of ingested microorganisms by CGD phagocytes. Prolonged survival of ingested bacteria and fungi in the leukocytes of these patients leads to abscess and granuloma formation and to the concentration 65] [66]

of these pathogens in the spleen, liver, and lymph nodes. Granuloma can obstruct the urinary and GI tracts but respond to short courses of glucocorticoids.[

67]

Chronic colitis characterized histologically by eosinophila, eosinophilic crypt abscesses, and pigmented macrophages with or without granuloma may occur.[ with colitis is often unresponsive to antimicrobial treatment but may improve with antiinflammatory agents such as 5-aminosalicylic acid, sulfasalazine, and corticosteroid. The clinical spectrum has been reviewed recently in the report on a national registry of 368 patients with CGD[ biochemical, and genetic features.

62]

; Segal et al[

68]

CGD

review clinical,

The majority of patients with CGD are male, suggesting an X-linked inheritance in most forms of the disease. The recognition of variants of CGD with autosomal inheritance and the identification of kindreds with milder forms of the disorder suggest that the etiology of the defective intracellular killing involves a complex enzyme system.[

69]

All patients have in common an inability to develop the normal respiratory burst on activation, usually measured as an inability to reduce the dye 70

71

nitroblue tetrazolium (NBT)[ ] or by flow cytometry with dihydrorhodamine dye.[ ] Chemiluminescence and superoxide generation are used at some centers where these tests are available to demonstrate defects in respiratory burst activity. Quantitation of neutrophil bactericidal capacity may be used to confirm a defect in killing of Staphylococcus, but this is a laborious test and is not universally available. Despite the universal finding of defective superoxide generation, the genetic alterations underlying the defect in bactericidal activity are pleiotropic. The most common biochemical defects causing CGD involve the cytochrome b558, or NADPH oxidase, enzyme system ( Figure 60-1 ). Abnormal flavoproteins and 72] [73] [74]

severe glucose-6-phosphate dehydrogenase (G6PD) deficiency may also cause CGD. Many individual gene mutations causing CGD have been reported,[ 75 76

and murine knockout models for the several forms of CGD have been generated.[ ] [ ] Studies in these animal models have demonstrated partial correction of superoxide production and variable improvement in host defense against infection after retroviral transfer of normal copies of the defective gene, laying the groundwork for gene therapy trials in humans. In the most common form of CGD, X91, the 91-kD glycoprotein subunit is absent, leading secondarily to absence of membrane expression of the 22-kD subunit with which it associates and usually to a complete lack of the enzyme on cell membranes.[

77]

X91 CGD is inherited as an X-linked defect and accounts for 70% of all

cases CGD. Mothers of affected boys have abnormal NBT reduction, with approximately half their neutrophils capable of reducing the formazan dye. The heterozygous carriers of the X91 CGD gene may have a broad variation (10% to 90%) in the percentage of neutrophils with normal oxidative metabolism because of skewed lyonization. [

62]

Figure 60-1 Neutrophil NADPH oxidase. The NADPH oxidase system includes the 91-kD and 22-kD membrane components of cytochrome b558 and the cytoplasmic subunits p67phox , p47phox , and p40phox required for the enzyme's function. The cytoplasmic subunits associate noncovalently in the cytoplasm. Activation phosphorylates these subunits, as indicated by -P, allowing p47phox to form a complex with the membrane components, the p91phox -p22phox flavocytochrome. This translocation is facilitated by dissociation of Rac from its inhibitor, rhoGDI, and its redistribution to the plasma membrane.

Box 60-3. Laboratory Evaluation of Nonspecific Immune Defense Factors Absolute granulocyte count Cell morphology Serum total hemolytic complement (CH50 , alternative pathway CH50 ) Nitroblue tetrazolium test Flow cytometry using dihydrorhodamine dye Chemotaxis assay Myeloperoxidase Flow cytometry for leukocyte adhesion molecules (CD11b)

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T CELL IMMUNODEFICIENCY Congenital deficiencies of T lymphocyte function usually present early in infancy, often by 3 months of age. If the T cell defect is profound, the presentation will be at an early age; if it is less severe, the child may do well initially, with the occurrence of infection somewhat delayed. In some children, associated anomalies may suggest T lymphocyte deficiency before the onset of infection. For example, most children with DiGeorge anomaly are identified in the newborn period because of the presence of associated cardiac disease, facial anomalies, and hypocalcemic tetany. The immune deficiency may occur early or become overt years later. Profound defects in T lymphocyte function, or defects arresting development of T cells early in ontogeny, will affect not only cell-mediated immunity but also will impair the development of B lymphocyte function (humoral immunity) resulting from the absence of T cell help and T cell–derived cytokines. The clinical syndromes resulting from these more profound immune defects are referred to as combined immunodeficiency diseases. Children with significant T cell impairment tend to grow poorly and often have failure to thrive during their first years of life ( Box 60-4 ). Recurrent infections, including those resulting from organisms of relatively low virulence in a normal host (opportunistic infections), then ensue. An early problem in the patient with T cell deficiency is infection with Candida albicans. Other fungal infections, severe viral diseases, and infection with opportunistic pathogens such as Pneumocystis

carinii or Mycobacterium avium-intracellulare should prompt an evaluation for disorders in T cell function. Infants with severe T cell deficiency may present with GVHD, either after transfusion of lymphocyte-containing blood products or as a result of intrapartum or prenatal maternal-fetal transfusion. Erythroderma and severe seborrheic dermatitis may be seen in children with severe T cell and combined immunodeficiency diseases. In many cases, these skin manifestations lead to the diagnosis of immunodeficiency. Severe Combined Immunodeficiency Disease The severe combined immune deficiency diseases (SCIDs) are disorders of host defense in which there is significant impairment of both humoral and cellular immune function. Box 60-4. Clinical Characteristics of T Cell Deficiencies Onset of symptoms in early infancy (4–5 months) Recurrent infections with fungal, viral, and mycobacterial pathogens Infections caused by opportunistic organisms (e.g., Pneumocystis carinii) Failure to thrive, often fatal in childhood Fatal infections after live-virus vaccines or bacille Calmette-Guérin vaccination Graft-versus-host disease from transfusion of blood products Increased incidence of malignancy

Children with SCID tend to present in the first few months of life with failure to thrive, chronic diarrhea, seborrheic dermatitis or erythroderma, persistent mucocutaneous candidiasis, and severe bacterial infections. GVHD from maternal-fetal transfusion may occur. Pneumonia may be a significant problem early and may be caused by P. carinii, CMV, or measles virus. Chronic viral infections, especially those caused by enterovirus, can lead to slowly progressive encephalitis. Infection with vaccine strain polioviruses may be fatal. 90

Lymphopenia is present in about 90% of children with SCID, [ ] but a normal lymphocyte count does not exclude a combined immunodeficiency disease. In most patients with SCID, immunoglobulin quantitation reveals very low levels of all isotypes, with IgA and IgM nearly undetectable and IgG below 200 mg/dl. Rarely,

SCID patients may have elevated serum immunoglobulin isotypes. The diagnosis of pediatric AIDS must be excluded, and risk factors in the child and parents must be examined. HIV-specific antibody should be tested in the child's mother if the likely route of infection is perinatal transmission; this is especially true if the child is under 15 months of age. Polymerase chain reaction (PCR) for HIV genes and virus culture from peripheral blood can assist in the diagnosis of HIV in the young child. The response to mitogens and allogeneic cells in vitro is typically abnormal in patients with SCID but may not be universally depressed. The presence of some response to in vitro stimulation is not necessarily correlated with milder clinical disease. Proliferative responses to antigens are usually abnormal and are a more sensitive indication of impairment. Advanced testing of cytokine elaboration by stimulated lymphocytes, and studies of signal transduction may help to delineate the underlying defect. The majority of patients with SCID are male, and in SCID patients with normal purine salvage pathway enzyme levels and normal expression of MHC antigens, males outnumber females by a ratio of 4:1,[

91]

suggesting X-linked inheritance in a large proportion. Approximately 20% of children with SCID have deficiency in

adenosine deaminase or purine nucleoside phosphorylase, which are inherited as autosomal recessive traits.[

92]

The patient with bare lymphocyte syndrome, who has 93

a defect in the expression of MHC antigens, is also inherited in an autosomal recessive manner and accounts for an additional 2% to 5% of SCID cases.[ ] Despite the high proportion of affected males, relatively few (20% to 30%) have a family history of immune deficiency. In a study of 16 women without a family history of immunodeficiency disease who had a son with SCID, lymphocytes from 7 of the 16 demonstrated nonrandom X-inactivation, implying that the genetic defect 94

responsible for the SCID was located on the X chromosome in these families.[ ] The majority of boys with X-linked severe combined immunodeficiency disease (XSCID) had an elevated proportion of B lymphocytes in their peripheral blood; significantly fewer with autosomally transmitted SCID demonstrated high B lymphocyte numbers. The presence or absence of B lymphocytes in peripheral blood serves as a useful marker in the classification of SCIDs, as described next. Severe Combined Immunodeficiency Phenotypes The investigation of the child with presumed combined T cell/B cell deficiency begins with the quantitation of T, B, and

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NK lymphocyte numbers using flow cytometry. Based on the results of the flow cytometry, the phenotype of the immunodeficiency can be categorized as T cell positive (T+ ) or negative (T− ), B cell positive (B+ ) or negative (B− ), and NK cell positive (NK+ ) or negative (NK− ). These phenotypes can more easily be associated with a more limited number of genetic defects, and the further characterization of the child's immunodeficiency can be delineated accordingly. The characterized causes of human SCID are grouped according to the presence or absence of T, B, and NK lymphocytes ( Table 60-4 ). Approximately 70% of human SCID cases are T− B+ , and 20% to 30% are T− B− . T− B− NK− SCID

Reticular Dysgenesis.

95]

Reticular dysgenesis is an extremely rare condition characterized by deficiency in myeloid and lymphoid cell lines but intact erythrocyte production. [ for less than 2% of SCID in

TABLE 60-4 -- Characterized Causes of Severe Combined Immunodeficiency Disease (SCID) Disease

It accounts

96 humans.[ ] *

Serum Ig

B Cells

T Cells

NK Cells

Associated Features

Reticular dysgenesis

↓↓↓

↓↓↓

↓↓↓

↓↓↓

Granulocytopenia Thrombocytopenia

ADA deficiency

↓↓

Prog ↓

Prog ↓

Prog ↓

RAG-1/RAG-2 deficiency

↓↓↓

↓↓↓

↓↓↓

+

Omenn's syndrome

↓↓ ↑IgE

↓

↓

+

Exudative maculopapular erythema, pachyderma Chronic diarrhea Adenopathy Hepatosplenomegaly

Navajo SCID

↓↓↓

↓↓↓

↓↓↓

+

Recurrent orogenital ulcers

X-linked SCID

↓↓↓

↑↑

↓↓↓

↓↓↓

Jak-3 deficiency

↓↓↓

↑↑

↓↓↓

↓↓↓

PNP deficiency

+ to ↓

+

Prog ↓

Prog ↓

↓-↓↓↓

+

↓↓

+

T− B− NK− SCID

T− B− NK+ SCID

T− B+ NK− SCID

T− B+ NK+ SCID TCR/CD3

+

IL-7Rαdeficiency

↓to +

+ to ↑

Low serum uric acid

T± B+ NK+ SCID ZAP-70 deficiency

+

+

CD4+/ CD8↓↓

+

Palpable lymph nodes Normal thymus

Type 1 BLS (TAP-2 defect)

+

+

CD4+/ CD8↓↓

+

MHC class I deficiency Progressive susceptibility to infection

Type 2 BLS

+ to ↓↓

+

↓CD4 ↑CD8

+

Defect in one of several trans-activating genes: RFX5, RFXANK, RFXAP, others

↓↓↓, Extremely decreased; ↓↓ very decreased; Prog ↓, progressively decreased; +, ↑, increased; ↓, decreased; ↑↑, very increased. Ig, Immunoglobulin; NK, natural killer; ADA, adenosine deaminase; RAG, recombinase activating gene; Jak-3, Janus kinse 3; PNP, purine nucleoside phosphorylase; TCR, T cell antigen receptor; IL-7Rα, interleukin-7 alpha-chain receptor; ZAP-70, zeta-chain-associated protein 70; BLS, bare lymphocyte syndrome; TAP-2, transporter of antigen peptides type 2; MHC, major histocompatibility complex. * Autosomal recessive inheritance, except X-linked SCID.

The molecular basis of this disease is unknown. Although T, B, and NK cells are greatly reduced in number in the blood of affected individuals, the T cells can be activated by mitogens.[

97]

Several reports of successful treatment with BMT suggest that this is the current treatment of choice for reticular dysgenesis, but that 95]

conditioning may be required to ensure engraftment. [ Adenosine Deaminase Deficiency.

The most common cause of T− B− NK− SCID is deficiency of the enzyme adenosine deaminase (ADA). ADA deficiency accounts for about 15% of all SCID cases. [96]

These children usually have severe lymphopenia, often with fewer than 500 cells/mm3 in peripheral blood.[ 99 pathway.[ ]

98]

ADA catalyzes the irreversible deamination of

adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine in the purine salvage Deficiency of the enzyme leads to intracellular accumulation of deoxyadenosine, which is phosphorylated by a series of kinases to deoxy-adenosine triphosphate (ATP) ( Figure 60-2 ). Although

1054

Figure 60-2 Pathways of purine metabolism. In adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiencies the nucleotide degradation pathway is blocked. Adenosine and deoxyadenosine are generally formed from the degradation of nucleic acids and adenine nucleotides. These compounds are either deaminated (ADA) or phosphorylated. Deamination by ADA enzyme leads to inosine and eventually to the production of uric acid. In ADA deficiency the block in deamination of adenosine and deoxyadenosine leads to the accumulation of these nucleotides. Deoxyadenosine is preferentially phosphorylated (kinase), which leads to an increase in deoxy–adenosine triphosphate (dATP). The accumulation of dATP is lymphocytotoxic in which DNA synthesis is blocked through the inhibition of ribonucleotide reductase. In addition, the buildup of adenosine leads to the accumulation of S-adenosylhomocysteine (SAH). SAH is a potent inhibitor of methylation reactions important in DNA synthesis. Deoxyadenosine is also an important inhibitor of the enzyme SAH hydrolase. IMP, Inosine monophosphate. Similarly, the absence of PNP enzyme leads to the accumulation of deoxyguanosine and deoxyinosine and eventually to the accumulation of their respective triphosphate compounds, particularly, deoxy–guanosine triphosphate (dGTP). A similar mechanism of lymphocytotoxicity has been suggested for PNP deficiency as in ADA deficiency.

Figure 60-3 Signal transduction pathways in T lymphocytes associated with combined immunodeficiency disease. After the binding of antigen (Ag) associated with self-major histocompatibility complex (MHC) to the T cell antigen receptor (TCR), phosphorylation (-P) by the src-family kinase, p56lck of the CD3 ζ chain occurs. The phosphorylated CD3 ζ chain then binds to and activates the zeta-chain-associated protein 70 (ZAP-70) kinase, and subsequently, phospholipase C is activated. This activation in turn leads to release of intracellular calcium and the activation of protein kinase C. Binding of interleukin-2 to its receptor (IL-2R) leads to activation of the protein tyrosine kinase, p56lck and the Janus kinases Jak-1 and Jak-3. Binding of interleukin-7 to its receptor (IL-7R) leads to phosphorylation of Jak3, as does the binding of interleukin-12 to its receptor (IL-12R). Interferon-γ binding to its type 1 receptor (IFN-γR1) activates Jak-1, and IFN-γ binding to the IFN-

γR2 activates Jak-2. The Janus kinases then activate the signal transducer and activator of transcription (STAT) proteins. The STAT proteins in turn activate nuclear factor kappa B (NF-κB) essential modulator (NEMO, also known as IKK-γ) to phosphorylate I-κB, the inhibitor of NF-κB. The unbound NF-κB now is able to relocate to the nucleus and promote gene activation. Asterisks (*) denote deficiencies of proteins associated with combined immunodeficiency disease.

Box 60-5. Tests for Cellular or T Cell Immunity

Screening Tests Absolute lymphocyte count Chest radiograph for thymus shadow in newborns Delayed skin hypersensitivity to recall antigens Quantification of T cell subsets

Advanced Testing Lymphocyte proliferative responses to mitogens, antigens, and allogeneic cells (MLC) Lymphocyte-mediated cytotoxicity: NK and ADCC activity Production of cytokines Functional response to cytokines Signal transduction studies Molecular analysis for specific defects MLC, Mixed-lymphocyte count; NK, natural killer; ADCC, antibody-dependent cell-mediated cytotoxicity.

Evaluation

The finding of a total lymphocyte count below 1500/mm3 in the adult or less than 2500/mm3 in the child should be considered significant and raise the suspicion of T cell immunodeficiency ( Box 60-5 ). Delayed-type hypersensitivity (DTH) skin testing remains the major screening test of cellular immunity but must be interpreted with caution, especially in the young child, who may have had limited exposure to antigens, and the malnourished patient. In most infants with suspected cellular immunodeficiency, in vitro testing is required. More advanced testing of T cell function includes the analysis of lymphocyte subsets using flow cytometry and in vitro lymphocyte proliferation assays to mitogens (e.g., PHA, ConA), recall antigens (e.g., tetanus, Candida proteins), and allogeneic cells in mixed-lymphocyte culture (MLC). With the identification of gene mutations for many of the SCID syndromes and other T cell deficiencies, molecular studies can be extremely helpful in making a diagnosis.

B CELL IMMUNODEFICIENCY Unlike the patients with severe combined T cell deficiencies, in whom onset is at 4 or 5 months of age, patients with severe B cell deficiencies usually do not have problems with infections until 7 to 9 months of age ( Box 60-6 ). The later onset of infectious problems in this group of patients occurs because they are protected until this time by the maternal antibodies that have passed through the placenta during the third trimester of pregnancy. These patients usually have infections with encapsulated bacterial organisms such as streptococci, pneumococci, and Haemophilus influenzae type B. The sites of infection, as discussed previously, include otitis media, meningitis, septicemia, sinusitis, pneumonia, abscess, osteomyelitis, and urinary tract. Occasionally these patients may have problems with fungal or viral pathogens. Males with infantile XLA have an unusual susceptibility to enteroviruses and may develop a chronic enteroviral encephalomyelitis.[ severe growth failure is not

1062

Box 60-6. Clinical Characteristics of B Cell Deficiencies Onset of symptoms delayed until age 7–9 months after maternal antibodies wane Recurrent infections with encapsulated bacteria Chronic/recurrent sinusitis, otitis media, pneumonia, bronchiectasis Lymphadenitis and abscess formation Hematogenous spread causing meningitis, septicemia, osteomyelitis Few problems with fungal or viral pathogens (except enteroviruses)

205]

Generally,

Little growth failure; survival into adulthood with treatment Hypoplasia of peripheral lymph nodes and nasopharyngeal lymphoid tissues (XLA) Lymphoid hyperplasia/hepatosplenomegaly (CVID) Increased incidence of allergy and autoimmune diseases XLA, X-linked agammaglobulinemia; CVID, common variable immunodeficiency.

Box 60-7. Antibody Deficiency Disorders

Absent B Cells with Early-Onset Profound Hypogammaglobulinemia X-linked agammaglobulinemia (Bruton's disease): Btk gene mutations Igα defect: mutations in mb-1 that encodes Igα (CD79a) Surrogate light chain: mutations in gene that encodes surrogate light chain BLNK defect (B cell linker protein): splice defect of BLNK gene Mu (•) heavy chain: mutations or deletions in the heavy-chain-region genes

Hyper-IgM Syndromes *

X-linked hyper-IgM syndrome : mutations in CD40 ligand (TNFSF5) gene

Autosomal recessive immunodeficiency with hyper-IgM: mutations in activation-induced cytidine deaminase (AID) gene Nuclear factor kappa B (NF-κB) essential modulator (NEMO): mutations in IKK-γ gene

Isolated Immunoglobulin Deficiencies IgM deficiency Kappa (κ)-chain deficiency IgA deficiency IgG subclass deficiency Common Variable Immunodeficiency Transient Hypogammaglobulinemia of Infancy

Selective Antibody Deficiency Poor antibody responses to polysaccharide antigens

* T cell deficiency.

seen in patients with B cell deficiency as in T cell-deficient patients. Patients with antibody deficiency can survive into adulthood and can lead normal lives with the use of replacement IVIG therapy. Patients with severe B cell deficiency, such as infantile XLA, typically have a paucity of lymphoid tissue, including tonsils, adenoids, and peripheral lymph nodes ( Box 60-7 ). In contrast, patients with CVID often have lymphoid hypertrophy and hepatosplenomegaly. [ increased, particularly in patients with IgA deficiency and

21 206 CVID.[ ] [ ]

21]

The incidence of allergy and autoimmune disease is

X-Linked Agammaglobulinemia XLA is a X-linked recessive B cell deficiency originally described in 1952 by Colonel Ogden Bruton (Bruton's disease). Worldwide, the incidence appears to be approximately 5 to 10 cases per 1 million population. Infections occur predominantly in the sinopulmonary tract (60% of patients), including otitis media, chronic 17

sinusitis, and pneumonia.[ ] Other types of infections include pyoderma (25%), chronic conjunctivitis (8%), gastroenteritis (35%), arthritis (20%), meningitis/ encephalitis (16%), and less often, osteomyelitis (3%) and septicemia (10%). The most common pathogens are Haemophilus influenzae and Streptococcus 207

pneumoniae. Untreated boys experience repeated pulmonary tract infections, leading eventually to bronchiectasis. Cuccherini et al[ ] reported two XLA patients who had bacteremia and skin/bone infections with an unusual organism related to Flexispira/ Helicobacter species. Infections with Giardia lamblia and Pneumocystis carinii also may be seen in XLA.[

17]

Because cellular immunity is intact, most viral infections, fungal infections, and tuberculosis do not seem to be a problem in patients with XLA. Exceptions to this include viral hepatitis, disseminated polio, and chronic enteroviral encephalitis. Physical findings relate to the occurrence of repeated bacterial infections of susceptible target organs, such as the middle ear, sinuses, and lungs. There is a paucity of lymphoid tissues (e.g., adenoids, lymph nodes, spleen), unlike in patients with CVID, who often have lymphoid hyperplasia. Unusual complications in XLA include arthritis, a dermatomyositis-like syndrome, and meningoencephalitis. Chronic meningoencephalitis may occur in association with dermatomyositis or independently. 205]

Both are manifestations of chronic enterovirus infections, including the echoviruses and occasionally coxsackievirus.[

Arthritis in XLA is caused by acute bacterial infection in fewer than half the cases. The arthritis tends to affect the large joints. Sedimentation rates may be normal, 208

and serologic tests are negative. In some patients the joint inflammation is caused by infections with enteroviruses or Ureaplasma urealyticum.[ ] Symptoms usually improve or resolve with IVIG therapy. Patients with XLA are highly susceptible to poliovirus infection; vaccine-associated poliomyelitis has been reported in XLA.[

10]

Unlike patients with CVID, autoimmune disorders do not seem to be a frequent problem in XLA patients. Although a predisposition to various cancers seems to be common with many types of immune deficiencies, it is less clear whether XLA patients have the same predisposition.[ reported that 4.2% of registry patients with malignancy were XLA patients.[

210]

209]

The primary immunodeficiency registry

Lymphoreticular and GI malignancies were more common.

1063

XLA patients have a total absence or marked deficiency of serum immunoglobulins and fail to make antibodies to even potent protein antigens. Circulating B cells or 211]

surface membrane immunoglobulin-positive lymphocytes are extremely low (less than 2%) or absent.[ even increased in

211 number.[ ]

However, pro-B cells in the bone marrow are normal or

T lymphocytes and other lymphoid subpopulations and DTH skin testing to recall antigens are normal. The response of peripheral

blood mononuclear cells to mitogens and allogeneic cells is normal. Lymphoid tissues show absence of plasma cells, lymphoid follicles, and germinal centers. 17

Early diagnosis, broad-spectrum antibiotics, and replacement therapy with IVIG have changed the outcome of XLA patients.[ ] Infections, especially chronic enteroviral infections and chronic pulmonary disease, are still the two major complications of XLA. With the availability of IVIG in the early 1980s, however, the 212

management of this disease has been made much easier. In a retrospective study of 31 patients with XLA, Quartier et al[ ] reported that early IVIG replacement therapy and nadir serum IgG levels greater than 500 mg/dl were important in preventing severe acute bacterial infections and bronchiectasis. Trough serum IgG levels greater than 800 mg/dl may be necessary to prevent chronic sinusitis and enteroviral infections. 213

Obligate carriers show selective use of the normal X chromosome in B-lineage cells,[ ] whereas other cell types show lyonization of the X chromosome. The gene responsible for XLA has been identified as a cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk), which is expressed mainly in lymphocytes of the B lineage. [214]

Btk is critical in B lymphocyte signal transduction pathways and B cell differentiation. A number of distinct mutations of the Btk gene have been described in 209

patients with XLA, most involving the kinase domain.[ ] Some patients with Btk mutations may not present until later in life,[ of Btk mutations. In fact, the block in B cell differentiation may be “leaky,” resulting in some immunoglobulin synthesis.

215]

which may reflect different types

Agammaglobulinemia with Absent B Cells

Approximately 10% of patients with agammaglobulinemia and absent B cells are girls. Females with a clinical presentation indistinguishable from XLA have been described by several groups.[

209]

Any mutation of a gene involved in the early stages of B cell differentiation could result in an XLA phenotype. Yel et al[ 217]

described three families with a mutation in the mu heavy chain resulting in agammaglobulinemia. Minegishi et al[

216]

reported a mutation in the λ5/14.1 gene 218

encoding the pre−B cell receptor surrogate light chain that resulted in a B cell deficiency and agammaglobulinemia. Minegishi et al[ ] identified a 2-year-old girl with absent B cells and early-onset hypogammaglobulinemia who had a homozygous splice defect in Igα, a transmembrane proteins that forms the Igα/Igβ signal transduction unit of the pre−B cell receptor complex. These females who present with absent B cells and agammaglobulinemia appear to have a more severe disease 217 219

than the boys with Btk mutations.[ ] [ ] A subgroup of patients with CVID may also present with profound hypogammaglobulinemia and greatly reduced numbers of B cells. Molecular analysis for mutations in Btk is necessary to distinguish these CVID patients from those with XLA. More details can be found in the review by Conley.[

219]

Hyper-Immunoglobulin M Deficiency Syndrome X-linked hyper-IgM immunodeficiency syndrome (hyper-IgM, XHIGM) primarily affects boys and is characterized by severe recurrent bacterial infections with 220

decreased serum levels of IgG, IgA, and IgE but elevated IgM.[ ] Recurrent bacterial infections of the sinopulmonary tract usually begin in the first or second year of life. The clinical history of infection often resembles that of patients with XLA. Stomatitis and mouth ulcers may occur in association with the neutropenia. 220 221

Pneumocystis carinii has been reported in patients with this disease, which suggests a T cell deficiency.[ ] [ ] Other opportunistic infections include CMV, cryptococci, and mycobacteria. The susceptibility to opportunistic organisms and the frequent occurrence of autoimmune disease (e.g., thrombocytopenia, hemolytic 220] [222]

anemia, neutropenia, nephritis, arthritis) and the hyperimmunoglobulinemia distinguish these patients from those with XLA.[

222

Diarrhea is a frequent finding, affecting more than 50% of XHIGM patients. Cryptosporidium infection may result in sclerosing cholangitis.[ ] Hepatitis B and hepatitis C viral infections produce chronic hepatitis in hyper-IgM patients. Unlike patients with XLA, those with XHIGM have marked hypertrophy of the lymphoid 223

tissues, including the tonsils, lymph nodes, and spleen. However, the lymph nodes are poorly organized with absence of the germinal centers.[ ] Proliferation of IgM-producing plasma cells with extensive invasion of the GI tract and liver may occur in the second decade of life. Patients also have an increased risk of 223]

malignancies, predominantly lymphomas. An increased incidence of liver and biliary tumors is a unique feature of XHIGM.[

222

Serum levels of IgM are greatly increased and may reach levels in excess of 1000 mg/dl; early in life, however, the IgM may be normal.[ ] After antigen exposure these patients can produce IgM and IgD antibody, but the secondary IgG response is usually diminished or absent. Germinal centers are absent and follicles are rarely seen, but plasma cells can be identified in the lymphoid tissue. Surface immunoglobulin-positive lymphocytes in the peripheral blood are primarily positive for IgM; IgA-bearing and IgG-bearing lymphocytes are decreased or absent. T lymphocyte numbers and mitogen responses are normal. Patients with X-linked hyper-IgM 224]

syndrome lack CD40 ligand on T cells as a result of mutations in the gene for CD40 ligand. [

Supportive care, use of prophylactic antibiotics for P. carinii, and recognition and treatment of other opportunistic infections are important interventions. Parenteral nutrition may be necessary for patients with severe GI disturbances. Treatment consists of IVIG replacement therapy. The autoimmune neutropenia responds well to IVIG and granulocyte-macrophage colony-stimulating factor (GM-CSF). BMT has been used in the treatment of several children with XHIGM. Other Hyper-IgM Phenotypes 225

Several female patients have been described with findings that suggest an autosomal form of hyperimmunoglobulinemia M disease.[ ] These patients express CD40L normally, and the surface expression of CD40 on B cells is also normal. Recent molecular studies have shown that the defect in the autosomal variant of the hyper-IgM syndrome involves mutations in the activation-induced cytidine deaminase (AID) gene.[

226]

Characteristic features include lymphoid hyperplasia

1064

with marked follicular hyperplasia, enlarged germinal centers with highly proliferating B cells, defective immunoglobulin variable region gene somatic mutation generation, and defective immunoglobulin class switch and recombination. Recently, another rare form of X-linked hyper-IgM syndrome has been described, associated with ectodermal dysplasia characterized by the absence or hypoplasia of 227

hair, teeth, and sweat glands.[ ] Unlike other patients with hyper-IgM, these patients did not have a history of opportunistic infections. This disorder is related to mutations in the gene that encodes nuclear factor kappa B (NF-κB) essential modulator (NEMO, or IKKγ), which is required for activation of the transcription factor NF-κB. Zonana et al[

228]

described more of a dysgammaglobulinemia in these patients than a hyper-IgM phenotype.

Transient Hypogammaglobulinemia of Infancy Transient hypogammaglobulinemia of infancy (THI) involves an abnormal delay in the onset of immunoglobulin synthesis in the infant such that the normal physiologic hypogammaglobulinemia that occurs between 2 and 4 months of age is exaggerated. [ occasionally extend into the second or third year of life. less often

229 pneumonia.[ ]

[230]

229]

This exaggerated “physiologic” hypogammaglobulinemia may

Patients usually have recurrent upper respiratory tract infections, including otitis media, sinusitis, and

Serum IgG and IgA levels are usually low, but IgM is normal or increased. Circulating immunoglobulin surface positive lymphocytes are 231]

normal. Antibody responses to protein antigens are normal, but the response to viral respiratory agents is usually diminished. [ 229

By definition, THI is a self-limited disorder, with recovery between 18 and 36 months of age.[ ] These children must be differentiated from patients with primary immune deficiency diseases, particularly those with mild CVID, through long-term follow-up and reevaluation of immunoglobulins and B cell responses. Common Variable Immunodeficiency CVID is a heterogenous group of disorders involving both B cell and T cell immune function with the predominant manifestation of hypogammaglobulinemia. Other names for this entity include acquired hypogammaglobulinemia, adult-onset hypogammaglobulinemia, and dysgammaglobulinemia. CVID is characterized by recurrent bacterial infections, decreased serum immunoglobulin levels (at least two immunoglobulin isotypes more than 2 standard deviations [SD] below normal for age), and abnormal antibody responses. These patients may present in early childhood, during adolescence, or as young adults. In most patients, onset of symptoms is in the second and third decade of life. In a large study the average age of onset of symptoms was 25 years and the average age at diagnosis 28 years. [

232]

In a

6 Bodian,[ ]

subsequent study by Cunningham-Rundles and mortality over 25 years was 24%, primarily from lymphoma (18%) and chronic pulmonary disease (11%). The mean age at death was 45.5 years in females and 40 years in males. The patients who died were more likely to have lower levels of IgG at diagnosis and poorer T cell proliferative function to PHA. Twenty-year survival after diagnosis of CVID was 64% for males and 67% for females versus 92% to 94% for the general population. The most frequent presenting infections in adults with CVID involve the respiratory tract, including recurrent otitis media, chronic sinusitis, and recurrent pneumonia, often with resulting bronchiectasis.[

21]

The bacterial pathogens involved are similar to those described in XLA. Mycoplasma hominis and Ureaplasma 208]

urealyticum have been implicated in various infections in CVID patients and associated with arthritis. [

The GI tract is affected in approximately half of patients

21 232 diarrhea.[ ] [ ]

with CVID, who present with malabsorption or chronic These symptoms can be related to a number of underlying abnormalities, including lactose intolerance, protein-losing enteropathy, or superimposed infection of the small bowel, either with bacteria (e.g., Campylobacter, Yersinia, the parasite Giardia lamblia) or flora of the large bowel (e.g., small bowel bacterial overgrowth syndrome). Atrophic gastritis with achlorhydria may lead to pernicious anemia. Chronic gastroenterologic disease is often associated with nodular lymphoid hyperplasia, characterized by hypertrophy of the Peyer's patches in the small bowel, diffuse lymphoid infiltration, and loss of villi. Hypertrophy of other lymphoid tissues, including peripheral lymph nodes, spleen, and occasionally the liver, is also seen. [ Rarely, hepatosplenomegaly may be severe enough to result in secondary neutropenia or thrombocytopenia. Autoimmune disorders occur frequently in CVID (approximately 22% of patients) and include rheumatoid arthritis, autoimmune hematologic disorders (e.g., hemolytic anemia, idiopathic thrombocytopenic purpura, pernicious anemia), autoimmune neurologic diseases (e.g., Guillain-Barré syndrome), chronic active

21]

hepatitis often related to hepatitis C virus, and autoimmune endocrinopathies, particularly involving the thyroid.[ to 13%) in CVID during the fifth and sixth decades of

232 233 life.[ ] [ ]

21]

The incidence of malignancy is increased (11%

The majority of these malignancies (e.g., non-Hodgkin's lymphoma) involve the GI tract and 234]

lymphoid tissues. Another clinical feature of patients with CVID is noncaseating granulomatous lesions infiltrating the liver, lymph nodes, lungs, and skin.[ lesions are often confused with sarcoidosis.

These

Serum immunoglobulin levels are greatly diminished in CVID, although extreme variability may exist in the degree of hypogammaglobulinemia. Specific antibodies are usually lacking, and isohemagglutinin titers are usually diminished. The proportions of circulating B cells in the peripheral blood are usually normal, but a subset of patients may lack circulating B lymphocytes.[

6] [232]

T cell function can be quite variable; it is normal in half of patients and depressed in the other half, with

absent DTH to recall antigens, low numbers of circulating peripheral blood T cells, and depressed in vitro responses to mitogens and specific antigens.[

6] [232]

Several mechanisms have been proposed to explain the immune abnormalities in CVID patients, including an intrinsic B cell defect, excessive T suppressor activity, deficient T helper function, cytokine deficiencies, and suboptimal T-B cell interactions through deficient expression of the CD40 ligand. Eisenstein et al[ demonstrated that CVID T cells have diminished production of IL-2, IL-4, IL-5, and IFN-γ. A signaling defect may be intrinsic to the B cells[ expressed CD40 ligand on T cells and the CD40 receptor on B cells.

[237]

236]

235]

or between a poorly

Whether these abnormalities are a cause or consequence of CVID remains to be determined.

1065

The number of immune deviations described in patients with CVID underscores the heterogenous nature of the immune defect(s) in this syndrome. Family members 238

of CVID patients have an unusually high incidence of IgA deficiency,[ ] autoimmune diseases, autoantibodies, and malignancy. Patients and families with CVID or IgA deficiency have an unusually high frequency of an extended MHC haplotype: HLA-A1, B8, tumor necrosis factor-alpha 5 (TNF-α5), HSP70-7.5, C2a, Bf-0.4, G11-15, C4A-deleted (null allele for C4A complement gene), C4B-Sf, DQB1*0201, and DR3.[ chromosome 6 may be involved in the pathogenesis of CVID and IgA deficiency.

239]

One or more genes within the MHC class III region on

Immunodeficiency with Thymoma 240

Immunodeficiency with thymoma was first described by Jeunet in 1954.[ ] It is a disorder of adults, typically between the ages of 40 and 70. This immune deficiency presents with recurrent sinopulmonary infections. Affected individuals have hypogammaglobulinemia, which may affect all major immunoglobulin isotypes. A thymoma may be discovered during the initial investigation of hypogammaglobulinemia by the detection of a mediastinal mass on routine chest 241]

radiographs.[ 241 benign.[ ]

Occasionally the thymoma predates the hypogammaglobulinemia. The thymic tumors are predominantly of the spindle cell type and are usually

The clinical symptoms are similar to those found in patients with CVID. In contrast to CVID, however, associated disorders include aregenerative (pure red cell) anemia, agranulocytosis, and myasthenia gravis. These conditions may improve after thymectomy; however, the immunodeficiency persists. Infections typically associated with T cell abnormalities may be seen in this disease, including CMCC, CMV infection, herpes zoster, and P. carinii pneumonia.

Immunoglobulin A Deficiency Selective deficiency in serum IgA (A Def) is one of the most common B cell deficiencies, with an approximate incidence of 1 in 400 to 2000 individuals in the general population. IgA deficiency is defined as a serum IgA concentration below 7 mg/dl, with normal serum IgM and IgG. A Def may occur in association with the administration of drugs such as phenytoin, sulfasalazine, hydroxychloroquine, and d-penicillamine. A Def has also been associated with partial deletion of the long arm of chromosome 18 (18q syndrome) and a ring chromosome 18.[

242]

Many individuals with A Def do not have symptoms. The variability in clinical expression may be related to two factors. First, A-Def patients who tend to be 243

relatively asymptomatic appear to have a compensatory increase in secretory monomeric IgM in their saliva, upper respiratory tract secretions, and GI fluid. [ ] Second, the association of IgG2/IgG4 or IgG4 subclass deficiencies with A Def may predispose IgA-deficient patients to more severe and recurrent sinopulmonary infection than in those with isolated A Def.[

244] [245]

Symptoms of A Def include sinopulmonary infections and GI involvement with giardiasis, nodular lymphoid hyperplasia, ulcerative colitis, Crohn's disease, or a spruelike disease. An increased frequency of autoimmune disorders has also been associated with A Def, including arthritis, a lupuslike illness, autoimmune 243 246

endocrinopathies, chronic active hepatitis, and autoimmune hematologic disorders.[ ] [ ] A-Def patients are at risk for the development of anti-IgA antibodies on receipt of blood products. Caution must be exercised in the administration of IVIG for replacement of IgG subclass deficiency in A-Def patients because most of these preparations contain small amounts of IgA. However, this risk does not appear to be a problem in those patients with partial A Def, that is, IgA levels more than 2 SD below the normal range for age but greater than 7 mg/dl. 247]

Some atopic children have a transient A Def in which the serum IgA returns to normal between 1 and 5 years of age.[

The peripheral blood B cells of A-Def

248]

patients coexpress IgA, IgM, and IgD, an immature phenotype similar to the IgA-bearing B cells found in cord blood.[

Studies of T cell function have been

normal in most patients with A Def. As discussed, IgA deficiency shares with CVID the inheritance of a restricted MHC extended haplotype. [ pathogenesis of A Def is still unknown but may share a common etiology with CVID because these two disorders share many immune

239] [249]

The

249 aspects.[ ]

Immunoglobulin G Subclass Deficiencies Considerable controversy surrounds the biologic significance of IgG subclasses and the clinical significance of an isolated IgG subclass outside the normal range. Because healthy individuals without recurrent infections may have an abnormally low serum IgG subclass concentration, there is question as to whether IgG subclass deficiency represents a true immunodeficiency disease.[

250]

The first suggestion that IgG subclass deficiency might represent a new primary immunodeficiency

251 Oxelius[ ]

disorder was the 1974 report by of a mother and two children with recurrent sinopulmonary infections. These individuals were found to have normal serum immunoglobulin concentrations but low IgG2 and IgG4. In addition, they could not make antibodies to polysaccharide antigens such as H. influenzae type B. Antibody responses to protein antigens such as tetanus toxoid were normal. Several groups have reported homozygous deletions of parts of the constant region genes 252] [253]

that result in the absence of one or more of the IgG subclasses. [

Deficiency in an IgG subclass is defined as a serum IgG subclass concentration that is more than 2 SD below the normal mean for age. The age at which each of the IgG subclasses reaches adult levels varies. Gm allotype also influences serum concentrations of certain IgG subclasses, particularly IgG2 and IgG3.[

254]

In adults,

255 deficiency.[ ]

deficiencies in IgG3 subclass are most common, whereas in children, IgG2 is the most prevalent IgG subclass IgG subclass deficiency may be seen in conjunction with other primary immunodeficiency disorders, such as AT and A Def. IgG subclass deficiency occurs in approximately 18% of IgA-deficient patients. [256]

An IgG subclass deficiency might occur as an isolated immune defect or two or more IgG subclass deficiencies may coexist, such as IgG2 and IgG4 deficiency.

The most frequent clinical problem associated with IgG subclass deficiency is recurrent infections of the upper and lower respiratory tracts. Pathogens are generally limited to bacteria and respiratory viruses. IgG2 deficiency occurs either as an isolated entity or in association with IgG4 deficiency.[ in the response to

257]

Because IgG2 is important

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polysaccharide antigens, these patients typically have infections with H. influenza or S. pneumoniae. Patients may be unable to produce specific antibodies after 250

immunization with purified polysaccharide antigens (e.g., Pneumovax). Some individuals with IgG2 subclass deficiency may be asymptomatic,[ ] which may result in part from a shifting of the antibody response to another IgG subclass or immunoglobulin isotype, which compensates for the selective IgG2 subclass deficiency. In young children (age 3 to 4 years), low IgG2 serum levels and absent antibody responses to polysaccharide antigens are more frequently a developmental abnormality, with eventual correction of the immune perturbation as these children age. 257

IgG3 deficiency has been associated with recurrent upper and lower respiratory tract infections and may occur in combination with IgG1 deficiency.[ ] Several studies have suggested that IgG3 is especially important in the primary response to viral respiratory agents. Also, IgG3 is the predominant antibody response in Moraxella catarrhalis, an organism frequently isolated from patients with chronic sinusitis. IgG4 deficiency occurs in the general population at a rate of 258]

approximately 10% to 15%. The clinical significance of IgG4 deficiency has been debated. Heiner et al[ severe recurrent respiratory tract infections and bronchiectasis.

have described selective IgG4 deficiency in patients with

Selective Antibody or Antigen-Specific Antibody Deficiency Ambrosino et al[

259]

described patients who have abnormal responses to immunization with polysaccharides such as Haemophilus influenzae type B (HiB) capsular 260

antigen or to the pneumococcal polysaccharide antigens but who have normal serum immunoglobulin and IgG subclass concentrations. Granoff et al[ ] described a group of children who developed HiB infections despite prior immunization with HiB vaccine. These patients also failed to respond adequately to reimmunization 261

with HiB. Herrod et al[ ] described a similar group of children with poor responses to HiB vaccine. However, patients immunized to HiB-conjugate vaccine responded normally. Because the antibody response to the conjugate vaccine falls principally within the IgG1 subclass instead of the IgG2 subclass, this suggests different mechanisms of response to these two vaccines.[

262]

Similar observations have been made for selective antibody deficiency after immunization with a pneumococcal polysaccharide vaccine in children presenting with 263 264

recurrent acute sinusitis.[ ] [ ] These studies suggest that a selective antibody deficiency to polysaccharide antigens may be the basis underlying increased susceptibility to infection in these children. Selective antibody deficiency appears to be a disorder of young children, most between 3 and 9 years of age, and is seldom identified in adolescents. This abnormality may therefore reflect a maturational delay of the humoral immune system. Evaluation In the evaluation of patients with suspected B cell deficiency, serum immunoglobulin concentrations should be measured by quantitative techniques, including nephelometry ( Box 60-8 ). Values in children must be compared with normal laboratory values for age. Immunoelectrophoresis (IEP) is semiquantitative and should not be used to evaluate the patient with suspected antibody deficiency. IEP should only be used to examine serum for paraproteins, such as those found in Waldenström's macroglobulinemia or multiple myeloma. IgG subclass quantitation may be helpful, although debate continues over the utility of these measurements. [265]

A careful history and physical examination are important to determine the clinical significance of an IgG subclass deficiency. In addition, the measurement of functional or specific antibodies is important to determine the clinical relevance of an IgG subclass deficiency. Patients may have normal total serum immunoglobulins and normal IgG subclasses yet fail to make specific antibodies to bacterial or common viral pathogens. Therefore the assessment of specific antibody formation after vaccine administration is an important part of the laboratory evaluation in patients with suspected B cell deficiency. Isohemagglutinins are naturally occurring IgM antibodies to the ABO blood group substances. By 1 year of age, 70% of infants have positive 266

isohemagglutinin titers depending on their blood type.[ ] Responses to protein antigens generally fall in the IgG1 subclass, whereas the immune response to the polysaccharide antigens resides within the IgG2 subclass. With the conjugated vaccines for HiB and pneumococcal polysaccharides, antibody responses occur 262

primarily in the IgG1 rather than IgG2 subclass.[ ] Therefore these conjugate vaccines may not be helpful in the functional evaluation of an IgG2 subclass deficiency or a selective polysaccharide antibody deficiency. Fortunately, the vaccine for pneumococcal polysaccharide antigens is still available for evaluating patients for a selective or antigen-specific antibody deficiency. Because a common complaint of many of these patients is recurrent upper respiratory tract infections, their serum can be

Box 60-8. Laboratory Tests for Evaluation of B Cell Immune Function

Screening Tests Quantitative serum immunoglobulins IgG subclasses Specific antibodies • Isohemagglutinins (IgM antibodies to A and B blood group antigens) • Tetanus/diphtheria (IgG1) • Pneumococcal and meningococcal polysaccharides (IgG2) • Viral respiratory agents (IgG1, IgG3) • Other vaccines: hepatitis B, measles-mumps-rubella (MMR), typhoid

Advanced Testing In vitro B cell immunoglobulin production Regulation of immunoglobulin synthesis CD40 ligand–CD40 interactions Molecular analysis for gene deletions or mutations

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tested for the presence of antibodies to common respiratory viral agents such as influenza A and B, mycoplasma, respiratory syncytial virus, adenovirus, and 231

267

parainfluenza viruses.[ ] These antibodies fall into both the IgG1 and IgG3 subclasses.[ ] Molecular analysis for genetic abnormalities, especially those that involve the early stages of B cell maturation in those patients who present with absent B cells, can be very helpful in diagnostic evaluation.

Treatment Although a detailed discussion is beyond the scope of this presentation, the approaches used in the prevention and treatment of patients with B cell deficiency involve several basic principles. Supportive care, including antibiotics and good pulmonary hygiene measures to improve the mobilization of secretions, are very important. In patients with lung disease, pulmonary function testing should be performed at least once every 6 months. High-resolution chest computed tomography (CT) may also be useful for the early detection of bronchiectasis. Sputum cultures should also be obtained routinely in patients with bronchiectasis to track respiratory flora. As discussed earlier, diarrhea is a frequent symptom in patients with antibody deficiency. Stool examinations for ova and parasites and bacterial cultures should be obtained, with special attention to identification of G. lamblia, Campylobacter, and Yersinia. Giardia responds to metronidazole, as does bacterial overgrowth of the small bowel. Patients receiving chronic antibacterial therapy are at risk of Clostridium difficile overgrowth with toxin-induced diarrhea. Blood chemistries, including hepatitis screens and liver function tests, should be obtained on a regular basis. Blood counts and differentials should be determined at least every 6 months, especially in patients with CVID who develop autoimmune cytopenias. 268

Dosage regimens for IVIG replacement therapy range from 200 to 600 mg/kg every 3 to 4 weeks. Roifman et al[ ] showed that doses of 600 mg/kg every 4 weeks achieved serum IgG trough levels of more than 500 mg/dl. These higher doses may be necessary for patients with chronic sinopulmonary infections and bronchiectasis. Serum IgG trough levels need not be measured with each infusion. After a dose change or change in product, equilibration of the serum IgG level may take 3 to 6 months. The patient's clinical status, including improvement in pulmonary function, decrease in missed days of work or school, and antibiotic use, provides a more important indicator of successful replacement therapy. A number of commercial preparations are available in the United States. The secondgeneration and third-generation commercial products have greatly reduced adverse effects. Most adverse reactions are related to the infusion rate and can be controlled by careful monitoring and slowing the rate when necessary. Other symptoms unrelated to rate can often be controlled by pretreatment with acetaminophen, nonsteroidal antiinflammatory drugs (NSAIDs), and antihistamines. Renal failure as a complication of IVIG therapy has been reported, especially in patients receiving high-dose IVIG (e.g., ITP); less than 5% of the patients had primary immunodeficiency. IVIG products with sucrose as a stabilizer were most frequently associated with changes in renal function, with histologic findings consistent with osmotic injury of the proximal tubules. Several comprehensive review articles provide further information on IVIG therapy in patients with primary or secondary immunodeficiency.[

269] [270] [271] [272]

GENETIC ANALYSIS OF X-LINKED IMMUNODEFICIENCY DISORDERS AND PRENATAL DIAGNOSIS A number of primary immunodeficiency diseases are the result of defects in genes encoded on the X chromosome ( Figure 60-4 ). This finding has made it possible to provide genetic analysis of the X-linked immunodeficiency disorders and approaches for prenatal diagnosis. A careful family history, examining particularly the maternal side for males who might be affected by an immune defect, is important. However, a negative family history does not rule out an X-linked disorder. New 273]

mutations can occur, and at least one third of the cases represent the first manifestation of a new mutation on the X chromosome.[

Random inactivation of one X chromosome in a normal female occurs by a process called lyonization, such that a population of normal cells consists of an equal mixture of both X chromosomes (one from the maternal, the other from the paternal X). In contrast, if an X-linked gene defect involves lymphocyte differentiation, as in patients with X-linked SCID, immunologic testing in the obligate carrier is completely normal because the chromosome with the mutant gene remains inactive. Only lymphocytes with the normal X chromosome survive. On the other hand, if the gene defect involves an end-stage differentiated cell, such as the neutrophils of mothers with children who have X-linked CGD, a population of cells in the obligate carrier will express both the normal and the abnormal X chromosome. This

finding is illustrated with the NBT test or flow cytometry with dihydrorhodamine dye, which shows that approximately half the maternal neutrophils cannot reduce the NBT dye.

Figure 60-4 Map locations of X-linked immune deficiencies. IPEX, Immune dysregulation, polyendocrinopathy, X-linked syndrome.

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Chapter 61 - Human Immunodeficiency Virus and Allergic Disease

Mary E. Paul William T. Shearer

1

Acquired immunodeficiency syndrome (AIDS) was first reported to the Centers for Disease Control (CDC) in 1981.[ ] The human immunodeficiency virus (HIV) was identified as the causative agent of AIDS in 1983.[

2] [3]

CLINICAL FEATURES OF HIV INFECTION Since the early 1980s, HIV infection has been increasingly recognized as a problem of pandemic proportions affecting diverse groups of individuals. HIV type 1 (HIV-1) infection is usually a chronic disease, with patients living years before the deterioration of the immune system causes the patient to become vulnerable to opportunistic infections (OIs). HIV-1 infection is often less chronic in children than in adults. In the past, infected individuals often presented with OIs (e.g., PCP) before HIV-1 infection was identified. Now that physicians and at-risk persons in the community are more aware of transmission risk, HIV-1 infection is more often identified early and OIs prevented. Prophylaxis for OIs has resulted in increased survival for patients with HIV-1 infection. Because individuals are living longer with HIV-1 infection, clinicians have noted that HIV-infected patients have an increased propensity for symptoms of allergic disease, such as skin rash, rhinitis, sinusitis, and wheezing. The relationship among symptoms of hypersensitivity in patients with HIV infection, allergic disease, and changes in the immune system that are apparent in HIV-1-infected individuals is under investigation. 4

In 2002 the estimated worldwide number of HIV-infected individuals was 42 million.[ ] In North America the number of individuals living with HIV infection and AIDS was approximately 980,000 in 2002.

Diagnosis Diagnosis of HIV-1 infection in children 18 months of age and older and in adults can be established by a positive HIV enzyme immunoassay (EIA) confirmed by a positive Western blot. Both assays test for antibody to antigens of HIV-1. A negative EIA does not exclude very early infection, when antibody production has not yet begun. The EIA is very sensitive and specific, but false-positive results can occur. Infection is often confirmed by the HIV-1 Western blot ( Figure 61-1 ). An indeterminate Western blot should be repeated. An indeterminate Western blot can mean early infection or the presence of a cross-reacting antibody due to a process other than HIV infection.[

5]

Although the measure of HIV-1-specific antibody production is appropriate for diagnosis of infection in most adults, infants less than 18 months of age who are exposed to HIV-1 perinatally will likely have acquired HIV-specific antibodies passively from their mothers. To define infection accurately in this population, direct measures of HIV are used. Culture of plasma or of peripheral blood mononuclear cells provides a highly specific and sensitive direct measure of HIV if the specimen is processed correctly. Use of polymerase chain reaction (PCR) is routine in the diagnosis of HIV infection in infants. In PCR, amplification and molecular hybridization allow detection of genomic material (DNA, RNA). The DNA PCR has the advantage of faster results and is more cost-effective than the culture for most clinicians. For adults who are newly infected and do not yet have antibody to HIV, one of the above methods for direct detection of HIV can be used. Measure of HIV antigen, 5

the p24 antigen, is not recommended due to low sensitivity early in infection.[ ] Also, in infants infected by their mothers perinatally, measure of the p24 antigen is less sensitive than other direct measures, even when acid dissociation is used to free the p24 antigen from immune complexes.[

6]

Classification System Natural history studies and clinical experience show a wide variation in HIV disease manifestations. Patients vary from being asymptomatic to having severe conditions characterized by severe immunodeficiency, OIs, and malignancy. Management decisions hinge on the individual's past clinical history and current 7 8

immunologic status. CD4+ T lymphocyte count and risk for OIs are strongly associated with the risk for infection increasing as the CD4+ count decreases.[ ] [ ] The Centers for Disease Control and Prevention (CDC) has published guidelines based on these studies for classification of HIV-infected and AIDS patients, with criteria 9

for adolescents and adults ( Tables 61-1 and 61-2 and Boxes 61-1 and 61-2 ). [ ] Use of this categorization is helpful when determining need for prophylaxis against OIs, for public health surveillance, and for reporting in the medical literature. The CDC definition of AIDS includes both immunologic and clinical criteria. The clinical conditions in category C disease in adults make this the clinical category for AIDS

1074

Figure 61-1 Diagnosis of HIV-1 infection in children 18 months of age and older using HIV-1 enzyme immunoassay and Western blot.

TABLE 61-1 -- Classification System for HIV Infection (AIDS)

*

Clinical Categories

CD4+T Cell Categories (1) ≥500/•l

(A) Asymptomatic, Acute (Primary) HIV or PGL A1

†

(B) Symptomatic, Not (A) or (C) Conditions B1

‡

(C) AIDSIndicator Conditions C1

§

(2) 200–499/•l

A2

B2

C2

(3) 400•IU/ml) is one of the diagnostic criteria of this condition, and IgE levels can be used to follow the course of the disease.[

80]

When the disease is successfully treated with glucocorticoids, serum IgE levels fall, and conversely, disease exacerbations often are heralded by an

increase in serum IgE concentrations.[

79] [80]

81]

An association also appears to exist between disease activity and levels of anti-Aspergillus IgE antibodies.[

A gross elevation of serum IgE may also be helpful in distinguishing tropical pulmonary eosinophilia from some other causes of pulmonary symptoms associated

82]

with eosinophilia, such as Churg-Strauss syndrome and Wegener's granulomatosis [

(see Chapter 63 ).

Lung Function Evaluation of the relationship between total serum IgE levels and lung function is complicated by the associations between asthma and IgE concentrations and between cigarette smoking and IgE concentrations.[ 84] [85] [86]

function with advancing age.[

83]

Several studies suggest that higher total serum IgE levels are associated with a more rapid decline in lung

In a study of 10 pairs of monozygotic adult twins who were discordant for smoking, serum IgE levels were significantly

increased and airflow measurements significantly decreased in the smoking twins.[

87]

The relationship between total serum IgE levels and the decline in FEV1 was

evaluated in 1533 adults over 35 years old as part of the Tucson Epidemiological Study of Airways Obstructive Disease. Subjects had been observed over periods of up to 20 years. A significant inverse association was found between IgE levels and lung function, was independent of smoking and asthma status, and was also statistically distinct from age except in current smokers over age 55 years. The effect was small in nonasthmatic subjects but larger in both current and neversmoking asthmatic subjects. [

84]

Somewhat different results came from a study in the United Kingdom of individuals 65 years and older who were evaluated for pulmonary function, methacholine 85

responsiveness, skin test reactivity to common allergens, and total serum IgE levels and then were followed for 4 years.[ ] In the 212 subjects who completed the study, multivariable analysis demonstrated that male gender was the most important predictor of FEV1 decline, whereas methacholine responsiveness also tended to be associated with FEV1 decline. When the analysis was restricted to individual categories by smoking status, age was the only significant factor in never-smokers, whereas both skin test reactivity and methacholine responsiveness were significant predictors of accelerated FEV1 decline in former and current smokers combined. Information is conflicting about an inverse relationship between IgE levels and FEV1 in smokers, but IgE level does not seem to be significantly related to the rate of decline in FEV1 . [

88] [89]

In a study of the genetics of asthma and atopy, total serum IgE levels were found to be different among current, previous, and never-smoking individuals after 90

considering gender, personal or family history of asthma, and socio-occupational class.[ ] Evidence also suggested that passive exposure to environmental tobacco smoke was associated with increased IgE in women who were first-degree relatives of asthmatic patients. Cord Blood Most infants, at least in developed countries, are not stimulated to produce IgE in utero, and maternal IgE does not normally cross the placenta. Therefore the total IgE concentration in cord serum has been postulated to provide an estimate of an infant's basal genetic potential for IgE production and thus an estimate of the infant's risk of developing allergic disease (see Chapter 65 ). 51]

Measuring serum IgE concentrations less than 1•IU/ml is technically difficult and therefore somewhat imprecise.[

However, the population mean, or median, cord

9] [10] [11] [91]

IgE concentration was between 0.1 and 0.5•IU/ml in 22 of 30 studies (73%) and between 0.2 and 0.35•IU/ml in 12 of 30 studied populations (40%).[ [92] [93] [94] [95] [96] [97] [98] [99]

The relatively small range of IgE values among these studies, from many laboratories in multiple countries, suggests that the IgE standards and the assays are largely consistent. A number of factors may influence cord blood IgE concentrations ( Box 62-2 ). Cord blood IgE concentrations could be falsely elevated by admixture with maternal blood during delivery. This possibility is supported by studies showing correlations between maternal and cord blood IgE concentrations.[

suggest that admixture of maternal and fetal blood is a relatively uncommon event, occurring in less than 5% of detect contamination of cord blood in many studies may have been too

11 93 94 95 96 97 births,[ ] [ ] [ ] [ ] [ ] [ ]

Box 62-2. Factors Evaluated for Effect on Cord Blood IgE Concentration

Factors Associated with Increased Levels of Cord Blood IgE Maternal atopy[

9] [112]

Atopy in first-degree relatives[ Elevated maternal IgE levels[

10] [11] [102]

9] [93]

10] [96] [278] [279] [280]

Male gender[

95] [278] [280]

Black or Hispanic ethnicity[

9]

Progesterone administration[ Maternal parasitosis[ Maternal smoking[

14] [95] [278]

98] [102]

Factors Not Affecting Levels of Cord Blood IgE Prenatal oral contraceptives[

9]

92] [93] [94]

Most studies

but the criteria used to

9]

Albuterol administration[ Pregnancy diet[

281] [282] [283]

Maternal smoking[

9] [99] [279]

Gestational age from 34 to 42 weeks[

96] [98]

Type of delivery (spontaneous, cesarean, vacuum extraction)[ Maternal use of corticosteroids[

98]

98]

Factors Associated with Decreased Levels of Cord Blood IgE 92]

Maternal hepatitis B carrier status[ Maternal alcohol consumption[

284]

1092

liberal.[

100]

101]

The rate of contamination may also be influenced by the technique used to obtain the cord blood.[ 10] [91]

Conflicting reports surround the effect of gestational age on IgE concentrations,[

to find any effect of gestational age within the range of 34 to 42 weeks' gestation.

but two large studies encompassing 5305 and 6401 cord blood samples failed

[91] [98]

10 91 98 boys.[ ] [ ] [ ]

Cord blood IgE concentrations appear to be higher in boys than in girls, 102

with a correspondingly higher prevalence of early allergic disease in Magnusson[ ] reported that maternal cigarette smoking increased the concentration of cord blood IgE without reducing the predictive value of the cord blood IgE measurements for subsequent allergic disease. Others, however, have 9] [99]

failed to find an effect of maternal cigarette smoking on cord blood IgE levels.[ 95]

have ascariasis [

but lower in mothers who are hepatitis B carriers.[

disease may also be related to cord blood IgE concentration.

[97] [103]

92]

Cord IgE concentrations have also been reported to be higher when mothers 96]

Cord blood IgE concentrations may also vary seasonally.[

Parental history of allergic

A number of early studies suggested that cord blood IgE concentrations were predictive of allergic disease during childhood, but subsequent studies have failed to 104 105 106 107 108

10

demonstrate a significant relationship.[ ] [ ] [ ] [ ] [ ] In an early large study, Croner et al[ ] found that infants born with cord blood IgE concentrations greater than 1.3•IU/ml had a 70% risk of allergic disease compared with a 5% risk in infants with lower IgE concentrations. Subsequent reports from this study validated the prognostic role of the cord blood IgE measurements, with risks of 57.5% and 12.5%, respectively, for the high and low IgE groups at age 7 years and 109] [110]

risks of 67% and 27.6% at 11 years.[

Children with asthma evaluated at ages 12 to 14 years showed no relationship between cord blood IgE or family history 111]

of asthma and the presence or severity of asthma. [ disease.

Cord blood IgE measurements have little prognostic value for childhood asthma or other forms of allergic

Because atopy is hereditary, and if cord blood IgE levels were related to atopic risk, an association between cord blood IgE concentration and family atopic history should exist. Such a relationship could confound the predictive value of cord blood IgE. Studies examining this relationship have not produced consistent results. A study of Swedish neonates did find that infants with a positive atopic family history were more likely (38/528, 7.2%) to have elevated cord IgE levels (>1.3•IU/ml) 10]

than those with negative family histories (27/703, 3.8%).[ serum IgE are related to cord blood IgE

Other studies, however, have found that maternal but not paternal histories of allergy or elevations of

9 112 levels.[ ] [ ]

The results of studies comparing the risk of allergic disease predicted by either family history or cord blood IgE have been inconsistent. At the 11-year follow-up of a cohort of 1654 Swedish children, a family history was a more sensitive screen for allergic disease than cord blood IgE levels of more than 0.9•IU/ml (45% vs. 26%, 109]

respectively) but the specificity of the cord blood IgE level was higher than that of the family history.[

Overall, the efficiency of the cord blood IgE level (72%)

112 Magnusson[ ]

was higher than that for family history (64%). In contrast, found that a cord blood IgE level of more than 1.2•IU/ml had a significantly better positive predictive value (72.2%) than a history of allergy in a first-degree relative (29.8%). The method used to obtain the family history may result in significant variations in the reported prevalence of allergic disease.[

113]

Detection of Allergen-Specific IgE When the first allergen-specific IgE immunoassay was commercially introduced, a scoring system of 0 to 4+ was used to indicate the range from “undetectable” through increasing quantities of IgE. Many current assays now provide allergen-specific IgE values in mass units of IgE per volume (e.g., ng/ml, IU/ml, kilo allergen 114 115 116

units per liter [kaU/L]) by interpolating from a standard curve of known IgE concentrations.[ ] [ ] [ ] Reports in mass units have many advantages, but it has been difficult to convince physicians to adopt and request results in this format. It is hoped this situation will change in the future. The value of quantitative assays for allergen-specific IgE has been slowly emerging. Certain levels of food-specific IgE predict positive responses to double-blind placebo-controlled food challenges well enough to eliminate the need for the food challenge.[

117] [118] [119]

A similar study with inhalant allergens suggested that the 120

quantity of allergen-specific IgE could be used to distinguish symptomatic from asymptomatic but sensitized individuals using pollens and dust mite allergens.[ ] Interestingly, the values of specific IgE found to discriminate best between symptomatic and asymptomatic individuals with pollen allergens and dust mites (10.7 and 8.4•IU/ml, respectively) are similar to the specific IgE values found to discriminate food-sensitive individuals who would respond to a food challenge from those who would not respond (6 to 32•IU/ml for egg, milk, peanut and fish, but 65 and 100•IU/ml for soybean and wheat, respectively).[

117] [119] [120]

These levels of

specific IgE that best discriminated allergic individuals clinically are well above the minimal detectable level of IgE in most current assays (typically, >0.35•kaU/L 121]

or >0.35•IU/ml), suggesting that in vitro tests have adequate sensitivity in most clinical situations.[

Other potential uses for quantitative specific IgE have been reported. One study showed that levels of mite-specific IgE reflect the ongoing levels of mite allergen exposure in a person's home, suggesting that measurement of mite-specific IgE could be used as an indicator of whether adequate mite control measures were being 122]

performed.[

A potential limitation to the clinical use of in vitro measurement of allergen-specific IgE is cross-reactivity among allergens. A major source of cross-

reactivity is carbohydrate determinants[ specificity.

123]

(see Chapter 36 ). In the future these carbohydrates might be eliminated from allergens, leading to improved diagnostic

Many factors must be considered during the development of assays for allergen-specific IgE (see Chapter 37 ). For the practicing physician the most important variable in measuring allergen-specific IgE is the quality of the laboratory performing the assays. When coded serum samples were submitted through normal 121

channels to six different commercial laboratories, the results of 12,708 tests varied widely. [ ] Two laboratories using the same commercial assay system reported results that closely approximated an ideal system in terms of reproducibility and quantitation. The results from the other four laboratories were highly variable, with coefficients of variation (CVs) ranging from 5% to 49%. Some laboratories were not even able to identify serial dilutions of the same sample. This study suggests that some commercial assays for specific IgE can produce highly consistent results, when properly performed, but that unacceptable levels of variation exist for specific IgE testing among at least some commercial laboratories in the United States.

1093

Specific IgE concentrations vary with a person's age, the degree and duration of the most recent allergen exposure, the degree and duration of exposure to cross36] [124] [125]

reactive allergens, and immunotherapy.[

In contrast, sera from children with atopic dermatitis often have very high total concentrations of IgE, which 126

may produce nonspecific binding and lead to false-positive results in some assays.[ ] When multiple weak, positive results are obtained from a serum with an elevated total IgE level, the only method of determining the validity of the results is to perform inhibition experiments to see whether the IgE is allergen specific. 35 36 124

Allergen-specific IgE concentrations also tend to rise as a result of pollen exposure.[ ] [ ] [ ] The levels of specific IgE typically peak approximately 4 weeks after a seasonal pollen exposure and then gradually fall to a nadir before the next pollen season. These seasonal changes are not usually of sufficient magnitude to shift an assay result from negative to positive, but seasonal changes could shift a borderline sample to positive. Similarly, IgE antibody levels rise after insect stings in some individuals regardless of whether the sting provoked allergic symptoms.[ been documented with both insect venom and drug-specific

127] [128]

129 130 IgE.[ ] [ ]

A general trend of declining allergen-specific IgE concentrations with time has

IgE levels also tend to decline during immunotherapy, but part of the decline observed in

some studies may have been the result of IgG antibody formation blocking the detection of IgE antibodies.[

36] [124]

An important issue in clinical allergy is the degree of association between positive tests for allergen-specific IgE (skin or in vitro) and clinical manifestations of allergic disease. When large populations of normal individuals are tested for allergen-specific IgE using skin tests or in vitro immunoassays, many asymptomatic individuals have positive tests. In the pioneering work of Hagy and Settipane,[

131] [132] [133]

students beginning their first year of college were skin-tested and

followed. Of the 1243 students evaluated as freshmen, 989 were reassessed 3 years later as seniors. A total of 614 had no histories of allergic symptoms as freshmen, but 108 had at least one positive skin test to a panel of 15 common allergens, and 19 of these students had developed rhinitis symptoms.[ reactive the skin test, the greater was the risk that symptoms would positive skin tests and the risk of developing allergic symptoms

132 133 develop.[ ] [ ]

134 persisted.[ ]

133]

The more strongly

Even when the students were followed for 23 years, the association between

In a study of 985 Swedish teenagers, 181 (18%) had both symptoms of allergic disease 135

and positive skin tests, but 250 (25%) had positive skin tests and no history of symptoms. A total of 165 (17%) had symptoms but no positive skin tests.[ ] Results were similar in a cross-sectional study of 2295 Dutch adults ages 20 to 70 who were questioned about nasal symptoms and tested by skin and in vitro tests for IgE 136

antibodies to nine common allergens.[ ] Associations between nasal symptoms to indoor and outdoor allergen exposures and positive tests for specific IgE antibodies were highly significant but not absolute. The prevalences of at least one positive skin test were 39.1%, 43.7%, and 55.4% in individuals with indoor-only, outdoor-only, and both indoor and outdoor symptoms, respectively, but 15.5% of those without nasal symptoms had at least one positive skin test. A study of 477 blood donors found that 119 (24.9%) claimed to be allergic, but allergen-specific IgE was detected in only 54.5% of these individuals; in the 358 137

donors without complaints of allergic disease, specific IgE was detected in 12.3%.[ ] These results clearly indicate that a positive test for specific IgE cannot be used as the sole basis for a diagnosis of allergic disease and that even when allergic symptoms and a positive test are present, allergic disease is not absolutely confirmed. The physician must still consider the strength of the history, physical examination findings, results of IgE tests, and possibilities of alternative explanations for the patient's problems before making a diagnosis. Correlation of In Vitro and In Vivo Tests.

The results of in vitro immunoassays for allergen-specific IgE antibodies usually correlate well with the results of in vivo tests such as skin tests and bronchial or conjunctival challenge. The results correlate best when relatively pure and potent allergens are studied. Even under the best of circumstances, in vitro tests usually fail to detect a modest number of skin test–positive individuals. The number not detected depends on the criteria used for classifying the skin test as positive or 138 139 140 141 142 143 144

negative but generally ranges from 5% to 40%.[ ] [ ] [ ] [ ] [ ] [ ] [ ] Although this false-negative rate may not be important in many clinical settings, it may be extremely important in the evaluation of people with a history of life-threatening reactions such as those induced by insect stings, drugs, foods, or natural 145] [146]

rubber latex. In these individuals the extra sensitivity of the skin tests may be necessary to detect the potentially fatal sensitivities.[

Finding a positive in vitro

57 occur.[ ]

test in a skin test–negative person is less common but can If a probable allergen can be clearly identified by history, a positive in vitro test for the same allergen greatly increases the probability that the causative allergen has been identified. If the in vitro test to the allergen is negative, however, skin testing should be performed before concluding that the patient is not sensitive. Thus, when the risk of missing an allergic sensitivity is very high for the patient, both skin tests and in vitro assays should be strongly considered. Several studies have compared skin tests with in vitro immunoassays or compared two or more in vitro tests using receiver operating characteristic (ROC) analysis. [147] [148] [149]

The value of ROC analysis is the ability to see the relationship between the sensitivity and specificity of a test as a continuous curve rather than as one or more discrete points. ROC analysis curves provide an indication of the total amount of information provided by a test, allowing a clearer decision as to whether one test is actually more informative than another.

Allergens Available for Immunoassay.

In vitro assays for allergen-specific IgE have been developed to a variety of allergens. The primary problems limiting the availability of allergens for in vitro assays are the difficulty of adequately presenting the allergen on the solid phase and finding a sufficient number of truly positive sera to validate the assay. Most proteins can be easily coupled to cellulose supports using cyanogen bromide activation of the support, and many proteins passively adhere to the plastic microtiter wells used for enzyme-linked immunosorbent assay (ELISA). Some therapeutic agents (e.g., insulin, chymopapain) are large enough proteins to be directly coupled to solid supports. Other therapeutic agents (e.g., antibiotics) are more difficult to present on solid phases because they are small haptens rather than complete antigens. For penicillin the penicilloyl moiety can be conjugated to a carrier (e.g., polylysine), which in turn can be coupled to the solid phase.[ develop appropriate conjugates for detecting some IgE

150] [151]

It may be possible to

1094

152 153

antibodies to sulfonamide antibiotics and neuromuscular blocking agents.[ ] [ ] Another difficult group of allergenic haptens are industrial chemicals, such as toluene diisocyanate and platinum (see Chapter 73 ). Again, these small molecules must be conjugated onto suitable carrier proteins before being bound to a solid phase.[

154]

Substances such as opiate derivatives or iodinated radiographic contrast media may produce adverse reactions, which resemble IgE-mediated reactions, by non-IgEmediated mechanisms. Unfortunately, a few laboratories have offered IgE assays “specific” for these agents. It is incumbent on physicians ordering tests for allergen146] [155]

specific IgE to know whether the substance used in the test produces IgE-mediated disease and to know the quality of the laboratory performing the test. [ [156]

Indications for Measuring Specific IgE Antibodies.

Once the clinical decision has been made to test a patient for allergen-specific IgE, the physician must decide whether in vivo skin tests or in vitro immunoassays are 143

in the patient's best interests[ ] ( Box 62-3 ). When an individual is highly sensitive, both skin tests and immunoassays are likely to detect IgE to the provoking allergens. In individuals with histories of anaphylaxis, the most compelling advantage of in vitro assays is freedom from the risk of inducing an anaphylactic reaction. In vitro assays may also be more convenient. It may be difficult to withdraw a medication known to interfere with skin test response from the patient, or the patient may not have an adequate area of normal skin. Other medical problems may interfere with skin testing, as with hospitalized patients experiencing nonhemolytic 157

transfusion reactions.[ ] In some uncooperative individuals, such as mentally impaired adults, skin testing may be hazardous to both the patient and the person attempting to perform the tests, making in vitro tests preferable. In some circumstances it may be more convenient to transport a serum sample than to transport the patient for testing. When individuals with high levels of total IgE from parasitic disease are tested, in vitro analysis may be more reliable. As discussed later, high levels of total IgE may saturate mast cells, leading to false-negative skin tests even when

Box 62-3. Advantages of Immunoassays and Skin Tests for AllergenSpecific IgE

Immunoassays Lack of risk for allergic reaction Results not affected by drugs Results not affected by condition of skin Greater patient convenience Easier-to-document quality control

Skin Tests Greater sensitivity Wider selection of allergens Immediately available results Less time and reagent expense per test

allergen-specific IgE is easily detectable in serum.[

158]

146] [155] [156]

A final advantage of in vitro tests is ease of standardization for quality control purposes.[

Despite the advantages, two major considerations limit the use of in vitro immunoassays for allergen-specific IgE in the United States. The first limitation is the rather consistent finding that in vitro tests are not as sensitive as skin tests for detecting allergen-specific IgE.[

138] [139] [140] [141] [142]

The second limitation is that on

143 costs.[ ]

a per-test basis, skin tests have lower time and reagent The importance of diagnostic sensitivity depends on the clinical circumstances, but the increasing pressures for containment of medical costs will probably ensure the continued use of skin tests in most clinical situations. Another potential problem with in vitro assays is the detection of cross-reactive IgE antibodies. Many individuals allergic to pollens have detectable IgE antibodies to foods, and 20% to 30% of those with food-specific IgE antibodies do not report symptoms related to foods.[

159] [160]

Pollen-food cross-reactions may result from

proteins such as profilin, which is a highly conserved molecule among plants,[

161] [162]

123]

or from cross-reactive carbohydrate determinants[

(see Chapter 36 ). 163]

Cross-reactive antibodies between foods and natural rubber latex allergens also complicate the interpretation of in vitro tests for latex-specific IgE antibodies.[ [164] [165]

INFECTIOUS AND PARASITIC DISEASE Serum IgE levels have been measured in many infectious and parasitic diseases. The changes noted in some infections appear to be the result of IgE antibodies specific for the infectious agent, whereas in other diseases, changes in IgE appear to be nonspecific ( Box 62-4 ). Viral Infection The presence of IgE specific for the respiratory syncytial virus (RSV) was highly associated with wheezing and other signs of lower respiratory tract involvement 166]

during primary RSV infections in children.[ episodes during a 4-year follow-up

Peak IgE-RSV titers during acute infection were also significantly associated with the risk of recurrent wheezing

167 study.[ ]

Studies of serum IgE concentrations during other viral infections have produced variable results. Studies of mononucleosis related to Epstein-Barr virus (EBV) 168

infection have shown an initial rise in IgE concentration for 7 to 10 days, followed by a decline and finally a return to baseline levels over weeks to months.[ ] A study of acute viral upper and lower respiratory tract infections showed no consistent pattern of serum IgE changes, but there was a trend toward lower IgE levels in 169]

the convalescent sera compared with those during the acute illness.[

The pattern of lower IgE concentrations during convalescence was much clearer in a study of

170 measles.[ ]

childhood The mean serum IgE level at initial presentation of measles was 258•IU/ml in 182 Peruvian children, compared with a mean of only 82.5•IU/ ml during the second week after the appearance of the skin rash in children without complications. IgE levels in children with measles-related pneumonia were similar to those with uncomplicated disease, whereas children who developed measles encephalitis had the highest mean levels (540•IU/ml) in the first week after the onset of rash and a slower decline in their IgE levels.[

170]

Experimentally induced infections with rhinovirus in adults with allergic rhinitis

1095

Box 62-4. Nonallergic Diseases Associated with Increased and Decreased Total Serum IgE Levels Increased IgE levels

Parasitic diseases Ascariasis[

32] 285]

Visceral larva migrans[ Capillariasis[

202]

Paragonimiasis[ Fascioliasis[

286]

286]

Schistosomiasis[ Hookworm[ Trichinosis[ Filariasis[

286]

286]

203]

287]

Strongyloidiasis[

288]

289]

Echinococcosis[ Onchocerciasis[ Malaria[

Infections

291]

290]

79]

Allergic bronchopulmonary aspergillosis[ Systemic candidiasis[ Coccidioidomycosis[

292]

293]

192]

Leprosy[

168]

Epstein-Barr virus mononucleosis[ Cytomegalovirus mononucleosis[

294]

169]

Viral respiratory infections[

HIV type 1 infections (AIDS)[ Pertussis[

172] [179] [180] [181]

196]

Tuberculosis[

194]

Cutaneous diseases Bullous pemphigoid[

295] 296]

Chronic acral dermatitis[

Streptococcal erythema nodosum[ Other dermatoses[

297]

298]

Other diseases 250] [252] [253] [299]

Nephrotic syndrome[

254]

Drug-induced interstitial nephritis[ Liver disease,[

255] [256] [257]

but not hepatitis C[

301]

Cystic fibrosis[

Kawasaki's disease[

302] 303]

Infantile polyarteritis nodosa[

304]

Primary pulmonary hemosiderosis[ Guillain-Barré syndrome[ Burns[

305]

305]

Rheumatoid arthritis[

306] 245] [246] [247]

Bone marrow transplantation[

88] [258] [259]

Cigarette smoking[ Alcoholism[

256] [257]

Fanconi's anemia[ Kimura's disease[

307]

224] [225]

Neoplastic diseases Hodgkin's disease[ IgE myeloma[

231] [232]

230]

Bronchial carcinoma[

241]

300]

Immunodeficiency diseases (see Chapter 59 ) Wiskott-Aldrich syndrome Hyper-IgE syndrome Thymic hypoplasia (DiGeorge's syndrome) Cellular immunodeficiency with immunoglobulins (Nezelof syndrome) Selective IgA deficiency

Decreased IgE Levels 183]

Familial IgE deficiency, recurrent sinopulmonary infections[ 178]

Human T cell lymphotropic virus type 1 infections[ Primary biliary cirrhosis[

308]

acutely produced a significant increase in total serum IgE levels (p 3 episodes during the previous year) at ages 6, 8, 11 and 13 years compared with children without LRI. The relative risk for any wheezing decreased over time from 4.2 at age 6 years to 2.8 at 8 years. Children with croup but not wheeze did not show an increased risk for episodes of wheezing, whereas children with other LRI, of whom 80% had wheezing LRI, showed a significantly increased risk of having current wheezing at age 6 years or 8 years compared with children without LRI. However, other LRI was not significantly associated with current frequent wheezing after age 6 years. RSV was more frequently identified in children with croup accompanied by wheezing and other LRI than in those with croup without wheezing. 203

Mechanisms have been proposed to describe the potential relationship between viral respiratory infections and the subsequent development of asthma.[ ] Current hypotheses involve the ability of viral infections to enhance airway inflammation, a component of the asthma phenotype; to enhance absorption of aeroallergens; and 203 204

to influence T helper cell type 1/type 2 (Th1/Th2) responses.[ ] [ ] A study of 79 children with documented RSV infection indicated that some respiratory viruses can stimulate an IgE-specific response. The level of this response and the level of histamine and leukotriene C4 in nasal secretions were associated with the degree of 205

airway obstruction in lower airway disease.[ ] Moreover, when 38 infants with RSV infection were followed prospectively for 48 months, only 20% with undetectable RSV-specific IgE titers developed episodes of wheeze, compared with 70% with high RSV IgE antibodies. This finding suggests that the ability to 206]

generate a virus-specific IgE response may be a marker for individuals with a genetic susceptibility to develop BHR.[ of IgE is a marker or a cause of subsequent asthma has not been

However, whether virus-specific generation

204 established.[ ]

Another similar mechanistic hypothesis suggests that viral respiratory infections increase subsequent AHR and the pattern of airway response to inhaled allergen.

Results from a small group, examined before and after rhinovirus infection, indicated that 8 of 10 subjects experienced airway obstruction 4 to 8 hours after antigen 207

inhalation (late-phase reaction) during rhinovirus infection, when only one of the 10 subjects had a late-phase reaction before infection.[ ] Furthermore, five of the seven available for testing still had evidence of airway obstruction 4 weeks after rhinovirus inoculation. These observations suggest that viral respiratory infections may have an extended effect on factors involved in the pathogenesis of asthma.[

204]

The role of viral respiratory antigens in the etiology of asthma, however, remains unclear and may be more indirect than the previous studies suggest. The inflammatory response of the airways is mediated by T helper (Th) lymphocytes, which may be classified in Th1 or Th2 subgroups on the basis of the cytokines that they generate when activated. Viral antigens have been shown to stimulate the production of cytokines associated with Th1 lymphocytes. However, studies of asthmatic airways report the presence of cytokines associated with the Th2 rather than the Th1 profile. Some have suggested as an explanation that viruses do not 208

205 206

elicit a pure Th1 response. [ ] This is supported by the virus-specific IgE production seen in infants with previous viral infection,[ ] [ ] since IgE synthesis requires the presence of cytokines produced by Th2 lymphocytes. Another possibility is that Th1 cytokines may up-regulate the inflammatory functions of effector 208]

cells in virus-induced asthma.[

An opposing hypothesis postulates that the rising prevalence of asthma seen in developed countries is caused by the overall decrease in viral and bacterial infections 1 145 209

experienced in childhood as a result of improved immunization and public health.[ ] [ ] [ ] These infections promote activation of the Th1 profile, which results in increased IgE levels, but not in asthma. The decline in early-childhood infections may be resulting in an increase in the population of activation of the Th2 lymphocyte profile associated with asthma, although relevant longitudinal data are not available. Prematurity Premature birth has been associated with the development of symptoms consistent with asthma and other long-term pulmonary sequelae in a number of studies. The basis for these sequelae is uncertain. The pulmonary injury might be acquired during mechanical ventilation of preterm infants with respiratory distress syndrome (RDS), from the RDS itself, or from some other facet of prematurity. Prematurity has been examined as a risk factor for asthma in both cohort studies of affected children and cross-sectional studies ( Table 64-8 ). 210

In one of the earliest reports, Northway et al[ ] considered the first possibility, that asthma is a long-term consequence of bronchopulmonary dysplasia (BPD). BPD is a syndrome of chronic lung disease in premature infants who are mechanically ventilated for at least 1 week as a treatment for RDS. The clinical diagnosis requires the presence of symptoms of persistent respiratory distress during infancy, dependence on supplemental oxygen, and abnormal chest radiographs. [

210]

Northway et al

[211]

studied adolescents and young adults born between 1964 and 1973 who had BPD in infancy and compared their long-term pulmonary outcomes to two control groups ( Table 64-8 ). They found that most subjects with a history of BPD in infancy had pulmonary dysfunction. Moreover, the increase in airway reactivity was not associated with a more frequent family history of asthma in this sample or with an increased prevalence of atopy. These findings suggest that lung injury resulting from mechanical ventilation of premature infants has a role in the pathogenesis of persistent pulmonary dysfunction similar to asthma. 212

Bertrand et al[ ] investigated the role of RDS in prematurity in the pathogenesis of AHR in subjects who did not have BPD as infants. The group with a history of RDS had evidence of more hyperinflation and airway obstruction compared with controls. However, results from the histamine challenge to determine AHR and

familial aggregation of AHR were inconclusive. Airway reactivity was elevated in both cases and controls, in addition to the mothers and siblings of both cases and controls. The authors suggest that the elevated incidence of AHR in mothers of both groups supports the hypothesis that there may be an association between onset of premature labor and airway reactivity. Because no comparison group was established for mothers of full-term children, however, this assertion cannot be affirmed from the study.

1149

213] [214]

Some researchers have investigated the effect of very low birth weight (VLBW) ( females (prepubertal)

Rates are linked to hospitalizations. [16] [17] [18]

Females > males (postpubertal)

Obesity may increase prevalence overall and incidence before menarche. [18] [19]

Blacks > Caucasians

Recent increase in prevalence is greatest in Caucasians.[

18]

Urban > rural

Specific U.S. cities have greatest risk. [18] [20] 15]

Coastal > inland

Related to dust mite exposure.[

School age > preschool

Preschoolers have greatest increase in prevalence.[

by a twofold increase in positive exercise bronchoprovocation challenge.[

14]

2]

Similarly, between 1982 and 1992, Australian children ages 8 to 10 years experienced an 15]

increased prevalence of asthma diagnosis, medication use, wheezing, and bronchial responsiveness to histamine challenge.[ prevalence may relate to increased urban living, proximity to coastal regions, and obesity ( Table 68-2 ).

The force driving this increase in

Incidence 21

Only a few studies have evaluated the incidence of childhood asthma. [ ] Several studies noted that boys had an overall increased incidence of asthma, regardless of age group or study location (United Kingdom, Australia, or the United States). In Australia the incidence was greater in 8-year-old than 12-year-old children. In the United States, however, the age group up to 4 years had the greatest incidence compared with the groups 5 to 9 years and 10 to 14 years old. Less information is available regarding the remission rate of asthma. A 10% remission rate was measured in 7- to 8-year-old children living in Sweden and was associated with negative allergy skin tests.[

22]

In the southwestern United States, allergic sensitization to Alternaria at age 6 years has been associated with only a 9% asthma remission rate at 23]

age 11 years versus a 39% remission rate if sensitization to this mold was not present during this period.[ Severity

Of all children with asthma, only 10% had severe asthma in one study, but this small subset accounted for 27% of school days missed, 35% of hospitalizations, and 77% of inpatient days.[

24]

Almost 30% of asthmatic children have some limitations in activity, compared with 5% of children without asthma. African-American 25]

children seem to have more severe functional limitations and more hospitalizations than their Caucasian counterparts.[ with increased morbidity were inadequate knowledge of asthma and its treatment and parental smoking in the

In Hispanic children, two factors associated

26 home.[ ]

Office visits continue to be the clinical venue where the majority of childhood asthma management is provided, and the number of visits listing asthma as the primary diagnosis doubled between 1979 and 1994. This represents an increase from 25.3 to 50.3 per 1000 for children up to 4 years old and 22.5 to 51.5 per 1000 for children 5 to 14 years old. Emergency department (ED) visits for asthma between 1992 and 1995 remained stable.[ Risk Factors

2]

It is highly doubtful that a single cause for asthma will be found. As a result, a discussion of risk factors for childhood asthma must account for events or exposures related to the de novo development of asthmatic symptoms in genetically predisposed individuals, as well as factors that exacerbate the disease once it has already been identified in an individual patient. For example, exposure to high levels of house-dust mite antigen in the first year of life may not lead to an acute episode of 27

asthma as it may in older children, but it may increase the risk of developing asthma by the time the child is 5 years of age.[ ] Moreover, exposure to endotoxin during infancy may protect the child from developing allergic disease and asthma; in contrast, once asthma is established, endotoxin exposure may increase both acute and chronic lower airway inflammation and overall asthma control.[

28]

Perinatal Factors 18]

About 30% of children with asthma develop symptoms within their first year of life, 50% by age 2, and 80% by the time they start school.[

Prenatal and perinatal 29

risk factors for the development of asthma in inner-city African-American children have been evaluated using obstetric, perinatal, and pediatric records.[ ] Asthmatic children had significantly lower birth weights and gestational ages than nonasthmatic children and were more likely to have required oxygen supplementation and positive-pressure ventilation (PPV) after birth. Mothers of asthmatic children were more likely to have smoked during pregnancy, to have gained less weight during pregnancy, and to have had no prenatal care. The strongest independent predictors of asthma, in descending rank of odds ratios (ORs), were maternal history of asthma (OR 9.7), lack of prenatal care (OR 4.7), history of bronchiolitis (OR 4.7), low maternal weight gain (OR 3.4), PPV at birth (OR 3.3), and maternal smoking during pregnancy (OR 2.8). Anatomic and Physiologic Factors

A number of anatomic and physiologic peculiarities in early life predispose to wheezing illnesses in infants and young children. The most important of these are a 18

disproportionate narrowing of the peripheral airways and decreased static elastic recoil properties of the lung.[ ] Peripheral airway conductance is low in the first 5 years of life, then undergoes a fourfold increase on the basis of “catch-up growth,” resulting in an increase in the cross-sectional diameter of the smaller airways. Several other factors, including the more compliant rib cage in infants, the decreased collateral ventilation, and the absence of fatigue-resistant skeletal muscle in the diaphragm, may contribute to the development of obstructive airway symptoms in infants and young children. [

18]

Finally, the relatively small airway diameter in 18]

relationship to edema, mucus secretion, and smooth muscle constriction poses geometric challenges to maintaining airway patency in this age group.[ Viral Upper Respiratory Tract Infections

In infants and children, viruses are the predominant microorganism associated with infection-induced wheezing.[

30] [31]

1227

Lower respiratory tract infections in the first 3 years of life, with or without documented pneumonia, significantly increased the risk of a child developing asthma

32

when evaluated prospectively at both age 6 and 11 years of age, with most infections caused by viruses.[ ] Risk factors related to the development of symptomatic wheezing during viral illnesses relate to both virus-specific and host-specific characteristics. For example, infection with respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infancy, at least in part because of trophism for the lower airway and its ability to cause primary or reinfection even in the presence of neutralizing antibody (virus-specific factors). Although many children infected with RSV recover completely after their first infection, others continue to 33 34 35

wheeze for years and may exhibit persistent alterations in lung function.[ ] [ ] [ ] Although some investigators have concluded that RSV infections induce these long-term sequelae, others have provided evidence that decreased levels of lung function are present at birth in these at-risk children, and this abnormality predisposes them to wheezing and airway obstruction during viral respiratory infections in early life (host-specific factors).[ to cytokine production may also be associated with an increased risk of RSV bronchiolitis.

4] [36]

In addition, genetic factors related

[7]

30 37

Beyond infancy, viruses associated with episodes of wheezing in children include rhinovirus, parainfluenza virus, influenza virus, and coronavirus. [ ] [ ] Using polymerase chain reaction (PCR) to detect viral antigen in school-age children, prospective evaluations of increasing asthmatic symptoms and reductions in home 38

peak expiratory flow rates have clearly demonstrated a temporal relationship with rhinovirus upper respiratory tract infections.[ ] Moreover, a subsequent time-trend analysis using this same data set also noted a relationship among these episodes, hospital admissions for asthma within the community, and the times of year during which children were in school versus on vacation or holiday.[

31]

A number of other host-specific factors may influence either the initial response or the persistence of a given response after viral respiratory tract illnesses. These include a family history of asthma, serum IgE levels, and exposure to passive smoking. For example, children that have transient wheezing (before age 3 years but not at 6 years) are more likely to have mothers who smoked but not mothers with asthma, normal serum IgE levels, no skin test reactivity, and diminished lung functions at 1 and 6 years of age. In contrast, children who wheezed persistently (before 3 and at 6 years) were more likely to have a history of maternal asthma, elevated serum IgE, and normal lung function in the first year of life but diminished at age 6 years.[

6]

In contrast to the overwhelming evidence that viral infections can trigger acute episodes of wheezing and can be linked epidemiologically with asthmatic 39]

exacerbations, certain viral and other microbial infections also may play a role in reducing the incidence of atopy and asthma.[

For example, children who have 40

experienced natural infections with hepatitis A virus (HAV) or other enteric pathogens have a significantly lower prevalence of atopic disorders.[ ] Attendance in day care, which greatly increases exposure to common viral infections, has been associated with reduced rates of atopy, if the day care exposure happens at an early 41 42

age.[ ] [ ] Some have attributed these findings to an effect on the critical balance between the expression of T helper type 1 (Th1) and type 2 (Th2) cytokines. According to this “hygiene hypothesis,” early exposure to microbial agents that produce an exuberant Th1 response (e.g., HAV or recurrent viral infections) would 43] [44]

down-regulate the subsequent Th2 allergic response to other environmental stimuli.[ role in both the initiation and the induction of atopy and

Thus the type of virus and the timing of exposure to it may play a critical

45 asthma.[ ]

Socioeconomic Factors

Lower socioeconomic class and the inherent living conditions are particularly problematic in relationship to asthma in the United States. Although poverty itself, independent of concurrent factors such as urban living, is not a risk factor for developing asthma,[

20]

its presence raises important considerations in caring for

children with asthma. Children living in poverty are more likely to use neighborhood health centers or hospital clinics than physician offices; moreover, when they are acutely symptomatic, these children are four times more likely to use an emergency department as a primary care source than are children from higher 46]

socioeconomic environments.[

Finally, sensitization to cockroach allergen is more prevalent in inner-city children, and sensitization with ongoing exposure is

closely associated with symptomatic asthma. [

47]

Hygiene Hypothesis The increasing prevalence of asthma in westernized cultures, coupled with evidence that factors such as childhood infections, day care attendance, and microbe 48 49

exposure may be protective, led to the initial development and later expansion of the “hygiene hypothesis.”[ ] [ ] The immune system of the newborn infant may be significantly influenced by the type, amount, and timing of various environmental exposures that modulate immunoinflammatory responses and the ultimate expression of various allergic diseases, including asthma. In this regard, environmental factors that could enhance Th1 responses and associated epidemiologically with a reduced incidence of allergy and asthma include (1) infection with HAV or other enteric pathogens,[ 48] [50] [51]

greater number of older siblings or day care attendance early in life,[ polymorphisms for the major endotoxin receptor concomitant alterations in gastrointestinal flora.

52 CD14.[ ]

[49]

40]

(2) increased exposure to infections as a result of a

(3) increased environmental endotoxin exposure, and (4) certain genetic

Restoration of Th1/Th2 balance may also be impeded by frequent oral antibiotic administration with

Immune “imprinting” may actually begin in utero, and maternal influences may be related to transplacental

transfer of allergens and cytokines, potentially contributing to Th1/Th2 cytokine balance.[

53]

Although these observations have generated intense interest in the

intrauterine-neonatal, genetic, and environmental influences, conflicting results have prevented any firm conclusions at present.[

54]

Day Care

Day care attendance has complex effects on the development of recurrent wheezing and asthma. In the first 3 to 4 years of life, day care attendance has been linked to an increased incidence of lower respiratory illnesses and recurrent wheezing[

55]

but appears to protect against asthma later in childhood. Interestingly, these 51]

protective effects appear to be maximized if the child attends day care in the first 6 months of life. [

In addition to the timing of the infection, evidence

1228

indicates that the route of exposure may also be influential, because food-borne or enteric infections may have greater effects on the subsequent development of allergic sensitization than do respiratory infections.[

56]

Endotoxin

It has been proposed that exposure to microbes or microbial products in the environment may be even more important than the number of clinical infections per se in

helping the immune system to mature. [

57]

This concept is supported by the finding that exposure to a farming environment in early childhood, and having exposure 58 59 60 61

to high levels of lipopolysaccharide (LPS) is associated with reduced rates of allergy and asthma.[ ] [ ] [ ] [ ] Collectively, these findings suggest that prolonged exposure to common microbial products may protect against the development of allergies and asthma. If these concepts are confirmed, determining the relevant mechanisms, discussed later in regard to Th1/Th2 cytokine balance, could lead to novel approaches to the development of secondary prevention strategies. Pet Exposure

Traditional dogmatic statements regarding cat and dog allergens have emphasized that the presence of an animal in the home of a child at risk for developing allergic diseases increases the child's risk of allergic sensitization. This thinking has also been applied to children at risk for developing asthma. Ironically, recent studies suggest that early-life exposure to pets may actually protect against developing asthma in later life.[ linked to a decreased risk of recurrent wheezing in children with no family history of asthma. incidence of asthma in newborns followed until 7 years of

64 age.[ ]

[63]

62]

For example, dog allergen exposure in early life has been

For cat-exposed individuals, early exposure does not increase the

In fact, the presence of two or more cats or dogs during the first year of life may protect against 65

not only asthma but also allergic sensitization to non–animal-based allergens.[ ] Unlike mite allergens, greater levels of cat dander exposure during adolescence may induce a modified Th2 response (increased allergen-specific IgG4 level) that is associated with a reduction in cat sensitization (IgE antibody formation) and no 66

67

increase in asthma prevalence.[ ] Conversely, cat exposure has also recently been related to an increased risk of asthma in Germany after reunification.[ ] These findings indicate that the timing and amount of allergen exposure, as well as the physiochemical nature of the allergen itself, may significantly influence allergic sensitization and possibly asthma as well. Hospitalization Hospitalization rates for asthma in urban environments such as New York City correlate with low median family income, percentage of minorities in the population, and percentage of children under age 18 years.[

68]

Hospitalization rates for children with asthma gradually increased by 1.4% per year from 1980 to 1999 but may 25]

have leveled off after 1996 (21.6 per 10,000 in 1980–81, 31.7 per 10,000 in 1995–96 and 26.9 per 10,000 in 1998–99). [ in rates occurred in children up to 4 years of age and among black

2 25 children.[ ] [ ]

During these periods the greatest increase

Also, among children 5 to 14 years of age, boys were 1.3 times more likely to be

hospitalized, whereas the opposite gender preponderance was noted in the population 15 to 24 years of age (females 2.1 times more likely to be hospitalized).[

69]

Mortality Between 1980 and 1993, asthma accounted for 3850 deaths among patients up to 24 years of age. Asthma death rates increased by an average of 3.4% per year from 25

1980 to 1998, reaching a peak of 3.8 per 1 million children in 1996. [ ] Childhood asthma deaths then declined 18% in 1997, only to rise again in 1998 to 3.5 per 1 million children. Black non-Hispanic children had the highest asthma death rates and the greatest increase over time (an increase of 4.1 to 11.7 per 1 million between 1985 to 1986 and 1995 to 1996). Mortality in black non-Hispanic children has been consistently higher compared with white non-Hispanic children (4.1 times higher 25]

during 1985–86 and 4.6 times higher during 1997–98). Adolescents have asthma mortality rates that are approximately twice those of younger children.[

Risk factors for fatal asthma in children are similar to those in adults.[ severe exacerbations of asthma, as follows: 1. 2. 3. 4.

70]

Four general risk factors seem to be the most important in influencing outcomes related to

Current use of, or withdrawal from, systemic corticosteroids Hospitalization or emergency care visit(s) for asthma in the past year History of psychiatric disease or psychosocial problems History of noncompliance with asthma medications

Other factors associated with asthma deaths in children include ethnicity and socioeconomic status related to poor access to and interaction with health care facilities 18

71

72

and providers,[ ] passive smoke exposure,[ ] and deaths resulting from anaphylaxis after inadvertent ingestion of foods to which the patient is allergic.[ ] Previous near-fatal episodes of asthma also increase the risk for subsequent death from asthma, and many of these individuals have a history of prior intubation and mechanical ventilation. In children ages 5 to 12 years admitted over a 10-year period (1984–1994) at the University of California Davis Medical Center, 13 of 300 73

children admitted for asthma were subsequently intubated.[ ] Significant risk factors associated with intubation (ranked by descending ORs) are second-hand smoke exposure, psychosocial problems, family dysfunction, upper respiratory infection, little formal education, prior asthma ED visit(s) in past year, prior hospitalization for asthma in past year, crowding, low socioeconomic status, steroid dependence, parental history of asthma or allergy, and language barrier.

PATHOPHYSIOLOGY The pathophysiologic factors believed to contribute to the reversible airflow obstruction in asthma are reviewed in depth elsewhere in this textbook; however, the demonstration that airway inflammation exists even in patients with mild asthma underscores the importance of better defining its origin and its fluctuations with disease patterns or severity over time. To this end, learning more about its contribution to infantile wheezing and childhood asthma would provide important insight into the natural history of asthma. In one of the first biopsy studies in children, Cutz et al[

74]

reported significant airway inflammatory changes in nonfatal

1229

asthma. Lung biopsies from two children with bronchial asthma in remission were compared to those of two children dying in status asthmaticus. Bronchial changes, such as goblet cell hyperplasia, mucus plugging, and increased collagen deposition beneath the epithelial basement membrane, were comparable in the two groups. Interestingly, the only differences were the presence of a larger number of submucosal eosinophils and more extensive denudation of the epithelium in fatal asthma. In a recent study of airway structure that compared adolescents/young adults and older adults who had fatal asthma with control subjects, airway wall characteristics differed among these three groups. Increased airway wall/ smooth muscle area and increased airway narrowing were seen in the older patients compared with the younger patients, suggesting that airway remodeling occurs with aging. However, the young asthmatic patients had more connective tissue deposition, greater intraluminal obstruction, and thicker subepithelial collagen than similarly aged control patients, suggesting that these changes begin early in life.[

75]

Reinforcing this

76

finding, Cokugras et al[ ] evaluated bronchial biopsy samples from asthmatic children 5 to 14 years old without recent respiratory infections or inhaled antiinflammatory treatment. Electron and light microscopic examination of biopsies showed thickening and hyalinization of the basement membrane, “overactive fibroblasts,” loss of cilia, lymphocyte accumulation, and evidence of mast cell degranulation. Although few in number, biopsy studies in children demonstrate earlyonset inflammatory changes consistent with the concept that asthma has its origins early in life. Because of obvious ethical issues and technical factors, premortem information regarding asthmatic inflammation using invasive techniques is not as abundant in children as in adults. Within the past few years, however, a number of interesting observations have emerged. Using a variety of techniques, including sampling of peripheral blood, nasal washings, sputum induction, and bronchoalveolar lavage (BAL), a spectrum of abnormalities have been described in children with a variety of airway diseases that are characterized by wheezing and airway obstruction, including asthma. 77

Hoekstra et al[ ] noted that the number of blood eosinophils, serum eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), and urinary EDN were significantly higher in asthmatic children (ages 4–14 years) compared with controls. Nocturnal peak expiratory flow rates and 1-second forced expiratory 78

volume were significantly related to urinary EDN. Zimmerman et al[ ] observed that serum ECP levels were higher in symptomatic versus asymptomatic asthmatic children (mean age 12 years), and that the initiation of corticosteroid therapy in the symptomatic group increased pulmonary function while decreasing serum ECP 79

levels. Schauer et al[ ] found similar numbers of eosinophils and serum ECP levels among 24 nonatopic children born prematurely (15 of whom were labeled as having hyperresponsive airways based on a histamine PC60 sGaw [specific conductance] 30/min

Respiratory Arrest Imminent

Symptoms

Drowsy or confused

Signs Respiratory rate

Guide to rates of breathing in awake children: Age

Normal rate

1250•g

Fluticasone

88–264•g

88–176•g

264–660•g

176–440•g

>660•g

>440•g

DPI: 50, 100, or 250• 100–300•g • g/inhalation

100–200•g

300–600•g

200–400•g

>600•g

>400•G

Triamcinolone acetonide 100•• g/ puff

400–800•g

1000–2000•g

800–1200•g

>2000•g

1200•g

MDI: 44, 110, or 220••g/puff

400–1000•g

From National Institutes of Health: NAEPP guidelines for the management and diagnosis of asthma. National Asthma Education and Prevention Program, NIH Pub No 02-5075, Bethesda, Md, 2002, NIH. CFC, Chlorofluorocarbon; HFA, hydroxyfluoroalkane; DPI, dry powder inhaler; MDI, metered-dose inhaler. * Children ≤12 years of age.

adjunct to a beta agonist.[

179]

Most, but not all,[

180]

studies evaluating the use of intravenous theophylline in the treatment of acute exacerbations have been unable to 181] [182] [183]

demonstrate any additional benefit to aggressive intervention with beta agonists[ [180] [184]

; its use in hospitalized asthmatic children also remains controversial.

Although magnesium sulfate may be of benefit in children failing bronchodilator and corticosteroid therapy, double-blind placebo-controlled studies have

both supported [

185] [186] [187]

and refuted [

188]

its efficacy.

Hospital Management

Despite similar histopathologic changes noted in status asthmaticus in both adult and pediatric patients, considerations particular to infants and young children are relevant to the pathogenesis and management of children in status asthmaticus. Besides the anatomic and physiologic features of early life that predispose to airway narrowing (see earlier discussion), the unique susceptibility of infants and young children to fluid and acid-base disturbances during various diseases is well known to pediatricians. In childhood status asthmaticus, respiratory acidosis is frequently complicated by the development of a metabolic acidosis, which is observed infrequently in adults. In particular, there is a trend toward larger base deficits in young children. [

18]

The administration of sodium bicarbonate to patients in status 189]

asthmaticus to correct the metabolic acidemia seems rational, provided alveolar ventilation is sufficient to eliminate the carbon dioxide produced.[ potential for development of hypokalemia from frequent β agonist administration also needs to be

Further, the

190 evaluated.[ ]

During acute asthmatic episodes, hypoxemia is the first ABG abnormality to be noted. As such, oxygen saturation should be monitored frequently and oxygen administered to achieve acceptable levels of saturation. Because ABG determinations are uncomfortable for children, the use of pulse oximetry as a surrogate indicator of hypoxemia has been evaluated. Some groups have concluded that oxygen saturation greater than 92% is associated with a low risk for respiratory failure, [191]

whereas others have argued that the sensitivity and specificity of saturation in this range are inadequate to predict the potential for eventual adverse outcomes.

[192]

If objective measurements of pulmonary function (e.g., PEF, FEV1 ) are 40% or less of predicted values after adequate bronchodilator intervention, the patient

likely is hypercarbic; therefore ABGs should be obtained to evaluate the individual prospectively for development of respiratory failure and need for rapid intubation. 18]

Medical therapy involves the use of frequent or continuous β agonist nebulization treatments[ corticosteroids at regular intervals[ asthma is controversial.

193]

(with or without ipratropium bromide) and oral or intravenous

(see Figure 68-2 and Table 68-5 ). As mentioned previously, the use of intravenous beta agonists in the management of acute

In addition to relieving the acute airway obstruction that resulted in ED or hospital intervention by appropriate medical monitoring and therapeutic intervention, one of the most important aspects of this event should be the prevention of recurrent asthma exacerbations. Prevention can be accomplished most successfully by ensuring that patients use the time in the medical facility to optimize their asthma education ( Table 68-7 ). Trained clinical personnel should review the names and purposes of the various asthma medications, teach proper inhaler technique and the use of objective monitoring devices, schedule follow-up visits, and construct a mutually satisfactory action plan that includes age-appropriate maintenance and intervention strategies.

1240

Figure 68-1 Management of asthma exacerbations: home treatment.

Figure 68-2 Emergency management of asthma exacerbations in children. ICU, Intensive care unit; ER, emergency room; PEF, peak expiratory flow rate; FEV1 , forced expiratory volume in 1 second; PO, oral(ly); IV, intravenous(ly); q20min, every 20 minutes; PE, physical examination; RR, respiratory rate.

TABLE 68-7 -- Hospital and Emergency Department Discharge Checklist for Patients with Asthma Exacerbations Intervention

Dose/Timing

Education/Advice

Inhaled medications (MDI + spacer/holding chamber Select agent, dose, and frequency (e.g., albuterol 2 puffs, q3-4h prn; inhaled corticosteroid, medium or DPI) β2 agonist dose)

Teach purpose Teach technique Emphasize need for spacer/holding chamber with MDIs

Corticosteroids

Check patient performance

Oral medications

Select agent, dose, and frequency (e.g., prednisone 20•mg bid for 3–10 days)

Teach purpose Teach side effects

Peak flow meter

Measure am and pm PEF and record best of three efforts each time

Teach purpose Teach technique

Follow-up visit

Make appointment for follow-up care with primary clinician or asthma specialist

Distribute peak flow diary

Action plan

Before or at discharge

Advise patient of date, time, and location of appointment within 7 days of hospital discharge Instruct patient on simple plan for actions to be taken for symptoms, signs, and PEF values suggesting recurrent airflow obstruction

From National Institutes of Health: NAEPP guidelines for the management and diagnosis of asthma. National Asthma Education and Prevention Program, NIH Pub No 02-5075, Bethesda, Md, 2002, NIH. MDI, metered-dose inhaler; DPI, dry powder inhaler; prn, as needed; PEF, peak expiratory flow; q3-4h, every 3 to 4 hours; bid, twice daily. 70 116

panels as a means to achieve and maintain control of asthmatic symptoms[ ] [ ] ( Table 68-8 ). These various steps represent the level of symptoms before initiation of asthma therapy and classify asthma into four levels of severity: intermittent, mild persistent, moderate persistent, and severe persistent.

TABLE 68-8 -- Stepwise Approach for Managing Asthma in Teenagers/Adults and in Children Over 5 Years Old: Clinical Features Before Treatment Symptoms

†

Nighttime Symptoms

Lung Function FEV1 /PEF≤ 60% predicted

Step 4 Severe Persistent

Continual symptoms Limited physical activity Frequent exacerbations

Frequent

Step 3 Moderate Persistent

Daily symptoms Daily use of inhaled short-acting β2

>1 time a week

PEF variability >30% FEV1 /PEF>60% to 30%

agonist Exacerbations affect activity Exacerbations>2 times a week: may last days Step 2 Mild Persistent

Symptoms >2 times a week but 2 times a month

FEV1 /PEF ≥80% predicted PEF variability 20%–30%

Step 1 Mild Intermittent

Symptoms ≤2 times a week Asymptomatic and normal PEF between exacerbations Exacerbations brief (from a few hours to a few days); intensity may vary

≤2 times a month

FEV1 /PEF ≥80% predicted PEF variability 1 night/week

• Preferred treatment: - Low-dose inhaled corticosteroids and long acting inhaled β2 agonists. or - Medium-dose inhaled corticosteroids. • Alternative treatment: - Low-dose inhaled corticosteroids and either leukotriene receptor antagonist or theophylline.

Step 2 Mild Persistent

> 2× /week but < 1× /day >2 nights/month

If needed (particularly in patients with recurring severe exacerbations): • Preferred treatment: - Medium-dose inhaled corticosteroids and long-acting β2 agonists. • Alternative treatment: - Medium-dose inhaled corticosteroids and either leukotriene receptor antagonist or theophylline.

Step 1 Mild Intermittent

≤2 days/week ≤2 nights/month

• Preferred treatment: - Low-dose inhaled corticosteroids (with nebulizer or MDI with holding chamber with or without face mask or DPI). • Alternative treatment (listed alphabetically): - Cromolyn (nebulizer is preferred or MDI with holding chamber) or leukotriene receptor antagonist. • No daily medication needed.

Quick Relief All Patients

• Bronchodilator as needed for symptoms. Intensity of treatment will depend upon severity of exacerbation. - Preferred treatment: Short-acting inhaled β2 agonists by nebulizer or face mask and spacer/holding chamber - Alternative treatment: Oral β2 agonist • With viral respiratory infection - Bronchodilator every 4–6 hours up to 24 hours (longer with physician consult); in general, repeat no more than once every 6 weeks - Consider systemic corticosteroid if exacerbation is severe or patient has history of previous severe exacerbations • Use of short-acting β2 agonists >2 times a week in intermittent asthma (daily, or increasing use in persistent asthma) may indicate the need to initiate (increase) long-term control therapy.

↓ Step-down Review treatment every 1 to 6 months; a gradual stepwise reduction in treatment may be possible.

Note • The stepwise approach is intended to assist, not replace, the clinical decision making required to meet individual patient needs. • Classify severity: assign patient to most severe step in which any feature occurs • There are very few studies on asthma therapy for infants. • Gain control as quickly as possible (a course of short systemic corticosteroids may be required); then step-down to the least medication necessary to maintain control. • Provide parent education on asthma management and controlling environmental factors that make asthma worse (e.g., allergens and irritants). • Consultation with an asthma specialist is recommended for patients with moderate or severe persistent asthma. Consider consultation for patients with mild persistent asthma.

↑Step-up If control is not maintained, consider step-up. First, review patient medication technique, adherence, and environmental control. • Minimal use of short-acting inhaled β2

Goals of Therapy: Asthma Control • Minimal or no chronic symptoms day or night • Minimal or no exacerbations • No limitations on activities; no school/ parent's work missed

agonist (1 night/week

>60% to 30%

• Preferred treatment: - Low-to-medium dose inhaled corticosteroids and long-acting inhaled β2 agonists. • Alternative treatment (listed alphabetically): - Increase inhaled corticosteroids within mediumdose range or - Low to medium-dose inhaled corticosteroids and either leukotriene modifier or theophylline. If needed (particularly in patients with recurring severe exacerbations): • Preferred treatment: - Increase inhaled corticosteroids within mediumdose range and add long-

acting β2 agonists • Alternative treatment: - Increase inhaled corticosteroids within mediumdose range and add either leukotriene modifier or theophylline.

Step 2 Mild Persistent

> 2x /week but < 1x /day >2 nights/month

≥80% 20–30%

• Preferred treatment: - Low-dose inhaled corticosteroids. • Alternative treatment (listed alphabetically): - Cromolyn, leukotriene modifier, nedocromil, or sustained release theophylline to serum concentration of 5– 15•g/ml.

Step 1Mild Intermittent

≤2 days/week ≤2 nights/month

≥80% 2 times a week in intermittent asthma (daily, or increasing use in persistent Asthma) may indicate the need to initiate (increase) long-term control therapy.

↓ Step-down Review treatment every 1 to 6 months; a gradual stepwise reduction in treatment may be possible.

Note • The stepwise approach is intended to assist, not replace, the clinical decision making required to meet individual patient needs. • Classify severity: assign patient to most severe step in which any feature occurs (PEF is % of personal best; FEV1 is % predicted). • Gain control as quickly as possible (a course of short systemic corticosteroids); then step-down to the least medication necessary to maintain control. • Provide education on selfmanagement and controlling environmental factors that make asthma worse (e.g., allergies and irritants). • Refer to an asthma specialist if there are difficulties controlling asthma or if step 4 care is required. Referral may be considered if step 3 care is required.

↑ Step-up If control is not maintained, consider step-up. First, review patient medication technique, adherence, and environment control.

• Maintain (near) normal pulmonary function • Minimal use of short-acting inhaled β2 agonist ( paternal) history of asthma.[ ] In a clinical index 137

developed to define future asthma risk in young children,[ ] a child having recurrent wheezing with one major (parental history of asthma or history of child having atopic dermatitis) or two of three minor criteria (allergic rhinitis, wheezing apart from colds, and peripheral eosinophilia >4%) had a 4.3 to 9.8 times greater risk of having asthma during later school years (6–13 years old). Active asthma was present in 76% of children with a “positive” index, and asthma was not present in 95% of children with a “negative” index. When evaluating possible relationships between the presence of childhood asthma symptoms at age 7 years and the presence of similar symptoms at age 25 years, a slightly different risk profile emerges. More than 1400 men and women were surveyed in 1991–1993 at ages 29 to 32 years in Tasmania, Australia, for self-reported 279

asthma or wheezy breathing in the previous 12 months.[ ] Risk factors measured at age 7 years that independently predicted current asthma as an adult included female gender, history of atopic dermatitis, presence of low mid-expiratory flow rates, maternal or paternal history of asthma, presence of childhood asthma with the first episode after age 2 years, or having more than 10 attacks. Finally, data obtained from a cohort of 378 asthmatic children in Melbourne, Australia, evaluated every 7 years from age 7 through 35 years provided information linking asthma and other atopic conditions. The presence of an atopic condition in childhood increased the risk of severe asthma in later life, whereas severe asthma in childhood predicted an increased risk of atopic eczema or allergic rhinitis in later life.[

280]

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151. Bousquet J, Van Cauwenberge P, Khaltaev N: Allergic rhinitis and its impact on asthma, J Allergy Clin Immunol 108:S147, 2001. 152. Sheikh S, Goldsmith LJ, Howell L, et al: Lung function in infants with wheezing and gastroesophageal reflux, Pediatr Pulmonol 27:236, 1999. 153. Ayres JG, Miles JF: Oesophageal reflux and asthma, Eur Respir J 9:1073, 1996. 154. Harding SM: Gastroesophageal reflux and asthma: insight into the association, J Allergy Clin Immunol 104:251, 1999. 155. Sheikh S, Stephen T, Howell L, et al: Gastroesophageal reflux in infants with wheezing, Pediatr Pulmonol 28:181, 1999. 156. Vijayaratnam V, Lin CH, Simpson P, et al: Lack of significant proximal esophageal acid reflux in infants presenting with respiratory symptoms, Pediatr Pulmonol 27:231, 1999.

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157. Russell G: Childhood asthma and growth: a review of the literature, Respir Med 88(suppl A):31, 1994. 158. Allen DB: Growth suppression by glucocorticoid therapy, Endocrinol Metab Clin North Am 25:699, 1996. 159. Silverstein MD, Yunginger JW, Reed CE, et al: Attained adult height after childhood asthma: effect of glucocorticoid therapy, J Allergy Clin Immunol 99:466, 1997. 160. Allen DB, Bronsky EA, LaForce CF, et al: Growth in asthmatic children treated with fluticasone propionate. Fluticasone Propionate Asthma Study Group, J Pediatr 132:472, 1998. 161. Volovitz B, Amir J, Malik H, et al: Growth and pituitary-adrenal function in children with severe asthma treated with inhaled budesonide, N Engl J Med 329:1703, 1993. 162. Nicolaizik WH, Marchant JL, Preece MA, et al: Endocrine and lung function in asthmatic children on inhaled corticosteroids, Am J Respir Crit Care Med 150:624, 1994. 163. Pedersen S, Steffensen G, Ekman I, et al: Pharmacokinetics of budesonide in children with asthma, Eur J Clin Pharmacol 31:579, 1987. 164. Childhood Asthma Management Program (CAMP) Research Group: Long-term effects of budesonide or nedocromil in children with asthma, N Engl J Med 343:1054, 2000. 165. Agertoft L, Pedersen S: Effect of long-term treatment with inhaled budesonide on adult height in children with asthma, N Engl J Med 343:1064, 2000. 166. Tinkelman DG, Reed CE, Nelson HS, et al: Aerosol beclomethasone dipropionate compared with theophylline as primary treatment of chronic, mild to

moderately severe asthma in children, Pediatrics 92:64, 1993. 167. Doull IJ, Freezer NJ, Holgate ST: Growth of prepubertal children with mild asthma treated with inhaled beclomethasone dipropionate, Am J Respir Crit Care Med 151:1715, 1995. 168. Simons FE, Canadian Beclomethasone Dipropionate-Salmeterol Xinafoate Study Group: A comparison of beclomethasone, salmeterol, and placebo in children with asthma, N Engl J Med 337:1659, 1997. 169. Verberne AA, Frost C, Roorda RJ, et al: One year treatment with salmeterol compared with beclomethasone in children with asthma, Am J Respir Crit Care Med 156:688, 1997. Treatment 170. Ellis EF: Asthma in infancy and childhood. In Middleton E Jr, Reed CE, Ellis EF et al, editors: Allergy: principles and practice, ed 4, St Louis, 1993, Mosby, p 1225. 171. Ploin D, Chapuis FR, Stamm D, et al: High-dose albuterol by metered-dose inhaler plus a spacer device versus nebulization in preschool children with recurrent wheezing: a double-blind, randomized equivalence trial, Pediatrics 106:311, 2000. 172. Dewar AL, Stewart A, Cogswell JJ, et al: A randomised controlled trial to assess the relative benefits of large volume spacers and nebulisers to treat acute asthma in hospital, Arch Dis Child 80:421, 1999. 173. Schuh S, Johnson DW, Callahan S, et al: Efficacy of frequent nebulized ipratropium bromide added to frequent high-dose albuterol therapy in severe childhood asthma, J Pediatr 126:639, 1995. 174. Qureshi F, Pestian J, Davis P, et al: Effect of nebulized ipratropium on the hospitalization rates of children with asthma, N Engl J Med 339:1030, 1998. 175. Ducharme FM, Davis GM: Randomized controlled trial of ipratropium bromide and frequent low doses of salbutamol in the management of mild and moderate acute pediatric asthma, J Pediatr 133:479, 1998. 176. Zorc JJ, Pusic MV, Ogborn CJ, et al: Ipratropium bromide added to asthma treatment in the pediatric emergency department, Pediatrics 103:748, 1999. 177. Craven D, Kercsmar CM, Myers TR, et al: Ipratropium bromide plus nebulized albuterol for the treatment of hospitalized children with acute asthma, J Pediatr 138:51, 2001. 178. Goggin N, Macarthur C, Parkin PC: Randomized trial of the addition of ipratropium bromide to albuterol and corticosteroid therapy in children hospitalized because of an acute asthma exacerbation, Arch Pediatr Adolesc Med 155:1329, 2001. 179. Schuh S, Reisman J, Alshehri M, et al: A comparison of inhaled fluticasone and oral prednisone for children with severe acute asthma, N Engl J Med 343:689, 2000. 180. Ream RS, Loftis LL, Albers GM, et al: Efficacy of IV theophylline in children with severe status asthmaticus, Chest 119:1480, 2001.

181. Littenberg B: Aminophylline treatment in severe, acute asthma: a meta-analysis, JAMA 259:1678, 1988. 182. Fanta CH, Rossing TH, McFadden ER Jr: Treatment of acute asthma: is combination therapy with sympathomimetics and methylxanthines indicated? Am J Med 80:5, 1986. 183. Huang D, O'Brien RG, Harman E, et al: Does aminophylline benefit adults admitted to the hospital for an acute exacerbation of asthma? Ann Intern Med 119:1155, 1993. 184. Strauss RE, Wertheim DL, Bonagura VR, et al: Aminophylline therapy does not improve outcome and increases adverse effects in children hospitalized with acute asthmatic exacerbations, Pediatrics 93:205, 1994. 185. Ciarallo L, Sauer AH, Shannon MW: Intravenous magnesium therapy for moderate to severe pediatric asthma: results of a randomized, placebo-controlled trial, J Pediatr 129:809, 1996. 186. Gurkan F, Haspolat K, Bosnak M, et al: Intravenous magnesium sulfate in the management of moderate to severe acute asthmatic children nonresponding to conventional therapy, Eur J Emerg Med 6:201, 1999. 187. Ciarallo L, Brousseau D, Reinert S: Higher-dose intravenous magnesium therapy for children with moderate to severe acute asthma, Arch Pediatr Adolesc Med 154:979, 2000. 188. Scarfone RJ, Loiselle JM, Joffe MD, et al: A randomized trial of magnesium in the emergency department treatment of children with asthma, Ann Emerg Med 36:572, 2000. 189. Ostrea EM Jr, Odell GB: The influence of bicarbonate administration on blood pH in a closed system: clinical implications, J Pediatr 80:671, 1972. 190. Gern JE, Lemanske RF Jr: β-Adrenergic agonist therapy. In Bush RK, editor: Immunology and allergy clinics of North America, Philadelphia, 1993, Saunders, p 839. 191. Carruthers DM, Harrison BD: Arterial blood gas analysis or oxygen saturation in the assessment of acute asthma? Thorax 50:186, 1995. 192. Mayefsky JH, el-Shinaway Y: The usefulness of pulse oximetry in evaluating acutely ill asthmatics, Pediatr Emerg Care 8:262, 1992. 193. Barnett PL, Caputo GL, Baskin M, et al: Intravenous versus oral corticosteroids in the management of acute asthma in children, Ann Emerg Med 29:212, 1997. 194. Beausoleil JL, Weldon DP, McGeady SJ: β2 -Agonist metered dose inhaler overuse: psychological and demographic profiles, Pediatrics 99:40, 1997. 195. Agertoft L, Pedersen S: Effects of long-term treatment with an inhaled corticosteroid on growth and pulmonary function in asthmatic children, Respir Med 88:373, 1994. 196. Panhuysen CI, Vonk JM, Koeter GH, et al: Adult patients may outgrow their asthma: a 25-year follow-up study, Am J Respir Crit Care Med 155:1267, 1997.

197. Oswald H, Phelan PD, Lanigan A, et al: Childhood asthma and lung function in mid-adult life, Pediatr Pulmonol 23:14, 1997. 198. Pedersen S: Do inhaled corticosteroids inhibit growth in children? Am J Respir Crit Care Med 164:521, 2001. 199. Allen DB, Mullen M, Mullen B: A meta-analysis of the effect of oral and inhaled corticosteroids on growth, J Allergy Clin Immunol 93:967, 1994. 200. Ferguson AC, Spier S, Manjra A, et al: Efficacy and safety of high-dose inhaled steroids in children with asthma: a comparison of fluticasone propionate with budesonide, J Pediatr 134:422, 1999. 201. Hollman GA, Allen DB: Overt glucocorticoid excess due to inhaled corticosteroid therapy, Pediatrics 81:452, 1988. 202. Shapiro G, Mendelson L, Kraemer MJ, et al: Efficacy and safety of budesonide inhalation suspension (Pulmicort Respules) in young children with inhaled steroid-dependent, persistent asthma, J Allergy Clin Immunol 102:789, 1998. 203. Baker JW, Mellon M, Wald J, et al: A multiple-dosing, placebo-controlled study of budesonide inhalation suspension given once or twice daily for treatment of persistent asthma in young children and infants, Pediatrics 103:414, 1999. 204. Scott MB, Skoner DP: Short-term and long-term safety of budesonide inhalation suspension in infants and young children with persistent asthma, J Allergy Clin Immunol 104:200, 1999. 205. Suissa S, Ernst P, Benayoun S, et al: Low-dose inhaled corticosteroids and the prevention of death from asthma, N Engl J Med 343:332, 2000. 206. Simons FE, Soni NR, Watson WT, et al: Bronchodilator and bronchoprotective effects of salmeterol in young patients with asthma, J Allergy Clin Immunol 90:840, 1992. 207. Meijer GG, Postma DS, Mulder PG, et al: Long-term circadian effects of salmeterol in asthmatic children treated with inhaled corticosteroids, Am J Respir Crit Care Med 152:1887, 1995. 208. Lenney W, Pedersen S, Boner AL, et al: Efficacy and safety of salmeterol in childhood asthma, Eur J Pediatr 154:983, 1995. 209. Becker AB, Simons FER: Formoterol, a new long-acting selective beta- 2-adrenergic receptor agonist: double-blind comparison with salbutamol and placebo in children with asthma, J Allergy Clin Immunol 84:891, 1989. 210. Foucard T, Lonnerholm G: A study with cumulative doses of formoterol and salbutamol in children with asthma, Eur Respir J 4:1174, 1991. 211. Akpinarli A, Tuncer A, Saraclar Y, et al: Effect of formoterol on clinical parameters and lung functions in patients with bronchial asthma: a randomised controlled trial, Arch Dis Child 81:45, 1999. 212. Von Berg A, Berdel D: Formoterol and salbutamol metered aerosols: comparison of a new and an established beta-2-agonist for their bronchodilating efficacy in the treatment of childhood bronchial asthma, Pediatr Pulmonol 7:89, 1989. 213. Bartow RA, Brogden RN: Formoterol: an update of its pharmacological properties and therapeutic efficacy in the management of asthma, Drugs 55:303, 1998.

214. Verberne AA, Hop WC, Creyghton FB, et al: Airway responsiveness after a single dose of salmeterol and during four months of treatment in children with asthma, J Allergy Clin Immunol 97:938, 1996. 215. Russell G, Williams DA, Weller P, et al: Salmeterol xinafoate in children on high dose inhaled steroids, Ann Allergy Asthma Immunol 75:423, 1995. 216. Primhak RA, Smith CM, Yong SC, et al: The bronchoprotective effect of inhaled salmeterol in preschool children: a dose-ranging study, Eur Respir J 13:78, 1999. 217. Simons FE, Gerstner TV, Cheang MS: Tolerance to the bronchoprotective effect of salmeterol in adolescents with exercise-induced asthma using concurrent inhaled glucocorticoid treatment, Pediatrics 99:655, 1997. 218. Lipworth B, Tan S, Devlin M, et al: Effects of treatment with formoterol on bronchoprotection against methacholine, Am J Med 104:431, 1998. 219. Greening AP, Wind P, Northfield M, et al: Added salmeterol versus higher-dose corticosteroid in asthma patients with symptoms on existing inhaled corticosteroid, Lancet 344:219, 1994. 220. Woolcock A, Lundback B, Ringdal N, et al: Comparison of addition of salmeterol to inhaled steroids with doubling of the dose of inhaled steroids, Am J Respir Crit Care Med 153:1481, 1996.

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221. Faurschou P, Steffensen I, Jacques L, et al: Effect of addition of inhaled salmeterol to the treatment of moderate-to-severe asthmatics uncontrolled on high-dose inhaled steroids, Eur Respir J 9:1885, 1996. 222. Van den Berg NJ, Ossip MS, Hederos CA, et al: Salmeterol/fluticasone propionate (50/100••g) in combination in a Diskus inhaler (Seretide) is effective and safe in children with asthma, Pediatr Pulmonol 30:97, 2000. 223. Bisgaard H: Long-acting beta(2)-agonists in management of childhood asthma: a critical review of the literature, Pediatr Pulmonol 29:221, 2000. 224. Lemanske RF Jr, Sorkness CA, Mauger EA, et al: Inhaled corticosteroid reduction and elimination in patients with persistent asthma receiving salmeterol: a randomized controlled trial, JAMA 285:2594, 2001. 225. Verberne AA, Frost C, Duiverman EJ, et al: Addition of salmeterol versus doubling the dose of beclomethasone in children with asthma, Am J Respir Crit Care Med 158:213, 1998. 226. Pauwels RA, Löfdahl CG, Postma DS, et al: Effect of inhaled formoterol and budesonide on exacerbations of asthma, N Engl J Med 337:1405, 1997. 227. Heuck C, Heickendorff L, Wolthers OD: A randomised controlled trial of short term growth and collagen turnover in asthmatics treated with inhaled formoterol and budesonide, Arch Dis Child 83:334, 2000.

228. Lazarus SC, Boushey HA, Fahy JV, et al: Long-acting beta2-agonist monotherapy vs continued therapy with inhaled corticosteroids in patients with persistent asthma: a randomized controlled trial, JAMA 285:2583, 2001. 229. Stelmach I, Jerzynska J, Brzozowska A, et al: Double-blind, randomized, placebo-controlled trial of effect of nedocromil sodium on clinical and inflammatory parameters of asthma in children allergic to dust mite, Allergy 56:518, 2001. 230. Tasche MJ, Uijen JH, Bernsen RM, et al: Inhaled disodium cromoglycate (DSCG) as maintenance therapy in children with asthma: a systematic review, Thorax 55:913, 2000. 231. Nassif EF, Weinberger MM, Thompson R, et al: The value of maintenance theophylline in steroid-dependent asthma, N Engl J Med 304:71, 1981. 232. Pearlman DS, Ostrom NK, Bronsky EA, et al: The leukotriene D4 -receptor antagonist zafirlukast attenuates exercise-induced bronchoconstriction in children, J Pediatr 134:273, 1999. 233. Pearlman DS, Lampl KL, Dowling PJ Jr, et al: Effectiveness and tolerability of zafirlukast for the treatment of asthma in children, Clin Ther 22:732, 2000. 234. Nathan RA, Bernstein JA, Bielory L, et al: Zafirlukast improves asthma symptoms and quality of life in patients with moderate reversible airflow obstruction, J Allergy Clin Immunol 102:935, 1998. 235. Kemp JP, Dockhorn RJ, Shapiro GG, et al: Montelukast once daily inhibits exercise-induced bronchoconstriction in 6- to 14-year-old children with asthma, J Pediatr 133:424, 1998. 236. Knorr B, Matz J, Bernstein JA, et al: Montelukast for chronic asthma in 6- to 14-year-old children: a randomized double-blind trial. Pediatric Montelukast Study Group, JAMA 279:1181, 1998. 237. Bisgaard H, Nielsen KG: Bronchoprotection with a leukotriene receptor antagonist in asthmatic preschool children, Am J Respir Crit Care Med 162:187, 2000. 238. Knorr B, Franchi LM, Bisgaard H, et al: Montelukast, a leukotriene receptor antagonist, for the treatment of persistent asthma in children aged 2 to 5 years, Pediatrics 108:E48, 2001. 239. Simons FE, Villa JR, Lee BW, et al: Montelukast added to budesonide in children with persistent asthma: a randomized, double-blind, crossover study, J Pediatr 138:694, 2001. 240. Milgrom H, Fick RB Jr, Su JQ, et al: Treatment of allergic asthma with monoclonal anti-IgE antibody, N Engl J Med 341:1966, 1999. 241. Busse W, Corren J, Lanier BQ, et al: Omalizumab, anti-IgE recombinant humanized monoclonal antibody, for the treatment of severe allergic asthma, J Allergy Clin Immunol 108:184, 2001. 242. Milgrom H, Berger W, Nayak A, et al: Treatment of childhood asthma with anti-immunoglobulin E antibody (omalizumab), Pediatrics 108:E36, 2001. 243. Custovic A, Green R, Taggart SC, et al: Domestic allergen in public places. II. Dog (Can f 1) and cockroach (Bla g 2) allergens in dust and mite, cat, dog, and

cockroach allergens in the air in public buildings, Clin Exp Allergy 26:1246, 1996. 244. Peat JK, Tovey E, Toelle BG, et al: House dust mite allergens: a major risk factor for childhood asthma in Australia, Am J Respir Crit Care Med 153:141, 1996. 245. Hill DJ, Thompson PJ, Stewart GA, et al: Eliminating house dust mites in the domestic environment. Melbourne House Dust Mite Study, J Allergy Clin Immunol 99:323, 1997. 246. Huss K, Adkinson NF Jr, Eggleston PA, et al: House dust mite and cockroach exposure are strong risk factors for positive allergy skin test responses in the Childhood Asthma Management Program, J Allergy Clin Immunol 107:48, 2001. 247. Wilson NW, Robinson NP, Hogan MB: Cockroach and other inhalant allergies in infantile asthma, Ann Allergy Asthma Immunol 83:27, 1999. 248. Almqvist C, Larsson PH, Egmar AC, et al: School as a risk environment for children allergic to cats and a site for transfer of cat allergen to homes, J Allergy Clin Immunol 103:1012, 1999. 249. Perzanowski MS, Ronmark E, Nold B, et al: Relevance of allergens from cats and dogs to asthma in the northernmost province of Sweden: schools as a major site of exposure, J Allergy Clin Immunol 103:1018, 1999. 250. Lonnkvist K, Hallden G, Dahlen SE, et al: Markers of inflammation and bronchial reactivity in children with asthma, exposed to animal dander in school dust, Pediatr Allergy Immunol 10:45, 1999. 251. Almqvist C, Wickman M, Perfetti L, et al: Worsening of asthma in children allergic to cats, after indirect exposure to cat at school, Am J Respir Crit Care Med 163:694, 2001. 252. Klucka CV, Ownby DR, Green J, et al: Cat shedding of Fel d 1 is not reduced by washings, Allerpet-C spray, or acepromazine, J Allergy Clin Immunol 95:1164, 1995. 253. Schwartz J, Timonen KL, Pekkanen J: Respiratory effects of environmental tobacco smoke in a panel study of asthmatic and symptomatic children, Am J Respir Crit Care Med 161:802, 2000. 254. Holberg CJ, Wright AL, Martinez FD, et al: Child day care, smoking by caregivers, and lower respiratory tract illness in the first 3 years of life, Pediatrics 91:885, 1993. 255. Ross RN, Nelson HS, Finegold I: Effectiveness of specific immunotherapy in the treatment of asthma: a meta-analysis of prospective, randomized, doubleblind, placebo-controlled studies, Clin Ther 22:329, 2000. 256. Adkinson NF Jr, Eggleston PA, Eney D, et al: A controlled trial of immunotherapy for asthma in allergic children, N Engl J Med 336:324, 1997. 257. Abramson MJ, Puy RM, Weiner JM: Is allergen immunotherapy effective in asthma? A meta-analysis of randomized controlled trials, Am J Respir Crit Care Med 151:969, 1995. 258. Cools M, Van Bever HP, Weyler JJ, et al: Long-term effects of specific immunotherapy, administered during childhood, in asthmatic patients allergic to either

house-dust mite or to both house-dust mite and grass pollen, Allergy 55:69, 2000. 259. Des Roches A, Paradis L, Menardo JL, et al: Immunotherapy with a standardized Dermatophagoides pteronyssinus extract.VI. Specific immunotherapy prevents the onset of new sensitizations in children, J Allergy Clin Immunol 99:450, 1997. 260. Miller BD, Wood BL: Psychophysiologic reactivity in asthmatic children: a cholinergically mediated confluence of pathways, J Am Acad Child Adolesc Psychiatry 33:1236, 1994. 261. Fritz GK, Yeung A, Wamboldt MZ, et al: Conceptual and methodologic issues in quantifying perceptual accuracy in childhood asthma, J Pediatr Psychol 21:153, 1996. 262. Meijer AM, Griffioen RW, van Nierop JC, et al: Intractable or uncontrolled asthma: psychosocial factors, J Asthma 32:265, 1995. 263. Sandberg S, Paton JY, Ahola S, et al: The role of acute and chronic stress in asthma attacks in children, Lancet 356:982, 2000. 264. Weil CM, Wade SL, Bauman LJ, et al: The relationship between psychosocial factors and asthma morbidity in inner-city children with asthma, Pediatrics 104:1274, 1999. 265. Liu C, Feekery C: Can asthma education improve clinical outcomes? An evaluation of a pediatric asthma education program, J Asthma 38:269, 2001. 266. Wilson SR, Latini D, Starr NJ, et al: Evaluation of parents of infants and very young children with asthma: a developmental evaluation of the Wee Wheezers program, J Asthma 33:239, 1996. Prevention 267. Gern JE, Lemanske RF Jr: Pediatric allergy: can it be prevented? In Vassallo J, editor: Immunology and allergy clinics of North America, Philadelphia, 1999, Saunders, p 233. 268. Stein RT, Holberg CJ, Sherrill D, et al: Influence of parental smoking on respiratory symptoms during the first decade of life. Tucson Children's Respiratory Study, Am J Epidemiol 149:1030, 1999. 269. Arshad SH, Tariq SM, Matthews S, et al: Sensitization to common allergens and its association with allergic disorders at age 4 years: a whole population birth cohort study, Pediatrics 108:E33, 2001. 270. Figueroa-Munoz JI, Chinn S, Rona RJ: Association between obesity and asthma in 4–11 year old children in the UK, Thorax 56:133, 2001. 271. Infante-Rivard C, Amre D, Gautrin D, et al: Family size, day-care attendance, and breastfeeding in relation to the incidence of childhood asthma, Am J Epidemiol 153:653, 2001. 272. Wright AL, Sherrill D, Holberg CJ, et al: Breast-feeding, maternal IgE, and total serum IgE in childhood, J Allergy Clin Immunol 104:589, 1999. 273. Haby MM, Peat JK, Marks GB, et al: Asthma in preschool children: prevalence and risk factors, Thorax 56:589, 2001.

274. Wright AL, Holberg CJ, Taussig LM, et al: Maternal asthma status alters relation of infant feeding to asthma childhood, Adv Exp Med Biol 478:131, 2000. 275. Warner JO, ETAC Study Group: A double-blinded, randomized, placebo-controlled trial of cetirizine in preventing the onset of asthma in children with atopic dermatitis: 18 months' treatment and 18 months' posttreatment follow-up, J Allergy Clin Immunol 108:929, 2001. 276. Martinez F, Cline M, Burrows B: Increased incidence of asthma in children of smoking mothers, Pediatrics 89:21, 1992. 277. Bjorksten B, Sepp E, Julge K, et al: Allergy development and the intestinal microflora during the first year of life, J Allergy Clin Immunol 108:516, 2001. 278. Kalliomaki M, Salminen S, Arvilommi H, et al: Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial, Lancet 357:1076, 2001. Prognosis 279. Jenkins MA, Hopper JL, Bowes G, et al: Factors in childhood as predictors of asthma in adult life, BMJ 309:90, 1994. 280. Wolfe R, Carlin JB, Oswald H, et al: Association between allergy and asthma from childhood to middle adulthood in an Australian cohort study, Am J Respir Crit Care Med 162:2177, 2000.

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Chapter 69 - Asthma in Adults: Evaluation and Management

Nizar N. Jarjour

*

1 2

Asthma is a clinical syndrome characterized by airway obstruction, inflammation, and hyperresponsiveness.[ ] [ ] The major symptoms of asthma are paroxysms of dyspnea, wheezing, and cough, which may vary from mild and almost undetectable to severe and unremitting. Airway obstruction in asthma is typically reversible, but not always completely so. Bronchial hyperresponsiveness (BHR) is a cardinal feature of asthma that manifests as spontaneous fluctuations in the severity of

obstruction, substantial improvements in the severity of obstruction after use of bronchodilators or corticosteroids, or increased obstruction caused by specific or 2] [3]

nonspecific agonists.[

Airway inflammation, with the participation of complex cellular (e.g., mast cell, eosinophil, neutrophil, T lymphocyte) and chemical (e.g., 4

leukotriene, histamine, cytokine, chemokine) mediators, is a fundamental pathophysiologic feature of asthma.[ ] Evaluation and management of asthma in the adult can be approached systematically.

EVALUATION The diagnosis of asthma is suggested by a history of episodic or fluctuating respiratory symptoms supported by physical findings and laboratory or radiographic 1] [5]

abnormalities and is confirmed by objective measurement of airway obstruction and reversibility.[ History of Illness

2

The cardinal symptoms of asthma are the combination of shortness of breath, wheezing; chest tightness; and cough.[ ] Chronic cough can be the sole presenting 6

manifestation of asthma.[ ] Patients with cough-variant asthma can develop the complete clinical syndrome of asthma. However, some atopic patients with chronic 7

cough do not respond to bronchodilators and do not progress to a full clinical picture of asthma.[ ] Dyspnea as an isolated symptom can be attributed to asthma in at 8

least 15% of patients evaluated for this complaint.[ ] As with many other diseases, asthma has a circadian rhythm with a characteristic increase in symptoms (e.g., 9

cough, dyspnea, and wheezing) during sleep. Frequent nocturnal awakening can lead to disturbed sleep and daytime fatigue.[ ] The frequency of nocturnal asthma is 1

key indicator of asthma severity.[ ] Patients with severe persistent asthma have frequent nighttime symptoms, but even patients with mild intermittent asthma may have occasional and nocturnal exacerbations (typically less than twice per month). In asthma, airway narrowing causes increased work of breathing to overcome the added bronchial resistive load. Dyspnea is perceived when that effort exceeds a certain threshold.[ temporal

10]

Thereafter the severity of dyspnea increases proportionally to further increases in resistive load[

13 adaptation.[ ]

11]

12]

and lung hyperinflation,[

Reduced chemosensitivity to hypoxia and blunted perception of dyspnea may predispose patients to fatal asthmatic

1 ). Anxiety, anger, depression, and cognitive disturbance may intensify respiratory symptoms. [

15]

but subject to

14 attacks[ ]

( Figure 69-

These findings point out the risks of estimating the severity of 1] [2] [5]

asthma based on symptoms alone and the need for objective measurements of pulmonary function to quantify asthma severity.[

The management of the asthma depends on assessments of predisposing, inducing, enhancing, and triggering factors; frequency and severity of attacks; responses to previous interventions; and associated illness. The history relevant to each of these factors should be obtained from the patient and supplemented, when appropriate, by observations of spouse, family, and previous health care providers.[ Risk Factors

1] [16] [17]

Asthma in the adult may have had onset in childhood or may appear at any age.[ and immunoglobulin E (IgE) 31]

air pollutants,[

21 22 23 24 antibodies,[ ] [ ] [ ] [ ] 32]

including diesel exhaust.[

prevalence and severity of

aeroallergen

18] [19]

25 26 exposures,[ ] [ ]

Risk factors for asthma include family history of allergy or asthma,[ viral respiratory

27 28 illnesses,[ ] [ ]

and exposures to tobacco

20]

atopy

29 30 smoke[ ] [ ]

and

In the United States, low socioeconomic groups, African Americans, and Hispanic Americans are at increased risk for

33 34 asthma.[ ] [ ]

The course of illness varies from patient to patient, ranging from intermittent nuisance symptoms that remit spontaneously; to acute, severe, and even fatal exacerbations; to chronic persistent and sometimes progressive illness with disability and corticosteroid dependence.[

1] [5]

Factors in Asthma Exacerbation and Severity Identified factors may result in asthma exacerbations or contribute to its severity ( Box 69-1 ). Patients frequently, but

* This chapter, originally written by David A. Mathison, M.D., appeared in the last edition of this book and has been updated here by Nizar N. Jarjour, M.D.

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Figure 69-1 Mean (±SE) perception of dyspnea (Borg score) during breathing at six levels of resistance in 12 normal subjects, 11 patients with asthma but no nearfatal attacks, and 11 patients with near-fatal asthma. (From Kikuchie Y, Okabe S, Tanura G, al: N Engl J Med 330:1332, 1994.)

(From Kikuchie Y, Okabe S, Tanura G, al: N Engl J Med 330:1332, 1994.) not always, recognize the association between these factors and exacerbation of their asthma symptoms.[

35]

The physician should carefully review the potential

contribution of each factor and determine the need for further testing and treatment, particularly in patients with severe or refractory asthma.[

36]

Aeroallergens

Asthma, when earmarked by immediate hypersensitivity reactions to aeroallergens is called allergic or extrinsic asthma, although multiple immunologic mechanisms, including complex cellular and chemical mediators, are involved.[

1] [2]

Aeroallergen-provoked asthma is more frequently a problem for children and

young adults than for older adults, but it may occur at any age. The inhaled substances most likely to produce allergic asthma include house-dust mite,[ 39]

cockroach,[

40]

dog,[

cat, and other animal proteins[

44 conditioners;[ ]

23]

and fungal spores within the house;[

41]

automobile air and various organic and a few inorganic dusts and fumes in the household that may be relevant to a person's asthma

42]

pollens[

26] [43]

and mold spores [

45 workplace.[ ]

37] [38]

outdoors; mold spores from

Other aeroallergens occasionally encountered in the

Box 69-1. Provoking Factors of Asthma Aeroallergens Respiratory tract infections (mostly viral) Aeroirritants/pollutants Meteorologic factors Stress Exercise Aspirin sensitivity Sulfite/SO2 sensitivity Occupational exposure

include allergens of plant origin, such as the seed hair of kapok trees used as stuffing for pillows or mattresses, cottonseed contaminants of inexpensive cotton stuffing in upholstery and cushions, lycopodium dusting powders, psyllium laxatives, and jute carpets. Fish and other airborne food molecules generated during cooking can produce asthmatic symptoms in highly sensitive patients. Community outbreaks of asthma may be associated with localized allergens, such as insect 46]

particles[

or soybean dust.[

47]

Wind-borne pollen grains are 14 to 60••m in average diameter, ordinarily impinge on the eyes, and are entrapped in the nose, producing hay fever symptoms in the IgE-susceptible person. A subset of these patients develops lower airway symptoms typical of asthma in addition to hay fever. Fragments of pollens, mold spores, and vertebrate animal and arthropod proteins less than 5••m in diameter may penetrate directly to the tracheobronchial tree.[

48] [49] [50]

Dust mite, pet, and cockroach sensitivity should be considered as prime factors in patients with perennial symptoms of asthma and rhinitis, especially when occurring 51]

at nighttime or on awakening.[

Apart from asthmatic symptoms occurring during generalized anaphylactic reaction [

hypersensitivity reactions to food rarely contribute to asthma in Respiratory Infections

53 adults.[ ]

52]

to ingested food, immediate

In normal subjects, consequences of viral respiratory infection may include persistence of small airway obstruction for weeks and transient airway hyperreactivity. [54] [55] [56]

57]

Similar consequences in asthmatic and potentially asthmatic subjects lead to exacerbation [ [58]

include alterations in autonomic nervous system function; damage to airway epithelium; infection; sensitization to allergen or virus-related enhancement of cytokine production airway inflammation and

[59]

or appearance of asthma. Underlying mechanisms may

production of virus-specific IgE antibody; and enhanced

or mast cell/basophil mediator release, leading to expression of allergen-induced

27 60 BHR.[ ] [ ]

Acute respiratory viral infections precipitate wheezing in some adult patients with asthma, although the frequency is lower and more difficult to document than in school-age children.[

61]

typically cause asthma

Influenza, respiratory syncytial virus (RSV), and rhinovirus are the agents implicated. [

63 64 exacerbations,[ ] [ ]

62]

Bacterial infections of the lower airway do not

However, increasing evidence indicates that mycoplasma respiratory infections can exacerbate asthma;[

65] [66]

whereas

[67]

chlamydial respiratory infections may exacerbate or initiate asthma. When asthma patients who had evidence of recent chlamydial infection (by serology or culture) were treated with an antibiotic against Chlamydia pneumoniae, they had improved asthma symptoms and increased pulmonary function testing, and in a 65 68 69

70

small subset of these patients, the pulmonary symptoms resolved completely.[ ] [ ] [ ] Using bronchoscopy, bronchoalveolar lavage (BAL), and biopsy[ ] to determine the presence of chlamydia or mycoplasma infections, patients with and without evidence of Mycoplasma pneumoniae or C. pneumoniae were treated with clarithromycin (500•mg) or placebo twice daily for 6 weeks. Forced expiratory volume in 1 second (FEV1 ) values improved in clarithromycin-treated patients compared with the placebo group. However, the improvement was significant only in the PCR-positive group, raising the question of persistence of

1259

70]

these organisms in the lower airway, which might lead to chronic airway inflammation and persistent symptoms.[ Aeroirritants and Pollutants

Aeroirritants aggravate asthma in patients of all ages. Perhaps the greatest nemesis is tobacco smoke,[ “allergic” to smoke despite that IgE reactivity to smoke does not account for the

71 reaction.[ ]

29] [30]

so much so that many patients consider themselves

Environmental tobacco smoke (ETS) is a potential contributor to asthma

[72]

73

exacerbations and, at least in infants, to new onset asthma. Other aeroirritants in the home may include fumes from gas cooking[ ] and heating appliances; dusts and fumes from chemical cleaning (especially oven cleaning), painting, and gardening aids; fresh newsprint; smoke from fireplaces; aromatic terpenes from evergreen Christmas trees; volatile organic compounds[

74]

75]

; hair sprays and other aerosols; and perfumes and colognes.[

Although asthma is among the disorders 76]

noted by occupants of homes insulated with urea-formaldehyde foam and mobile homes constructed with formaldehyde-treated plywood or particle board,[ 77]

formaldehyde at concentrations typically found in such households does not provoke bronchospasm in the majority of asthmatic subjects,[ does not produce

78 BHR.[ ]

and exposure usually

The role of air pollution—the atmospheric accumulation of substances injurious to humans—is difficult to evaluate.[

31] [79]

Diesel exhaust particles (DEPs) can 32

enhance human IgE production and modulate cytokine production and have been implicated in the worldwide increased prevalence of allergic asthma.[ ] Endotoxin is also an important factor contributing to asthma exacerbations. In fact, asthma severity correlates better with levels of endotoxin in house dust than with mite 80

allergen levels in house dust mite–sensitive asthmatic patients.[ ] The major aeropollutants from combustion of fossil fuels and photochemical oxidation of the combustion products, sulfur dioxide, ozone, and oxides of nitrogen, are known to increase bronchial reactivity under experimental conditions with concentrations at, 81]

or only slightly greater than, peak levels recorded at times in industrialized urban areas.[ vulnerability to another

82 aeropollutant[ ]

or to aeroallergens,

[83]

Increased bronchial reactivity from one aeropollutant may enhance

infectious agents, and meteorologic changes,[

84]

and vice versa. Sulfur dioxide at a concentration of

0.5 parts per million (ppm), in the upper range experienced in photochemical smog, or in volcanic eruption, incites bronchoconstriction in asthmatic subjects,[ 86]

especially during exercise.[

85]

Ozone during exercise at 0.12 ppm, the National Ambient Air Quality Standard (NAAQS), a level exceeded at least 4 days annually in

most metropolitan United States cities, may or may not produce bronchospasm in asthmatic subjects[

87]

; ozone at 0.2 ppm or greater (Southern California Stage I 81]

Smog Alert) during intermittent exercise causes decreases in forced expiratory volume in 1 second (FEV1 ) and symptoms in persons with asthma,[ asthma severity correlates with personal ozone exposure.[ response to subsequent allergen

88 89 exposure.[ ] [ ]

43]

and daily

Ozone has been shown to increase asthma symptoms, enhance airway inflammation, and increase airway

Nitrogen dioxide at 0.3 ppm, six times the NAAQS, does not have an effect on exercising individuals with stable

90 asthma.[ ]

91] [92] [93]

Exposure to acid fog can also increase the frequency of asthma exacerbation and worsen airway inflammation.[ of pollutants and asthma symptoms might be difficult to confirm, even when it seems evident to the patient.

The relationship between levels

Meterologic Factors

Meteorologic factors include ionization of the atmosphere, barometric pressure, temperature, wind velocity, and humidity; climate is the result of the interaction of these factors. No single factor or combination of factors has a significantly consistent direct effect on large populations of asthmatic patients. However, indirect effects on air pollution, as during periods of thermal inversion; levels of pollen and spore aeroallergens, as during thunderstorms;[ as exercise in cold-dry ambient

96 air,[ ]

94] [95]

and combined effects, such

may be of consequence to the individual patient with asthma.

Emotional Factors

Behaviors that alter breathing, such as vigorous laughter, shouting, or crying, may trigger bronchoconstriction, especially in those with significant airway hyperresponsiveness (AHR) or during exacerbations. Hyperventilation, including that associated with emotional stress, may provoke bronchospasm.[ cooling and cholinergic pathways may be factors common to all these triggers. of the inflammatory response to other stimuli such as

[98]

97]

Airway

Moreover, chronic stress can contribute to asthma exacerbation by up-regulation

99 100 101 aeroallergens.[ ] [ ] [ ]

Nocturnal and Morning Asthma Sleep disruption by nocturnal or early-morning wheezing and dyspnea is a common and troublesome problem for asthmatic patients, particularly those with

persistent disease.[

102]

The majority of in-hospital sudden deaths and episodes of ventilatory arrest from asthma occur at night; therefore, nocturnal worsening is an

important manifestation of asthma that reflects disease severity or instability.[

103]

In diurinally active patients with asthma, as well as healthy individuals, pulmonary 104]

function reaches a nadir about 4 am and a peak about 4 pm. In shift workers, asthma continues to be related to sleep rather than solar time.[

Therefore the sleep-

wake cycle act to synchronize lung function, which then follows a consistent circadian pattern. However, it does not seem to be related to a specific sleep stage.[

105]

106

Nocturnal worsening of asthma may be quantitated by overnight fall in pulmonary function.[ ] However, the true severity of nocturnal asthma is underestimated when this quantification is based on pulmonary function obtained at bedtime and on waking. Although the exact cause of nocturnal asthma is not yet fully defined, several mechanisms have been proposed: 1. Enhanced airway inflammation at night. Direct evaluation of airway inflammation by bronchoscopy has shown increased BAL neutrophil and eosinophil 107]

numbers,[

increased oxidative metabolism of airway cells,[

interleukin-5 (IL-5) generation at night

[110]

108]

and higher interleukin-1 (IL-1) levels,[

109]

and increased sputum eosinophils, as well as

in patients with nocturnal asthma. Transbronchial biopsy studies suggest that airway inflammation (eosinophils,

lymphocytes) is more prominent in the distal (small) airway than the proximal (large) airway.[

111] [112] 106]

2. Increased bronchial hyperresponsiveness at night. This has been noted both to nonspecific stimuli[

and

1260

to allergen, with increased occurrence of late-phase response (LPR), enhanced fall in FEV1, and longer duration of LPRs at night compared to daytime challenge. [

113][114][115] In

addition, early responses and LPRs to inhaled antigen challenge at nighttime occur at lower antigen doses than with daytime

114 challenge.[ ]

3. Circadian rhythms. These include exaggeration of normal nocturnal decreases in airway caliber,[ 118 119 epinephrine[ ] [ ]

113] [116]

118 histamine.[ ]

and decreases in endogenous cortisol[ 120 hydrocortisone[ ]

117]

and

121 adrenalin[ ]

plasma with a concomitant rise in plasma However, infusion of exogenous or sufficient to prevent the nighttime fall in levels of these hormones failed to abolish nocturnal asthma. Others have described decreased β-adrenergic receptor density and responsiveness, [

122]

reduced glucocorticoid receptor–binding affinity,[

123]

and increased platelet activation.[

124]

125]

4. Increased vagal tone[

5. Mucus retention. This is related to posture and impaired mucociliary clearance at night.[ 6. Airway cooling and drying[

126]

127]

7. Waning effect of pharmacologic agents taken before sleep[

128]

8. Gastroesophageal reflux with aspiration of gastric contents or reflex bronchoconstriction[ 131

129] [130]

9. Congestive heart failure with paroxysmal nocturnal dyspnea[ ] 10. Sleep apnea. In patients with known sleep apnea, asthma improves after successful therapy of sleep apnea with nasal continuous positive airway pressure 132]

(nasal CPAP).[

Physical Examination Examination of the chest in patients with mild intermittent asthma is typically normal when performed between exacerbations. In more severe disease and during attacks, the physical signs of asthma reflect, and are about proportional to, the degree and duration of airway obstruction and include tachypnea, tachycardia, 133]

exaggeration of normal inspiratory fall of systolic blood pressure (pulsus paradoxus), [

134]

hyperinflation of the chest[

with flattened diaphragmatic excursion, use

135

of accessory muscles of respiration,[ ] diaphoresis, hyperresonance of percussion note, prolongation of expiration, and inspiratory and expiratory musical-sonorous rhonchi and wheezes heard on auscultation. Absence of wheezing on auscultation does not always mean lack of airway obstruction, because patients with very severe obstruction may have minimal airflow, in whom auscultation may reveal a “silent chest.” “Wheezing” may also be misleading. The glottic aperture decreases during induced bronchoconstriction, [ widen or contract in asthmatic subjects during patients with vocal cord

139 dysfunction.[ ]

137 exercise,[ ]

may narrow with forced expirations in nonasthmatic

138 subjects,[ ]

136]

may

and narrows during inspiration in

When the glottic aperture decreases sufficiently, glottic and tracheal expiratory sounds may transmit to the chest, mimic 138 139

bronchial wheezes, and falsely lead toward diagnosis of asthma.[ ] [ ] When glottic closure is suspected as a cause for noisy breathing in a patient with known asthma, auscultation over the neck may reveal typical stridor with a louder inspiratory component. Patient exhaustion with difficulty maintaining speech, full use of accessory muscles of respiration, diaphoresis, and cyanosis may reflect extremely severe asthma and impending ventilatory arrest. Physical examination in asthma should also include the nose, for obstruction by anatomic deformity, mucosal edema, or polyps, and the oropharynx, for redundance or laxity of the soft palate or fauces, hypertrophy of lymphoid tissue, or presence of mucus or exudate draining from the nasopharynx. Pulmonary Function Tests Pulmonary function tests (PFTs) can be used to establish the diagnosis of asthma, to quantify the severity of the disease, and to monitor the course of the disease and response to therapy. Spirometry

The diagnosis of asthma is confirmed by spirometry demonstrating airflow limitation and its improvement after inhalation from a bronchodilator. The simplest and 140

most reliable spirometers are conventional volumetric devices. Transducers have proved to be comparably accurate.[ ] Display of flow-volume or volume-time curves and measurements of forced vital capacity (FVC), FEV1 , maximum midexpiratory flow (MMEF), or peak expiratory flow (PEF) rates provide the essential information. Greater than 12% improvement of FEV1 after inhalation of an ordinary dose of inhaled β2 -adrenergic agonist generally supports the diagnosis of asthma [141]

142]

( Figure 69-2, A to C ). Greater than 20% diurnal (evening to morning) fall in PEF is also indicative of asthma.[

However, neither the presence of obstruction 143]

nor its reversibility with bronchodilators is pathognmonic for asthma because asthma, chronic bronchitis, and emphysema overlap. [ clinical diagnosis that is confirmed by objective findings on

1 spirometry.[ ]

Therefore, asthma remains a

The configuration of the forced expiratory-inspiratory flow-volume loop can provide clues in the differential diagnosis of asthma. Patients with obstruction or collapse of peripheral airways, as in emphysema, have a rapid fall of flow rates and a marked prolongation of expiration ( Figure 69-2, D and F ). When the patient is inexperienced or incapable of performing a peak forced expiratory movement, there is irregularity in the initial effort-related portion of the recording ( Figure 69-2, G ). In patients with variable extrathoracic airway obstruction (e.g., vocal cord dysfunction), there is a “cutoff” of the inspiratory limb of the flow-volume loop ( Figure 69-2, H ), whereas in fixed airway obstruction, there is a cutoff of both expiratory and inspiratory peaks ( Figure 69-2, E ). Bronchoprovocation—Nonspecific Airway Hyperresponsiveness

Because of the paroxysmal nature of the airway obstruction in asthma, it may not be possible to demonstrate reversible airway obstruction when the patient is in remission or when symptoms and signs are minimal or atypical. In such patients the nonspecific AHR of asthma may be identified (or excluded) by provocative challenge with inhaled methacholine.[

144]

144] [145]

Airway responsiveness to methacholine can be reproducibly measured by a simple and standardized method.[

Symptoms of asthma, decreased airflow in large and small airways,[

146]

1261

Figure 69-2 Flow-volume spirometric measurements of patients with respiratory symptoms. A through D, Full (A) and partial (B to D) reversal of airway obstruction in separate patients after inhalation of aerosol of albuterol (POST) compared with baseline (PRE) and 95th percentile predicted for subjects of comparable age and height (NORMAL) for patient in D, POST 2, After 3-week course of daily high-dose corticosteroid and bronchopulmonary toilet regimen. E, Patient with fixed obstruction to expiratory (EX) and inspiratory (IN) airflow. F, Patient with severe obstruction to airflow both before (PRE) and after (POST) inhalation of aerosol of albuterol. G, Patient unable to adequately perform, in part because of cough, a smooth forced expiratory maneuver. H, Patient with variable, extrathoracic airway obstruction secondary to vocal cord dysfunction. All panels: Flow (L/sec), ordinate, and Volume expired (L), abscissa from total lung capacity (0); ↓ arrows indicate 1-second forced expiratory volumes in liters.

Figure 69-3 Severity of asthma exacerbation. (From National Heart, Lung, and Blood Institute: NAEP guidelines for diagnosis and management of asthma, NIH Pub No 97-4051, 1997.)

(From National Heart, Lung, and Blood Institute: NAEP guidelines for diagnosis and management of asthma, NIH Pub No 97-4051, 1997.)

1264

Figure 69-4 Peak expiratory flows in liters per minute—ordinate time days/hours—abscissa before (•) and after (•) inhalation of β2 -adrenergic from metered-dose inhaler by asthmatic patient with “morning dipping” pattern of airflow obstruction.

Box 69-2. Compounding and Confounding Conditions in Asthma That May Necessitate Modification of Treatment Bronchopulmonary aspergillosis and other mycoses Rhinosinusitis and nasal polyposis Vocal cord dysfunction Adrenergic aerosol overuse Mucoid impaction of bronchi Chronic obstructive bronchopulmonary disease Obesity Deconditioning Cardiovascular disease

Gastroesophageal reflux disease Pregnancy Premenstrual state Sleep disorders Allergic angiitis and granulomatosis Exogenous Cushing's syndrome Corticosteroid resistance Pharmacotherapy • Histamine releasers • β-Adrenergic blockers • Cholinesterase inhibitors • Angiotensin-converting enzyme inhibitors Psychologic factors Socioeconomic factors

(see Figure 69-2, H ). Laryngoscopy, especially when performed while the patient is symptomatic, can confirm the diagnosis. About half of patients with the combination of laryngeal dysfunction and asthma have psychologic factors, such as somatization with or without depression, conversion, and histrionic personality traits. Treatment must be directed to the psychologic factors, if present, and to altering the dysfunctional laryngeal behavior by speech therapy.[ Adrenergic overuse, especially of nebulized isoproterenol in high concentration in the 1960s in the United Kingdom [ and

257 Canada,[ ]

256]

229] [231]

and fenoterol in the 1980s in New Zealand

were recognized as contributing to increased death rates from asthma. More selective β2 bronchodilators that are currently used have an improved 258

259

the margin of safety, so that at least 8 inhalation[ ] or as many as 50 inhalations[ ] from a metered-dose cartridge may be taken for an acute exacerbation. Nonetheless, overuse of adrenergic agonists may be accompanied by reduced bronchodilatory effect, increased nonspecific AHR, decreased arterial Pao2 , cardiac stimulation, skeletal muscle tremor, central nervous system stimulation, tolerance to nonbronchodilator actions[ bronchoconstrictor

261 stimulus,[ ]

and delay in more effective

260]

and protective effects against a

262 treatment.[ ]

Chronic obstructive bronchopulmonary disease—chronic bronchitis and/or emphysema—is seen among middle-age and older adults who have been smokers.

Superimposed variable airway obstruction, BHR, and eosinophilic inflammation of asthma assume proportionately greater consequence in

1267

such individuals than in those without COPD. A trial of corticosteroid treatment is sometimes required to determine the presence and degree of reversible airway obstruction (i.e., asthma) in these patients (see Figure 69-2, D ).[

263]

Obesity reduces lung volume proportional to the degree of the overweight state, compounds problems of breathing disorders during sleep, and increases ventilatory impairment in asthma. Corticosteroid treatment may in turn compound the tendency to weight gain. Sedentary deconditioned individuals likewise have impaired ventilatory and cardiovascular responses that reduce their tolerance for airway obstruction at the same time that asthma may restrict their ability to exercise. Obesity is a key risk factor for the development of obstructive sleep apnea, which in turn leads to worsening of asthma control.[ Hypertension and other cardiovascular diseases occur at expected or slightly greater than expected[ rises during an asthma exacerbation in proportion to the severity of the

267 attack[ ]

265] [266]

132] [264]

frequency in adult patients with asthma. Blood pressure

and falls with relief of airway obstruction even when adrenergic agents are used.

[268]

However, adrenergic agents and theophylline may precipitate or compound cardiac arrhythmia or hypertrophic obstructive cardiomyopathy. Treatment of hypertension or other disorders with β-adrenergic blocking agents, especially nonselective drugs (e.g., propranolol) that block both β1 cardiovascular and β2 269

bronchial receptors, have the potential for exacerbating existing asthma or provoking bronchospasm in individuals with a potential for asthma.[ ] Furthermore, cough, which can be the main symptom of asthma, can also be a side effect of treatment with angiotensin-converting enzyme (ACE) inhibitors given for control of hypertension or congestive heart failure.[

270]

Gastroesophageal dysfunction reflex disease (GERD) is found more frequently in asthma patients than in healthy controls. Prednisone-related increase in gastric 129]

acidity and reduced lower esophageal sphincter (LES) tone secondary to theophylline can further aggravate GERD.[ [271]

Reflux may contribute to nocturnal attacks

by vagal reflex or, in the case of regurgitation and aspiration, by direct irritative effects. Reflux can be detected by intraesophageal pH monitoring.[

Occasionally, GERD accounts for the majority of asthma symptoms and may be corrected by surgery.[

273]

272]

A trial of a proton-pump inhibitor is warranted when 274]

GERD is present in asthma patients, especially those with suboptimal control, because asthma can improve dramatically with this therapy.[ 275]

Premenstrual increase of asthma is reported by a significant minority of ovulatory women.[ be

278 severe.[ ]

As with other premenstrual symptoms, the biologic explanations are

Although unrecognized in some patients,[

276] [277]

it occasionally can

279 unclear.[ ]

Sleep disorders include snoring, hypopneas, and apneas from obstruction of the pharyngeal airway (especially in obese subjects) by the tongue from waning of genioglossus contraction or from central neurogenic defect or a combination of both.[

280]

Brief periods of apnea occur, but infrequently in healthy people and more

281 282

283

frequently in chronic stable asthma.[ ] [ ] Snoring is also more prevalent in asthmatic subjects.[ ] Individuals with prolonged and recurrent apneas-hypopneas (generally more than 10 episodes per hour), arousals with disruption of sleep, and daytime drowsiness are described as having sleep apnea syndrome. Although the frequency of sleep apnea syndrome is not greater in subjects with bronchopulmonary diseases, including asthma, the nocturnal respiratory distress of sleep apnea 132]

may be confused with, or compound, asthma.[ breathing, and gas exchange are assessed.

Definitive diagnosis of the sleep apnea syndrome requires overnight polysomnography, during which sleep stage,

284

Allergic angiitis and granulomatosis (Churg-Strauss syndrome) is a rare complication of asthma[ ] characterized by granulomatous vasculitis of blood vessels of various types and sizes (small- and medium-size muscular arteries, veins, and venules) with frequent involvement of pulmonary vessels (in contrast to classic polyarteritis nodosa), peripheral blood eosinophilia (at least 1000/mm3 ), and eosinophilic tissue infiltrates. This disorder should be considered in the asthmatic patient with refractory bronchospasm and pulmonary infiltrates. Patients receiving leukotriene modifiers reportedly may develop Churg-Strauss syndrome when their 285 286

oral corticosteroid dose is tapered.[ ] [ ] It is unclear whether these patients developed Churg-Strauss as a result of antileukotriene therapy, or if they had ChurgStrauss syndrome that was misdiagnosed as severe asthma, and the reduction of oral corticosteroids allowed for the emergence of the Churg-Strauss syndrome. Exogenous Cushing's syndrome occurs in proportion to dose and duration of systemic corticosteroid treatment and to individual susceptibility. Features of exogenous Cushing's syndrome may include (1) fluid retention and hypertension; (2) weight gain with central obesity, rounding of facies, prominence of dorsal and ventral fat pads, and abdominal protuberance; (3) thinning of the skin with susceptibility to ecchymoses and mucocutaneous yeast and fungal infections; (4) increase in lanugo hairs; (5) posterior subcapsular cataract; (6) hyperglycemia and diabetes mellitus; (7) myopathy (but not at doses of corticosteroids usually administered in chronic 287

288

289

290

severe asthma[ ] ); (8) low serum osteocalcin[ ] and osteoporosis with fractures[ ] ; (9) reduction of serum testosterone levels with impotence[ ] ; (10) decreased levels of immunoglobulin G (IgG) with retained capacity to produce specific antibody; and (11) psychic changes, especially affective and psychotic disorders. [

291]

Antihistamines have potential drying effect on bronchial mucus. For this reason, at the time of their introduction, there had been caution regarding their use in asthma. However, concern was more theoretic than practical.[

292]

There is no evidence that H1 -blocking antihistamines are harmful in asthma, and some of the

newer preparations have been reported to be of benefit in asthma. [

293]

294]

Pharmacologic agents that may exacerbate asthma include nonselective β-adrenergic receptor blocking agents such as propranolol[ 296 metipranolol[ ]

297 betaxolol[ ]

and sotalol[

298 adenosine[ ]

295]

(used for 299

cardiovascular disease) and timolol, levobunolol, and (but less likely ) used for glaucoma; and ropafenone[ ] antiarrhythmics; intravenous dipyridamole; and mast cell degranulators, including iodinated radiocontrast dye and opiates, especially codeine and morphine. ACEIs 300]

occasionally provoke cough but typically no other asthma symptoms.[

Psychologic problems are often seen in patients with chronic illnesses, including asthma.[ panic disorder), [305]

[302] [303]

304 depression,[ ]

301]

Asthma patients have been reported to suffer from anxiety (especially 99]

and stress, as with marital/family dysfunction; emotional, sexual, physical, or substance abuse; or financial problems.[

Associated behaviors, such as hyperventilation[

306]

15] [307]

and anger, may trigger or intensify the asthmatic attack[

or, in the case of denial, impair recognition

of disease and lead to delays in treatment and fatal outcomes.[

308] [309]

1268

310

Psychologic problems are recognized to be a key factor contributing to asthma severity. [ ] Clinical clues to the presence of psychologic problems are disparity between subjective and objective (i.e., either too much or too little of symptoms for degree of measured airways obstruction) and failure to adhere to or respond as expected to a prescribed regimen for asthma. In these circumstances or when the history indicates a psychologic problem, consulting with a psychiatrist or behavioral psychologist can help evaluate and manage these disorders. Failing to address psychologic problems in these patients can make it difficult to achieve optimal control of asthma.[

311]

Socioeconomic factors that affect asthma reflect low socioeconomic status[

33] [312]

and may include increased environmental exposure to dust mites or cockroaches;

[51] [313]

impaired access to health insurance and care; inability to enact environmental changes to reduce aeroallergen exposures; inability to purchase asthma medications; inability to cope with chronic illness; and susceptibility to unorthodox or unproven diagnostic and treatment approaches. When these factors are operative, assistance from public and private social resources may have a favorable effect on asthma control.

MANAGEMENT Goals and Principles The goals for management of adults with asthma vary according to the stage and severity of the disease, the resources of the health care facility, and the expectations 1 5

of the patient and the treating physicians.[ ] [ ] According to the severity of an exacerbation (see Figure 69-3 ), patients with status asthmaticus with respiratory failure (or impending respiratory failure) require intensive care. Those with a poor response to intensified ambulatory or emergency care require hospitalization. Patients with persistent asthma with a relapsing course may benefit from specialist care, whereas those with intermittent mild attacks usually require only primary or self-care resources.[

1]

At each level of care, the goals of therapy should be to relieve the patient's symptoms, improve bronchopulmonary function as quickly as possible with minimal risk and side effects from medications, and help the patient achieve higher levels of function and independence in management of the disease. Ultimate goals include normal activities of daily living, including restful sleep and exercise, and prevention of exacerbations of asthma.[

1] [2]

Several general principles apply to the treatment of adult asthma, as follows: 1. Symptoms and physical signs are subjective observations[

314]

that may lead to underestimation of the severity of asthmatic relapse and overestimation of

1 5

response to treatment. Spirometry, oximetry, or ABG measurements are therefore essential to the optimal management of asthma.[ ] [ ] 2. The severity of repeated attacks for an individual tends to be similar. A history of a previous hospitalization or a life-threatening attack (especially one with unconsciousness or necessitating intubation), noncompliance with asthma medication plan, recent withdrawal from systemic corticosteroids, and 1 5 315 316

socioeconomic or psychologic problems merit careful consideration in management.[ ] [ ] [ ] [ ] 3. Recovery time from initiation or change of treatment to restoration of full remission approximately equals the duration of relapse. For example, increase of asthma severity of hours' duration clears over hours, that of days' duration clears over days, that of weeks' duration clears over weeks, and that of years' duration improves over months with therapy. 4. Continue treatment efforts until normal function, including aerobic exercise, is attained or until individual optimal reversal of airway obstruction and rehabilitation are achieved. 5. When the patient fails to improve as expected, reconsider the possibilities for provoking factors (see Box 69-1 ), different diagnosis, and confounding conditions (see Box 69-2 ). Acute Asthma: Emergency and Urgent Care The assessment, treatment, and disposition of adult patients with acute asthma exacerbation have been detailed in the 1995 Global Initiative for AsthmaI (GINA)[

5]

1

and the National Asthma Education and Prevention Program (NAEPP) panel report.[ ] NAEPP guidelines for classification of asthma exacerbation severity are shown in Table 68-3 on p. 1232 . The following steps are suggested based on these guidelines. Initial Assessment

1. Obtain the essential history: the provoking stimulus, time of onset, duration, and subjective severity of the attack; drugs taken and response; severity of prior attacks; and compounding conditions. 2. Perform limited physical examination to assess the severity of the attack: use of accessory muscles of respiration, difficulty in maintaining speech, and degree of fatigue; respiratory and pulse rates; blood pressure and pulsus paradoxus; chest aeration; prolongation of expiration; and location and nature of adventitious sounds. 3. Measure pulmonary function: patients with severe exacerbation have PEF less than 50% of predicted or personal-best values. In patients with PEF or FEV1 less than 30% after initial treatment (or if the patient is so breathless, exhausted, obtunded, or anxious that he or she is unable to perform simple spirometry or oximetry 1] [317]

measures less than 90% saturation), ABGs should be obtained.[ Initial Treatment

4. Administer a β2 -adrenergic bronchodilator. If breathing is unlabored, administer short-acting bronchodilator by inhalation using metered-dose inhaler (MDI) or nebulizer aerosol. In mild attacks (PEF >50%) the β2 -adrenergic therapy can be repeated up to three times over an hour. β-Adrenergic inhalers can be used every

few minutes for at least 8 inhalations[

258]

and perhaps as many as 50 inhalations,[

259]

unless there are side effects. The addition of anticholinergic therapy enhances

the bronchodilator effect of β-adrenergic therapy, particularly in patients with severe exacerbation.[ the lung and facilitate coordination.

318] [319]

The use of spacer device may improve drug delivery to

1

Systemic corticosteroids are recommended for most patients with acute asthma exacerbations.[ ] Corticosteroids result in faster improvement in symptoms and lung 320

321

322]

function,[ ] decrease the need for hospitalization,[ ] and reduce the risk of relapse after an emergency department (ED) visit.[ used in acute asthma exacerbation because of the lower density of helium. This allows for

Heliox (80:20 helium/oxygen) is

1269

decreased airway turbulence, work of breathing, dyspnea, and pulsus paradoxus while improving PEF.[ asthmatics while on mechanical ventilation, with improvement in gas

323] [324]

Heliox can also be used in patients with status

325 exchange.[ ]

Repeat Assessment

5. Review symptoms and physical signs within 20 to 30 minutes after administration of the initial and/or repeated β2 -adrenergic bronchodilators, and remeasure pulmonary function, O2 saturation, or ABGs. If there is no improvement in pulmonary functions or ABGs, the patient is in status asthmaticus. If pulmonary function improves, an additional measurement should be made 60 minutes after the last administration of the β2 -adrenergic to make certain there has been significant (FEV1 1]

or PEFR of 50% of predicted) and sustained effect.[ Disposition

6. Categorize the response to therapy (see Figure 69-2 ). Patients with good response (PEF >70%, no distress) can be discharged home, whereas those with poor response (PEF 42•mm Hg) need to be admitted to the hospital. 7a. Patients with life-threatening or severe asthma with a poor response to initial treatment (status asthmaticus) need hospitalization and, if there is respiratory failure or impending respiratory failure, patients should be admitted to an ICU. 7b. Disposition of patients with moderate exacerbation and incomplete response/relief of airway obstruction after initial treatment varies according to the degree of response and the individual's past history of asthma. Patients may need to be hospitalized if they have (1) a history of marked bronchial lability or previous lifethreatening asthma with unconsciousness, seizure, or need for intubation; (2) severe persistent asthma with a relapsing course for weeks or months despite intensified

corticosteroid treatment as outpatients; (3) prior emergency or urgent care visits for the current relapse; (4) prior hospitalization in the previous year; or (5) serious psychiatric disease or psychologic or socioeconomic problems. Patients not hospitalized should be started or continued on corticosteroids and treated as described next. 7c. Patients with a good response who have persisting but mild airway obstruction after initial therapy should be preliminarily assessed regarding provoking factors (see Box 69-1 ) and compounding conditions (see Box 69-2 ) and treated accordingly. Patient should be educated about medications, action plans, and follow-up. 322

They may be given a 5-day to 10-day tapering course of oral corticosteroid, aimed at reducing the risk of relapse[ ] by relieving airway inflammation. These patients should be continued on established regimens of bronchodilators and discharged for follow-up within a few days by primary physician or specialist. 326]

Facilitated referral to an asthma specialist can reduce relapses in ED visits.[

7d. Patients who have had a short-lived, isolated acute attack, precipitated by an evident stimulus with an excellent response/complete relief of symptoms and normalization of bronchopulmonary function with β2 -adrenergic bronchodilators, may be discharged and followed up by a primary care provider. Most of these patients, however, should be considered for a course of oral corticosteroids. [

1]

Status Asthmaticus: Critical Care Intensive care with respiratory support, if necessary, is appropriate for asthmatic adults who are unresponsive to a β2 -adrenergic agents, have elevated or rising Paco2 ,[

327]

and show respiratory muscle fatigue or exhaustion.[

176] [328]

Respiratory rates greater than 30 breaths/min, paradoxical respiratory efforts, or sustained

tachycardia, especially in the elderly patient, may portend respiratory failure. Monitoring and General Measures 329

The patient with severe respiratory impairment is at risk of sudden death from respiratory arrest,[ ] asphyxia, and cardiac arrhythmia brought on by hypoxemia and potentially compounded by β2 -adrenergics and methylxanthine treatment. A chest radiograph should be taken to evaluate the presence of an alternative diagnosis (e. g., pneumonia, congestive heart failure) and to exclude the presence of complications (e.g., pneumothorax, pneumomediastinum, pneumonia, atelectasis) that may 212]

require additional treatment. [

Continuous monitoring of oximetry, ECG, respiratory rate and amplitude, and repetitive measurements of blood pressure, ABGs, 177

and serum electrolytes are indicated.[ ] Sputum or tracheal aspirate should be obtained for analysis for eosinophils (if a question regarding the diagnosis of asthma) and to evaluate the presence of neutrophils and microbes (Gram's stain and culture) in patients who might have a lower respiratory tract infection. The results may help guide the selection of antibiotic therapy when such therapy is indicated. An intravenous (IV) catheter should be placed to correct any dehydration. However, overvigorous fluid therapy may favor pulmonary edema by increasing microvascular hydrostatic pressure and decreasing plasma colloid osmotic pressure in the face of the more negative pleural pressure.[ may need to be substantial when β2 agonists have depressed serum

331 332 levels.[ ] [ ]

333 Magnesium[ ]

and phosphate

[334]

330]

Supplements of potassium

should be given intravenously when

indicated by measurement of reduced serum level. Humidified oxygen should be continued via nasal cannula, Venturi, or nonrebreather mask at a flow and concentration sufficient to normalize Pao2 . Pharmacotherapy

Aerosolized selective β2 -agonist bronchodilator by inhalation is administered continuously or at 20-minute to 120-minute intervals, with careful attention to side effects, including tachycardia, arrhythmia, hypokalemia, and tremor or central nervous system irritability. Adrenergic agents should be administered with caution in elderly patients and those with cardiac disease[ restored by corticosteroid

335] [336]

until metabolic or respiratory acidosis and hypoxemia have been corrected and adrenergic responsiveness

337 therapy.[ ]

Intravenous corticosteroid therapy should be instituted immediately, although the desired effects of restoration of adrenergic responsiveness and reduction of inflammation occur over hours to days. [ to 8 hours.

[1]

337]

Typical doses that are effective in the majority of patients are 40 to 60•mg of methylprednisolone or equivalent every 6

Generally, there is no advantage for doses of corticosteriod greater than 120 to

1270

180•mg/day. This dose is continued for 48 hours and then reduced to 60 to 80•mg/day until the patient is improved to the point of responsiveness to adrenergic bronchodilator agents (FEV1 or PEFR >70% of personal best or predicted). Daily, or twice daily if initial values are abnormal, serum potassium phosphorus and glucose level measurements are appropriate during IV corticosteroid treatment. Several days of high-dose corticosteroid are remarkably well tolerated by the majority of asthmatic patients; however, acute psychosis or other side effects may complicate such treatment.[ Although IV aminophylline or theophylline has been used in acute severe asthma,[

339] [340] [341]

338]

1

it is generally not recommended[ ] due to minimal added benefit and

342 343 344

increased risk of toxicity.[ ] [ ] [ ] For patients who have been taking methylxanthine drugs before hospitalization, serum theophylline level should be checked to rule out theophylline toxicity. Common side effects related to theophylline include nausea, vomiting, agitation, headaches, confusion, seizures, and hypercalcemia. [345] [346] [347]

Anticholinergic-antimuscarinic bronchodilator therapy,[

348]

with ipratropium up to 500••g via nebulizer[

349]

or 36 to 72••g by MDI and repeated at 30 minutes for 350]

three doses, then every 2 to 4 hours as needed, can enhance the bronchodilator effect of β2 -adrenergic agents. The added bronchodilation is usually small,[

but

this improvement may be critical in forestalling the need for intubation and mechanical ventilation in an occasional patient with a marginal lung function and limited response to β2 -adrenergic therapy.

172

173

Lactic[ ] or non–anion-gap[ ] (related to excessive renal bicarbonate excretion) metabolic acidosis indicates severe attack. Patients with associated respiratory acidosis (CO2 retention) should be evaluated for mechanical ventilatory assistance (invasive or noninvasive) without delay because they are at risk for respiratory arrest. Sedative, narcotic, or hypnotic drugs can be safely administered to the asthmatic patient in status asthmaticus only when adequate mechanical ventilation is in progress or at hand. Mechanical Ventilation

Tracheal intubation and mechanically controlled ventilation[

351] [352]

are indicated for exhaustion, progressive hypercapnia, and CO2 narcosis (usually a Paco2 >60•

mm Hg after initial treatment) or actual respiratory or cardiac arrest. Goals are to improve oxygenation, provide adequate ventilation and minimize risk of barotrauma. Mechanical ventilation is continued until airway resistance decreases, hypercapnia is relieved, and the patient is able to resume the work of breathing. Potential complications of intubation and ventilation include trauma to laryngeal structures, hypotension, arrhythmia, barotrauma (e.g., subcutaneous emphysema, pneumomediastinum, pneumothorax) and nosocomial bronchopulmonary infection.[ asthmaticus was as high as 38% in patients treated in the 1960s and 1970s.[ long-term mortality remains high, about 10% at 1 year and 20% at 6

354]

353]

The mortality of patients requiring mechanical ventilation in status

More recent reports indicate significantly lower mortality,[

351] [353] [355]

However,

356 years.[ ]

To provide mechanical ventilatory support while minimizing the complications of intubation and invasive mechanical ventilation, noninvasive mechanical ventilation through a nasal mask has been advocated. Several studies have shown that noninvasive mechanical ventilation is effective, better tolerated, and associated with fewer 357

complications compared with intubation and mechanical ventilation.[ ] Disadvantages of nasal mechanical ventilation include pressure necrosis of the nasal ridge, claustrophobia, and increased risk for aspiration. Most patients with acute severe asthma can be managed with noninvasive mechanical ventilation; if that fails, however, intubation and mechanical ventilation become necessary. Status Asthmaticus: Hospital Care General Measures and Pharmacotherapy

Patients evaluated and treated for acute asthma in the ED or urgent care setting and found unresponsive to β2 -adrenergic therapy (status asthmaticus) but without exhaustion, acidemia, or hypercapnia or who have marked bronchial lability or who relapse after initial treatment and disposition, as well as those treated over several days as an outpatient for severe asthma with corticosteroid but without response, are at risk of developing respiratory failure and its complications. Hospitalization, chest radiograph, aerosol β2 -adrenergic therapy IV or oral therapy with corticosteroid, hydration, electrolyte replacement, and oxygen therapy, as outlined for the critical care of status asthmaticus (see Figure 69-4 ), are indicated and should be continued until subjective and objective spirometric evidence of reversal of airway obstruction (FEV1 >70% predicted or personal best) is attained. Frequently, such patients will improve rapidly over 24 to 72 hours and may be discharged to primary or specialist care for adequate outpatient medical therapy to prevent rehospitalization.[ require critical care measures.

358]

Occasionally, such a patient will deteriorate and

Clearance of Airway Secretions

Chest physical therapy is generally not recommended in asthma patients. The exception might be those patients who do not show significant clinical improvement despite full pharmacotherapy and have evidence of retained secretions on chest radiography, keeping in mind that therapy itself can worsen respiratory distress in patients with asthma. In patients with significant mucus plugging (lobar or multiple segmental) who fail to respond to chest physical therapy, the use of therapeutic 208] [359]

bronchoscopy with BAL has been reported.[

However, no controlled study has been done to confirm the reported benefits in these anecdotal reports. The

bronchoscopy procedure itself in these patients can be associated with asthma exacerbation with added morbidity.[

360]

Mucolytic (e.g. N-acetylcysteine, potassium 361]

iodide) agents are generally not recommended in patients with acute severe asthma because these drugs may induce bronchoconstriction.[ ultrasonic aerosols may provoke bronchospasm and are best avoided in severe asthma.

Likewise, large-volume

Persistent Asthma: Specialist Care Evaluation and long-term ambulatory treatment of the adult asthmatic patient who has needed critical care or hospitalization and/or has a chronic or relapsing course with severe or moderate exacerbations and significant morbidity, as reflected by excessive absences from school or work, is best undertaken by an experienced physician.[

1] [362]

Initial evaluation should include complete history, a careful examination, and assessment of available data. Additional testing (e.g. blood

1271

tests, PFTs) should be arranged as needed. Formulation of the diagnosis should cover severity and provoking or contributing factors. The treatment plan should be tailored to the individual, including education and counseling regarding the disease and the expected prognosis.[

1]

Environmental Controls/Avoidances

When it is established that an individual's asthma is induced or exacerbated by airborne allergens or irritants localized in the home or workplace, avoidance of these stimuli can be an effective treatment and in some patients allows complete resolution of disease.[

161] [363] [364]

House-dust mite exposure may be reduced by removal of colonized carpets or pads and upholstered furniture; encasement of mattress; regular hot-water washings of 51] [365]

bed coverings and pillows; and treatment of nonremovable mite reservoirs with anacaricide.[ of house-dust mites. However, most of these measures are often too difficult to

Reducing the indoor humidity levels can also discourage growth

366 accomplish.[ ]

Cats, dogs, and the reservoirs of their allergens in litter boxes, carpets/pads, and upholstered furniture are best removed from the residence. When it is not feasible to remove the cat, airborne cat protein can be reduced by placing the litter box out of residence and regularly bathing the cat.[

367]

If the reduction is of insufficient

magnitude to reduce the asthma, pet removal from the residence is mandatory for the sake of the asthmatic patient. Obvious collections of mold spores in water-contaminated walls, wall coverings, draperies, carpets, and floors may be removed and recurrence prevented by eliminating sources of moisture.[

368]

Cigarette, cigar, and pipe smokes; paint and varnish fumes; aerosolized sprays of chemicals used for oven and other household cleaning; and outdoor air pollution can be avoided by the individual as necessary to prevent aggravation of asthma. Central[

369]

370]

or room-unit[

air conditioning can reduce indoor levels of outdoor pollens and mold spore allergens. High-efficiency particulate air (HEPA) air

cleaners may reduce indoor pet, mite, or roach allergens, provided that the reservoirs of these in carpets or pads are removed.[

367]

Diet and Nutrition 371

Maimonides, in the twelfth century, expounded on the role of diet in the treatment of asthma but recognized that diet alone could not fully cure the disorder.[ ] To this day, suggestions regarding diet for the asthmatic individual are offered in the lay and scientific literature; to date, none has been confirmed to be beneficial. In the 1980s, fish oil–enriched diets that contain eicosapentaenoic acid and docosahexaenoic acid analogs of arachidonic acid and eicosapentaenoic acid ester administration were studied in an animal model of asthma[

372]

and in subjects with persistent asthma. In asthma, alterations were observed in leukocyte phospholipid 373] [374]

composition, and LPR to inhaled allergen was attenuated; however, no changes were noted in the clinical course of disease.[ The rare asthmatic patient with systemic anaphylactic sensitivity to food[

52] [53]

needs to avoid the foods to which he or she has such a reaction.

The asthmatic patient dependent on oral corticosteroids needs to exercise and, if overweight, restrict calorie intake. In addition, steps should be taken to ensure sufficient calcium intake to retard development of osteoporosis. Ascorbic acid has been reported to reduce bronchial reactivity to methacholine in some asthmatic patients;[

375]

however, vitamin C and other antioxidant therapy have not been shown to have efficacy for treatment of asthma.[

evidence suggests that flavonoid may have a protective effect in

374] [376]

Limited epidemiologic

377 asthma.[ ]

Medications

A stepwise approach to the long-term management of asthma has been suggested in the NAEP recommendations ( Table 69-1 ) as well as the GINA guidelines. 378] [379] [380]

Corticosteroid for antiinflammatory effect in the airways[

is the mainstay of pharmacotherapy for persistent asthma. Initial parenteral therapy in the ED,

urgent care, or hospital setting is followed by daily single (morning or mid afternoon)[

381]

or divided high doses of prednisone, 40 to 80•mg/day (or equivalent), until

subjective and objective evidence of normalized, or best previously achieved ventilatory function, is noted.[

382]

For prednisone courses of 40•mg up to 10 days,

383 necessary.[ ]

tapered withdrawal is not Premature tapering from prolonged or high-dose corticosteroids risks asthma relapse, and if repeatedly so, a “roller coaster” effect takes place: relapse, partial relief with corticosteroids, corticosteroid withdrawal, and relapse. An occasional asthmatic patient has seemingly persisting airway obstruction, despite weeks of daily, high doses of oral corticosteroids and a full regimen of other 384 385 386

medications. The terms corticosteroid-dependent and corticosteroid-resistant asthma have been applied to such patients.[ ] [ ] [ ] The majority of these individuals will have improvement of their course and less need for corticosteroids—indeed, one-quarter or one-third discontinue corticosteroids—when managed more closely at centers with multidisciplinary expertise in asthma treatment. [

387] [388] [389] [390] [391]

The rare individual in this category will have incomplete 392

absorption or unusually rapid metabolism of corticosteroids administered by mouth and will require an alternative route or higher dose.[ ] Others may have a decreased ability of the activated nuclear-located glucocorticoid receptor to bind to glucocorticoid-responsive elements in the promotor region of steroid-responsive genes.[

393]

390]

Alternative antiinflammatory medications, including combined methylprednisolone-troleandomycin,[ 396

397

gold,[

391]

388] [394]

methotrexate,[

395]

dapsone,[

398

cyclosporin, [ ] and IV immunoglobulin, [ ] have been administered to such patients with limited benefits.[ ] An attempt can be made to taper the oral dose rapidly over 7 to 14 days and thereafter switch to maintenance treatment with the least but fully effective dose and route. Patients who have an asthma exacerbation while receiving maintenance corticosteroids (e.g., after allergen or irritant exposure during an acute respiratory viral infection) should be treated with higher corticosteroid dose to prevent significant exacerbation and need for acute asthma care.[

322]

Patients on a long-term daily dose of corticosteroids need a calorie/sodium-restricted diet to minimize weight gain, fluid retention, and hypertension. In addition, they need optimal calcium and vitamin D intake and weight-bearing exercise to reduce tendencies to osteoporosis[ risk for

289 fracture[ ]

is still a concern, however, so monitoring of bone

401 density[ ]

399]

and myopathy. [

400]

Osteoporosis with an increased

and treatment with calcitonin or biphosphonate may be appropriate.[

402]

1272

TABLE 69-1 -- Long-Term Management of Asthma: Treatments in Stepwise Approach Treatment Long-Term Control

Quick Relief

Preferred treatments are in bold print Education

STEP 4 Severe Persistent

Daily medications: • Antiinflammatory: inhaled corticosteroid (high dose) and • Long-acting bronchodilator: either long acting inhlaed β2 agonist, sustained-release theophylline, or long-acting β2 -agonist tablets and • Corticosteroid tablets or syrup long term (2•mg/kg/day, generally do not exceed 60•mg per day).

• Short-acting bronchodilator: inhaled β2 agonists as needed for symptoms. • Intensity of treatment will depend on severity of exacerbation. • Use of short-acting inhaled β2 agonists on a daily basis, or increasing use, indicates need for additional long-termcontrol therapy • Short-acting bronchodilator: inhaled β2 agonists as needed for symptoms. • Intensity of treatment will depend on severity of exacerbation. • Use of short-acting inhaled β2 agonists on a daily basis, or increasing use, indicates need for additional long-term control therapy.

STEP 3 Moderate Persistent

Daily medications • Either Antiinflammatory: inhaled corticosteroid (medium dose) or Inhaled corticosteroid (lowmedium dose) and add a longacting bronchodilator, especially for nighttime symptoms either long-acting inhaled β2 agonist, sustainedrelease theophylline or long-

Steps 2 and 3 actions+ • Refer to individual education/ counseling.

acting β2 agonist tablets. • If needed Antiinflammatory: inhaled corticosteroids (mediumhigh dose) and Long-acting bronchodilator, especially, for nighttime symptoms: either long-acting inhaled β2 agonist, sustained release theophylline or longacting β2 agonist tablets

STEP 2 Mild Persistent

One daily medication: • Antiinflammatory: either inhaled corticosteroid (low doses) or cromolyn or nedocromil (children usually begin with a trial of cromolyn or nedocromil). • Sustained-release theophylline to serum concentration of 5–15••g ml. is an alternative but not preferred, therapy. Zafirlukast or zileuton may also be considered for patients ≥12 years of age although their position in therapy is not fully established. • No daily medication needed.

• Short-acting bronchodilator: inhaled β2 agonists as needed for symptoms. • Intensity of treatment will depend on severity of exacerbation. • Use of short-acting inhaled β2 agonists on a daily basis, or increasing use, indicates need for additional long-term control therapy

Step 1 actions plus: • Teach self monitoring. • Refer to group education if available. • Review and update selfmanagement plan.

STEP 1 Mild Intermittent

• Short-acting bronchodilator: inhaled β2 agonists as needed for symptoms. • Intensity of treatment will depend on severity of exacerbations. • Use of short-acting inhaled β2 agonists more than 2 times a week indicate need to initiate long-term control therapy.

↓Step down Review treatment every 1 to 6 months; a gradual stepwise reduction in treatment may be possible.

• Teach basic facts about asthma. • Teach inhaler spacer/holdingchamber technique. • Discuss roles of medications. • Develop self-management plan. • Develop action plan for when and how to take rescue actions, especially for patients with a history of severe exacerbations • Discuss appropriate environment control measures to avoid exposure to known allergens and irritants

↑Step up If control is not maintained, consider step up. First, review patient medication technique, adherence, and environment control. (avoidance of allergens or other factors that contribute to asthma severity).

• The stepwise approach presents general guidelines to assist clinical decisionmaking; it is not intended to be specific prescription. Asthma is highly variable; clinicians should tailor specific medication plans to the needs and circumstances of individual patients. • Gain control as quickly as possible; then decrease treatment to the least medication that is necessary to maintain control. Gaining control may be accomplished by either starting treatment at the step most appropriate to the initial severity of the condition or starting at a higher level of therapy (e.g., a course of systemic corticosteroids or higher dose of inhaled corticosteroids). • A rescue course of systemic corticosteroids may be needed at any time and at any step. • Some patients with intermittent asthma experience severe and life-threatening exacerbations separated by long peroids of normal lung function and no symptoms. This may be especially common with exacerbations provoked by respiratory infections. A short course of systemic corticosteriods is recommended. • At each step, patients should control their environment to avoid or control factors that make their asthma worse (eg., allergens, irritants): this requires specific diagnosis and education. • Referral to an asthma specialist for consultation or comanagement is recommended if there are difficulties achieving or maintaining control of asthma or if the patient requires step 4 care. Referral may be considered if the patient requires step 3 care.

1273

In general, lower doses of inhaled corticosteroids (ICS) are given for the patients who are not taking oral corticosteroids, whereas high-doses are initiated in those requiring oral corticosteroid therapy. In either group, stepping up or stepping down therapy should be done based on the patient's clinical status and pulmonary function. ICS treatment offers the advantage of fewer systemic side effects[ 405 needed.[ ]

403]

but requires coordination for inhaler use,[

404]

with assistance of a spacer device if

However, ICS therapy is more expensive than oral corticosteroid treatment and may produce local side effects, including oropharyngeal candidiasis,

which may be reduced by spacer device, and dysphonia, which may preclude continued use.[

406]

Patients who receive parenteral or oral corticosteroids or ICS at high dose may develop suppression of the hypothalamic-pituitary-adrenal (HPA) axis with adrenal 407 408

insufficiency[ ] [ ] proportionate to the intensity and duration of treatment. Patients who have received greater than 10•mg of prednisone for longer than 2 weeks have at least a partial HPA insufficiency. To prevent acute adrenal crisis, these patients should receive parenteral hydrocortisone (50–100•mg), or equivalent of other corticosteroids, every 8 hours when they have surgery, major illness, asthma exacerbation, or other stress. The HPA reserve can be measured with a rapid adrenocorticotropic hormone (ACTH) test; a peak plasma cortisol level of 20••g/dl (550•nmol/L) or greater 30 to 60 minutes after parenteral administration of 0.25• 409]

mg of ACTH (cosyntropin), is reflective of normal adrenal function.[

β2 -Agonist bronchodilators may be used on a regular basis,[

410]

although there may be small risk of increasing AHR,[

411]

for patients with a continuous tendency to

bronchospasm or on an as-needed basis to prevent or relieve bronchospasm. The aerosol route by MDI is preferred for those able to coordinate effective inhalation 405

from the device, assisted by a spacer device if necessary,[ ] because the action is immediately effective and, if not overused, without significant side effect; alternatively, a small-volume nebulizer may be used. Long-acting inhaled β2 -adrenergic therapy (salmeterol or formoterol) can provide bronchodilator action for almost 12 hours. Because the onset of action is slow, however, patients should continue to use albuterol for quick relief. Oral theophylline,[

340]

inhaled cromolyn, or nedocromil treatment sometimes has a corticosteroid-sparing effect in the corticosteroid-treated patient and thus may

merit trial in such patients. Gastroesophageal and neuropsychologic side effects[ corticosteroid and may preclude continued methylxanthine treatment.

412]

occur with increased frequency in patients treated with methylxanthine and

Leukotriene modulators, synthesis inhibitors (zileuton) and receptor antagonists (zafirlukast and montelukast), offer another direction in asthma therapy[ [415]

416]

Studies in patients with mild-to-moderate disease show a small but significant improvement of asthma control with zafirlukast,[ 418]

zileuton. [

montelukast,[

413] [414]

417]

and

Typically this improvement, especially in FEV1 , is less than that seen with ICS but can be seen sooner. Leukotriene-modifying drugs can prevent

exercise-induced and antigen-induced asthma, in addition to improving airway inflammation. These agents are currently recommended as a third-line drug after inhaled β-adrenergics and corticosteroids. H1 antihistamines are effective for treatment of allergic rhinitis, conjunctivitis, and urticaria but ineffective for asthma. For several decades there was concern that these agents, because of their known drying effects in the upper respiratory

1274

tract, might act similarly in the tracheobronchial tree and compound mucus retention in asthma and therefore be dangerous. In practice, this has not been a problem of consequence, and these agents may be used safely for treatment of allergic disorders in the asthmatic patient. Anticholinergic agents have no established role in the management of persistent asthma.[ bronchodilation over β2 -adrenergic agonists and in patients with COPD[

421]

420 theophylline,[ ]

419]

Ipratropium, 36••g 4 times daily by MDI, does slightly increase

but the increase may not justify the added expense. There is a role for chronic use of ipratropium

and in occasional asthmatic patients who do not tolerate beta agonists.

Antibiotic treatment of respiratory or other infection for the asthmatic patient should follow the principles and criteria of such treatment for nonasthmatic individuals or, if applicable, patients with chronic bronchitis or sinusitis.[ outcome.[

423]

422]

Empiric administration of an antibiotic for acute asthma does not favorably influence patient

Prophylactic immunizations with pneumococcal and influenza vaccines are indicated for asthmatic patients with persistent disease.

Immunotherapy 163 164 424 425 426 427

The role of immunotherapy in the management of allergic asthma is controversial.[ ] [ ] [ ] [ ] [ ] [ ] The patient with persistent asthma and ongoing problems with an unavoidable aeroallergen exposure may elect to undertake such treatment when every effort is being made to spare corticosteroid or other therapy. The use on a monoclonal antibody against IgE have been recently investigated and found to be of benefit in patients with moderate-to-severe asthma.[ and other novel therapies might soon be available to patients with asthma, especially those who do not respond to the standard ICS and β-agonist

428] [429]

This

430 therapy.[ ]

Compounding and Confounding Conditions 431

Hypertension in the asthmatic patient may reflect the asthma and its treatment with adrenergic or corticosteroid drugs,[ ] overweight state, obstructive sleep disorder, and other mechanisms found in nonasthmatic patients. Each of these possibilities should be addressed before embarking on pharmacotherapy for the hypertension. Drugs that have potential for compounding asthma include the nonselective β-adrenergic receptor-blocking agents such as propranolol; the α-β blocker labetalol; and to a lesser extent the selective β2 -adrenergic blockers. The ACE-inhibiting drugs might aggravate asthma; however, cough rather than bronchospasm is associated with the use of these drugs.[

300] [432]

Angina and hypertrophic cardiomyopathy may be compounded, especially in elderly patients, by adrenergic agents used to treat asthma. These agents and methylxanthine drugs may individually or together potentiate arrhythmias, but these theoretic risks often are not of practical consequence.[

433]

The rare patient with

both obstructive cardiomyopathy and persistent asthma should be managed with a calcium channel blocker and corticosteroids alone or with only judicious use of an inhaled β2 -adrenergic bronchodilator. Inhaled anticholinergic drugs provide an alternative to bronchodilators in these patients. Anesthesia

Optimal reversal of airway obstruction before general anesthesia and surgery is key to preventing intraoperative and postoperative asthma exacerbations. Several 434

days of high-dose oral or parenteral corticosteroid,[ ] intensified bronchopulmonary toilet measures, measurements of spirometry, and if indicated, plasma theophylline and electrolyte levels are appropriate to ensuring an optimal state. Atropine used to dry upper respiratory secretions may have a salutary bronchodilator 435

436

effect. The neuromuscular blocking agents alcuronium and d-tubocurarine may provoke bronchoconstriction[ ] ; pancuronium bromide[ ] and succinylcholine are usually tolerated by the asthmatic patient. Halothane, enflurane, isoflurane, and ketamine general anesthetics are inherently bronchodilators and therefore not likely to provoke bronchoconstriction.[ with halothane.

436]

However, there is risk of ventricular tachycardia or fibrillation if aminophylline is given beforehand or if epinephrine is used

Activity and Rehabilitation

Regular aerobic physical exercise, up to 1 hour daily, 3 to 5 days per week, as tolerated without limitation by bronchospasm and initiated at a time when asthma has been stabilized with a pharmacologic regimen, may benefit the patient with asthma by improving cardiorespiratory and musculoskeletal conditioning, retarding the 437

development or progression of osteoporosis, and lessening tendencies to anxiety and depression associated with chronic disease.[ ] Counseling by an exercise physiologist in a graduated program individualized according to functional aerobic capacity, as determined by exercise testing, is appropriate for the deconditioned 438

individual, especially if there is coexisting heart disease. [ ] Cautions against aeropollutant and aeroallergen exposures and instructions in the use of inhalations from a β-adrenergic bronchodilator before exercise may also apply. 439

The patient with disability related to asthma[ ] or severe limitations from associated chronic bronchitis or emphysema, exogenous Cushing's syndrome, cardiovascular disease, and depression or somatization or other psychic state with excess disability may benefit from an individualized rehabilitative program, 311] [440] [441]

including respiratory, physical, exercise, behavioral, and psychologic therapies. [ Patient Education

Education of the patient, spouse, and family about asthma should include general information regarding the disease process, therapy, and monitoring. Education fosters a partnership among the patient, family, and provider[

1] [442]

and improves prospects for compliance with therapy.[

(1) the reasons for and proper use of medications, including patient demonstrations of competence in using inhaler

443]

In addition, education should focus on

404 devices[ ]

; (2) the complications of the disease

and treatment; (3) the use of the peak flowmeter and keeping a record of PEF measurements and a diary of symptoms and medication usage, when appropriate[ [445] [446]

444]

; (4) expectations for control of the disease and functional level of performance; and (5) contingency action plan for exacerbation, including criteria for

initiating additional medicines and accessing additional care.[

447]

The goals of education are to reduce disease morbidity through greater self-reliance and patient

responsibility for an active role in managing the disease. Educational efforts may be undertaken by a physician, nurse, or trained educator. Self-management programs,[ Education

448]

books, or interactive computer/ Internet-based programs may facilitate the educational process but may not fully substitute for human involvement.

1275

efforts have been shown to improve asthma management,[ reduce overall costs in management of the

449]

increase adherence to medication regimens, decrease utilization of emergency services, [

450]

and

451 disease.[ ]

Intermittent Asthma: Primary Care The majority of asthma patients, at some time in their lives, have isolated or recurring mild attacks, with a clear precipitating factor(s) and rapid response to optimal therapy. The principles of evaluation and management outlined previously can be applied in the degree appropriate to the morbidity of the individual patient. Pharmacotherapy

A selective β2 -adrenergic bronchodilator delivered from pressurized-cartridge metered-dose dispenser is the primary treatment for relief or prevention of intermittent asthma. The patient must be instructed in the proper synchronization of inhalation and breath holding of the aerosol, allowing several minutes between inhalations, 404

and in the use of a spacing device, if necessary, to ensure penetration of the aerosol to the central bronchial airways.[ ] As-needed use of 2 to 4 inhalations of a βadrenergic drug can be given at 4-hour to 8-hour intervals; before exercise or exposure to known aeroallergen or aeroirritant; at bedtime; at onset of acute upper respiratory illness; or to relieve unanticipated exacerbations. Inhaled corticosteroid is the cornerstone of controller therapy for persistent asthma. ICS therapy reduces airway inflammation and AHR and controls symptoms with side effects. In patients who continue to have symptoms despite moderate ICS doses, adding a long-acting inhaled beta agonist (LABA) is generally superior to increasing the ICS dose.[

452]

The advantage of using a combined inhalation of LABA and ICS has scientific justification [

453]

that has been confirmed in clinical

454 455 456 trials.[ ] [ ] [ ] 340]

Sustained-release theophylline administered at bedtime may prevent nocturnal or morning attack.[ [414] [415] [457]

Referral to Specialist

Leukotriene modulators provide another alternative.[

1] [413]

Referral to a physician with a specialized interest and knowledge in the diagnosis and management of asthma is indicated when (1) the diagnosis of asthma is considered as an explanation for cough, dyspnea, or wheezing but not confirmed by physical findings, laboratory or pulmonary function tests, or therapeutic trials of bronchodilator medication; (2) evaluation and management of an allergic component is desired; (3) there is significant morbidity, as indicated by hospitalization, loss of time from school or work, disruption of sleep/fatigue, or limited exercise tolerance; (4) side effects compromise pharmacotherapy; (5) the patient fails to respond to multiple medications, including systemic corticosteroids, or is dependent on doses of corticosteroids that produce side effects; (6) the patient overuses inhaled bronchodilators; (7) associated medical or psychologic conditions compound and perpetuate the asthma; or (8) the patient or family needs education regarding the disease and its optimal management.[

1] [326]

Other practitioners that may be of assistance in the management of asthma include the rhinosinusoidal surgeon, psychiatrist, psychologist, nutritionist, exercise physiologist, speech therapist, and nurse practitioner or clinical educator. The asthma specialist can help determine the need for such assistance. The American Academy of Allergy, Asthma and Immunology (800-822-ASMA or www.aaaai.org), American Lung Association (800-LUNG-USA or www.lungusa. org/asthma/), Asthma and Allergy Foundation of America (800-7-ASTHMA or www.aafa.org), Allergy and Asthma Network/Mothers of Asthmatics (800-878-4403 or www. aanma.org), and National Asthma Education and Prevention Program of the National Heart, Lung, and Blood Institute (301-251-1222 or www.nhlbi.nih. gov) provide educational aids and forums for patients with asthma.

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363. Mathison DA, Stevenson DD, Simon RA: Asthma and the home environment, Ann Intern Med 97:128, 1982. 364. Luskin AT: Environmental control: key to asthma management—developing a program that patients will “buy into,” J Respir Dis 16:253, 1995. 365. Platts-Mills TAE: Allergen avoidance at home: what really works? J Respir Dis 10:53, 1989. 366. Marks GB, Tovey ER, Green W, et al: House dust mite allergen avoidance: a randomized controlled trial of surface chemical treatment and encasement of bedding, Clin Exp Allergy 24:1078, 1994. 367. De Blay F, Chapman MD, Platts-Mills TA: Airborne cat allergen (Fel d 1): environmental control with the cat in situ, Am Rev Respir Dis 143:1334, 1991. 368. Kozak PP, Gallup J, Cummins LH, et al: Factors of importance in determining the prevalence of indoor molds, Ann Allergy 43:88, 1979. 369. Hirsch DJ, Hirsch SR, Kalbfleisch JH: Effect of central air conditioning and meteorologic factors on indoor spore counts, J Allergy Clin Immunol 62:22, 1978. 370. Solomon WR, Burge HA, Boise JR: Exclusion of particulate allergens by window air conditioners, J Allergy Clin Immunol 65:305, 1980. 371. Maimonides M: Treatise on asthma, Philadelphia, 1963, Lippincott. 372. Lee TH, Austen KF, Leitch AG, et al: The effects of a fish-oil-enriched diet on pulmonary mechanics during anaphylaxis, Am Rev Respir Dis 132:1204, 1985. 373. Thien FC, Mencia-Huerta JM, Lee TH: Dietary fish oil effects on seasonal hay fever and asthma in pollen-sensitive subjects, Am Rev Respir Dis 147:1138, 1993. 374. Troisi RJ, Willett WC, Weiss ST, et al: A prospective study of diet and adult-onset asthma, Am J Respir Crit Care Med 151:1401, 1995. 375. Mohsenin V, Dubois AB, Douglas JS: Effect of ascorbic acid on response to methacholine challenge in asthmatic subjects, Am Rev Respir Dis 127:143, 1983. 376. Malo JL, Cartier A, Pineau L, et al: Lack of acute effects of ascorbic acid on spirometry and airway responsiveness to histamine in subjects with asthma, J Allergy Clin Immunol 78:1153, 1986. 377. Shaheen SO, Sterne JA, Thompson RL, et al: Dietary antioxidants and asthma in adults: population-based case-control study, Am J Respir Crit Care Med 164:1823, 2001. 378. Wilson JW, Djukanovic R, Howarth PH, et al: Inhaled beclomethasone dipropionate downregulates airway lymphocyte activation in atopic asthma, Am J Respir Crit Care Med 149:86, 1994. 379. Bentley AM, Hamid Q, Robinson DS, et al: Prednisolone treatment in asthma: reduction in the numbers of eosinophils, T cells, tryptase-only positive mast cells, and modulation of IL-4, IL-5, and interferon-gamma cytokine gene expression within the bronchial mucosa, Am J Respir Crit Care Med 153:551, 1996. 380. Kelly EA, Busse WW, Jarjour NN: Inhaled budesonide decreases airway inflammatory response to allergen, Am J Respir Crit Care Med 162:883, 2000. 381. Pincus DJ, Szefler SJ, Ackerson LM, et al: Chronotherapy of asthma with inhaled steroids: the effect of dosage timing on drug efficacy, J Allergy Clin Immunol

95:1172, 1995. 382. McFadden ER Jr: Dosages of corticosteroids in asthma, Am Rev Respir Dis 147:1306, 1993. 383. O'Driscoll BR, Kalra S, Wilson M, et al: Double-blind trial of steroid tapering in acute asthma, Lancet 341:324, 1993. 384. Cypcar D, Busse WW: Steroid-resistant asthma, J Allergy Clin Immunol 92:362, 1993. 385. Woolcock AJ: Corticosteroid-resistant asthma: definitions, Am J Respir Crit Care Med 154:S45, 1996. 386. Spahn JD, Landwehr LP, Nimmagadda S, et al: Effects of glucocorticoids on lymphocyte activation in patients with steroid-sensitive and steroid-resistant asthma, J Allergy Clin Immunol 98:1073, 1996. 387. Dykewicz MS, Greenberger PA, Patterson R, et al: Natural history of asthma in patients requiring long-term systemic corticosteroids, Arch Intern Med 146:2369, 1986. 388. Erzurum SC, Leff JA, Cochran JE, et al: Lack of benefit of methotrexate in severe, steroid-dependent asthma: a double-blind, placebo-controlled study, Ann Intern Med 114:353, 1991. 389. Alvarez J, Surs W, Leung DY, et al: Steroid-resistant asthma: immunologic and pharmacologic features, J Allergy Clin Immunol 89:714, 1992. 390. Nelson HS, Hamilos DL, Corsello PR, et al: A double-blind study of troleandomycin and methylprednisolone in asthmatic subjects who require daily corticosteroids, Am Rev Respir Dis 147:398, 1993. 391. Szefler SJ, Kamada AK, Hughes D, et al: Alternative treatments for asthma: assessing the need, J Asthma 29:91, 1992. 392. Hill MR, Szefler SJ, Ball BD, et al: Monitoring glucocorticoid therapy: a pharmacokinetic approach, Clin Pharmacol Ther 48:390, 1990. 393. Adcock IM: Steroid resistance in asthma: molecular mechanisms, Am J Respir Crit Care Med 154:S58, 1996. 394. Mullarkey MF, Lammert JK, Blumenstein BA: Long-term methotrexate treatment in corticosteroid-dependent asthma, Ann Intern Med 112:577, 1990. 395. Berlow BA, Liebhaber MI, Dyer Z, et al: The effect of dapsone in steroid-dependent asthma, J Allergy Clin Immunol 87:710, 1991. 396. Szczeklik A, Nizankowska E, Dworski R, et al: Cyclosporin for steroid-dependent asthma, Allergy 46:312, 1991. 397. Mazer BD, Gelfand EW: An open-label study of high-dose intravenous immunoglobulin in severe childhood asthma, J Allergy Clin Immunol 87:976, 1991. 398. Busse WW, McGill K, Jarjour NN: Current management of asthma patients with corticosteroid resistance, Am J Respir Crit Care Med 154:S70, 1996. 399. Reid IR, Veale AG, France JT: Glucocorticoid osteoporosis, J Asthma 31:7, 1994. 400. Perez T, Becquart LA, Stach B, et al: Inspiratory muscle strength and endurance in steroid-dependent asthma, Am J Respir Crit Care Med 153:610, 1996.

401. Johnston CC Jr, Slemenda CW, Melton LJ III: Clinical use of bone densitometry, N Engl J Med 324:1105, 1991.

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402. Liberman UA, Weiss SR, Broll J, et al: Effect of oral alendronate on bone mineral density and the incidence of fractures in postmenopausal osteoporosis. Alendronate Phase III Osteoporosis Treatment Study Group, N Engl J Med 333:1437, 1995. 403. Utiger RD: Differences between inhaled and oral glucocorticoid therapy, N Engl J Med 329:1731, 1993. 404. Barron EN Jr: Proper technique for using inhalers in asthma, N Engl J Med 316:951, 1987. 405. Newhouse MT: Pulmonary drug targeting with aerosols: principles and clinical applications in adults and children, Am J Asthma Allergy Pediatricians 7:23, 1993. 406. Hanania NA, Chapman KR, Kesten S: Adverse effects of inhaled corticosteroids, Am J Med 98:196, 1995. 407. Toogood JG: Inhaled glucocorticoids: benefits and risks. In Szefler SJ, Leung DYM, editors: Severe asthma: pathogenesis and clinical management, New York, 1996, Marcel Dekker, p 207. 408. Oelkers W: Adrenal insufficiency, N Engl J Med 335:1206, 1996. 409. May ME, Carey RM: Rapid adrenocorticotropic hormone test in practice: retrospective review, Am J Med 79:679, 1985. 410. Drazen JM, Israel E, Boushey HA, et al: Comparison of regularly scheduled with as-needed use of albuterol in mild asthma. Asthma Clinical Research Network, N Engl J Med 335:841, 1996. 411. O'Byrne PM, Kerstjens HA: Inhaled beta 2-agonists in the treatment of asthma, N Engl J Med 335:886, 1996. 412. Schraa JC, Dirks JF: The influence of corticosteroids and theophylline on cerebral function: a review, Chest 82:181, 1982. 413. Holgate ST, Bradding P, Sampson AP: Leukotriene antagonists and synthesis inhibitors: new directions in asthma therapy, J Allergy Clin Immunol 98:1, 1996. 414. Smith LJ: Leukotrienes in asthma: the potential therapeutic role of antileukotriene agents, Arch Intern Med 156:2181, 1996. 415. Horwitz RJ, McGill KA, Busse WW: The role of leukotriene modifiers in the treatment of asthma, Am J Respir Crit Care Med 157:1363, 1998. 416. Spector SL, Smith LJ, Glass M: Effects of 6 weeks of therapy with oral doses of ICI 204,219, a leukotriene D4 receptor antagonist, in subjects with bronchial asthma. ACCOLATE Asthma Trialists Group, Am J Respir Crit Care Med 150:618, 1994.

417. Malmstrom K, Rodriguez-Gomez G, Guerra J, et al: Oral montelukast, inhaled beclomethasone, and placebo for chronic asthma: a randomized, controlled trial. Montelukast/Beclomethasone Study Group, Ann Intern Med 130:487, 1999. 418. Israel E, Cohn J, Dube L, et al: Effect of treatment with zileuton, a 5-lipoxygenase inhibitor, in patients with asthma: a randomized controlled trial. Zileuton Clinical Trial Group, JAMA 275:931, 1996. 419. Johns KA, Buse WW: Anticholinergic drugs: what role in asthma? J Respir Dis 10:35, 1989. 420. Rebuck AS, Gent M, Chapman KR: Anticholinergic and sympathomimetic combination therapy of asthma, J Allergy Clin Immunol 71:317, 1983. 421. Braun SR, McKenzie WN, Copeland C, et al: A comparison of the effect of ipratropium and albuterol in the treatment of chronic obstructive airway disease, Arch Intern Med 149:544, 1989. 422. Anthonisen NR, Manfreda J, Warren CP, et al: Antibiotic therapy in exacerbations of chronic obstructive pulmonary disease, Ann Intern Med 106:196, 1987. 423. Graham VA, Milton AF, Knowles GK, et al: Routine antibiotics in hospital management of acute asthma, Lancet 1:418, 1982. 424. Platts-Mills TA: Allergen-specific treatment for asthma. III, Am Rev Respir Dis 148:553, 1993. 425. Creticos PS: The role of immunotherapy in allergic rhinitis/allergic asthma, Allergy Proc 16:297, 1995. 426. Norman PS: Is there a role for immunotherapy in the treatment of asthma? Yes, Am J Respir Crit Care Med 154:1225, 1996. 427. Ramirez NC, Ledford DK: Immunotherapy for allergic asthma, Med Clin North Am 86:1091, 2002. 428. Bush RK: The use of anti-IgE in the treatment of allergic asthma, Med Clin North Am 86:1113, 2002. 429. Babu KS, Holgate ST: The role of anti-IgE therapies in the treatment of asthma, Hosp Med 63:483, 2002. 430. Hansel TT, Barnes PJ: Novel drugs for treating asthma, Curr Allergy Asthma Rep 1:164, 2001. 431. Sanders BP, Portman RJ, Ramey RA, et al: Hypertension during reduction of long-term steroid therapy in young subjects with asthma, J Allergy Clin Immunol 89:816, 1992. 432. Packard KA, Wurdeman RL, Arouni AJ: ACE inhibitor-induced bronchial reactivity in patients with respiratory dysfunction, Ann Pharmacother 36:1058, 2002. 433. Ofori CS, Gradman AH: Asthma therapy and the heart: what to watch for, J Respir Dis 14:861, 1993. 434. Kabalin CS, Yarnold PR, Grammer LC: Low complication rate of corticosteroid-treated asthmatics undergoing surgical procedures, Arch Intern Med 155:1379, 1995. 435. Yeung ML, Ng LY, Koo AW: Severe bronchospasm in an asthmatic patient following alcuronium and d-tubocurarine, Anaesth Intensive Care 7:62, 1979.

436. Fung DL: Emergency anesthesia for asthma patients, Clin Rev Allergy 3:127, 1985. 437. Varray A, Prefaut C: Importance of physical exercise training in asthmatics, J Asthma 29:229, 1992. 438. Garfinkel SK, Kesten S, Chapman KR, et al: Physiologic and nonphysiologic determinants of aerobic fitness in mild to moderate asthma, Am Rev Respir Dis 145:741, 1992. 439. American Thoracic Society: Guidelines for the evaluation of impairment/disability in patients with asthma, Am Rev Respir Dis 147:1056, 1993. 440. Ries AL, Kaplan RM, Limberg TM, et al: Effects of pulmonary rehabilitation on physiologic and psychosocial outcomes in patients with chronic obstructive pulmonary disease, Ann Intern Med 122:823, 1995. 441. Clark CJ, Cochrane LM: Physical activity and asthma, Curr Opin Pulm Med 5:68, 1999. 442. Sheffer AL: The National Asthma Education Program attacks asthma, J Allergy Clin Immunol 87:468, 1991. 443. Bender B, Milgrom H: Compliance with asthma therapy: a case for shared responsibility, J Asthma 33:199, 1996. 444. Ignacio-Garcia JM, Gonzalez-Santos P: Asthma self-management education program by home monitoring of peak expiratory flow, Am J Respir Crit Care Med 151:353, 1995. 445. Chan-Yeung M, Chang JH, Manfreda J, et al: Changes in peak flow, symptom score, and the use of medications during acute exacerbations of asthma, Am J Respir Crit Care Med 154:889, 1996. 446. Malo JL, L'Archeveque J, Trudeau C, et al: Should we monitor peak expiratory flow rates or record symptoms with a simple diary in the management of asthma? J Allergy Clin Immunol 91:702, 1993. 447. Strunk RC: Identification of the fatality-prone subject with asthma, J Allergy Clin Immunol 83:477, 1989. 448. Bailey WC, Richards JM Jr, Brooks CM, et al: A randomized trial to improve self-management practices of adults with asthma, Arch Intern Med 150:1664, 1990. 449. Kotses H, Bernstein IL, Bernstein DI, et al: A self-management program for adult asthma. Part I. Development and evaluation, J Allergy Clin Immunol 95:529, 1995. 450. Bolton MB, Tilley BC, Kuder J, et al: The cost and effectiveness of an education program for adults who have asthma, J Gen Intern Med 6:401, 1991. 451. National Asthma Education and Prevention Program Task Force: Cost-effectiveness, quality of care, and financing of asthma care, Am J Respir Crit Care Med 154:S81, 1996. 452. Heyneman CA, Crafts R, Holland J, et al: Fluticasone versus salmeterol/ low-dose fluticasone for long-term asthma control, Ann Pharmacother 36:1944, 2002.

453. Barnes PJ: Scientific rationale for inhaled combination therapy with long-acting beta2-agonists and corticosteroids, Eur Respir J 19:182, 2002. 454. Walters EH, Bjermer L, Faurschou P, et al: The anti-inflammatory profile of inhaled corticosteroids combined with salmeterol in asthmatic patients, Respir Med 94(suppl F):26, 2000. 455. Lalloo U: Symbicort: controlling asthma in adults, Respir Med 96(suppl A):16, 2002. 456. Lazarus SC, Boushey HA, Fahy JV, et al: Long-acting beta2-agonist monotherapy vs continued therapy with inhaled corticosteroids in patients with persistent asthma: a randomized controlled trial, JAMA 285:2583, 2001. 457. Drazen JM, Israel E, O'Byrne PM: Treatment of asthma with drugs modifying the leukotriene pathway, N Engl J Med 340:197, 1999.

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Chapter 70 - Asthma Guidelines and Outcomes

Louis-Philippe Boulet

The process of retrieving information and translating knowledge into “best practice” has been ongoing in medicine since time immemorial, although this unstructured process has been tainted by individuals' backgrounds and beliefs and at times by elements of “local folklore.” As reported by Guo, the famous Chinese physician 1

Bian Que had already produced guidelines for the practice of medicine a few centuries before the “Common Era.”[ ] There has, however, been an exponential increase in medical knowledge in the past century, particularly in the last decades, making the translation of knowledge into care more difficult. For example, a Medline search on the keyword “asthma” brought 715 references in 1966, 1186 in 1980, and 3718 in 2000. This makes the physician's task of regularly updating his or her knowledge more demanding than before, even with the current available aids such as reviews, congress and symposia, and academic detailing. This fact, combined with the evidence that disease-related morbidity is often preventable by optimal care and with the increasing demands of patients, health organizations, and governments, has provided incentives to develop more effective ways to guide the practitioner.

Clinical practice guidelines have become an integral part of the medical environment. Although they have frequently been criticized over the past two decades, improvements in their mode of production, the fact that most of them are now evidence based, and the development of dissemination strategies and tools facilitating 2 3 4

their integration into practice make them a useful means of optimizing care.[ ] [ ] [ ] Although these guidelines should remain flexible and their recommendations individualized, they provide a common template not only for physicians but also for other health professionals, patients, and public and health authorities to articulate educational initiatives and interventions. The terms clinical practice guidelines and consensus reports are generally used to describe documents that include recommendations about best practice for a 5

particular condition, with the goal of improving quality of care. Standards are perceived as more rigid, and position statements less binding.[ ] The American Thoracic Society describes guidelines as systematically developed statements to assist practitioner and patient decisions about appropriate health care for specific 4

clinical circumstances.[ ] Structured guidelines have been produced in the past by individuals, small groups of experts or committees formed by medical societies, 6] [7] [8]

although they have not necessarily been of the same type as more recent ones.[

The main goal of clinical practice guidelines is to improve health care in order to decrease the morbidity and mortality associated with common health conditions. This has been done primarily by providing health care professionals with the scientific evidence supporting a series of recommendations on clinical practices in order to guide therapeutic decisions, namely those that are most effective and have the least potential for untoward effects.[

5]

This chapter reviews the origin, content, and evaluations of asthma guidelines thus far produced and discusses their implementation into current practice and the asthma outcomes targeted by the recommendations or used to assess their effects. The similarities and differences between the main guidelines are stressed as are the areas of uncertainty and those where additional research is required. Briefly mentioned are more local guidelines and those published in languages other than English or French. Furthermore, this chapter does not review all the evidence supporting the recommendations mentioned; a review of such evidence has already been 9

published for the first series of guidelines.[ ] For the others, refer to the original documents. Most recent guidelines are “evidence-based” and include a list of studies supporting the recommendations.

ORIGIN OF ASTHMA CONSENSUS REPORTS The increase in mortality and morbidity associated with asthma observed in industrialized countries during the 1970s and the following decade gave rise to several task forces and consensus conferences, initially in Australia and New Zealand, Canada, the United Kingdom, and the United States. Their goal was to define the ideal 10 11 12 13 14 15 16 17 18

approach for the evaluation and management of asthma and to produce guidelines for clinicians[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ( Table 70-1 ). These reports were produced under the auspices of national medical societies and health institutes. Although not specified as formal guidelines, some of these reports, such as those of the American College of Allergy Asthma Immunology and the American Academy of Allergy, Asthma, and Immunology, developed practice parameters for the diagnosis and treatment of asthma.[

19]

It was considered that current asthma care was not optimal, with deficiencies such as underestimation of disease severity,

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TABLE 70-1 -- Asthma Consensus Statements and Expert Opinion Guidelines Year

Author

1979

†

*

Publication

Type

Topic

Eggleston PA

Allergy Clin Immunol

Consensus

Asthma exercise challenge guideline

1985

Anonymous

Rev Respir Dis

Expert opinion

Asthma: BAL in pathogenesis

1985

Anonymous

Chest

Expert opinion

Asthma: BAL in pathogenesis

1986

AAAI

J Allergy Clin Immunol

Consensus

Asthma non-bronchodilator

1986

Clinton, JE

Ann Emerg Med

Expert opinion

Asthma CXR in ED guideline

1989

Warner JO

Arch Dis Child

Consensus

Asthma guideline

1989

Woolcock A

Med J Austr

Consensus

Asthma guideline

1990

Hargreave FE

J Allergy Clin Immunol

Consensus

Asthma guideline

1990

BTS

BMJ

Consensus

Asthma guideline

1991

NAEP

Allergy Clin Immunol

Consensus

Asthma guideline

1992

Anonymous

Lakartidningen

Consensus

Asthma guideline

1992

Warner JO

Arch Dis Child

Consensus

Follow-up pediatric guideline

1992

Cartier A

Rev Mal Respir

Review

Asthma guideline

1992

NIH, NHLBI

International Consensus Report on the Diagnosis and Management of Asthma

Consensus

Asthma guideline

1992

SAPS

S Afr Med J

Consensus

Asthma guideline

1993

AAAI

Practice Parameters for the Diagnosis of Asthma

Consensus

Asthma guideline

1993

ATS

Am Rev Respir Dis

Consensus

Disability in patients with asthma

1993

BTS

Thorax

Consensus

Asthma, revised

1994

Bergner A

Clin Ther

Expert opinion

Asthma guideline

1994

Goldstein RA

Ann Intern Med

Consensus

Asthma guideline, national

1994

Kibbe DC

Jt Comm J Qual Improv

Consensus

Quality improvement, asthma example

1994

SAPS

S Afr Med J

Consensus

Asthma guideline

1995

Cockcroft DW

Ann Allergy Asthma Immunol

Review

Asthma guideline in ED

1995

Fireman P

Allergy Proc

Review

Asthma β2 agonist guideline safety

1995

NHLBI/WHO

Global Strategy Asthma Management & Prevention

Consensus

Asthma guideline

1996

Beveridge R

CMAJ

Consensus

Emergency asthma management

1996

NEAGDG

BMJ

Consensus

Asthma guideline

1996

Ernst P

Can Resp J

Consensus

Asthma guideline

1997

NIH, NHLBI

Guidelines for the Diagnosis and Management of Asthma Expert Panel Report II

Consensus

Asthma guideline

1997

BTS

Thorax

Consensus

Asthma guideline

1998

Warner JO

Pediatr Pulmonol

Consensus

Pediatric asthma guideline

1999

Boulet LP

CMAJ

Consensus

Asthma guideline

2000

Dahl R

Respir Med

Consensus

Asthma guideline

2001

Boulet LP

Can Resp J

Consensus

Update on asthma

AAAI, American Academy of Allergy and Immunology; ATS, American Thoracic Society; BTS, British Thoracic Society; CXR, chest radiograph; ED, emergency department; NAEP, National Asthma Education Program; NEAGDG, North of England Guideline Development Group; NHLBI, National Heart, Lung, and Blood Institute; NIH, National Institutes of Health; SAPS, South African Pulmonary Society, WHO, World Health Organization. * Partial list, modified with permission from Hackner D, Tu G, Weingarten S, et al: Chest 116:1046, 1999. † First author.

insufficient use of objective measures of airflow obstruction, and insufficient use of some asthma medications, particularly antiinflammatory agents.[

20] [21] [22] [23]

[24]

It was also concluded that a large proportion of asthma-related morbidity and mortality could be avoided by improving general asthma care. Another incentive to optimize asthma management was the observation that direct and indirect costs for asthma were high and on the rise, resulting in an increasing burden on patients and health care systems alike.[

25] [26]

Initially originating from “national” sources, the process has been rapidly “internationalized.” In this regard, the authors of the first report in Canada in 1990 included 14 27]

participants from other countries, conferring on it an “international flavor,” although the first “official” international report was published in June 1992.[ ] [ was followed by the Global Initiative for Asthma (GINA), which followed the American National Heart, Lung, and Blood Institute (NHLBI)/World Health

This

28

Organization (WHO) 1993 Workshop Report on Global Strategy for Asthma Management and Prevention published in January 1995.[ ] Following its first publications (1991) on asthma guidelines, the American NHLBI, in conjunction with the WHO, published a report on asthma management and prevention as a

1285

19 28

result of the work of an international panel of experts.[ ] [ ] The report stressed the fact that such guidelines should be successfully applied to all countries, taking into consideration the cultural and economic context of each. More options were offered than in previous reports, considering potential economic barriers. Among GINA's accomplishments are the production of guideline reports (workshop report, practical guide, pocket guide, patient materials) and their translation in many languages; workshops and symposia; and a Website (www.ginasthma.com) offering copies of guidelines, slides presentations, and patient materials. The GINA guidelines have been revised regularly, and various publications have reported some of their components.[ were published in 1997 (expert Panel Report and

36 37 1998.[ ] [ ]

32 2).[ ]

The British guidelines were revised in 1993 and

33 34 35 1995.[ ] [ ] [ ]

29] [30] [31]

Updates of the NHLBI guidelines

The Canadian guidelines were updated in 1995

Most guidelines are currently under revision.

Guidelines on specific issues or subgroups of asthmatic patients have generally been included in the global consensus reports, although some have been produced specifically for children, asthma during pregnancy, and asthma in the elderly patient.[ republished in 1992 and

40 41 1998.[ ] [ ]

42

10] [38] [39]

The 1989 International Pediatric Guidelines were revised and

Guidelines on emergency treatment of asthma are discussed later.

In 1999, Hackner et al[ ] produced a review of clinical practice guidelines published in peer-reviewed journals since 1974 in the area of pulmonary medicine (see Table 70-1 ). A total of 271 articles were identified, including 115 consensus statements and expert opinion guidelines. Close to one third (30.3%) were related to asthma. This review showed that the number of pulmonary guidelines published had markedly increased in the preceding years but that few had been properly

evaluated. In addition, it indicated that the many published guideline reports had, in turn, led to the development of local practice guidelines, mostly unpublished.[

42]

43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56] [57] [58] [59]

They also generated an extensive debate about their production, content, and integration into care.[

[60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89] [90] [91] [92] [93] [94]

CONTENT OF MAIN ASTHMA GUIDELINES This section focuses on general recommendations provided by the most recent national and international guidelines, particularly the most widely disseminated. [ [32] [35] [37]

28]

The other guidelines published have much in common with the guidelines discussed here; some of their content is mentioned, but refer to the original

documents for a more complete review of their recommendations.[

95] [96] [97] [98] [99] [100] [101] [102] [103] [104]

Most guidelines stress the fact that asthma is a major public health problem everywhere and that current progress in understanding the disease and therapeutic advances have led to a reassessment of strategies to improve its long-term management and reduce the burden of the disease. The most widely disseminated guidelines are quite similar with regard to their different sections and recommendations; thus, a lack of specific references in the following text means that the component being discussed is common to most such guidelines. Recommendations specific to particular guidelines are mentioned. Definition of Asthma All recent guidelines define asthma as a chronic inflammatory disorder of the airways characterized by respiratory symptoms such as dyspnea, wheezing, cough, chest tightness, and sputum production. The effect of airway inflammation is variable airflow limitation, reversible in most patients, associated with airway hyperresponsiveness. 105

Treatment can reduce inflammation and long-term symptoms, maximize airway function, and prevent asthma-related problems.[ ] Airway structural changes, also called remodeling, have not been part of the definition of asthma until recently, but they are now considered to contribute to the alterations in airway function observed in asthma.[

106] [107] [108]

Main Recommendations of Guidelines Although some differences exist between the various countries, most asthma management consensus guidelines are relatively similar in nature. The basic therapeutic principles include the need to adequately make the diagnosis of asthma, identify its causal factors and severity according to symptoms and measurement of degree of bronchial obstruction, rapidly control asthma ( Table 70-2 ), and ensure the maintenance of such control. Treatment should be based on long-term improvement of asthma to reduce the gravity of the disease and help prevent irreversible changes.[

32] [35] [37]

Triggering factors (except exercise) should be avoided and adequate

TABLE 70-2 -- Definition of Asthma Control

28]

37]

35]

Guideline

GINA[

Canadian[

BTS[

Daytime symptoms

Minimal, ideally no

0.35•IU/ml) ranged from 9% in “secretaries” to 18% in “health care workers” to 38% in “other machinists,” suggesting that glove use was not associated with latex sensitization in health care workers or in those with jobs in which latex glove use was common. However, this study was limited by the relatively low response rate of participants concerning present occupation and glove use, raising issues of reliability and reporting bias.[

40]

Patients with Myelodysplasia

Individuals with spina bifida are at increased risk of latex sensitization as a consequence of undergoing repeated neurologic, urologic, and orthopedic surgical procedures or from early, repeated contact with rubber bladder catheters and rubber gloves during removal of fecal impactions. The reported prevalence of latex sensitivity in patients with myelodysplasia has varied widely, from 18%[ with myelodysplasia preferentially produce IgE antibodies to Hev b

41]

43 1,[ ]

to 64%.[

42]

[43]

Hev b 3,

Compared with latex-sensitive health care workers, latex-sensitive individuals

and Hev b 7.[

18]

Incidence of Latex Sensitization and Latex Allergy In contrast to the many cross-sectional studies of the prevalence of latex sensitization, there are relatively few studies on the incidence of latex sensitization or latex 44]

allergy. Annual incidence rates for contact urticaria in health care workers have been estimated to be 1.9 per 10,000 workers by questionnaire survey [ 45 registry.[ ]

and

46 al[ ]

between 1.3 and 11.8 per 10,000 workers by review of a national In perhaps the most ambitious incidence study, Sussman et first determined that the baseline prevalence of latex sensitization among 1351 health care workers at two hospital sites was 12.1%. At one site, the use of moderate- to high-protein latex gloves was then continued, while at the other site, powder-free latex gloves were introduced, and the cohort members were restudied after 1 year. In only four persons did the latex skin test convert from negative to positive in 1 year, including two in the powdered glove group (1.0% incidence rate) and two in the powderfree glove group (0.9% incidence rate); only the former two persons were symptomatic. Archambault et al[

47]

prospectively studied 122 dental hygiene students at

the beginning and end of their 3-year training period and found a cumulative incidence rate of 6.4% for skin sensitization to latex. This rate was significantly higher 48

than that found in two comparison groups (animal health apprentices, 1%; pastry-making apprentices, 1.6%).[ ] Among the dental hygiene students, the cumulative incidence rates were 4.5% for latex-induced occupational asthma and 1.8% for latex-induced rhinoconjunctivitis.

CLINICAL MANIFESTATIONS Irritant Contact Dermatitis The most common reaction to latex products is the development of dry, irritated areas on the skin, especially on the hands of glove wearers. These reactions are not immunologic but are caused by the irritant effects of repeated hand washing, the use of detergents and sanitizers, or the alkaline pH of cornstarch-powdered gloves. Allergic Contact Dermatitis Contact dermatitis is most commonly produced by rubber gloves, shoes, sports equipment, and medical devices; it

1490

TABLE 82-1 -- Rubber Product Components That May Produce Contact Dermatitis Chemical Group

Chemical

Other Nonrubber Sources

Thiuram group

Dipentamethylene thiuram disulfide Tetramethyl thiuram disulfide Tetramethyl thiuram monosulfide Tetraethyl thiuram disulfide

Adhesives, disinfectants, fungicides, germicides, insecticides, animal repellents, oil, paints

Carbamate group

Zinc dimethyl dithiocarbamate Zinc diethyl dithiocarbamate Zinc dibutyl dithiocarbamate

Lawn and garden fungicides

Benzothiazole group

Mercaptobenzothiazole N, Cyclohexyl-2-benzothiazole sulfonamide 2,2′-Benzothiazyldisulfide 4-Morpholinyl-2-benzothiazyldisulfide

Veterinary fungicide products, corrosion inhibitors in petroleum products, antifreeze, cutting oils, special detergents, heavy-duty greases

Thiourea group

N,N′-Diphenylthiourea N,N′-dibutylthiourea N,N′-diethylthiourea Ethylene thiourea

Detergents, polyvinyl adhesive backings, photocopy papers, textiles, metal anticorrosive agents

Amine derivatives

Phenylparaphenylenediamine Isopropyl-paraphenylenediamine N, Phenyl-N′-cyclohexyl-p-phenylenediamine N,N′-Diphenyl-p-phenylenediamine

Hair dyes

From Pecquet C: Clin Rev Allergy 11:413, 1993. appears 1 or 2 days after contact with the offending product. The dermatitis is a cell-mediated delayed-type hypersensitivity reaction to low molecular weight 49

accelerators and antioxidants contained in the rubber product. A partial listing of these chemicals is shown in Table 82-1 .[ ] Thiuram compounds are the most common cause of contact dermatitis to rubber, most often via rubber gloves. Thiurams are derivatives of dithiocarbamates and frequently cross-react with the carbamate compounds. Isopropyl-paraphenylenediamine is responsible for most cases of contact dermatitis to black rubber products, such as tires and boots. The diagnosis of rubber contact dermatitis is based on the clinical history, the morphology of the skin lesions, and their distribution; a specific diagnosis is established by patch testing with rubber chemicals (see Chapter 87 ). Contact Urticaria Contact urticaria is the most common early manifestation of rubber allergy, particularly in latex-sensitive health care workers, of whom 60% to 80% report contact urticaria involving the hands.[

50] [51]

Symptoms are IgE mediated and are caused by natural rubber proteins. Symptoms appear 10 to 15 minutes after the gloves are 52]

donned and include redness, itching, and wheal-and-flare reactions at the site of glove contact.[

Symptoms are often mistakenly attributed to glove powder or hand 51] [52]

washing. In health care workers, the contact urticaria may be preceded by a cell-mediated contact dermatitis. [ Rhinitis and Asthma

Inhalation of latex allergen-coated cornstarch particles from powdered gloves can evoke rhinitis and asthma in latex-sensitive persons.[ been described not only in health care workers but also in workers employed in a rubber glove manufacturing

53 facility.[ ]

13] [14]

These reactions have

The majority of latex-sensitive individuals

are highly atopic, with personal histories of seasonal allergic rhinitis caused by pollens or allergic asthma caused by house dust mites or animal danders.[ of the 29 health care workers with latex-induced asthma reported by Hunt et al,[

50]

However,

14 had no preceding history of asthma, suggesting that latex-induced wheezing

may occur as an isolated phenomenon. Latex-induced occupational asthma may be severe enough to cause some individuals to discontinue work.[ Anaphylaxis

10]

54]

Latex-sensitive persons can experience anaphylaxis in a variety of medical care situations,[ rubber balloon catheters used for barium toy

61 balloons,[ ]

rubber-handled squash

balloon rectal catheter,

[58]

a urethral

58 enemas[ ]

62 racquets,[ ]

64 catheter,[ ]

[8]

and during intraabdominal surgery, or latex padding in children's play

and a latex-containing hair

55]

including contact with rubber bladder catheters,[

59 childbirth,[ ]

63 pits.[ ]

or dental surgery.

[60]

56]

57]

condoms,[

or

Anaphylaxis may also be triggered by

Fatal latex-induced anaphylaxis has been attributed to a rubber

65 adhesive.[ ]

DIAGNOSIS Skin Testing Skin testing with natural rubber latex is the diagnostic procedure of choice in Europe and Canada, where commercial extracts are available for this purpose. In the United States, there are no licensed commercial latex extracts available for diagnostic use. Allergists frequently perform puncture skin testing with extracts of finished rubber products, usually latex gloves, but these gloves vary widely in their allergen

1491

12] [66]

contents,[

and systemic reactions have been reported with the use of these nonstandardized preparations. [

67]

Skin testing with glove extracts, ammoniated

[68]

latex, or nonammoniated latex, when standardized for protein content, is safe and efficient. Measurement of Latex-Specific IgE Antibodies

Rubber-specific IgE antibodies may also be demonstrated by immunoassay, and this test is available through diagnostic reference laboratories, using one of three immunoassays licensed by the U.S. Food and Drug Administration (FDA). The performance characteristics of these assays vary somewhat; when both skin tests and immunoassays are performed in the same patient groups, only 50% to 90% of skin test-positive persons have latex-specific IgE antibodies measurable by 69

immunoassay.[ ] The source of the allergen material used for assays is somewhat controversial. In one collaborative study, solid-phase allergens prepared in different laboratories from two nonammoniated latex samples, two ammoniated latex samples, and three latex rubber gloves produced concordant results, indicating in this particular study that the glove extracts contained as complete a repertoire of allergens as the raw latex preparations.[ specific IgE may be improved by spiking latex with recombinant latex proteins.

70]

The sensitivity of assays for latex-

[71]

Challenge Studies 12] [72]

The allergenicity of powdered gloves has been tested by having patients don and remove powdered gloves inside a small provocation chamber.[

A fitted face

73]

shield and hood system has also been described that permits graded inhalation challenge studies with latex allergen-coated cornstarch particles.[ bronchial inhalation challenge tests have been used to document the allergenicity of crude latex or isolated rubber

Nasal and

17 74 75 proteins.[ ] [ ] [ ]

Rubber allergy may also be confirmed by the “use test,” in which the fingers of rubber gloves are cut off and applied to the wet fingers of persons with suspected 36 60

contact urticaria to rubber.[ ] [ ] After 30 minutes (sooner if intense itching occurs), the glove finger is removed and the reaction is graded. If the finger challenge test is negative, a further challenge may be conducted using the whole glove. The usefulness of this test is limited by the widely varying allergen contents of gloves from different manufacturers and from different lots.[

76]

MANAGEMENT Latex Avoidance Successful long-term management of latex allergy involves documentation of latex sensitization by skin test or immunoassay and education regarding rubber avoidance. Latex-sensitive persons with myelodysplasia and latex-sensitive health care workers should be provided with lists of substitute rubber-free products for daily personal or occupational use. These individuals should not assume that such rubber-free products are universally available; for example, they should be advised to carry with them nonlatex examination gloves when seeking medical or dental care. Rubber-free condoms are also available; in one study, condoms made from thermoplastic elastomers were as resistant to leakage and viral penetration as latex condoms.[

77]

The association between latex allergy and allergy to bananas, kiwi fruit, chestnuts, avocados, papayas, potatoes, or tomatoes was mentioned previously. Less frequently noted is concomitant allergy to other foods listed in Box 82-1 . Latex-sensitive persons should be queried about reactions to these foods and advised to be cautious when eating these foods for the first time. Conversely, if these foods have previously been eaten without symptoms, there is little reason for latex-allergic persons to exclude them from the diet. Medical Gloves Currently, all medical gloves marketed in the United States are subject to FDA approval. Regulatory criteria that help ensure the quality of glove products were established by the American Society for Testing and Materials (ASTM), [

78]

and these now serve as an FDA quality standard. Existing criteria cover such

characteristics as stress resistance, tensile strength, elongation, material properties, number of holes, glove dimensions, and sterility.[

79]

With the increasing awareness of latex allergy, a mod-ified Lowry assay was initially established by the ASTM to permit glove manufacturers to measure extractable total proteins in gloves.[

80]

The FDA recommended a limit of 200••g protein per square decimeter for latex gloves marketed in the United States. However, both an 81

66 82

indirect enzyme-linked immunosorbent assay (ELISA) using rabbit antisera[ ] and inhibition immunoassays using pooled human antisera to latex allergens[ ] [ ] provided more sensitive estimates of latex glove allergen contents. Subsequently, the ASTM established an inhibition ELISA with polyclonal rabbit antiserum for measuring latex antigens in rubber products[

83]

and recommended that rubber products contain less than 10••g/dm2 antigen by this assay. Recently, the preparation of

purified or recombinant latex allergens, along with their corresponding monoclonal antibodies, has permitted the development of individual capture ELISAs for Hev b 1, 3, 5, and 6.02.[

84]

In 1990, the FDA adopted a regulation in which the maximum allowable failure rate for standardized tests for leakage was set at 2.5% for unused surgical gloves and 4% for unused examination gloves.[ and 31% for latex gloves.

[86] [87]

85]

Studies of previously used and discarded gloves found leakage rates between 43% and 85% for vinyl gloves and between 9%

Moreover, in a study of the ability of examination gloves to exclude virus particles, polyethylene and polyvinyl gloves failed in

40% and 22% of cases, respectively, whereas latex gloves failed in fewer than 1% of cases.[

88]

In more recent comparative studies, latex, polyvinyl, and nitrile

[89]

(acrylonitrile butadiene) gloves were studied by the standard leakage test after simulated use. Failure rates ranged from 12% to 61% for polyvinyl glove brands, compared with 0% to 4% for latex or nitrile brand gloves. Collectively, these data suggest that substitution of vinyl gloves for latex gloves involves some sacrifice in barrier protection. Latex Aeroallergens Hospitals and clinics may contain high levels of latex aeroallergens, which can be measured with the use of high-volume air samplers (3•L/sec) or personal breathing zone samplers (4•L/min) equipped with polytetrafluoroethylene (Teflon)

1492

TABLE 82-2 -- Latex Aeroallergen Levels in Various Clinic and Hospital Areas Using High-Volume Air Samples (3 L/Sec)

Area Sampled

Latex Aeroallergen Concentration (ng/m3 )

Extensive use of powdered rubber gloves Operating room (n = 4)

96–208

Cystoscopy room

122

Orthodontics outpatient surgery

100

Dermatology outpatient surgery

78

Blood bank—donor center

46

Blood bank—component laboratory

38

Surgical pathology laboratory

37

Venipuncture room

30

Blood bank—crossmatch laboratory

16

Hematopathology laboratory

14

Allergy research laboratory

14

Minimal use of powdered rubber gloves Allergic clinic

1.8

Spirometry laboratory

0.6

Bone marrow transplant unit

0.6

Virus serology laboratory

0.3

Modified from Swanson MC, Bubak ME, Hunt LW, et al: J Allergy Clin Immunol 94:445, 1994. 90

filters.[ ] In our medical center, latex aeroallergen levels were highest (range, 14–208• ng/m3 ) in work areas where powdered rubber gloves were in frequent use ( Table 82-2 ) and lowest (0.3–1.8• ng/m3 ) in work areas where powder-free or synthetic gloves were in use. Latex allergen was airborne only when there was activity in the work area. The level became undetectable on weekends, when no work activity was ongoing. Operating rooms had concentrations in the higher ranges, even though some had functioning laminar flow ventilation systems. Large quantities of latex allergen were recovered from used laboratory coats and anesthesia scrub suits and from laboratory surfaces. Latex allergen concentrations in personal breathing zone samplers were highly variable, ranging from 8 to 974•ng/m3 in 90]

areas where powdered gloves were frequently used.[

After the use of high-allergen latex gloves was phased out, follow-up studies in the same work areas showed 91]

that latex aeroallergen levels had fallen to less than 3•ng/m3 .[

To confirm that powdered rubber gloves were the major contributor to latex aeroallergen levels, a 52-day, prospective, multiple crossover study was conducted in a 92

single operating room, during which either high-allergen or low-allergen gloves were used and latex aeroallergen levels were monitored daily.[ ] Latex aeroallergen levels during low-allergen glove use days (range, 0.1 to 3.5•ng/m3 ) were significantly lower than on high-allergen glove use days (2.2 to 56•ng/m3 ) ( Fig. 82-2 ). On designated high-allergen glove use days, latex aeroallergen levels were strongly associated with the total number of gloves used. “Latex-Safe” Medical Environments Because most IgE-mediated reactions to rubber have been described in health care workers or in latex-sensitive individuals undergoing medical or dental procedures, emphasis has been placed on providing “latex-safe” clinic and hospital environments. A cost analysis study suggested that health care facilities are likely to benefit 93]

from becoming latex-safe, even if latex-related disability rates among employees are low.[

The Task Force on Allergic Reactions to Latex of the American

Academy of Allergy and Immunology recommended that medical procedures performed on latex-sensitive persons be conducted in a latex-free environment, defined 94]

as one in which no latex gloves are worn by any personnel and no latex accessories are brought into direct contact with the patient.[

eliminating the use of powdered latex gloves, cleaning of carpets and upholstered furniture can eliminate reservoirs of latex allergens. all medical devices or medical device packagings containing natural rubber latex were required to be identified by the the preparation of latex-safe health care delivery environments. Based on

96 FDA,[ ]

In addition to reducing or

[95]

After September 30, 1998,

and this requirement has facilitated

Figure 82-2 Mean latex aeroallergen levels in a single operating room during a 12-hour sampling period for 52 consecutive days, including nonsurgery days and surgery days when either high-allergen-content gloves (“high glove day”) or low-allergen-content gloves (“low glove day”) were in use. (From Heilman DK, Jones RT, Swanson MC, et al: J Allergy Clin Immunol 98:325, 1996.)

(From Heilman DK, Jones RT, Swanson MC, et al: J Allergy Clin Immunol 98:325, 1996.)

1493

97

measures of latex aeroallergen levels in the work areas of 22 latex-sensitized health care workers, Baur and colleagues[ ] suggested a threshold limit for latex aeroallergens of 0.6•ng/m3 . However, information presently available is insufficient to establish formal threshold limits or to estimate a latex aeroallergen concentration that can induce sensitization. There are anecdotal reports of latex-sensitive individuals who experienced systemic allergic reactions preoperatively or postoperatively purportedly induced by latex allergens present in intravenous fluids[

98]

99]

or by injection of medications through latex ports in intravenous tubing.[

However, in contrast to dipped products made

from liquid rubber latex, dry rubber products generally have low levels of extractable protein, and extracts of these products usually do not produce positive skin tests 100]

in latex-sensitive persons.[

Recently, small quantities of latex allergens were measured in phenol–saline–human serum albumin diluent stored in vials with natural

rubber closures, and these solutions elicited positive intradermal skin tests in a minority of latex-sensitive persons. [ eliminate natural rubber from pharmaceutical vial closures.

101]

These data support a recommendation to

Detailed perioperative nursing plans have been published for management of latex-sensitive patients, and some management protocols recommend preoperative prophylactic administration of glucocorticoids and both histamine1 (H1 ) and H2 class antihistamines to such patients undergoing surgery.[

9] [99] [102]

In a

retrospective study of children with myelodysplasia who underwent surgery, the use of empiric latex-avoidance protocols was associated with a significant decline in 103]

intraoperative allergic reactions, but the critical elements within the protocol were not identified.[ persons with the use of these premedication and latex sensitive

108 children.[ ]

104 105 protocols,[ ] [ ]

Although surgery can be performed safely in latex-sensitive

several groups have reported the occurrence of life-threatening intraoperative anaphylactic reactions despite

9 99 106 107 avoidance.[ ] [ ] [ ] [ ]

Moreover, life-threatening allergic reactions may be the presenting factor of latex allergy in up to 30% of latex-

In a randomized, double-blind, placebo-controlled trial involving 17 latex-sensitive persons, immunotherapy with a standardized latex extract

was associated with a significant decline in global medication scores and in nasal, conjunctival, and cutaneous symptoms after 6 or 12 months.[ symptoms were not improved, and four of nine patients receiving active treatment experienced several systemic reactions to the injections.

109]

However, asthma

LONG-TERM FOLLOW-UP STUDIES In long-term follow-up studies, latex-allergic persons have benefited from reducing their occupational exposures to latex. In 36 latex-sensitive workers with welldocumented occupational asthma who reduced or avoided latex exposure, significant lessening of asthma and decreased bronchial hyperreactivity were noted after a median follow-up period of 56 months.[ 111 months.[ ]

110]

Latex-specific IgE levels decreased in six of seven health care workers who avoided powdered latex glove use for 12

Twenty latex-sensitized anesthesiologists (12 symptomatic, 8 symptomatic) who personally avoided using latex gloves for 10 to 15 months all became 112

asymptomatic, and 16 of 18 also showed a decline in latex-specific IgE.[ ] However, latex skin test titration end points did not change appreciably, suggesting that a longer period of latex glove avoidance or tighter environmental control measures may be necessary to achieve maximal immunologic improvement. In two crosssectional surveys of dental students and staff performed 5 years apart, after the school began purchasing low-protein, powder-free latex gloves, there was a significant reduction in both latex-related symptoms and positive latex skin test responses in the second survey.[

113]

Since 1994, with active efforts to reduce workplace exposure to latex allergens and the use of either non-latex gloves or powder-free, low-protein latex gloves, workers compensation claims related to latex-induced asthma have declined in Ontario, Canada.[

114]

In addition, during the same period, the number of new cases of

occupational allergy to latex declined substantially at two large Toronto teaching hospitals with approximately 8000 employees.[

115]

CONCLUSIONS Allergy to natural rubber latex is an important cause of occupational allergy among health care workers. Disposable latex gloves are the major allergen reservoir—

particularly powdered gloves, which contribute substantially to the latex aeroallergen levels measured in health care delivery settings. Diagnosis of latex allergy requires a history of exacerbation of cutaneous, respiratory, ocular, or systemic signs and symptoms after exposure to natural rubber products, together with evidence of latex sensitization by skin testing, measurement of latex-specific IgE antibodies, or challenge testing. Optimal management of latex allergy involves education concerning cross-reacting allergens and minimization of contact with dipped rubber products such as gloves, balloons, and condoms.

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16. Phillips ML, Meagher CC, Johnson DL: What is “powder free”? Characterization of powder aerosol produced during simulated use of powdered and powder free latex gloves, Occup Environ Med 58:479, 2001. 17. Czuppon AB, Chen Z, Rennert S, et al: The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex, J Allergy Clin Immunol 92:690, 1993. 18. Wagner B, Buck D, Hafner C, et al: Hev b 7 is a Hevea brasiliensis protein associated with latex allergy in children with spina bifida, J Allergy Clin Immunol 108:621, 2001.

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19. Akasawa A, Hsieh LS, Martin BM, et al: A novel acidic allergen, Hev b 5, in latex: purification, cloning, and characterization, J Biol Chem 271:25389, 1996. 20. Slater JE, Vedvick T, Arthur-Smith A, et al: Identification, cloning, and sequence of a major allergen (Hev b 5) from natural rubber latex (Hevea brasiliensis), J Biol Chem 271:25394, 1996. 21. Beezhold DH, Sussman GL, Liss GM, et al: Latex allergy can induce clinical reactions to specific foods, Clin Exp Allergy 26:416, 1996. 22. Sowka S, Wagner S, Krebitz M, et al: cDNA cloning of the 43-kDa latex allergen Hev b 7 with sequence similarity to patatins and its expression in the yeast Pichia pastoris, Eur J Biochem 255:213, 1998. 23. Wright HT, Brooks DM, Wright CS: Evolution of the multidomain protein wheat germ agglutinin, J Mol Evol 21:133, 1985. 24. Sowka S, Hsieh LS, Krebitz M, et al: Identification and cloning of Prs a 1, a 32-kDa endochitinase and major allergen of avocado, and its expression in the yeast Pichia pastoris, J Biol Chem 273:28091, 1998. 25. Chen Z, Posch A, Cremer R, et al: Identification of hevein (Hev b 6.02) in Hevea latex as a major cross-reacting allergen with avocado fruit in patients with latex allergy, J Allergy Clin Immunol 102:476, 1998. 26. Mikkola JH, Alenius H, Kalkkinin N, et al: Hevein-like protein domains as a possible cause for allergen cross-reactivity between latex and banana, J Allergy Clin Immunol 102:1005, 1998. 27. Gazelius B, Olgart L, Wrangsjö K: Unexpected symptoms to root filling with gutta-percha: a case report, Int Endod J 19:202, 1986. 28. Boxer MB, Grammer LC, Orfan N: Gutta-percha allergy in a healthcare worker with latex allergy, J Allergy Clin Immunol 93:943, 1994. 29. Costa GE, Johnson JD, Hamilton RG: Cross-reactivity studies of gutta-percha, gutta-balata, and natural rubber latex (Hevea brasiliensis), J Endod 27:584, 2001.

Epidemiology 30. Novembre E, Bernardini R, Brizzi I, et al: The prevalence of latex allergy in children seen in a university hospital allergy clinic, Allergy 52:101, 1997. 31. Ruëff F, Kienitz A, Schöpf P, et al: Frequency of natural rubber latex allergy in adults is increased after multiple operative procedures, Allergy 56:889, 2001. 32. Ownby DR, Ownby HE, McCullough JA, et al: The prevalence of anti-latex IgE antibodies in 1000 volunteer blood donors, J Allergy Clin Immunol 97:1188, 1996. 33. Lebenbom-Mansour MH, Oesterle JR, Ownby DR, et al: The incidence of latex sensitivity in ambulatory surgical patients: a correlation of historical factors with positive serum immunoglobulin E levels, Anesth Analg 85:44, 1997. 34. Salkie ML: The prevalence of atopy and hypersensitivity to latex in medical laboratory technologists, Arch Pathol Lab Med 117:897, 1993. 35. Poole CJ, Nagendran V: Low prevalence of clinical latex allergy in UK health care workers: a cross-sectional study, Occup Med (Oxf) 51:510, 2001. 36. Turjanmaa K: Incidence of immediate allergy to rubber gloves in hospital personnel, Contact Dermatitis 17:270, 1987. 37. Yassin MS, Lierl MB, Fischer TJ, et al: Latex allergy in hospital employees, Ann Allergy 72:245, 1994. 38. Liss GM, Sussman GL, Deal K, et al: Latex allergy: epidemiological study of 1351 hospital workers, Occup Environ Med 54:335, 1997. 39. Garabrant DH, Roth HD, Parsad R, et al: Latex sensitization in health care workers and in the US general population, Am J Epidemiol 153:515, 2001. 40. Wartenberg D, Buckler G: Invited commentary: assessing latex sensitization using data from NHANES III, Am J Epidemiol 153:523, 2001. 41. Meeropol E, Kelleher R, Bell S, et al: Allergic reactions to rubber in patients with myelodysplasia, N Engl J Med 323:1072, 1990. 42. Yassin MS, Sanyurah S, Lierl MB, et al: Evaluation of latex allergy in patients with meningomyelocele, Ann Allergy 69:207, 1992. 43. Yeang HY, Cheong KF, Sunderasan E, et al: The 14.6•kd rubber elongation factor (Hev b 1) and 24•kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy, J Allergy Clin Immunol 98:628, 1996. 44. Kujala V: A review of current literature on epidemiology of immediate glove irritation and latex allergy, Occup Med (Oxf) 49:3, 1999. 45. Jolanki R, Estlander T, Alanko K, et al: Incidence rates of occupational contact urticaria caused by natural rubber latex, Contact Dermatitis 40:329, 1999. 46. Sussman GL, Liss GM, Deal K, et al: Incidence of latex sensitization among latex glove users, J Allergy Clin Immunol 101:171, 1998. 47. Archambault S, Malo JL, Infante-Rivard C, et al: Incidence of sensitization, symptoms, and probable occupational rhinoconjunctivitis and asthma in apprentices starting exposure to latex, J Allergy Clin Immunol 107:921, 2001.

48. Gautrin D, Ghezzo H, Infante-Rivard C, et al: Incidence and determinants of IgE-mediated sensitization in apprentices: a prospective study, Am J Respir Crit Care Med 162:1222, 2000. Clinical Manifestations 49. Pecquet C: Allergic contact dermatitis to rubber: clinical aspects and main allergens, Clin Rev Allergy 11:413, 1993. 50. Hunt LW, Fransway AF, Reed CE, et al: An epidemic of occupational allergy to latex involving health care workers, J Occup Environ Med 37:1204, 1995. 51. Charous BL, Hamilton RG, Yunginger JW: Occupational latex exposure: characteristics of contact and systemic reactions in 47 workers, J Allergy Clin Immunol 94:12, 1994. 52. Turjanmaa K, Reunala T: Contact urticaria from rubber gloves, Dermatol Clin 6: 47, 1988. 53. Tarlo SM, Wong L, Roos J, et al: Occupational asthma caused by latex in a surgical glove manufacturing plant, J Allergy Clin Immunol 85:626, 1990. 54. Sussman GL, Tarlo S, Dolovich J: The spectrum of IgE-mediated responses to latex, JAMA 265:2844, 1991. 55. Yunginger JW: Latex-associated anaphylaxis, Immunol Allergy Clin North Am 21:669, 2001. 56. Nguyen DH, Burns MW, Shapiro GG, et al: Intraoperative cardiovascular collapse secondary to latex allergy, J Urol 146:571, 1991. 57. Wrangsjö K, Wahlberg JE, Axelsson IG: IgE mediated allergy to natural rubber in 30 patients with contact urticaria, Contact Dermatitis 19:264, 1988. 58. Ownby DR, Tomlanovich M, Sammons M, et al: Anaphylaxis associated with latex allergy during barium enema examination, AJR Am J Roentgenol 156:903, 1991. 59. Laurent J, Malet R, Smiejan JM, et al: Latex hypersensitivity after natural delivery, J Allergy Clin Immunol 89:779, 1992. 60. Axelsson JG, Johansson SG, Wrangsjö K: IgE-mediated anaphylactoid reactions to rubber, Allergy 42:46, 1987. 61. Axelsson JG, Eriksson M, Wrangsjö K: Anaphylaxis and angioedema due to rubber allergy in children, Acta Paediatr Scand 77:314, 1988. 62. Beuers U, Baur X, Schraudolph M, et al: Anaphylactic shock after game of squash in atopic woman with latex allergy, Lancet 335:1095, 1990. 63. Fiocchi A, Restani P, Ballabio C, et al: Severe anaphylaxis induced by latex as a contaminant of plastic balls in play pits, J Allergy Clin Immunol 108:298, 2001. 64. Martinez Jabaloyas JM, Broseta Rico E, Ruiz Cerda JL, et al: Extracorporeal shockwave lithotripsy in patients with urinary diversion, Acta Urol Esp 19:143, 1995. 65. Pumphrey RSH, Duddridge M, Norton J: Fatal latex allergy, J Allergy Clin Immunol 107:558, 2001 (letter). Diagnosis

66. Yunginger JW, Jones RT, Fransway AF, et al: Extractable latex allergens and proteins in disposable medical gloves and other rubber products, J Allergy Clin Immunol 93:836, 1994. 67. Kelly KJ, Kurup VP, Zacharisen M, et al: Skin and serological testing in the diagnosis of latex allergy, J Allergy Clin Immunol 91:1140, 1993. 68. Hamilton RG, Adkinson NF Jr: Natural rubber latex skin testing reagents: safety and diagnostic accuracy of nonammoniated latex, ammoniated latex, and latex rubber glove extracts, J Allergy Clin Immunol 98:872, 1996. 69. Hamilton RG, Biagini RE, Krieg EF: Diagnostic performance of Food and Drug Administration-cleared serologic assays for natural rubber latex-specific IgE antibody: the Multi-Center Latex Skin Testing Study Task Force, J Allergy Clin Immunol 103:925, 1999. 70. Hamilton RG, Charous BL, Adkinson NF Jr, et al: Serologic methods in the laboratory diagnosis of latex rubber allergy: study of nonammoniated, ammoniated latex, and glove (end-product) extracts as allergen reagent sources, J Lab Clin Med 123:594, 1994. 71. Lundberg M, Chen Z, Rihs HP, et al: Recombinant spiked allergen extract, Allergy 56:794, 2001. 72. Laoprasert N, Swanson MC, Jones RT, et al: Inhalation challenge testing of latex-sensitive health care workers and the effectiveness of laminar flow HEPAfiltered helmets in reducing rhinoconjunctival and asthmatic reactions, J Allergy Clin Immunol 102:998, 1998. 73. Kurtz KM, Hamilton RG, Schaefer JA, et al: Repeated latex aeroallergen challenges employing a hooded exposure chamber: safety and reproducibility, Allergy 56: 857, 2001. 74. Marcos C, Lázaaro M, Fraj J, et al: Occupational asthma due to latex gloves, Ann Allergy 67:319, 1991. 75. Pisati G, Baruffini A, Bernabeo F, et al: Bronchial provocation testing in the diagnosis of occupational asthma due to latex surgical gloves, Eur Respir J 7:332, 1994. 76. Turjanmaa K: Diagnosis of latex allergy, Allergy 56:810, 2001. Management 77. Ketering J: Efficacy of thermoplastic elastomer and latex condoms as viral barriers, Contraception 47:559, 1993. 78. American Society for Testing and Materials. Standard specifications for rubber surgical gloves. ASTM D-3577-88. Annual Book of ASTM Standards, Vol 9.02. Philadelphia, 1990, ASTM, p 344. 79. Fay MF, Sullivan RW: Changing requirements for glove selection and hand protection, Biomed Instrum Technol 26:227, 1992. 80. American Society for Testing and Materials. Standard test method for analysis of protein in natural rubber and its products: ASTM D-5712. Annual Book of ASTM Standards, Vol 14. Philadelphia, 1995, ASTM, p 302. 81. Beezhold D: LEAP: Latex ELISA for antigenic protein, a preliminary report, Guthrie J 61:77, 1992.

82. Palosuo T, Mäkinen-Kiljunen S, Alenius H, et al: Measurement of natural rubber latex allergen levels in medical gloves by an allergen-specific IgE-ELISAinhibition, RAST-inhibition, and skin prick testing, Allergy 53:59, 1998. 83. American Society for Testing and Materials. Standard test method for the immunological measurement of antigenic protein in natural rubber and its products: ASTM D6499. Annual Book of ASTM Standards, Vol 9.02. Philadelphia, 2000, ASTM, p 219. 84. Palosuo T, Ovod V, Kärkkäinen T, et al: The major latex allergens Hev b 6.02 (hevein) and Hev b 5 are regularly detected in medical gloves with moderate or high allergen content, J Allergy Clin Immunol 107:S321, 2001 (abstract). 85. Nightingale SL: New regulations to improve the quality control medical gloves, JAMA 265:1229, 1991. 86. Korniewicz DM, Kirwin M, Cresci K, et al: Leakage of latex and vinyl exam gloves in high and low risk clinical settings, Am Ind Hyg Assoc J 54:22, 1993.

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87. Olsen RJ, Lynch P, Coyle MB, et al: Examination gloves as barriers to hand contamination in clinical practice, JAMA 270:350, 1993. 88. Klein RC, Party E, Gershey EL: Virus penetration of examination gloves, Biotechniques 9:196, 1990. 89. Rego A, Roley L: In-use barrier integrity of gloves: latex and nitrile superior to vinyl, Am J Infect Control 27:405, 1999. 90. Swanson MC, Bubak ME, Hunt LW, et al: Quantification of occupational latex aeroallergens in a medical center, J Allergy Clin Immunol 94:445, 1994. 91. Hunt LW, Boone-Orke JL, Fransway AF, et al: A medical center-wide, multidisciplinary approach to the problem of natural rubber latex allergy, J Occup Environ Med 38:765, 1996. 92. Heilman DK, Jones RT, Swanson MC, et al: A prospective, controlled study showing that rubber gloves are the major contributor to latex aeroallergen levels in the operating room, J Allergy Clin Immunol 98:325, 1996. 93. Phillips VL, Goodrich M, Sullivan TJ: Health care worker disability due to latex allergy and asthma: a cost analysis, Am J Public Health 89:1024, 1999. 94. Task Force on Allergic Reactions to Latex: Committee report, J Allergy Clin Immunol 92:16, 1993. 95. Charous BL, Schuenemann PJ, Swanson MC: Passive dispersion of latex aeroallergen in a healthcare facility, Ann Allergy Asthma Immunol 85:285, 2000. 96. Food and Drug Administration: Natural rubber-containing medical devices: user labeling, Federal Register 62:51021, 1997. 97. Baur X, Chen Z, Allmers H: Can a threshold limit value for natural rubber latex airborne allergens be defined? J Allergy Clin Immunol 101:24, 1998.

98. Schwartz HA, Zurowski D: Anaphylaxis to latex in intravenous fluids, J Allergy Clin Immunol 92:358, 1993. 99. Kwittken PL, Becker J, Oyefara B, et al: Latex hypersensitivity reactions despite prophylaxis, Allergy Proc 13:123, 1992. 100. Yip E, Turjanmaa K, Ng KP, et al: Allergic responses and levels of extractable proteins in NR latex gloves and dry rubber products, J Nat Rubb Res 9:79, 1994. 101. Primeau MN, Adkinson NF Jr, Hamilton RG: Natural rubber pharmaceutical vial closures release latex allergens that produce skin reactions, J Allergy Clin Immunol 107:958, 2001. 102. Anonymous: American Association of Nurse Anesthetists latex allergy protocol, AANA J 61:223, 1993. 103. Birminghan PK, Dsida RM, Grayhack JJ, et al: Do latex precautions in children with myelodysplasia reduce intraoperative reactions? J Pediatr Orthop 16:799, 1996. 104. Tosi LL, Slater JE, Shaer C, et al: Latex allergy in spina bifida patients: prevalence and surgical implications, J Pediatr Orthop 13:709, 1993. 105. Holzman RS: Latex allergy: an emerging operating room problem, Anesth Analg 76:635, 1993. 106. Gold M, Swartz JS, Braude BM, et al: Intraoperative anaphylaxis: an association with latex sensitivity, J Allergy Clin Immunol 87:662, 1991. 107. Setlock MA, Cotter TP, Rosner D: Latex allergy: failure of prophylaxis to prevent severe reaction, Anesth Analg 76:650, 1993. 108. Kwittken PL, Sweinberg SK, Campbell DE, et al: Latex hypersensitivity in children: clinical presentation and detection of latex-specific immunoglobulin E, Pediatrics 95:693, 1995. 109. Leynadier F, Herman D, Vervloet D, et al: Specific immunotherapy with a standardized latex extract versus placebo in allergic healthcare workers, J Allergy Clin Immunol 106:585, 2000. Long-Term Follow-Up Studies 110. Vandenplas O, Jamart J, Delwiche JP, et al: Occupational asthma caused by natural rubber latex: Outcome according to cessation or reduction of exposure, J Allergy Clin Immunol 109:125, 2002. 111. Allmers H, Brehler R, Chen Z, et al: Reduction of latex aeroallergens and latex-specific IgE antibodies in sensitized workers after removal of powdered natural rubber latex gloves in a hospital, J Allergy Clin Immunol 102:841, 1998. 112. Hamilton RG, Brown RH: Impact of personal avoidance practices on health care workers sensitized to natural rubber latex, J Allergy Clin Immunol 105: 839, 2000. 113. Saary MJ, Kanani A, Alghadeer H, et al: Changes in rates of natural rubber latex sensitivity among dental school students and staff members after changes in latex gloves, J Allergy Clin Immunol 109:131, 2002.

114. Liss GM, Tarlo SM: Natural rubber latex-related occupational asthma: association with interventions and glove changes over time, Am J Ind Med 40:347, 2001. 115. Tarlo SM, Easty A, Eubanks K, et al: Outcomes of a natural rubber latex control program in an Ontario teaching hospital, J Allergy Clin Immunol 108: 628, 2001.

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Chapter 83 - Anaphylaxis and Anaphylactoid Reactions

Phillip L. Lieberman

1

The term anaphylaxis was first coined by Portier and Richet in 1902.[ ] While attempting to immunize dogs to the venom of the sea anemone, they unwittingly sensitized the animals, and the dogs unexpectedly reacted fatally to a previously nonlethal dose. The phenomenon was the opposite of prophylaxis, so they referred to it as anaphylaxis, meaning “against, or without, protection.” Today, anaphylaxis refers to a systemic, immediate hypersensitivity reaction caused by immunoglobulin E (IgE)-mediated immunologic release of mediators from mast cells and basophils. The term anaphylactoid reaction refers to a clinically similar event not mediated by IgE. In this chapter the terms anaphylaxis and anaphylactoid reaction are used interchangeably.

INCIDENCE AND CAUSATIVE AGENTS Overall Incidence 2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]

Although the exact incidence of anaphylaxis is unknown, there have been several reviews of this issue,[ [20] [21] [22] [23] [24]

which are summarized in Table 83-1 . These data probably underestimate the true incidence of anaphylaxis for a number of reasons.

Underreporting of anaphylactic events has been documented on a number of occasions. For example, only 4% to 8% of anaphylactic reactions were reported to the Dutch Inspectorate for Health Care in the Netherlands.[ underreported. Pumphrey and

22 Davis[ ]

20] [21]

11]

Klein and Yocum[

found that cases of anaphylaxis seen in a community emergency room were

reviewed the issue of underreporting in an editorial appearing in The Lancet. They cited the fact that in a study of fatal

anaphylaxis in the United Kingdom conducted between the years 1992 and 1997, only 33 of 67 fatal reactions to drugs were reported. [ underreporting has been consistent throughout the years and was noted as early as

22]

This pattern of

23 1979.[ ]

INCIDENCE FOR SPECIFIC AGENTS Drugs and diagnostic agents, along with foods, are the most frequent causes of anaphylaxis. In three series of patients with anaphylaxis presenting to allergists for evaluation,[

25] [26] [27]

foods were the most common cause in two[

also the most common cause. As noted in the

14 Danish[ ]

and

25] [26]

15 Dutch[ ]

and drugs in the third.[

27]

In the Olmstead County epidemiologic review,[

16]

foods were

studies, drugs were the most common culprits.

The drugs that most commonly cause anaphylactic events are antibiotics and nonsteroidal antiinflammatory drugs (NSAIDs), and the most likely foods are peanuts and shellfish. In an ongoing survey of anaphylactic episodes dating back to 1978 and involving 411 subjects, when the cause was determined, food was found to be 25] [28]

the most common culprit, most often shellfish. NSAIDs were the most common class of drug to cause episodes, followed by antibiotics.[ from Mayo Clinic, foods were also the most common offender; however, peanuts, rather than shellfish, were most often incriminated. patients from Spain, drugs caused anaphylaxis more frequently than foods Wang et al[

29]

[26]

In a similar report

In an evaluation of 179

27 did.[ ]

reviewed all cases of suspected drug-induced anaphylactic reactions reported in Sweden between 1972 and 1995. They found 1338 cases, yielding a 29

reporting rate of 7 cases per 1 million inhabitants per year.[ ] There were 51 deaths (3.8%). In contrast to previously cited evaluations, the most frequent offenders were dextrans (418) and radiocontrast agents (161). All in all, 201 different drugs were incriminated. Table 83-2 summarizes the incidence for the most common offenders.

SUMMARY OF INCIDENCE From a review of the aforementioned studies, it is obvious that no definitive conclusions can be drawn regarding the exact overall incidence of anaphylaxis worldwide. Attempts to do so result in extremely wide-ranging estimates.[ 1.

24]

Nonetheless, some overall assumptions can be made, and these are presented in Box 83-

FACTORS AFFECTING INCIDENCE Geographic location, economic status, race, age, gender, route of administration of antigen, constancy of administration of antigen, chronobiologic factors, and atopy 87]

have been evaluated for their effects on the incidence of anaphylaxis. Race exerts no effect.[

The data are less clear for geographic location, which was considered

87

88

to be irrelevant [ ] until a recent report from Great Britain challenged that assumption. Sheikh and Elves[ ] analyzed hospital discharge data over a 4-year span for all English National Health Service hospitals. Of the 32.5 million discharges from these hospitals, 2424 patients had a primary diagnosis of anaphylaxis. The overall admission-adjusted rate of anaphylaxis was 17 per 100,000

1498

TABLE 83-1 -- Summary of Studies Assessing the Overall Incidence of Anaphylaxis First Author (reference no.)

Date

Comment

2

1924

41 cases of fatal anaphylaxis were recorded between 1895 and 1923 in the medical literature

3

1936

68 cases of fatal anaphylaxis were recorded between 1923 and 1935

Davies ([ ] )

1977

British Committee on Safety of Medications; 140 cases, four fatalities between 1966 and 1975

Boston Collaborative Drug Surveillance

1973

0.87 deaths per 10,000 patients monitored

1977

Drug-induced anaphylaxis occurred in 12 (0.04%) of 32,812 continuously monitored patients

1978

Four cases per 10 million population in Ontario, Canada, in 1978

1992

Nine cases in 20,064 admissions to a university hospital in Germany

1996

24 cases of 55,000 patients seen in an emergency department serving a catchment area of 350,000

1989

Retrospective review over 13 years of admissions to a hospital with a catchment area of 48,000 demonstrating incidence of 3.2 cases per 100,000 inhabitants per year

Lamson ([ ] ) Vaughn ([ ] ) 4

5

Program ([ ] ) 6

Porter ([ ] ) 7

Orange ([ ] ) 8

Amornmarn ([ ] ) 9

Stewart ([ ] ) Sorenson ([

10]

)

Klein ([

11]

1995

Retrospective analysis of emergency room records demonstrating an incidence of 0.09%

)

2001

Retrospective analysis of emergency department admissions demonstrating 140 episodes out of a total of 38,685 admissions (incidence of 0.4%)

)

1998

Collaborative study of 9 institutions with 481,752 individuals assessed; 123 cases occurred between 1992 and 1995 (two fatalities)

)

Pastorello ([

12]

13]

Kaufman ([

14]

)

1995

30 cases of fatal drug-induced reactions occurred between 1968 and 1990 in a Danish study

15]

)

1996

All adverse drug reactions registered as anaphylaxis in The Netherlands recorded between 1974 and 1994 —936 cases were noted; authors concluded that 50 drug-associated anaphylactic deaths occur in The Netherlands per year (total population 13 to 15 million)

1999

Review of medical records of 1.3 million residents in Olmstead County, Minnesota; annual incidence rate of anaphylaxis was 21 per 100,000 person years; one fatality was recorded; authors concluded that the incidence is less than 1% and fatalities rare

2001 2001 2000

Data from Drug Programs Information Network, an administrative claims database for prescriptions dispensed in an outpatient setting in Manitoba, Canada, population base of 279,638 infants, children, and adolescents; epinephrine formulations were given to 1.2% of thispediatric population; data for the entire population of Manitoba (children and adults) and recorded a dispensing rate of epinephrine for outpatient therapy of 954 per 100,000 persons (8.95%)

Lenler-Petersen ([ Van der Klauw ([

Yocum ([

Black ([

16]

17]

)

), Simons ([

18]

19]

), Black ([

)

Neugut ([

24]

2001

)

Analysis of published literature; in the United States the authors estimated that 3.3 to 4.3 million Americans were at risk and a total of 1433 to 1503 were at risk for a fatality; they suggested that 12.4% to 16.8% of total U.S. population may suffer an anaphylactic reaction

emergency department discharges. These cases were evaluated for rural versus urban residence and for geographic location (north versus south England and east versus west England). The authors found that geographic location had statistically significant effects. Individuals living in rural areas, in the south, and in the west of England experienced an increased incidence of anaphylactic episodes. No definitive reason was given for these effects based on geographic location.[

88]

18

Economic status may play a role in the incidence of anaphylactic episodes, at least based on epidemiologic data obtained by Simons et al.[ ] They found that, in an urban population, EpiPens were prescribed far more frequently to individuals in the higher income group than to those of lower income. The rate for prescribing EpiPens was 1.3% of the population in the higher income group and 0.6% in the lower income group. This difference could not be attributed to the utilization of specialists' care.[

18]

Gender does seem to play a significant role in the incidence of anaphylaxis, both to specific agents and also, perhaps, in regard to the overall incidence. For example, anaphylactic

1499

TABLE 83-2 -- Reported Incidence of Anaphylaxis for the Most Common Causes Agent Antibiotics

Reference Nos. 12, 24, 29–35

Comment Arguably the most common cause of drug-induced anaphylaxis. Beta-lactams are the most frequently incriminated agent, accounting for as many as 22% of all drug-related episodes.[

12]

It has been estimated that nonfatal penicillin-induced 24]

anaphylaxis may affect 1.9 to 27.2 million Americans.[

Latex

24, 36–42

Incidence has risen over the past decade. Populations at risk are those experiencing multiple mucosal exposures to latex (e.g., multiple catheterizations, multiple surgeries) and health care workers. It has been estimated that the overall incidence of latex allergy in the United States ranges from 2.7 to 16 million.[

Perioperative anaphylaxis

43–60

24]

Reported incidence rates vary considerably and range from 1 in 4500 to 1 in 43] [45] [51] [52]

25,000 cases of general anesthesia.[ 51 (3.4%),[ ]

Mortality rate can be high

and anaphylactic deaths can account for as many as 4.3% of all 53]

deaths occurring during general anesthesia.[

The most common agents 45]

responsible are muscle relaxants, which account for 50% to 75% of reactions.[ [47] [48] [49] [54] [56] [57]

Radiocontrast agents

61–65

Since the introduction of lower osmolarity, the incidence of anaphylactoid reactions has declined.

Hymenoptera stings and bites

66–72

Incidence ranges from 0.4% to as high as 5%, placing 1.36 to 13.6 million Americans at risk.[

66] [67] [68] [70]

Estimates of fatalities go as high as 100 per

71 year.[ ]

Food

12, 25, 28, 73–77

73]

It is estimated that 2% of the U.S. population has food allergies,[

and that as

74 reactions.[ ]

many as 100 deaths per year are caused by food It has also been estimated, based on the incidence in Colorado, that approximately 1080 Americans (0.0004% of the U.S. population) have anaphylactic reactions to food 75]

each year.[

in children.[

Shellfish is probably the most common culprit in adults, peanuts

25] [26] [28]

It has also been reported that 1.1% of the U.S. population

may be allergic to tree nuts or peanuts.[ NSAIDs

12, 25, 28, 78–81

73]

Incidence varies depending on whether asthmatic subjects are included. NSAIDs 12] [25] [28]

are probably the second most common offender next to antibiotics.[

Antisera

82–84

At one time antisera were the most important cause of anaphylaxis. Their importance has greatly diminished with decreased use, but antiserum is still used to treat snake bites and for immunosuppression. Incidence in patients receiving 83]

antilymphocyte globulin may be as high as 2%, [

and for antivenom it ranges

82 10%.[ ]

from 4.6% to The incidence may decline further with the recent release of Crotalidae Polyvalent Immun Fab, a purified preparation of the Fab fragment obtained from sheep immunized with venom. To date, although urticaria has been reported, there have been no anaphylactic episodes with this agent.[ Idiopathic anaphylaxis

25, 28, 85–86

84]

The cause of anaphylaxis remains unidentified in as many as two thirds of 25 28

patients presenting to an allergist-immunologist for evaluation.[ ] [ ] A survey of 75 allergists in the United States found that they had encountered 633 cases; the authors extrapolated these data and estimated 20,592 to 47,024 cases in the 85]

U.S. population. At the time of their report, 1020 cases had been reported.[ NSAIDS, Nonsteroidal antiinflammatory drugs. 89]

reactions to intravenous muscle relaxants have been reported to occur more frequently in women.[

This may be related to the fact that the quaternary ammonium 18

ion found in muscle relaxants is also present in some cosmetics: Women may be more sensitized by previous exposure. Simons et al [ ] demonstrated a significant age-related gender effect on incidence. Anaphylaxis was more frequent in boys than in girls at younger ages (less than 15 years), but among older individuals there was a reversal of this situation, with women being more frequently affected than men. Their data were obtained by ascertaining the number of prescriptions 18

90

91

dispensed for EpiPens in Manitoba.[ ] A similar increased incidence in adult females has been reported for reactions to aspirin[ ] and to latex.[ ] The increased incidence of anaphylaxis to latex in women may be due to an increased exposure to latex gloves. In addition, insect sting anaphylaxis has been reported to occur more frequently in men,[

92]

and this also is probably a function of exposure. However, exposure cannot account for the recorded increased incidence of anaphylaxis 12 13 24 25 26 27 28

overall in women regardless of cause.[ ] [ ] [ ] [ ] [ ] [ ] [ ] The reason for this apparent increased susceptibility in women is unclear. It is well known that hormonal influences affect mast cell “releasability,” with enhanced releasability and higher mast cell counts (at least in animal models) occurring during the estrus phase of the menstrual cycle.[

93]

94] [95]

In addition, animal models have shown that progesterone can enhance the sensitivity to anaphylaxis.[

1500

Box 83-1. Summary Conclusions Regarding the Incidence of Anaphylaxis • No exact incidence can be established based on available data • Most studies indicate significant under-reporting, and therefore the true incidence is probably significantly higher than formally reported • Based on series of episodes evaluated by allergists/ immunologists, events of unknown cause (idiopathic) account for as many as 50% of cases • Foods are probably the most frequent offenders, with drugs close behind • The most commonly incriminated drugs are nonsteroidal antiinflammatory agents and antibiotics; reactions to radiocontrast agents appear to be diminishing in frequency • Perioperative episodes may be increasing, with muscle relaxants being the most common offenders in this setting and latex-induced events increasing

As will be discussed later, similar hormonal effects on anaphylaxis have been noted in humans experiencing progesterone-related anaphylactic episodes.[ 99

92

96] [97] [98]

100

The incidence and/or severity of anaphylaxis and anaphylactoid reactions to radiocontrast material,[ ] Hymenoptera,[ ] plasma expanders, [ ] and anesthetics[ has been reported to be greater in adults than children. Whether this apparent effect of age is due to an increased frequency of exposure, a heightened level of hypersensitivity, or both, is conjectural.

100]

The route of administration of the provocative agent can exert an effect on both frequency of occurrence and severity. Anaphylaxis has occurred after all routes of administration, including oral, subcutaneous, intramuscular, intravenous, intranasal, intraocular, cutaneous, intravaginal, intrarectal, and endotracheal. An anaphylactic event has even been reported to the inhalation of perfume.[ than ingested.

[99]

101]

Attacks appear to be more frequent and more severe when the agent is injected rather

This fact is of clinical relevance regarding the prevention of anaphylactic episodes.

The constancy of administration of antigen is of note. For example, in most patients with insulin allergy, the anaphylactic reaction does not occur as long as there is uninterrupted administration of the drug. Interruption of administration contributes to the pathogenesis in this instance.[

99]

In addition, the time elapsing between an original episode of anaphylaxis and the readministration of antigen is an important variable. The likelihood of a second episode decreases as the time interval between the original event and readministration increases.

102

Boleman et al[ ] examined the effect of chronobiologic factors on the incidence of anaphylaxis in patients receiving immunotherapy. They retrospectively reviewed 210 anaphylactic reactions that occurred after immunotherapy between 1996 and 2000. They found no relationship between time of day or the lunar cycle and the incidence of anaphylactic episodes. The significance of atopy as a risk factor appears to depend on several variables, including the antigen involved and the route of administration. Early investigations 103 104

found that anaphylaxis to penicillin was more common in atopic individuals,[ ] [ ] but more recent reviews have not confirmed this observation. In a multicenter cooperative study of penicillin allergy by the American Academy of Allergy, there was no correlation between penicillin reactivity and a personal or family history 105]

of atopy.[

In 1973, in a study involving 1433 subjects, there was no difference in the incidence of positive skin tests to penicillin between atopic and nonatopic

106 individuals.[ ]

In one review of 1030 penicillin-allergic subjects and 1344 patients with pollen allergy, it was found that the atopic subjects had a lower than normal

risk for immediate hypersensitivity to penicillin.[ [109]

107]

Similar observations have been made regarding anaphylactic reactions to insulin, [

[110]

108]

to Hymenoptera stings,

111 112 individuals.[ ] [ ]

and to muscle relaxants. On the other hand, the incidence of anaphylaxis to latex is clearly increased in atopic This probably relates to the nature of the antigen, its cross-reactivity with foods to which atopic individuals have been sensitized (e.g., bananas, avocados, chestnuts), and the route of 112

sensitization (i.e., inhalation of latex-coated powder from gloves). [ ] Indeed, reactions to food in general, in which the exposure to antigen is mucosal, are more frequent in atopic subjects. This is in keeping with experimental data showing that atopic individuals appear to be more prone to manufacture IgE antibody when 113]

antigen is administered topically than when it is administered by injection.[

Atopic subjects appear to be predisposed to anaphylaxis and anaphylactoid reactions in general, because they account for an inordinate percentage of cases in random series[

24] [25] [27]

and in series of cases of idiopathic anaphylaxis,[

114]

115]

exercise-induced anaphylaxis,[

and anaphylactoid reactions to radiographic contrast media.

[63]

It is unclear why atopic individuals exhibit such heightened predisposition. It is evident that increased levels of IgE and IgE mast cell interaction (as conventionally understood) are not sufficient alone to account for this phenomenon. Certainly only a minority of atopic individuals are prone to anaphylaxis. For example, most tolerate large amounts of allergen administered during immunotherapy. Also, many individuals have elevated amounts of IgE to bee venom, latex, and various foods but fail to develop systemic reactions on exposure. Additional factors are clearly necessary to account for the idiosyncrasy of anaphylactic events. Possible abnormalities include autonomic nervous system dysfunction (e.g., beta-blockade) and the phenomenon of basophil “hyperreleasability.” Basophil 116]

hyperreleasability is seen in many atopic states. Spontaneous basophil histamine release is enhanced (compared with controls) in patients with food allergy,[ 117]

atopic dermatitis,[

118]

and asthma and bronchopulmonary aspergillosis.[

causing mast cell and basophil

118 degranulation.[ ]

This in vitro phenomenon could reflect increased in vivo sensitivity to agents capable of

In atopic subjects, the time of year at which the antigen is administered may also play a role, as evidenced by the

increase in frequency of anaphylactic reactions to allergen immunotherapy during the pollen season.[

119]

Frequency of exposure is an important factor in terms of the incidence of anaphylactic reactions. This is self-evident in most instances but can be cryptic in others. For example, diabetic subjects treated with protamine-containing insulin are 40 to 50

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TABLE 83-3 -- Factors Affecting the Incidence or Severity of Anaphylaxis and Anaphylactoid Reactions Factor

Effect

Age

More frequent in adults than in childrenfor some agents, radiocontrast media, plasma expanders, and anesthetics—may be function of exposure frequency[

92]

[99] [100]

Socioeconomic status

Increased frequency associated with higher socioeconomic status based on EpiPen dispensing rates in one study[

Gender

18]

Reportedly more frequent in females for latex, aspirin, and muscle relaxants. In addition, in random surveys, appears to be more frequent overall for females. May be more frequent in males for Hymenoptera, perhaps a function of 12 24 25 26 27 92

exposure.[ ] [ ] [ ] [ ] [ ] [ ] An age-related effect was shown, with males being affected more frequently 19]

before age 15 to 16, and females more frequently afterward.[ Route of administration

Oral administration less likely to produce reaction and reaction usually less severe

Constancy of administration

Gaps in administration may predispose to reactions

Time since last reaction

The longer the interval, the less likely the recurrence for many allergens

Atopy

Risk factor for anaphylaxis as a result of ingested antigens, exercise anaphylaxis, idiopathic anaphylaxis, radiocontrast reactions, latex reactions; probably not risk 21] [22] [23] [24] [25] [26]

factor for insulin, penicillin, and Hymenoptera reactions[

[27] [64] [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114]

Geographic location

In most instances no known effect has been documented, but in one study geographic location and rural environment affected incidence.[

Race

No known effect

Chronobiology

No known effect of time of day or lunar cycle[

88]

102]

120

times more likely to have reactions to protamine when this agent is administered to reverse heparin anticoagulation.[ ] Also, previous administration of protamine for heparin neutralization can sensitize a patient to insulin preparations containing protamine. Reactions to these preparations can be confused with anaphylactic reactions to insulin per se, and the previous sensitization experience to protamine can be missed, resulting in delayed recognition of protamine as the responsible

agent.[

121] [122]

The effects of these factors on the incidence of anaphylaxis are summarized in Table 83-3 .

PATHOLOGY Knowledge of the pathologic findings in anaphylaxis is sparse compared with that of other diseases because of a relative lack of autopsied cases. There are three series[

123] [124] [125]

describing the pathology of anaphylactic deaths, with the largest, reported by Pumphrey and Roberts,[

123]

consisting of only 56 cases, with

histology performed in only 20 necropsies. This represents anaphylactic deaths due to all causes and is the most recently reported evaluation. In 1983, Mosbech[ reviewed the findings in 26 deaths caused solely by insect stings, and in 1972 Delage and Irey[ come from case reports, small series, and reviews of previously published

124]

125]

evaluated 43 anaphylactic deaths. The remainder of cases have

125 126 127 128 129 130 131 132 133 134 135 136 data.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]

A significant finding from these reports is that anaphylactic deaths may have no significant macroscopic pathologic findings. In 23 of the 56 cases reported by Pumphrey and Roberts,[

123]

there was nothing on macroscopic examination indicative of anaphylaxis. This finding may result from the fact that death can occur 123]

rapidly. In their series, death occurred within 1 hour after the onset of symptoms in 39 cases.[

123

When pathologic findings are identifiable macroscopically, they most often involve the respiratory tract. In the Pumphrey and Roberts series,[ ] the most common macroscopic findings were pharyngeal/laryngeal edema (occurring in 23 of 56 cases) and mucus plugging and/or hyperinflated lungs (15 cases). In the series reported 124

by Delage and Irey,[ ] upper airway edema was found in about 60% of deaths, and bronchial obstruction with hyperinflation occurred in about 50% of the cases. Bronchial obstruction was caused by a combination of bronchospasm, submucosal edema, and secretions. Upper airway edema was the result of accumulation of transudate in the submucosa. In the series reported by Mosbech,[

124]

preexisting arteriosclerotic heart disease was thought to have contributed to cardiovascular collapse in 5 of the 26 cases. This 135]

is consistent with the fact that, when more extensive histologic review is obtained, myocardial damage has been reported in the majority of cases.[

Other findings have been dilatation of the right ventricle; diffuse eosinophilic infiltration of the pulmonary vessels, lamina propria of the gastrointestinal tract, and sinusoids of the spleen; and congestion of the abdominal viscera.[

123] [124] [125]

Taking these observations as a whole, several conclusions can be drawn. First, in view of the lack of significant findings in a large number of instances (at least on 123

macroscopic pathologic examination), it can be assumed that anaphylactic deaths may be underreported.[ ] Second, the most frequently involved system appears to be the upper respiratory tract, with upper airway edema being the most frequent finding. This observation, coupled with the cardiovascular findings noted previously and case reports, indicates that death from anaphylaxis is usually the result of respiratory obstruction involving the upper or lower airway (or both) or cardiovascular collapse. When death is caused by cardiovascular collapse, especially if death occurs rapidly after the onset of symptoms, pathologic findings can be sparse.

Of more recent interest is the observation that postmortem determination of specific IgE and serum tryptase may be useful in establishing anaphylaxis as the cause of death in patients experiencing sudden death of unknown cause.[

137] [138] [139] [140] [141] [142] [143] [144] [145]

1502

In one study, IgE antivenom was assayed by the radioallergosorbent test (RAST) in postmortem blood samples taken from patients who died unexpectedly from 138

139

spring through autumn; 23.2% of sera showed significant levels of IgE anti-Hymenoptera venom. [ ] In a follow-up of that study,[ ] 68 remaining sera previously examined for the prevalence of IgE antivenom were analyzed for tryptase, and 9 (13%) demonstrated elevation of serum tryptase (>10•ng/ml). There was some degree of correlation between elevated tryptase levels and the previously assayed antivenom IgE levels. In three samples with markedly elevated antivenom IgE levels, one showed elevation of serum tryptase. In 17 samples with moderate elevations of antivenom IgE, three (18%) demonstrated elevated tryptase levels. In the 47 samples with no increase in venom-specific IgE, five (11%) demonstrated elevated tryptase levels. The specificity and sensitivity of specific IgE and tryptase levels have been demonstrated by their assay in patients who died from documented anaphylactic episodes. [137]

Mast cell–derived tryptase and specific IgE antibody levels were measured in sera obtained before or within 24 hours after death from anaphylaxis in 19 subjects. Serum tryptase levels were elevated in 9 of 9 Hymenoptera sting fatalities, 6 of 8 food-induced fatalities, and 2 of 2 deaths caused by diagnostic or therapeutic agents. In contrast, normal serum tryptase levels were found in 57 postmortem control patients. Serum IgE antibodies were elevated in 5 of the 9 Hymenoptera fatalities and 8 of the 8 fatal food reactions. Of note is the observation that serum tryptase should be drawn within 15 hours after death, because nonspecific elevations occur beyond this time.[

140]

An interesting observation was that serum tryptase levels were higher in those to whom the allergen was administered parenterally (Hymenoptera venoms and injections) than in those who encountered the allergen orally (i.e., in foods). The authors postulated that this difference was due to the fact that connective tissue mast cells are preferentially degranulated when the antigen is injected and mucosal mast cells are preferentially degranulated when the antigen is ingested.[ tissue mast cells contain more tryptase than mucosal mast cells

139]

Connective

137 138 do.[ ] [ ]

The implications of these findings are important in two regards. First, they imply that the incidence of anaphylactic death may be underestimated, and that a 139

significant proportion of unexplained sudden deaths may be caused by anaphylaxis. [ ] Second, they are of clinical importance because they offer a diagnostic modality with the potential of determining the cause of unexpected death. It is recommended that serum should be obtained before death (if possible) or within 15 140

hours after death for the analysis of tryptase and allergen-specific IgE.[ ] Sera should be frozen and stored at −20° C until assayed. Of note is the fact that pleural and pericardial fluids are not satisfactory for tryptase quantitation. The authors suggested that unconsumed portions of foods eaten by the victim before the episode can be saved to construct “custom” specific IgE antibody reagents.[

137]

However, it should be noted that postmortem elevation of the tryptase concentration is not a specific finding, diagnostic of anaphylactic death. There are reports

143 144 145

143

detailing nonanaphylactic deaths with elevated postmortem tryptase levels. [ ] [ ] [ ] Randall, Butts, and Halsey[ ] evaluated tryptase levels in postmortem blood samples from 49 autopsy cases, none of which exhibited evidence of death from anaphylaxis. They found that tryptase levels were greater than the normal serum threshold (1•ng/ml by their definition) in 31 of these cases. Levels were between 1 and 5•ng/ml in 24 cases, between 5 and 10•ng/ml in 2 cases, and greater than 10•ng/ml in 5 cases. One postmortem serum tryptase value was 106•ng/ml; in this instance death resulted from multiple trauma. Only 18 of the 49 specimens examined by these investigators had a tryptase concentration lower than 1•ng/ml (the upper limit for normal for 95% to 98% of healthy, normal individuals in their assay). They concluded that “an elevated postmortem tryptase obtained at autopsy cannot be used to establish the cause of death as anaphylaxis without additional 143

144

supporting data.”[ ] Similar conclusions were drawn by Edston et al,[ ] who measured mast cell tryptase and total IgE in postmortem sera from 41 infants younger than 1.5 years of age. They found that in 40% of these infants, who presumably died from sudden infant death syndrome, the tryptase concentration was greater than 10•ng/ml (their cutoff level). The only variable, in their series, that could be statistically correlated with high tryptase levels was the prone position at death. They concluded that the elevated tryptase concentration found in infants with sudden infant death syndrome could not be attributed to mast cell degranulation due to allergy but might hypothetically be explained by degranulation due to, for example, hypoxic stimuli. 145

Edston and van Hagge-Hamsten[ ] also evaluated beta-tryptase levels in 193 postmortem cases, including 176 deaths from nonanaphylactic causes, 10 unexplained deaths, and 7 deaths caused by anaphylactic or anaphylactoid reactions. Using a binary logistic regression model, they calculated the sensitivity and specificity of tryptase to establish a postmortem diagnosis of anaphylaxis and found 10•ng/ml to be the optimal cutoff value. They found that the sensitivity using this value was 86%, and the specificity was 88%. Deaths caused by trauma, sudden infant death syndrome, and heroin injection resulted in elevated tryptase concentrations in 35%, 35%, and 32% of the cases, respectively. Increased concentrations were found in 40% of unexplained deaths. Tryptase levels in heart blood were elevated in 22% of controls, whereas levels in femoral blood were elevated in only 10%. There was no correlation between increased levels and age or delay in postmortem sampling. The authors concluded that tryptase measurements are useful in confirming death resulting from anaphylaxis or anaphylactoid reaction, and that blood should be sampled from the femoral vessel. They found that tryptase measurement is a useful indicator of an anaphylactic death, but that the diagnosis cannot be established based solely on an elevated tryptase concentration, because elevated levels are demonstrated in a significant number of nonanaphylactic deaths as well. The individual in their study with the highest tryptase level (170•ng/ml) died from multiple fractures and organ damage resulting from trauma. Therefore, it appears that elevated postmortem tryptase levels are useful for confirmation of anaphylaxis as the cause of death but can also occur in nonanaphylactic deaths, probably because of mast cell degranulation related to hypoxemia or postmortem events. On the other hand, a postmortem tryptase concentration that is not elevated is a strong indication of death resulting from another cause.

PATHOPHYSIOLOGY Anaphylaxis and anaphylactoid events are the result of the activation of several pathways of inflammation ( Box 83-2 ).

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Box 83-2. Pathophysiologic Classification of Anaphylaxis and Anaphylactoid Reactions Anaphylaxis: IgE-mediated reaction Food Drugs Insect bites and stings Perhaps some cases due to exercise Anaphylactoid Direct release of mediators from mast cells and basophils Drugs Idiopathic Exercise Physical factors, such as cold, sunlight Disturbances in arachidonic acid metabolism Aspirin Nonsteroidal antiinflammatory drugs Immune aggregates Gammaglobulin IgG–anti-IgA Possibly dextran and albumin Cytotoxic Transfusion reactions to cellular elements

Miscellaneous and multimediator activity Non–antigen-antibody–mediated complement activation Radiocontrast material Possibly some cases of protamine reactions Dialysis membranes Activation of contact system Dialysis membranes Radiocontrast material

Anaphylactic reactions are defined as those mediated through antigen-induced IgE mast cell and basophil degranulation. The most common causes of these reactions are foods, drugs and biologic agents, and insect stings and bites. In addition, some cases of exercise-induced anaphylaxis (food-dependent) may be mediated through this mechanism.[

146]

Anaphylactoid reactions are clinically similar to anaphylactic episodes but have a different underlying pathophysiology. One of the most common mechanisms of production of anaphylactoid reactions involves the direct (nonantigen-IgE) release of mediators from mast cells and basophils. This occurs in anaphylactoid reactions to drugs and biologic agents, most cases of idiopathic anaphylaxis, the majority of cases of exercise-induced anaphylaxis, and probably with anaphylaxis to other physical factors, such as cold and sunlight. However, it is feasible that during some of these events an endogenous antigen is produced, which then causes IgEmediated degranulation of mast cells. Examples of drugs causing direct degranulation of mast cells are radiocontrast media[

61]

and opioids.[

147]

148

Recently, new insights have been gained into the mechanisms of production of idiopathic anaphylaxis. Dykewicz[ ] described two patients with a history of episodes of idio-pathic anaphylaxis (both of whom had chronic urticaria) who demonstrated positive intradermal autologous serum skin tests. One of these was a 21year-old woman who had experienced daily episodes of urticaria 3 weeks before the first of two anaphylactic events (bronchospasm, angioedema, generalized urticaria, and hoarseness). The other was a 32-year-old woman who had a 2-year history of urticaria and a 1-year history of recurrent episodes of anaphylaxis (six in total), some of which were hypotensive. For both of these patients, a leukotriene antagonist (zafirlukast) was of benefit in their therapy. The author concluded that in some cases of idiopathic anaphylaxis, autoantibodies to IgE might be operative in the production of symptoms, and that leukotriene antagonists may be of value in this subset of patients with idiopathic anaphylaxis.[ Grammer et al[

149]

148]

at Northwestern University have also investigated the possible mechanisms underlying episodes of idiopathic anaphylaxis. They analyzed flow

cytometric patterns in patients with acute idiopathic anaphylactic episodes and compared them with patterns in normal controls and in patients with idiopathic anaphylaxis in remission (with or without prednisone therapy). This study was conducted prospectively in nine patients with acute attacks of idiopathic anaphylaxis who had not yet received prednisone, nine such patients who had received prednisone, eleven patients with idiopathic anaphylaxis in remission, four patients with idiopathic urticaria, and five normal individuals. Patients with idiopathic anaphylaxis in remission were compared with those patients experiencing an acute event. The latter had a significantly higher percentage of CD3+ HLA-DR+ cells. Normal subjects had a significantly lower percentage of CD3+ HLA-DR+ cells than all other groups. The authors concluded that patients experiencing acute episodes had more activated T cells than did patients in remission, and they postulated a role for activated T cells in the pathogenesis of idiopathic anaphylaxis.[ for the histamine release in idiopathic anaphylaxis.[

148] [149]

This same group, in a separate study, found that the chemokine, MCP-I, was not responsible

150] [151]

A group of investigators from Spain evaluated mast cell releasability in patients with idiopathic anaphylaxis. Their patients showed a higher cutaneous response to 152]

codeine than did atopic patients (without anaphylaxis). They concluded that an increase in mast cell releasability might contribute to idiopathic anaphylaxis. [ 79 153 154

Most reactions to aspirin and other NSAIDs in asthmatics occur as the result of aberrant metabolism of arachidonic acid.[ ] [ ] [ ] In most instances, asthmatics who experience anaphylactoid reactions to aspirin will have similar reactions to any NSAID—that is, there is cross-reactivity with any drug that inhibits the cyclooxygenase pathway, implicating a class effect related to the drug's ability to inhibit cyclooxygenase activity. In some anaphylactoid reactions in nonasthmatics, 154

however, there may be no cross-reactivity, and the patient reacts to only one NSAID.[ ] This implies that there is perhaps an IgE-mediated mechanism underlying the latter form of reaction to an NSAID. In both types of reactions, the final common pathway responsible for symptoms may be similar, because there is evidence of mast cell degranulation (elevations of histamine and tryptase) in asthmatics who react to any form of NSAID and whose reactions are therefore related to the drug's ability to inhibit cyclooxygenase activity. Although the exact mechanism of production of the degranulation of mast cells and/or basophils in this syndrome has not

1504

been established, it is thought that the inhibition of production of prostaglandin E (which protects against mast cell degranulation) along with the overproduction of 155]

leukotriene C (which enhances mast cell degranulation) is responsible for the release of the contents of mast cells, basophils, or both.[

Immune aggregate anaphylaxis occurs when complement is activated by antigen-antibody complexes or molecular aggregates. This type of anaphylactoid reaction has been reported after administration of protamine,[ IgA

159 deficiency.[ ]

156]

dextran,[

157]

158]

and albumin, [

and after administration of transfusions or gammaglobulin to patients with

However, many of the reactions to plasma transfusion as well as those to protamine in such patients are thought to be anaphylactic in nature and

mediated via IgE anti-IgA. [

154] [160]

In cytotoxic anaphylactoid reactions, the antigen is cell-fixed. Such reactions occur during incompatible transfusions when there are complement-fixing antibodies to formed elements of the blood, such as red cells, white cells, and platelets. Complement activation is thought to play a role in the production of these events.

Finally, several agents (e.g., radiocontrast material) produce anaphylactoid reactions by activation of multiple inflammatory pathways. These include complement, 61]

clotting, clot lysis, and the contact (kallikrein-kinin) systems.[

BASOPHIL AND MAST CELL DEGRANULATION SYNDROMES It is likely that the majority of anaphylactic and anaphylactoid events involve basophil and mast cell degranulation. It is therefore important to be familiar with the mediators released TABLE 83-4 -- Mast Cell and Basophil Mediators and Their Roles in Producing Anaphylactic and Anaphylactoid Events Mediators

Pathophysiologic Activity

Clinical Correlates

Histamine and products of arachidonic acid metabolism (leukotrienes, thromboxane, prostaglandins, and platelet-activating factor)

Smooth muscle spasm, mucus secretion, vasodilation, increased vascular permeability, activation of nociceptive neurons, platelet adherence, eosinophil activation, eosinophil chemotaxis

Wheeze, urticaria, angioedema, flush, itch, diarrhea and abdominal pain, hypotension, rhinorrhea, and bronchorrhea

Neutral proteases: tryptase, chymase, carboxypeptidase, cathepsin G

Cleavage of complement components, chemoattractants for eosinophils and neutrophils, further activation and degranulation of mast cells, cleavage of neuropeptides, conversion of angiotensin I to angiotensin II

May recruit complement by cleaving C3, may ameliorate symptoms by invoking a hypertensive response through the conversion of angiotensin I to angiotensin II and by inactivating neuropeptides; also, can magnify response caused by further mast cell activation

Proteoglycans: heparin, chondroitin sulphate

Anticoagulation, inhibition of complement, binding phospholipase A2, chemoattractant for eosinophils, inhibition of cytokine function, activation of kinin pathway

Can prevent intravascular coagulation and the recruitment of complement; also can recruit kinins, increasing the severity of the reaction

Chemoattractants: chemokines, eosinophil chemotactic factors

Calls forth cells to the site

May be partly responsible for recrudescence of symptoms in late phase reaction or extension and protraction of reaction

from basophils and mast cells and their possible roles in anaphylaxis and anaphylactoid episodes. Table 83-4 briefly summarizes these mediators, their roles in the pathophysiology of anaphylaxis and anaphylactoid reactions, and the clinical correlates that result from their effects. The major pathophysiologic events caused by the release of these mediators include smooth muscle spasm (especially of the bronchi, coronary arteries, and

gastrointestinal tract), increased vascular permeability, vasodilation, stimulation of sensory nerve endings with reflex activation of vagal effector pathways and antidromic pathways, and myocardial depression. These effects result in the classic symptoms of flush; urticaria and angioedema; wheeze; fall in blood pressure with potential shock; gastrointestinal smooth muscle contraction with nausea, vomiting, and diarrhea; and myocardial ischemia. 161]

In addition, many of these mediators are capable of activating other inflammatory pathways. Mast cell kininogenase[ kinin system. Tryptase also has kallikrein activity and can activate the complement cascade and cleave

163 fibrinogen.[ ]

and basophil kallikrein[

162]

can activate the

Platelet-activating factor can activate clotting

164 coagulation.[ ]

and produce disseminated intravascular In addition, chemotactic agents, by calling forth eosinophils and other cells, have the capacity to prolong and intensify reactions. Other agents perhaps modify the pathophysiologic events. Heparin can inhibit clotting, plasmin, and kallikrein. It also modulates the effects of tryptase and has anticomplementary activity. Chymase is capable of converting angiotensin I to angiotensin II and therefore theoretically could enhance the compensatory response to hypotension. Cells, especially eosinophils, called forth to the site by chemotactic agents originally released

1505

from mast cells and basophils, can be responsible for protracted episodes of anaphylaxis and for a recrudescence in symptoms after an initial improvement (late phase response). For example, it has been demonstrated that eosinophils, probably through their release of major basic protein, can cause activation of mast cells and their degranulation, producing a secondary release of mediators.[

165]

In one report of a protracted case of anaphylaxis in a 6-year-old boy, caused by either thiopentone or

cisatracurium, there was associated elevation of eosinophil cationic protein levels.[

166]

By visualizing the summation effect of these mediators, it can be seen that biphasic, delayed, recurrent, and protracted anaphylactic and anaphylactoid events could occur as a result of their release. Case reports and series of such events have been described.[ produce the majority of the symptoms of reviewed in more detail.

171 172 173 anaphylaxis,[ ] [ ] [ ]

167] [168] [169] [170]

Because histamine infusion has been shown to

and because it has been the most intensively studied mediator, the effects of this agent are

Histamine The actions of histamine that are salient to anaphylaxis occur through activation of H1 and H2 receptors. The overall effect of histamine on the vascular bed is dilation. This produces flushing and a lowering of peripheral resistance, with a subsequent fall in systolic pressure. There is also an increase in vascular permeability because of the exposure of the permeable basement membrane secondary to separation of endothelial cells at the postcapillary venule level. Vasodilation is mediated by both H1 and H2 receptors. H2 receptors exert their effect by a direct action on the vascular smooth muscle. H1 receptors also exert some effect directly, but their major activity is produced indirectly by the stimulation of endothelial cells to manufacture nitric oxide (NO).[

172] [173]

Cardiac effects of histamine are mediated primarily through the H2 receptor, but the H1 receptor also plays a role. H2 receptor stimulation increases both the rate and

the force of atrial and ventricular contraction, probably by enhancing calcium influx. This increases cardiac oxygen need. H1 receptor activity increases the heart rate 174]

by hastening diastolic depolarization at the sinoatrial node. H1 receptor stimulation also produces coronary artery vasospasm.[

Histamine produces varying effects on extravascular smooth muscle. It causes smooth muscle contraction in the bronchial tree, mediated entirely via the H1 receptor. H1 receptor stimulation also causes modest contraction of the human uterus, and H2 receptor stimulation can produce uterine relaxation. The predominant effect of histamine on the gastrointestinal smooth muscle is contraction mediated via the H1 receptor. Glandular secretion is mediated by both H1 and H2 receptors. Glycoprotein secretion from goblet cells and bronchial glands is produced by stimulation of the H2 receptor, whereas stimulation of the H1 receptor increases mucus viscosity. Infusion of histamine into humans produces symptoms similar to those observed during anaphylaxis. For maximal inhibition of flushing, headaches, hypotension, and tachycardia, a combination of H1 and H2 receptor blockade is required.[

171]

This observation is of clinical importance.

The roles of other basophil and mast cell mediators have not been as clearly defined. However, as noted earlier, these agents are potentially strong candidates for production of the pathophysiologic events responsible for anaphylaxis and anaphylactoid events. Recruitment of Other Inflammatory Pathways From the previous discussion, it is clear that mast cell and basophil contents can activate a number of inflammatory pathways, including the kinin system, complement system, clotting, and clot lysis (disseminated intravascular coagulation) ( Box 83-3 ). There is strong in vivo evidence to indicate that these recruitment pathways play an important role in clinical events.[

175] [176] [177] [178] [179] [180]

One of the first demonstrations of the activation of other inflammatory pathways in human anaphylaxis occurred during controlled studies of immunotherapy for 175 176 177

insect hypersensitivity.[ ] [ ] [ ] In subjects who experienced only generalized urticaria to insect sting challenge, there were no changes in blood histamine levels or recruitment of other mediator pathways. However, in three patients who experienced shock, peak histamine levels rose dramatically and correlated with the severity of hypotension. In addition, two of those three patients had diminution in factor V, factor VIII, fibrinogen, and high-molecular-weight kininogen. In one of these patients, there were diminished levels of C4 and C3. A later evaluation of eight patients with previous reactions to wasp stings who were rechallenged with a sting demonstrated a relationship between levels of the anaphylatoxin, C3a, and the severity of the anaphylactic event. There was no change in C3a in one patient who showed no reaction, and only a slight rise in three patients with mild reactions. In contrast, C3a rose substantially in four patients with severe reactions. The presence of this cleavage product of C3 indicated activation of the complement cascade.[

178]

176 179

Other investigations have demonstrated activation of the kallikrein-kinin system.[ ] [ ] Kallikrein–C1-inhibitor complexes, factor XIIa–C1-inhibitor complexes, antigenic prekallikrein, and antigenic factor XII were measured in serial blood samples obtained from 16 subjects with a history of insect sting Box 83-3. Multimediator Recruitment Occuring During Anaphylaxis and Anaphylactic Events (Pathway Activated) Coagulation pathway[

175] [177]

Decreased factor V Decreased factor VIII Decreased fibrinogen Complement cascade[

175] [178]

Decreased C4 Decreased C3 Formation of C3a 176] [177]

Contact system (kinin formation)[

Decreased high-molecular-weight kininogen Formation of kallikrein-C1-inhibitor complexes and factor XIIa-C1inhibitor complexes

1506

176

anaphylaxis immediately after insect sting challenge.[ ] Peak levels of kallikrein–C1-inhibitor complexes and factor XIIa–C1-inhibitor complexes were found 5 minutes after the onset of symptoms. By 15 minutes, antigenic prekallikrein levels had decreased in patients with angioedema as a component of the anaphylactic event. These findings occurred only in subjects experiencing reactions. Therefore, activation of the contact system (kallikrein-kinin) was strongly related to the

development of angioedema after sting challenge. Mast cells secrete a number of cytokines during the degranulation process, and histamine itself can be responsible for the up-regulation of cytokine production. Lin 180

and Trivino[ ] evaluated levels of C-reactive protein (CRP) and interleukin-6 (IL-6) during anaphylactic episodes to determine whether they were related to clinical manifestations and also whether these acute phase reactants might be helpful in establishing the diagnosis. They examined 85 patients admitted to the emergency department with acute allergic syndromes including anaphylaxis, measuring histamine, CRP, and IL-6. The levels of IL-6 and CRP were highly correlated. IL-6 and histamine levels were both significantly correlated with the extent of erythema. However, histamine was not positively correlated with either IL-6 or CRP. Unexpectedly, histamine levels were negatively correlated with levels of CRP, with CRP levels being lower in patients with elevated histamine levels. Histamine levels correlated with the extent of urticaria, but IL-6 and CRP levels did not. A negative correlation was demonstrated between IL-6 concentration and the mean arterial blood pressure. The authors concluded that CRP and IL-6 were not surrogate markers for histamine release in mast cells and basophils. They did find, however, that IL-6 levels related to erythema and to lower arterial blood pressure. They postulated that IL-6 levels might correspond with a decrease in peripheral vascular resistance and speculated that CRP and IL-6 increases might be markers of a late phase response. This might explain the inverse correlation between CRP and histamine levels; as histamine declines, CRP and IL-6, manufactured by macrophages and monocytes recruited to the site, might be increasing. As previously noted, recruitment of other inflammatory pathways could be due to tryptase, mast cell kininogenase, and basophil kallikrein. In addition, the cellular and biochemical events (e.g., hypoxia, endothelial damage) that occur during shock could activate the contact system, clotting system, and complement system. Activation of these systems has been noted in severe hypotensive shock of other etiologies (e.g., cardiovascular shock, endotoxic shock). Induction of Nitric Oxide Synthesis One of the most interesting advances in the understanding of the pathophysiology of immediate hypersensitivity reactions, including anaphylaxis, is the identification of NO as a central mediator of these events. NO is a diffusable gas that is a messenger of both intracellular and intercellular signals. It has long been known that histamine and other mediators of anaphylaxis can act indirectly in an endothelium-dependent fashion to produce vascular permeability and smooth muscle relaxation. In the case of histamine, as noted, this effect is mediated through H1 receptors present on endothelial cells. Originally the nature of the substance mediating this effect was unknown, but the substance itself, in keeping with

Box 83-4. Effects of Nitric Oxide in Anaphylaxis

Potentially Detrimental Effects Vasodilation (peripheral vascular bed) Increased vascular permeability

Potentially Beneficial Effects Bronchodilation Vasodilation (coronary arteries) Decreased mast cell degranulation

181] [182] [183] [184]

its activity, was called endothelium-derived relaxing factor (EDRF). In 1988, it was first proposed that EDRF was NO.[

NO is synthesized from l-arginine through the activity of nitric oxide synthase (NOS). There are three isoforms of NOS. Two of these (cNOS) exist constitutively and the third (iNOS) is inducible. cNOS can be found in endothelium, myocardium, endocardium, skeletal muscle, platelets, and neural tissue. iNOS is found in macrophages, fibroblasts, neutrophils, and smooth muscle. cNOS is Ca2+ /calmodulin-dependent, and iNOS is Ca2+ -independent. iNOS is regulated at the transcriptional level. Mediators known to induce the synthesis of cNOS are capable of acting very rapidly, with increased levels of NO occurring within seconds after exposure. These mediators are the same as those seen in anaphylactic and anaphylactoid episodes, including histamine, platelet-activating factor, several of the leukotrienes, and bradykinin, as well as acetylcholine. NO synthesis via iNOS requires gene transcription and can take minutes to hours; however, once initiated in this fashion, the synthesis can continue in a protracted way for several hours, and the amount of NO produced exceeds that made through the activity of cNOS.[ The effects of NO are protean.[ damaging ( Box 83-4 ).

185] [186] [187]

182] [183]

Its effects in anaphylaxis are somewhat “schizophrenic” in that they have the potential to be both protective and

NO can relax bronchial smooth muscle while at the same time dilating vascular smooth muscle. In addition to the peripheral vasodilatation, it can enhance vascular

permeability. Its effect on smooth muscle thus can improve bronchospasm while worsening hypotension. In addition, NO can inhibit mediator release from mast cells. Therefore NO has the potential for both a deleterious and a beneficial effect during an anaphylactic episode; however, it appears as if the summation effects are adversarial, resulting in hypotension, loss of intravascular volume, and resultant hemoconcentration. At least in animal models of anaphylaxis, an NO synthesis 188]

inhibitor exerts a beneficial effect in shock,[

189]

and similar results have been seen in humans with hypotension due to cardiogenic shock.[

MECHANISMS INVOLVED IN THE PRODUCTION OF ANAPHYLACTIC SHOCK Many of the pathophysiologic events that occur during anaphylactic shock are easily explained by the action of the

1507

mediators cited earlier. For example, increases in airway resistance and falls in partial pressure of oxygen (Po2 ) could be attributed to the direct contractile effect of histamine and other mediators on the smooth muscle of the lung, despite the fact that histamine levels do not always correlate with the presence of wheezing. Similar explanations are evident for flush, urticaria, angioedema, and gastrointestinal symptoms. However, the mechanism of production of hypotension and shock is more complicated and deserves special mention. Much of the data regarding the mechanism of production of shock in human beings has been obtained from the evaluation of patients who experienced anaphylaxis or anaphylactoid reactions during anesthesia or cardiac catheterization. [

190] [191] [192] [193]

This has allowed for hemodynamic measurements during the event. 194 195

Although the hemodynamic changes occurring during anaphylaxis and anaphylactoid reactions can vary,[ ] [ ] the major factors causing cardiovascular abnormalities are universal and result from an initial loss of intravascular fluid and vasodilation, which may be followed shortly by vasoconstriction and then myocardial depression. Increased vascular permeability can produce a rapid and dramatic loss of intravascular volume. Fluid shifted to the extravascular space can result in a loss of 50% of vascular volume within 10 minutes.[ norepinephrine and

198 199 epinephrine;[ ] [ ]

200 201 202 203 204 agents[ ] [ ] [ ] [ ] [ ]

This loss of blood volume leads to compensatory mechanisms that involve the secretion of catecholamines, such as

; activation of the angiotensin system, with conversion of angiotensin I to angiotensin II and increased production of these

; and the production of endothelin-1, a potent vasoconstrictor peptide that was previously found to be elevated in patients with heart

failure, stroke, or hypotension.[ 205 agent.[ ]

196] [197]

205]

Increased levels of endothelin-1 indicate that that the endothelium responds to hypotension with increased production of this 200] [203]

On the other hand, attempts to correlate elevations of vasopressin and oxytocin with anaphylactic episodes have shown inconsistent results.[

These internal compensatory vasopressor responses can produce variable results. In some patients with anaphylactic and anaphylactoid episodes, the peripheral 194]

resistance is abnormally elevated (indicating maximal vasoconstriction) because of this response.[

In other subjects, despite the elevation of catecholamines,

systemic vascular resistance falls.[

198]

202 206

It has been suggested that failure to mobilize these compensatory mechanisms may predispose patients to anaphylaxis.[ ] [ ] In a study comparing baseline plasma angiotensin I and angiotensin II levels in Hymenoptera-sensitive patients and normal controls, the former group had significantly lower amounts. An inverse correlation between angiotensin levels and the severity of the anaphylactic episodes was noted: the lower the levels, the more severe the symptoms. In addition, patients with a history of anaphylaxis to Hymenoptera stings had significantly lower angiotensin II levels in leukocytes than do controls.[ successful immunotherapy induced an increase in leukocyte angiotensin II levels in these

207]

It is of interest that

207 patients.[ ]

Further hemodynamic changes that contribute to or occur as a result of the fall in arterial pressure include a decrease in cardiac output, decreases in pulmonary wedge pressure and pulmonary artery pressure, and an increase in pulmonary vascular resistance. These latter changes can result in “shock lung” TABLE 83-5 -- Dynamics of Cardiovascular Abnormalities in Anaphylactic Shock Parameter

At Onset of Reaction

Early Stage (minutes) with No Treatment

Prolonged Shock

Blood pressure

↓

↓↓

↓↓↓

Pulse

↑

↑

↑↑

Cardiac output

↑

↓

↓↓

Peripheral vascular resistance

↓

→↓

Intravascular volume

→↓

↓

*

→↑↓

*

↓↓↓

* Peripheral vascular resistance can vary, probably depending on the internal compensation response (see text).

or adult respiratory tract distress syndrome with pulmonary edema.[

208]

Falls in blood pressure can be correlated with elevations of histamine, tryptase,[ the presence of urticaria, flush, or wheeze.[

178] [209] [210]

175] [209]

and C3a.[

178] [210]

However, levels of these mediators do not correlate with

Angioedema may be related to the appearance of activation products of the contact (kinin–kallikrein) system.

[210]

Anaphylactic episodes are dynamic, and cardiovascular status can change during different stages of the event.[ the onset of shock, systemic vascular resistance can be

193 reduced,[ ]

193] [198] [199]

For example, during the initial phase, at

whereas during the later phases, with prolonged shock, systemic vascular resistance can rise,

presumably through the compensatory vasoconstrictor response or the administration of endogenous vasoconstrictive agents, or both. Cardiac output, which can initially be increased, characteristically declines as the event progresses. Central venous pressure may be normal during the early phases of the event and then should consistently fall with progression of the reaction. The same occurs with pulmonary capillary wall pressure. Table 83-5 summarizes the potential changes in these parameters during the various stages of shock. These changes are highly important from a therapeutic standpoint. The one consistent and most important finding in regard to the production of hypotension is the loss of effective intravascular volume due to fluid extravasation into the extravascular space. In addition, because of internal compensatory mechanisms (secretion of epinephrine, norepinephrine, and endothelin-1, production of angiotensin II), such patients with shock may be maximally vasoconstricted and therefore unresponsive to pressor agents. Fluid replacement and volume expanders, not vasoconstrictor agents, are the treatments of choice in such cases.

SIGNS AND SYMPTOMS 16 25 26 27 28 169 211 212 213 214 215 216 217

The clinical manifestations of anaphylaxis can best be ascertained by a review of published series.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] An analysis of several of these series, consisting of a total of 1784 patients, is summarized in Box 83-5 . This summary includes series of patients suffering exclusively from exercise-induced anaphylaxis, [

211]

patients with idiopathic anaphylaxis only,[

212]

series limited to pediatric patients,[

169] [217]

1508

Box 83-5. Frequency of Occurrence of Signs and Symptoms of Anaphylaxis *

Signs and Symptoms

Percentage of

Cutaneous

>90

••Urticaria and angioedema

85–90

••Flush

45–55

Cases

†

series of randomly

••Pruritus without rash

2–5

Respiratory

40–60

••Dyspnea, wheeze

45–50

••Upper airway angioedema

50–60

••Rhinitis

15–20

Dizziness, syncope, hypotension

30–35

Abdominal ••Nausea, vomiting, diarrhea, cramping pain

25–30

Miscellaneous

†

••Headache

5–8

••Substernal pain

4–6

••Seizure

1–2

Percentages are approximations (see text).

* Based on a compilation of 1784 patients reported in references [ [27] [28] [169]

213] [214] [215] [216] [217] [218] [219]

and [

25] [26] [27] [28] [214]

selected patients of all ages presenting to an allergy clinic for evaluation,[ records of 1255 Olmstead County (Minnesota) residents.[

16]

16] [25] [26]

213–219.

and a series of patients obtained by a retrospective chart review of the

Because of the heterogeneity of these reports (e.g., some report the frequency of multiple signs and

symptoms and others do not, some include insect stings and others do not, some are limited to pediatric cases, not all record the same set of signs and symptoms), an exact analysis is impossible. Nonetheless, from a review of these studies the typical patient with anaphylaxis emerges. Overall, the clinical similarities shared by patients in all of these series is striking. With rare exception, the most common manifestations are cutaneous. Next in frequency are symptoms referable to the respiratory tract. Hypotension occurred next most commonly, followed by gastrointestinal manifestations. 28 217

The largest published series of anaphylaxis comprised 411 patients, age 9 to 79 years, evaluated between January 1978 and August 2000.[ ] [ ] A detailed analysis of these subjects is helpful to establish a typical clinical profile of anaphylaxis. Fifty-one percent of the subjects had more than three anaphylactic episodes. Cutaneous symptoms were present in almost all subjects. The most common cutaneous manifestation was urticaria and edema—a finding echoing through most studies. Females predominated (247 females versus 164 males). Headache was a somewhat unique complaint, occurring in a significant percentage of subjects with exercise-induced anaphylaxis (30%), but reported rarely (5% or less) in other series.[

26] [27] [28]

However, there are exceptions to these prototypical clinical manifestations. For example, cardiovascular collapse with shock can occur immediately without any 218

219

cutaneous or respiratory symptoms.[ ] In fact, in a series of 27 severely affected patients treated by anesthesiologist-staffed ambulance and helicopter crews,[ ] only 70% of patients with circulatory and/or respiratory failure had cutaneous symptoms. Some 30% of these patients had gastrointestinal symptoms, and, of note, 85% had neurologic symptoms (seizures, impaired consciousness, muscle spasms). The relative paucity of cutaneous symptoms may have been due to the fact that data were recorded only from signs observed after the arrival of emergency personnel at the scene. Another possible explanation is the difference in severity between 219]

these cases and those in the series included in Box 83-5 . Of the 27 patients reported in this series, there were 2 deaths and 23 hospitalizations.[

220

In addition, although cutaneous symptoms (next to gastrointestinal manifestations) are the most common manifestations of food allergy of all types, [ ] doubleblind, placebo-controlled food challenges, for reasons that have not been determined, often show a lower incidence of cutaneous reactions than has been recorded for 221

random series as a whole. For example, Sampson[ ] reported on 100 children evaluated for food allergy by oral food challenge. Skin symptoms were the most frequent manifestations, but they varied in frequency according to the allergen employed and occurred surprisingly infrequently for some allergens. Skin symptoms occurred in 22 of 25 egg challenges, 19 of 21 milk challenges, 14 of 21 soy challenges, and 16 of 17 wheat challenges. This represents an overall incidence of approximately 84%, slightly lower than that seen in random series, with an exceedingly low incidence in subjects challenged with soy.[

221]

The signs and symptoms of anaphylaxis that occur during anesthesia and surgery may differ considerably from those noted in episodes outside the operating room. In 45

addition, there may be significant differences between operative anaphylactic episodes and anaphylactoid events.[ ] The incidence of cutaneous manifestations is approximately 75% for anaphylactic events and slightly higher for those that are anaphylactoid in nature. Cardiovascular collapse is also more common in anaphylactic events that occur during surgery than in those that occur outside of the operating room, and it is significantly more frequent and more severe in anaphylactic events than in anaphylactoid episodes. The same holds true for wheezing and bronchospasm. Overall, anaphylactic events during surgery are more severe than those caused by non–IgE-mediated mechanisms. There is no clearcut explanation for these observations. Table 83-6 summarizes the clinical manifestations of surgical anaphylactic and anaphylactoid events.

Symptoms usually begin within 5 to 30 minutes when antigen has been administered by injection. However, there can be a delay of an hour or more. When antigen has been ingested, symptoms usually occur within the first 2 hours but can be delayed for several hours. It should be noted, however, that the onset of symptoms can occur immediately after ingestion, and such rapidly occurring events can be fatal. There is believed to be a direct correlation between the immediacy of onset of symptoms and the severity of a given attack: the more rapid the onset, the more severe the episode. An episode can abate and then exhibit a recrudescence several hours after symptoms have disappeared. This has been termed biphasic anaphylaxis.[ attacks can be protracted, persisting for several Protracted shock and adult respiratory

222 days,[ ]

168]

TABLE 83-6 -- Surgical Anaphylactic and Anaphylactoid Events Clinical Feautures Cutaneous signs

Anaphylactic Reaction (n = 406)

Anaphylactoid Reaction (n = 177)

307 (75.6%)

152 (86%)

Erythema

262

150

Angioedema

27

7

Urticaria

8

2

278 (68.5%)

60 (34%)

Hypotension

74

36

*

Collapse

201

22

*

Cardiovascular signs

22 (2 deaths)

3

Arrhythmias

4

5

170 (62.4%)

44 (25%)

From Laxenaire MC: Ann Fr Anesth Reanim 18:796–809, 1999.

*

*

Cardiac arrest

* p < 0.05 anaphylactoid versus anaphylactic.

In addition,

and can be characterized by multiple recurrences interrupted by asymptomatic periods lasting hours.[

1509

Bronchospasm

167]

208

169

distress syndrome can occur despite appropriate therapy.[ ] The exact incidence of biphasic reactions is unknown. However, in a recent series, [ ] which was a 14year retrospective review of a total of 180 anaphylactic episodes in 106 pediatric patients, a biphasic event occurred in 6%. Three percent of the biphasic reactions were considered significant, with symptoms beginning from 1.3 to 28.4 hours after resolution of the initial episode. There was also a 1% rate of protracted anaphylactic reactions and a relatively high rate of fatal reactions (2%) in this series. Biphasic reactions occurred more frequently when the initial administration of epinephrine was delayed. Also of importance is the fact that the biphasic reactions did not seem to be suppressed by the administration of corticosteroid therapy. The authors suggested that a 24-hour period of observation, after resolution of the initial anaphylactic episode, would be beneficial for approximately 2% of children experiencing anaphylaxis. In each case the biphasic reaction involved the same organ system as the initial reaction, and in one case the biphasic reaction was more severe than the initial reaction.[

169]

Death can occur at any time during the course of protracted anaphylaxis. 223 224 225 226 227 228 229 230 231 232 233

The cardiac manifestations of anaphylaxis can be varied and profound. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] Characteristically, anaphylaxis is associated with a compensatory tachycardia that occurs in response to a decreased effective vascular volume. This has often been used as a sign to differentiate an 226 227]

anaphylactic episode from a vasodepressor reaction. However, bradycardia, presumably caused by increased vagal reactivity, can also occur in anaphylaxis.[ ] [ This is probably due to the Bezold-Jarisch reflex, a cardioinhibitory reflex that has its origin in sensory receptors in the inferoposterior wall of the left ventricle. Unmyelinated vagal C fibers transmit the reflex, which is activated by ischemia. Myocardial depression with decreased cardiac output as a result of contractile depression can occur and can persist for several days. This is thought to be due to hypoxemia.[

45] [222]

222] [223]

Coronary artery vasospasm has been documented with coronary angiography.[

Vasospasm can result in myocardial infarction.[

Electrocardiographic abnormalities include S-T segment elevation, flattening of T waves, inversion of T waves, and arrhythmias,[ from heart

233 block.[ ]

231] [234]

223]

including those resulting

Cardiac enzyme elevations also occur.

Arterial blood gas abnormalities usually consist of a fall in Po2 and Pco2 early in the course. If severe respiratory difficulty supervenes, the hypoxia worsens and an elevation of Pco2 may occur, along with a fall in pH that is probably due to a combination of carbon dioxide retention and metabolic acidosis.[

210]

It should be remembered that anaphylaxis can present with unusual manifestations that make diagnosis difficult. Syncope without other manifestations can occur and has been reported in episodes resulting from fire ant sting[ simply with spontaneous loss of diagnosis of anaphylaxis is

235 consciousness.[ ]

234]

and from mastocytosis.[

235]

Individuals experiencing syncope alone can present with a seizure[

234]

or

This form of presentation often results in unnecessary cardiovascular and neurologic evaluation before the

236 established.[ ] 237]

In toddlers and infants who present with anaphylactic episodes, the major manifestation may mimic foreign body aspiration.[

Rarely, anaphylaxis can cause adrenal hemorrhage. In such instances, hypotension is prolonged. It is important to consider this complication in patients in whom all

manifestations excluding hypotension resolve.[

238]

DIFFERENTIAL DIAGNOSIS The differential diagnosis of anaphylaxis and anaphylactoid events is presented in Box 83-6 . This classification includes conditions that should be considered by the physician who sees the patient during the acute event, as well as those conditions that should be considered when the patient is seen after the episode for the purpose of determining the cause of the event. Perhaps the most common condition mimicking anaphylaxis is the vasodepressor reaction. The mechanisms underlying the vasodepressor response have not been definitively clarified, but such reactions may be due to the activation of the Bezold-Jarisch reflex. This reflex is thought to be initiated by excessive venous pooling, with a resulting decrease in ventricular volume and an increase in ventricular inotropy. These events activate sensory receptors that respond to wall tension in the inferoposterior portions of the left ventricle, paradoxically increasing neural response through the vagus nerve. The result is vasodilation, bradycardia, hypotension, 239

and loss of consciousness.[ ] Characteristic features of vasodepressor reactions are hypotension, pallor, weakness, nausea, vomiting, and diaphoresis. In severe reactions, there is loss of consciousness. These reactions often are the result of a threatening event or emotional trauma. The characteristic bradycardia can be used as a differential diagnostic factor to distinguish these episodes from anaphylaxis. However, as previously noted, the Bezold-Jarisch reflex can be elicited in anaphylactic 226 227 240

events characterized by hypotension, and therefore bradycardia can also be a feature of anaphylaxis.[ ] [ ] [ ] Another important distinguishing factor is the fact that vasodepressor reactions are not accompanied by cutaneous manifestations such as urticaria, angioedema, or flush. Entities that produce flush should also be considered in the differential diagnosis. Flushing is a common phenomenon and can result from a variety of causes. Various 241]

agents can induce flush, including niacin, nicotine, catecholamines, and angiotensin-converting enzyme (ACE) inhibitors.[

1510

Flush is

Box 83-6. Differential Diagnosis of Anaphylaxis and Anaphylactoid Reactions Anaphylaxis and anaphylactoid reactions Anaphylaxis and anaphylactoid reactions to exogenously administered agents Physical factors Exercise Cold, heat, sunlight Idiopathic Vasodepressor reactions Flush syndromes Carcinoid Postmenopausal Chlorpropamide—alcohol Medullary carcinoma thyroid Autonomic epilepsy “Restaurant syndromes” Monosodium glutamate (MSG) Sulfites Scrombroidosis Other forms of shock Hemorrhagic

Cardiogenic Endotoxic Excess endogenous production of histamine syndromes Systemic mastocytosis Urticaria pigmentosa Basophilic leukemia Acute promyelocytic leukemia (tretinoin treatment) Hydatid cyst Nonorganic disease Panic attacks Munchausen's stridor Vocal cord dysfunction syndrome Globus hystericus Undifferentiated somatoform anaphylaxis Miscellaneous Hereditary angioedema “Progesterone” anaphylaxis Urticarial vasculitis Pheochromocytoma Hyperimmunoglobulin E, urticaria syndrome Neurologic (seizure, stroke) Pseudoanaphylaxis

Red man syndrome (vancomycin) Capillary leak syndrome

also seen in association with carcinoid syndrome, pancreatic tumors, medullary carcinoma of the thyroid, hypoglycemia, rosacea, pheochromocytoma, alcohol administration (with and without associated drugs), postmenopausal status, autonomic epilepsy, panic attacks, and systemic mastocytosis.[

242] [243]

Flush occurs in a “wet” and a “dry” form. In the wet form there is associated sweating. This form is mediated by sympathetic cholinergic nerves that supply sweat glands in the skin. This type of flush is characteristic in postmenopausal women. It also occurs after the ingestion of spicy foods containing capsaicin. Direct vasodilatation without stimulation of the sweat glands produces a “dry flush,” as is seen in the carcinoid syndrome. It is not surprising that carcinoid syndrome produces symptoms similar to anaphylaxis, because carcinoid tumors secrete histamine, kallikrein, neuropeptides, and 243

prostaglandins in addition to 5-hydroxytryptamine (serotonin).[ ] A similar mechanism underlies the flush caused by pancreatic tumors and by medullary carcinoma of the thyroid. These tumors also can secrete prostaglandins, histamine, substance P, and 5-hydroxytryptamine. Patients with medullary carcinoma of the thyroid have telangiectasia, a positive family history for the disease, and mucosal neuromas. Postmenopausal flush lasts approximately 3 to 5 minutes, can occur several times a day, is associated with sweating, and may occur intermittently. Episodes usually occur over a period of 5 months to 5 years. Postmenopausal flush is often aggravated by alcohol and stress. There are no respiratory tract symptoms, and there is no fall in blood pressure. Autonomic epilepsy is a rare condition thought to be caused by paroxysmal autonomic discharges. The blood pressure can fall or rise, and tachycardia, flush, and syncope can occur. In alcohol-induced flush, a nonelevated, intense erythema, mainly over the face and trunk, occurs within minutes after the subject drinks even small amounts of alcohol. Symptoms usually peak 30 to 40 minutes after ingestion and subside within 2 hours. There are two forms of alcohol-induced flushing. In one form the event is associated with the intake of drugs, and in the other alcohol alone is sufficient stimulus to produce the episode. The latter form is extremely common in Asians 241

241

(incidence, 47% to 85%)[ ] but also occurs in non-Asians (3% to 29%).[ ] Symptoms are worse in the form that is independent of drug ingestion. Both types can be associated with nausea, anxiety, lightheadedness, headache, and vomiting. In the drug-independent form, wheezing and conjunctivitis can be seen as well. A number of drugs have been incriminated in alcohol-induced flushing, the best recognized being the sulfonylurea hypoglycemic agent, chlorpropamide. Chlorpropamide is only rarely used today, and therefore the incidence of flush reaction to this agent has markedly declined. However, other drugs can also produce flushing, including disulfiram, tolbutamide, griseofulvin, nitromidazoles, and cephalosporins. Alcohol-induced flush has also been described in patients with Hodgkin's disease, other malignancies, hypereosinophilic syndrome, or mastocytosis and in patients who have undergone splenectomy. [

241]

In the drug-independent form of flushing, at least in the form seen in Asians, the reaction may be caused by an abnormal metabolism of alcohol. This reaction occurs in individuals with homozygous or heterozygous null alleles for the mitochondrial enzyme aldehyde dehydrogenase-2. In the absence of activity of this enzyme, aldehyde accumulates after alcohol ingestion. In patients with this disorder, the increased levels of aldehyde have been shown to cause mast cell degranulation. [

241]

In many instances, the cause of flushing cannot be determined and the patient is said to have an idiopathic flush reaction. This occurs more frequently in women than in men. It can be associated with palpitations, diarrhea, syncope, and hypotension. There is no wheezing or abdominal pain.[

243]

1511

A group of postprandial syndromes resembling anaphylaxis have been attributed to the ingestion of monosodium glutamate (MSG), sulfites, or histamine. [ are referred to as “restaurant syndromes.”

244]

These

Ingestion of MSG can produce chest pain, facial burning, flushing, paresthesias, sweating, dizziness, headaches, palpitations, nausea, and vomiting. Children can experience shivering and chills, irritability, screaming, and delirium. The occurrence of these symptoms has been referred to as the “Chinese restaurant syndrome.” The mechanism of production of this condition is unknown, but it is believed that MSG causes a “transient acetylcholinosis.” About 15% to 20% of the general population appears to be sensitive to small doses of MSG, but reactions can occur in any individual should the dose be large enough. Symptoms usually begin no later than 1 hour after ingestion, but they can be delayed in onset up to 14 hours. There may be a familial tendency to develop these reactions.[ 245]

Histamine poisoning, caused by the ingestion of histamine contained in spoiled fish, appears to be increasing in frequency.[

244]

Histamine is the major chemical

246

involved in the production of symptoms, but all symptoms are not caused by the uncomplicated ingestion of histamine alone.[ ] The ingestion of histaminecontaminated spoiled fish is more toxic than the ingestion of equal amounts of pure histamine administered by mouth, for reasons that are unclear. However, cisurocanic acid, an imidazole compound similar to histamine that is derived from histidine in spoiled fish, could account for this phenomenon. cis-Urocanic acid can 247

degranulate mast cells, thus perhaps augmenting the response to endogenous histamine.[ ] Histamine itself is produced by histidine-decarboxylating bacteria that cleave histamine from histidine in the spoiled fish. It is important to note that the fish are not contaminated when caught. The increase in histamine content occurs after death—on board the fishing vessel, at the processing plant, in the distribution system, or in the restaurant or home. Fish with elevated histamine levels can look and smell normal. Cooking does not destroy the histamine, nor does it alter the activity of cis-urocanic acid. 247

Scombroidosis is the most prevalent form of seafood-borne disease in the United States.[ ] It is probably underreported, because most episodes are mild. In addition, many episodes are reported as allergic reactions. It is most commonly produced by scombroid fish belonging to the family Scombroidae (e.g., tuna, mackerel) or Scomberesocidae (e.g., saury), but nonscombroid species, such as mahi-mahi, anchovies, and herring, can also cause the problem. The production of histamine and cis-urocanic acid is increased when fish are stored at elevated temperatures. These agents are formed by bacteria such as Morganella morganii, Klebsiella pneumoniae, and Hafniae alvei. The optimal temperature for amine production is about 30° C. However, once the bacterial

population is enlarged, ongoing histamine production can occur even at refrigerated temperatures ranging from 0° to 5° C. As expected, the features of scombroidosis are very similar to those of anaphylaxis and can include cardiovascular, gastrointestinal, cutaneous, and neurologic manifestations. Episodes can occur in outbreaks, and the morbidity can be as high as 100%. However, there appear to be very large variations in individual susceptibility. Episodes can occur from a few minutes to several hours after the ingestion of fish. The symptoms usually last for a few hours but on occasion can persist for several days. The typical signs and symptoms are similar to those of histamine toxicity and include urticaria, flush, angioedema, nausea, vomiting, diarrhea, and hypotension. Neurologic findings are also common, and occasionally wheezing can occur. The most common symptom overall is flushing of the face and neck, which is accompanied by a sensation of heat and discomfort. The rash most frequently takes the appearance of a sunburn rather than urticaria. These cutaneous manifestations are the most common symptoms, with gastrointestinal symptoms being second in frequency. Serious complications can occur in patients with preexisting cardiovascular or respiratory tract disease. Although histamine fish poisoning resembles food-induced anaphylaxis, there are several distinguishing features. First, many people dining at the same table can be affected (i.e., everyone who ingests significant quantities of the fish). Second, the cutaneous symptoms, as noted, are usually somewhat different, consisting of a prolonged flush with the absence of urticaria. Third, the absence of elevated concentrations of tryptase in the serum, in the presence of elevated plasma histamine, suggests this disorder. Patients taking isoniazid appear to have enhanced susceptibility to episodes of scombroidosis.[

244]

There are several syndromes characterized by excessive endogenous production of histamine. These include systemic mastocytosis and leukemias in which there is an overproduction of histamine-containing cells (acute promyelocytic leukemia, basophilic leukemia). Anaphylaxis can occur in such patients after relevant stimuli. For example, episodes can be precipitated in patients with systemic mastocytosis on ingestion of opiates and in patients with promyelocytic leukemia on treatment with tretinoin.[

248]

Nonorganic diseases also can mimic anaphylaxis. Such episodes can be involuntary, such as in panic attacks, undifferentiated somatoform anaphylaxis, and the vocal 249 250

251

cord dysfunction syndrome,[ ] [ ] or they can be consciously self-induced, as in Munchausen's stridor.[ ] Panic attacks are accompanied by tachycardia, flushing, gastrointestinal symptoms, and shortness of breath. Vocal cord dysfunction syndrome and Munchausen's stridor have similar presentations. The former is caused by an involuntary adduction of the vocal cords that occludes the glottal opening. There is a bunching together of the false vocal cords, which produces obstruction in both inspiration and expiration. The patient is unaware of the process. The term Munchausen's stridor was coined to describe patients who intentionally adduct their vocal cords and present to emergency rooms with self-induced manifestations of laryngeal edema. This entity occurs in psychologically disturbed individuals.[

251]

It can be distinguished from vocal cord dysfunction syndrome by laryngoscopy during the acute episode and by the fact that patients with

Munchausen's stridor can be distracted from their vocal cord adduction by asking them to perform maneuvers such as coughing.[

251]

Undifferentiated somatoform anaphylaxis is a term used to describe patients who present with manifestations that mimic idiopathic anaphylaxis but who lack objective confirmatory findings, do not respond to therapy, and exhibit psychological signs of an undifferentiated somatoform disorder.[

249]

Other entities traditionally listed in the differential diagnosis of anaphylaxis include hereditary angioedema, progesterone anaphylaxis, anaphylaxis associated with recurrent and chronic urticaria, pheochromocytoma, neurologic disorders,

1512

tracheal foreign body, the pseudoanaphylactic syndrome that occurs after the administration of procaine penicillin, and the “red man syndrome,” which can occur after the administration of vancomycin. Hereditary angioedema, which can cause laryngeal edema, abdominal pain, and an erythematous rash that may be confused with urticaria, should be included in the differential diagnosis. However, in most instances, there is no difficulty making the distinction between these two entities. 296 297

Some patients previously thought to have idiopathic anaphylaxis have been found to have progesterone-related anaphylactic episodes.[ ] [ ] Patients with this disorder experience anaphylactoid reactions to the infusion of luteinizing hormone-releasing hormone (LHRH) and after the intradermal injection of medroxyprogesterone. LHRH analog therapy is beneficial. The mechanism of production of this disorder is unknown, but it has been postulated that the increased level of progesterone associated with menses predisposes subjects to their anaphylactic events. This entity should be considered in women, usually older than 35 years of age, who present with recurrent episodes of anaphylaxis that exhibit a temporal relationship with the menstrual cycle.[ demonstrate an IgE anti-progesterone antibody, but others do

298]

Some women with this disorder

252 not.[ ]

The vast majority of patients with chronic idiopathic urticaria or recurrent episodes of urticaria are not considered to be at risk for the development of anaphylaxis or anaphylactoid episodes. However, in rare instances, anaphylaxis can occur in patients who previously had only recurrent episodes of acute urticaria [

148] [253]

254

or 253

chronic urticaria.[ ] A subject with recurrent episodes of acute urticaria that culminated in an episode of anaphylaxis also had hyperimmunoglobulinemia-E.[ ] A subject with chronic episodes of urticaria had an urticarial vasculitis that was associated with an episode of shock, leukopenia, and thrombocytopenia; in this instance 254

the reaction was thought to be caused by activation of the complement cascade.[ ] Also, as noted previously, in the subjects described by Dykewicz[ idiopathic anaphylaxis associated with a positive autologous serum intradermal test, the anaphylactic episodes were preceded by urticaria.

148]

who had

Occasionally, patients with pheochromocytoma can have symptoms suggestive of an anaphylactic reaction. Pseudoanaphylaxis is a term used to describe patients who develop syncope and neurologic symptoms after the administration of procaine penicillin. These reactions are caused by the procaine and not the penicillin. [

255]

Similar episodes have been reported after the administration of lidocaine.[

255]

Capillary leak syndrome can present as anaphylaxis. This is a rare, often fatal disorder characterized by angioedema, gastrointestinal symptoms, and shock with hemoconcentration. It is usually associated with a monoclonal gammopathy. Recurrent episodes can mimic idiopathic anaphylaxis.[

256]

THE LABORATORY IN DIFFERENTIAL DIAGNOSIS The laboratory might be helpful in some instances to make a diagnosis of anaphylaxis or to rule out other causes. If carcinoid syndrome or pheochromocytoma is

being considered, blood levels of serotonin and urinary 5-hydroxyindole acetic acid, catecholamines, and vanillylmandelic acid should be measured. Schwartz and Irani, and Lin et al conducted a series of studies that elucidated the relative roles of tryptase and histamine in establishing the diagnosis of anaphylaxis 142 257

142

and in aiding the differential diagnosis.[ ] [ ] They demonstrated that human mast cell tryptase exists in two forms, alpha-tryptase and beta-tryptase.[ ] Alphatryptase is secreted constitutively, and beta-tryptase is released only during degranulation episodes. This observation is useful in distinguishing between systemic anaphylaxis and mastocytosis, as discussed in the following paragraphs. If a patient is seen shortly after the onset of an anaphylactic episode, plasma and urinary histamine (or histamine metabolites) and serum tryptase determinations may be helpful. Plasma histamine levels begin to rise within 5 to 10 minutes and remain elevated for 30 to 60 minutes.[ patient is seen as long as an hour or more after onset of the might be

260 useful.[ ]

258 event.[ ]

258] [259]

Therefore, they are of little help if the

However, urinary histamine and its metabolites are elevated for a longer period and therefore

Serum tryptase levels peak 60 to 90 minutes after the onset of anaphylaxis and persist longer than plasma histamine levels do. Elevated tryptase

concentrations may be found as long as 5 hours after the onset of symptoms[

259]

and therefore may be useful in establishing the diagnosis.[

measure serum tryptase is between 1 and 2 hours, but no longer than 6 hours, after the onset of 10 minutes and 1 hour after the onset of

259 symptoms.[ ]

258] [261]

The best time to

The best time to measure plasma histamine is between

259 symptoms.[ ] 259

There may be a disparity between histamine and tryptase levels, with some patients exhibiting elevations of only one of these mediators.[ ] In one such instance, a patient exhibited a hypotensive reaction to thiopental in which plasma histamine was elevated 10 minutes after the onset of hypotension without concomitant elevations in beta-tryptase at 45 minutes.[

262]

The authors interpreted this as indicating the activation of basophils without mast cell degranulation, because basophils

contain histamine but only negligible amounts of tryptase.[

262]

In one recent study this disparity occurred more commonly than expected: plasma histamine levels 257

were elevated with significantly greater frequency than serum tryptase levels in a group of patients entering the emergency department with allergic reactions.[ ] Elevated concentrations of plasma histamine were observed in 42 of 97 adult emergency department patients, whereas only 20 patients demonstrated elevations of tryptase. [

257]

Histamine levels correlated better with clinical signs than tryptase levels did. Histamine levels correlated with urticaria, more extensive erythema,

abnormal abdominal findings, and wheezing. Total tryptase levels were increased more frequently only in those patients with urticaria.[

257]

Schwartz and Irani proposed a schema to distinguish patients with systemic mastocytosis from those who experience anaphylaxis in the absence of systemic 142

mastocytosis.[ ] Their schema rests on the observation that subjects with mastocytosis, because of their increased mast cell burden, constitutively produce large amounts of alpha-tryptase (compared with normal individuals), whereas patients who have anaphylactic episodes resulting from other causes have normal baseline levels of alpha-tryptase. During anaphylactic events, beta-tryptase is secreted in large amounts in both groups. Therefore the ratio of total tryptase (alpha

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plus beta) to beta-tryptase levels can be useful to distinguish episodes of anaphylaxis in patients with mastocytosis from those that occur in patients without this

142

disorder. In addition, constitutively elevated levels of alpha-tryptase are useful in establishing the diagnosis of mastocytosis.[ ] A ratio of total tryptase (alpha plus beta) to beta-tryptase of 10•ng/ml or less is indicative of an anaphylactic episode not related to systemic mastocytosis, whereas a ratio of 20 or greater is consistent 142]

with systemic mastocytosis.[

This distinction is made possible because of the fact that the immunoassay for tryptase using a B12 monoclonal antibody or a G4 142]

monoclonal antibody recognizes both alpha- and beta-tryptase, whereas an assay using a G5 monoclonal antibody recognizes only the beta-tryptase molecule.[

It might also be beneficial to obtain serum for the analysis of specific IgE against suspected antigens. In search of the culprit in patients with possible anaphylaxis to food, leftover or vomited food may be a useful source of antigen for the creation of a “custom” RAST reagent.[

137]

25

Once the diagnosis of anaphylaxis has been established, it should be recognized that the specific agent may not be identifiable in the majority of instances.[ ] The search for such an agent should include, when appropriate, tests for foods. Such tests have been reported to determine the offending agent in some cases previously designated idiopathic.[

263]

Tests should include not only foods but also spices and vegetable gum products.[

264]

Of course, a detailed and intense history is the most

important “test.” This history should be repeated after each episode. Occasionally a serial history reveals a previously undetermined cause.[

265]

PREVENTION AND MANAGEMENT Prevention Anaphylactic reactions are an unavoidable aspect of the practice of medicine. However, the incidence and severity of such reactions can be decreased by preventive measures ( Box 83-7 ).

Box 83-7. Measures to Reduce the Incidence of Anaphylaxis and Anaphylactic Deaths

General Obtain thorough history for drug allergy. Avoid drugs that have immunologic or biochemical cross-reactivity with any agents to which the patient is sensitive. Administer drugs orally rather than parenterally when possible.

Check all drugs for proper labeling. Keep patients in the office 20 to 30 minutes after injections.

For Patients at Risk Have patient wear and carry warning identification tags. Teach self-injection of epinephrine and caution patients to keep an epinephrine kit with them. Discontinue beta-adrenergic blocking agents, angiotensin-converting enzyme (ACE) inhibitors, ACE blockers, monoamine oxidase inhibitors, and certain tricyclic antidepressants when possible. Use preventive techniques when patients are required to undergo a procedure or take an agent that places them at risk. Such techniques include pretreatment, provocative challenge, and desensitization.

A thorough history for drug allergy should be taken for every patient. Proper interpretation of this history requires a knowledge of the immunologic and biochemical cross-reactivities among drugs. When a history of an allergic reaction to a drug is present, a substitute, non–cross-reactive drug should be administered whenever possible. Parenteral administration of medication usually produces more severe reactions than oral administration does. Therefore, drugs should be administered orally whenever possible. If parenteral administration is required, the patient should remain in the office for 20 to 30 minutes after the drug is given. Instances of anaphylaxis resulting from drug mislabeling are rare but do occur. Therefore, proper labeling of drugs is essential, and whenever a drug is suspected as the cause of an episode, the contents of the container should be checked against the label. Patients subject to anaphylaxis should wear a MedicAlert bracelet or necklace and should keep an identification card in their wallet or purse. Patients at risk for anaphylaxis should be supplied with kits for the self-injection of epinephrine and should be told to keep the kit with them at all times. Beta blockers, ACE inhibitors, angiotensin II receptor blockers, monoamine oxidase inhibitors, and some tricyclic antidepressants should not be taken by patients who are at risk for anaphylaxis or anaphylactoid episodes if other agents will suffice. These drugs decrease the effectiveness of epinephrine (beta blockers), interfere with endogenous compensatory hypotensive responses (ACE inhibitors and angiotensin II blockers),[ administration (monoamine oxidase inhibitors and some tricyclics).

266]

or affect the use of epinephrine by making the patient subject to side effects on its

If patients are required to take medications or diagnostic agents or to undergo procedures that are known to place them at risk for an anaphylactic episode, specific preventive measures, such as pretreatment, provocative challenges, or desensitization, should be instituted when appropriate. In specific instances, published preventive regimens are helpful.

Management of the Acute Event This discussion of the treatment of anaphylaxis and anaphylactoid episodes emphasizes office management. However, the treatment of prolonged attacks requiring hospitalization is also mentioned. The drugs used to treat anaphylaxis and their suggested dosage regimens are listed in Table 83-7 . Therapy may be divided into those procedures that should be performed immediately and those that may be initiated after further evaluation. These steps are listed in Box 83-8 . Rapid recognition with immediate treatment is essential. It is believed that prompt initiation of therapy prevents fatalities. It is important to stress that the steps seen in Box 83-8 are subject to the discretion of the physician managing the care of the patient, and variations in their sequence and performance depend on clinical judgment. In addition, the determination of when a patient should be transferred to an intensive care center is also dependent on the skill, experience, and assessment of the individual physician. Obviously, to initiate therapy, the medications and apparatus must be available. A number of different position statements have been issued regarding the equipment that should be available in the office for the management of anaphylaxis.

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TABLE 83-7 -- Drugs and Other Agents Used to Treat Anaphylaxis and Anaphylactoid Reactions Drug

Dose and Route of Administration

Comment

Epinephrine

1:1000 0.3–0.5•ml IM (adult); 1:1000 0.01•mg/kg or 0.1–0.3•ml IM (child)

Initial drug of choice for all episodes; should be given immediately; may repeat q 10–15•min

0.1•ml (0.1•mg) of 1:1000 aqueous epinephrine diluted in 10•ml normal saline IV (see text for details)

If no response to IM administration and patient in shock with cardiovascular collapse

25–50•mg IM or IV (adult); 12.5–25•mg PO, IM, or IV (child)

Route of administration depends on severity of episode

Antihistamines ••Diphenhydramine

••Ranitidine, cimetidine

4•mg/kg IV cimetidine 1•mg/kg IV ranitidine

Cimetidine should be administered slowly, because rapid administration has been associated with hypotension; doses shown are for adults; doses in children are less well established

100•mg-1•g IV or IM (adult), 10–100•mg IV (child)

Exact dose not established; other preparations such as methylprednisolone can be used as well; for milder episodes, prednisone 30–60•mg may be given (see text)

••Aerosolized beta-agonist (albuterol, metaproterenol)

Dose as for asthma (0.25–0.5•ml in 1.5–2•ml saline q4h, prn)

Useful for bronchospasm not responding to epinephrine

••Aminophylline

Dose as for asthma

Rarely indicated for recalcitrant bronchospasm; beta-agonist is drug of choice

Volume expanders Crystalloids (normal saline or Ringer's lactate)

1000–2000•ml rapidly in adults; 30•ml/kg in first hour in children

Rate of administration titrated against blood pressure response for IV volume expander; after initial infusion, further administration requires tertiary care monitoring; in patients who are beta-blocked, larger amounts may be needed

••Colloids (hydroxyethyl starch)

500•ml rapidly followed by slow infusion in adults

Vasopressors Dopamine

400•mg in 500•ml; dextrose 5% in water as IV infusion; 2–20•• g/kg/min

Corticosteroids ••Hydrocortisone

Drugs for resistant bronchospasm

Dopamine is probably the drug of choice; the rate of infusion should be titered against the blood pressure response; continued infusion requires intensive care monitoring

Drugs used in patients who are beta-blocked ••Atropine sulfate

0.3–0.5•mg IV; may repeat every 10 minutes to a maximum of 2•mg in adults

Glucagon is probably the drug of choice, with atropine useful only to treat bradycardia

••Glucagon

Initial dose of 1–5•mg IV followed by infusion of 5– 15••g/minute titrated against blood pressure

Ipratropium might be considered as an alternative or added to inhaled beta-adrenergics for wheezing

••Ipratropium IM, Intramuscular; IV, intravenous; PO, by mouth; q4h, every 4 hours; q10–15•min, every 10–15 minutes. 267

These include statements by the American Academy of Pediatrics Committee on Drugs [ ] ; the American Academy of Allergy, Asthma, and Immunology[ Joint Task Force on Practice Parameters of the American Academy of Allergy, Asthma, and Immunology, the American College of Allergy, Asthma, and Immunology, and the Joint Council[

269]

270]

; the World Health Organization[

268]

; the

; and the American Academy of Allergy, Asthma, and Immunology Board of Directors

271

(position statement).[ ] Box 83-9 is a summation of these lists with revisions. For the sake of completeness, more equipment has been listed in Box 83-9 than was suggested in recent position statements. Some of this equipment is not considered mandatory by the professional organizations that have issued parameters and guidelines of care. The initial step in the management of anaphylaxis is a rapid assessment of the patient's status, with emphasis on evaluation of the airway and the state of consciousness. If the airway is compromised, it should be secured immediately.

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Box 83-8. Therapy for Anaphylaxis Immediate action Assessment Check airway and secure if needed Rapid assessment of level of consciousness Vital signs Treatment Epinephrine Supine position, legs elevated Oxygen Tourniquet proximal to injection site Dependent on evaluation Start peripheral intravenous fluids H1 and H2 antagonists

Vasopressors Corticosteroids Aminophylline Glucagon Atropine Electrocardiographic monitoring Transfer to hospital Hospital management Medical antishock trousers Continued therapy with above-noted agents and management of complications

Blood pressure and pulse measurements should be obtained. The patient should be placed in the supine position with the feet elevated. However, modification of this Trendelenburg position may be necessary if the patient is wheezing. In addition, the increase in intrathoracic pressure produced by this position can reduce the 272

pressure gradient between the right atrium and the inferior vena cava, thus limiting the benefit of the increased venous return.[ ] If the antigen has been injected, a tourniquet may be placed proximal to the injection site; however, the efficacy of this procedure has not been established. Care should be taken to release the tourniquet every 5 minutes (for a minimum of 3 minutes) during therapy, and it should be left in place no longer than a total of 30 minutes. Oxygen should be started. During this time, an estimate of the patient's weight should be made to help guide dosage decisions. Epinephrine is the mainstay of therapy. Physicians not only use epinephrine in their office but also prescribe epinephrine for treatment outside the office in patients who are at risk for anaphylactic episodes (e.g., patients who are allergic to insect stings, patients with food allergy, patients with idiopathic anaphylaxis). Over the 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291

past several years, a number of studies[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] have uncovered a number of problems regarding the use of epinephrine by outpatients outside the office. These include the fact that many physicians, especially those in hospital emergency departments, fail to give patients who have experienced anaphylactic episodes a prescription for epinephrine on discharge.[ the proper use of an automatic epinephrine injector and

284]

Often physicians are not familiar with

Box 83-9. Equipment and Medication Needed for Treatment of Anaphylaxis in the Office

Primary Tourniquet 1- and 5-ml disposable syringes Oxygen tank and mask/nasal prongs Epinephrine solution (aqueous) 1:1000 (1-ml ampules and multidose vials) Epinephrine solution (aqueous) 1:10,000 (commercially available preloaded in a syringe) Diphenhydramine injectable Ranitidine or cimetidine injectable Injectable corticosteroids Ambu-bag, oral airway, laryngoscope, endotracheal tube, No. 12 needle Intravenous setup with large bore catheter IV fluids: 2000•ml crystalloid, 1000•ml hydroxyethyl starch Aerosol beta 2 bronchodilator and compressor nebulizer Glucagon Electrocardiogram Normal saline 10-ml vial for epinephrine dilution

Supporting

Suction apparatus Dopamine Sodium bicarbonate Aminophylline Atropine IV “setup” with needles, tape, and tubing Non-latex gloves

Optional Defibrillator Calcium gluconate Neuroleptics for seizures Lidocaine

therefore fail to instruct patients correctly.[ it in a timely drug in

285 fashion.[ ]

279]

Patients fail to carry their epinephrine automatic injector with them, and when they do have it, they fail to administer

The lack of availability of intermediate dosage ranges in automatic epinephrine injectors can result in the underdosing or overdosing of this

18 273 283 children.[ ] [ ] [ ]

In some instances, inhaled epinephrine is being used,[

290]

despite the fact that the number of inhalations required to achieve an 291

adequate dose and the taste of the preparation make it an impractical alternative to intramuscular administration.[ ] In sum, there is ample evidence that physicians underprescribe and do not adequately teach the proper technique for administration of epinephrine; and patients do not carry their epinephrine kits, fail to use them properly, and let them expire. In addition, because of the limited dosage range, children are at risk for both undertreatment and overtreatment. These problems mandate careful attention to the education of at-risk patients regarding the use of epinephrine, with

1516

emphasis on the necessity to keep epinephrine with them at all times and to administer it promptly and appropriately. Such efforts can bear positive results, based on data obtained from a continuing series of patients at risk for anaphylaxis spanning approximately two decades. The first report of these patients, in 1995, revealed that 47% of patients did not carry their EpiPen with them.[

25]

This finding initiated a more intense effort to educate patients regarding the use of epinephrine, and the

second report, appearing in 2001, revealed that 33% of patients failed to keep their EpiPen kits with them.[

28]

The second significant observation regarding epinephrine administration is that intramuscular administration is superior to subcutaneous administration. In previous recommendations regarding the administration of epinephrine, it was assumed that both routes were equally effective.[ [294]

experiments, initially conducted in an animal model and later in both children and adults, Simons et epinephrine produces significantly faster peak plasma levels than subcutaneous injection does.

295 296 al[ ] [ ]

292] [293]

In a series of elegantly designed

showed that intramuscular injection of

Also of note, based on the same studies, is the fact that the lateral aspect of the thigh (vastus lateralis muscle) is the preferred site of injection (rather than the deltoid 295 296

muscle): Epinephrine concentrations were significantly higher when administered in this area, whether by EpiPen or by syringe.[ ] [ ] This observation may be more important than originally realized, because epinephrine can have contradictory effects, with low plasma and tissue concentrations associated with the undesirable enhancement of the release of mediators and the paradoxic effect of vasodilatation and possibly hypotension.[

297]

286

Another clinically salient observation by Simons, Gu, and Simons [ ] relates to the use of outdated EpiPens. As stated previously, it is well known that patients fail to refill their EpiPen prescriptions in a timely fashion. Therefore a question arises as to whether there is benefit in the administration of an outdated EpiPen, and to what extent this may be helpful. Simons et al evaluated outdated EpiPens and found that the autoinjectors were still intact and functioning, although the plastic sheaths did show signs of wear. In most instances, the epinephrine was not discolored and no precipitates were visible. Nonetheless, the bioavailability of the drug was significantly reduced. The loss in bioavailability, as expected, correlated with the duration of time that had passed since the expiration date. However, regardless of these findings, the injectors still retained some degree of potency. There, the authors recommended that, as long as the epinephrine is not discolored and does not 286]

contain precipitants, it should be administered.[

Epinephrine should be administered simultaneously while the patient is being assessed. The dose and route of administration of epinephrine depend on the severity of the reaction. In almost all instances of office management, the intramuscular route can be used. The dose in adults is 0.3•ml to 0.5•ml of a 1:1000 solution (0.3 to 0.5• mg), and in children it is 0.01•mg/kg. The initial dose can be repeated two or three times as needed, at 10- to 15-minute intervals. If severe hypotension is present, epinephrine can be given intravenously. There is no established dose,[

298]

and numerous regimens have been suggested.[

272] [298]

[299] [300] [301]

Regardless of the dose and regimen used, care should be taken and the patient should be monitored for arrhythmias. The amount administered depends on the severity of the episode and should be titrated against the response. A suggested protocol is as follows. The intravenous preparation can be prepared by diluting 0.1•ml (0.1•mg) of a 1:1000 aqueous epinephrine solution in 10•ml of normal saline. This 10-ml dose can be infused over 5 to 10 minutes (resulting in a total dose of 298

100••g at a rate of 10 to 20••g/min) and repeated depending on the response.[ ] The dose can be increased in more critical situations: 1•ml (1.0•mg) of a 1:1000 solution of epinephrine can be diluted in 10•ml normal saline (for a concentration of 0.1•mg/ml) and a dose of 1 to 2•ml (0.1 to 0.2•mg) administered every 5 to 20

299

minutes as indicated.[ ] An alternative to mixing this more concentrated solution would be to keep a commercially available 1:10,000 (0.1•mg/ml) preparation on hand. A 1:10,000 preparation is available in a 10-ml prefilled syringe. A constant intravenous infusion can be started if needed. It should be emphasized that intravenous epinephrine should be given only under dire circumstances and only if cardiovascular monitoring is available. If intravenous access cannot be obtained, sublingual (rather than subcutaneous or intramuscular) injection has been suggested as an alternative route because of the rich vascularity of this area. The same dose used for intramuscular injection should be injected in the posterior third of the sublingual area. If the patient has an endotracheal tube in place, the intravenous dose 302

can be administered via a long catheter through the endotracheal tube into the area near the carina. It is absorbed rapidly and dispersed within 5 to 10 breaths.[ ] If the antigen responsible for the episode was injected, 0.3•ml of 1:1000 aqueous epinephrine (0.1 to 0.3•ml in children) can be injected into the original site to slow antigen absorption. After any of these procedures, further therapy is dictated by the clinical course and findings. Antihistamine therapy can be useful as adjunctive treatment with epinephrine. Although this therapy is not considered life saving, it can at times offer dramatic relief of symptoms, especially itching and urticaria.[

303] [304] [305] [306] [307] [308] [309] [310]

discussed, a combination H1 and H2 antagonist is superior to an H1 antagonist

Based on clinical trials[

308]

and the known effects of histamine as previously

303 304 305 306 307 308 309 310 alone.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ]

For example, in an emergency

department setting, in a study involving 91 adult patients, the combination of diphenhydramine and ranitidine was superior to diphenhydramine and saline for 309

resolution of cutaneous symptoms and tachycardia.[ ] Therefore therapy should be instituted with diphenhydramine and ranitidine or cimetidine in the dosages noted in Table 83-7 . Because the rapid infusion of cimetidine has been associated with falls in blood pressure, this drug should be administered slowly. The route of administration, as with epinephrine, depends on the severity of the episode. The intramuscular or intravenous route can be used for diphenhydramine and the intravenous route for the H2 antagonist. The exact role of corticosteroids in the management of anaphylaxis and anaphylactoid reactions has not been established. However, based on an extrapolation of their effect in other allergic diseases, their administration is indicated. Patients with severe anaphylactic episodes and those patients who have received systemic glucocorticoid therapy within the previous several months should be given corticosteroids. Perhaps the most salient theoretic rationale for the use of corticosteroids relates to their effects on the late phase reaction. Because anaphylaxis can be biphasic, a role for steroids in

1517

preventing such a recurrence can be postulated. There is no established dose or drug of choice. Suggested intravenous doses for severe episodes are shown in Table 83-7 . In addition, it is probably wise to give oral prednisone (30 to 60•mg) to patients who have experienced milder anaphylactic symptoms and been discharged from therapy. If wheezing unresponsive to epinephrine occurs, aerosolized beta-adrenergic agents and, if necessary, intravenous aminophylline can be used. The doses are the same as those used in the treatment of asthma. Aerosolized beta-adrenergics are the drug of choice. Aminophylline should be reserved for patients who are taking beta blockers and for those who have recalcitrant bronchospasm not responding to epinephrine and aerosolized beta-adrenergic agents.

Perhaps the most difficult-to-treat manifestation of anaphylaxis and the most threatening, except for acute upper airway obstruction, is profound, protracted hypotension. Hypotension can be severe and resistant to therapy. This was evident in a study of insect sting reactions in which patients were deliberately stung while 169]

in an intensive care unit. Severe, persistent hypotension occurred that was unresponsive to fluid therapy and large doses of intravenous epinephrine.[

As previously discussed, hypotension is caused by a shift of fluid from the intravascular to the extravascular space. The mainstay of treatment should be the restoration of intravascular volume. This is best accomplished by the rapid administration of large volumes of fluid. There is debate as to whether colloids or 311]

crystalloids should be used, and there are arguments to support both forms of therapy.[ the composition of the fluid itself but rather the rate of

312 administration.[ ]

However, the most important component of fluid therapy initially is not

Large volumes of crystalloid are often required; 1000 to 2000•ml of lactated Ringer's or 27]

normal saline solution should be given rapidly—in an adult, depending on the blood pressure, at a rate of 5 to 10•ml/kg in the first 5 minutes.[

Children should

301 hour.[ ]

receive up to 30•ml/kg of crystalloid solution in the first An alternative to crystalloid therapy is the colloid hydroxyethyl starch. Adults should receive rapid infusion of 500•ml, followed by slow infusion thereafter. In patients who have beta-adrenergic blockade resulting from the administration of a beta-adrenergic blocking agent, the volume of fluid required may be much greater (5 to 7•L) before stabilization occurs. If fluid therapy of this magnitude is indicated, the patient should be transferred to an intensive care unit. Further administration of fluid depends on cardiovascular monitoring, including central venous pressure, pulmonary artery wedge pressure, cardiac output, oxygen consumption, urinary output, and electrocardiographic monitoring. Although fluids are clearly the most important element, along with epinephrine, in the treatment of hypotension, the use of other vasopressors may also be indicated. However, their effectiveness can be diminished because hypotension can occur in patients who have increased peripheral resistance resulting from endogenous 192 194

compensatory mechanisms.[ ] [ ] In such patients the hypotension is, as noted, caused by fluid shift and perhaps decreased cardiac output. However, vasopressors used in this situation can be helpful. The drug of choice is dopamine, administered at a rate of 2 to 20••g/kg per minute. The rate should be titrated according to the blood pressure. Any patient requiring dopamine should be transferred to a tertiary care facility. 313]

The patient who has beta-adrenergic blockade presents a special problem in the treatment of anaphylaxis.[ 314 316 regimens.[ ] [ ]

317 hypotension,[ ]

Such patients can be resistant to standard therapeutic 315

They can experience refractory bradycardia, and relapsing manifestations.[ ] In such patients, both inotropic and chronotropic functions of the heart are suppressed, resulting in marked hypotension with bradycardia. Two drugs, atropine and glucagon, have been recommended for therapy in patients with beta-adrenergic blockade. Atropine is useful only for bradycardia; it does not exert a beneficial effect on the inotropic function of the heart. Atropine sulfate can be injected intramuscularly or subcutaneously in a dose of 0.3 to 0.5•mg every 10 minutes, to a maximum of 2•mg. Glucagon, a polypeptide hormone produced by the alpha cells of the pancreas, has both positive inotropic and chronotropic effects on the heart. The inotropic effect does not depend on catecholamines 318

or their receptors and is therefore unaltered by beta-adrenergic blockade.[ ] For this reason, glucagon is probably the drug of choice for patients who are betablocked. The dose of glucagon is 1 to 5•mg intravenously as a bolus, followed by an infusion of 5 to 15••g/min titrated against the clinical response. The cardiotonic 319

effects of glucagon are not associated with increased myocardial irritability.[ ] Cardiotonic effects are seen within 1 to 5 minutes and are maximal at 5 to 15 minutes after a single 5-mg bolus. Nausea and vomiting are the major limiting factors of therapy. If resistant bronchospasm, and not shock, is the problem, then aminophylline, in doses used for the treatment of asthma, becomes the drug of choice. However, aminophylline must be used with caution if hypotension is present. In addition to the medications mentioned, military anti-shock trousers (MAST) provide rapid redistribution of intravascular fluid. Inflation of the MAST suit redirects fluid from the venous circulation of the legs and abdomen to the upper circulation. This redistribution of the intravascular volume can cause an immediate rise in

blood pressure in the upper extremities.[

311]

Because of the possibility of the occurrence of a biphasic episode, the patient should be observed, after the symptoms have subsided, before being discharged. There is no established period of observation, but 2 hours appears reasonable for mild episodes and perhaps as long as 24 hours for severe episodes.[

169]

FATALITIES 25 26 27 28 275

6 24 320

Anaphylactic deaths are relatively rare,[ ] [ ] [ ] [ ] [ ] but many are preventable, and a significant percent are iatrogenic.[ ] [ ] [ ] Therefore it is important to consider which characteristics place a patient at risk for death to learn what might be done to better prevent fatal episodes. Reid et al and Lockey et al pointed out 320] [321]

several features that tend to place a patient at risk, [

and these have been confirmed by other investigators.[

321]

However, signs and symptoms can be

variable, and there is often no indication that death is imminent; neither do the signs and symptoms predict the cause of death. [ be learned from these deaths. 275 320

320]

Nonetheless, several lessons can

322

The presence of asthma is clearly a risk factor.[ ] [ ] In the series by Pumphrey,[ ] the median time from onset to cardiac arrest was faster for injected than for ingested antigens (30 minutes for foods, 15 minutes for venom, 5 minutes for iatrogenic reactions caused by injections). This is in keeping with the

1518

320

data collected by Lockey et al,[ ] which showed that the time before onset of the reaction in 22 of 30 patients who died from anaphylaxis to immunotherapy was 30 minutes or less. However, it is clear from their article that the onset of symptoms after antigen injection can occur later, after 30 minutes, and did so in 10% of this series.[

320]

It is also clear that, although the early use of epinephrine is recognized to prevent death,[

322]

some patients die despite receiving it.[

275]

As previously

322 Pumphrey.[ ]

noted, there is underuse of epinephrine in the treatment of anaphylactic episodes. This was clearly seen in the series reported by Only 20% of those patients receiving epinephrine were given the drug before cardiac arrest occurred. This in part was due to the rapid onset of symptoms, but lack of availability also 277 278

accounted for the failure of timely administration. As in other series,[ ] [ ] it was also noted that epinephrine was underutilized by patients at risk: It was given to 62% of those with fatal reactions, but only 14% received the drug before cardiac arrest occurred. 322

In the series reported by Pumphrey,[ ] different manifestations occurred in patients who died from insect sting reactions, compared with those who died from anaphylaxis to food substances. In venom anaphylaxis, shock was an important component; upper airway compromise also occurred. In anaphylaxis to food, the main difficulty was respiratory, with lower respiratory tract symptoms predominating. This finding may have been related to the fact that patients with anaphylaxis to foods were atopic and therefore may have had asthma as well.[

322]

322

In the series of fatalities reported by Pumphrey,[ ] there was only one biphasic reaction, although 28% of the fatalities had protracted problems and complications. Death occurred from 3 hours to as long as 30 days after the onset of symptoms. Finally, death can occur regardless of the responsible agent. In the initial series of subjects with idiopathic anaphylaxis no fatalities were noted, but more recently it was observed that idiopathic episodes can be fatal.[

323]

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262. Sprung J, Schoenwald PK, Schwartz LB: Cardiovascular collapse resulting from thiopental-induced histamine release, Anesthesiology 86:1006, 1997. 263. Stricker WE, Anorve–Lopez E, Reed CE: Food skin testing in patients with idiopathic anaphylaxis, J Allergy Clin Immunol 77:516, 1986. 264. Yeates HM, Jenson KK: Chronic anaphylaxis caused by ingestion of vegetable gum products, J Allergy Clin Immunol 87:274, 1991 (abstract). 265. Seligman MJ, Witkin S: Not so idiopathic anaphylaxis, J Allergy Clin Immunol 91:155, 1993. Management of the Acute Event 266. Kemp SF, Lieberman P: Inhibitors of angiotensin II: potential hazards for patients at risk for anaphylaxis, Ann Allergy Asthma Immunol 78:527, 1997. 267. The American Academy of Pediatrics Committee on Drugs: Anaphylaxis, Pediatrics 51:136, 1973. 268. American Academy of Allergy and Immunology: Position statement, J Allergy Clin Immunol 77:271, 1986. 269. The diagnosis and management of anaphylaxis (position statement of the Joint Task Force on Practice Parameters), J Allergy Clin Immunol 101(suppl):S483, 1998. 270. World Health Organization position paper on allergen immunotherapy: therapeutic vaccines for allergic diseases, Allergy 53(suppl):S20, 1998. 271. American Academy of Allergy, Asthma, and Immunology Board of Directors: Guidelines to minimize the risk from systemic reactions caused by immunotherapy with allergenic extracts (position statement), J Allergy Clin Immunol 93: 811, 1994.

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Chapter 84 - Mastocytosis Syndromes

Dean D. Metcalfe

Mastocytosis is a disease whose clinical features include pruritus, urtication, flushing, nausea, vomiting, diarrhea, abdominal pain, vascular instability, and headache. The most remarkable pathologic feature of mastocytosis is mast cell hyperplasia in the skin, gastrointestinal (GI) tract, bone marrow, liver, spleen, and lymph nodes. Mastocytosis may occur at any age. The prevalence of the disease is unknown, and familial occurrence is unusual.

Mastocytosis may be further classified based on clinical presentation, pathologic findings, and prognosis. A revised classification system for mastocytosis developed 1

in a consensus conference is shown in Box 84-1 .[ ] Patients with cutaneous mastocytosis (CM) have the best prognosis followed by those with indolent systemic mastocytosis (ISM). Patients with systemic mastocytosis with an associated clonal, hematologic non–mast cell lineage disease (SM-AHNMD), aggressive systemic mastocytosis (ASM), or mast cell leukemia (MCL) experience more rapid and complex courses. Patients with CM or ISM may experience progressive difficulties, but their condition may be managed for decades with medications that offer largely symptomatic therapy. In SM-AHNMD, examination of the peripheral blood and bone marrow leads to the diagnosis of one of several hematologic disorders; survival of these patients is determined by the course of the hematologic disorder. Patients with ASM or MCL have a poor prognosis, as do those with mast cell sarcoma (MCS). Patients with ASM experience a rapid increase in the number of mast cells. Medical management may be difficult, and prognosis is less favorable. MCL is rare, with the most fulminant behavior. In this form of mast cell disease, numerous immature mast cells are found in peripheral blood smears. MCL is distinguished from the other disease types by its clinical and pathologic picture. MSC is exceedingly rare, and a leukemia phase may occur.

ETIOLOGY AND PATHOGENESIS Paul Ehrlich first reported mast cells more than 100 years ago; he described cells with prominent cytoplasmic granules. These cells are now known to contain histamine and other inflammatory mediators and to play a critical role in acquired immediate hypersensitivity responses. They also appear to function in innate immunity. Mast Cell Origin 2 3

Human mast cells develop from a bone marrow–derived hematopoietic precursor cell (CD34+, Kit+) that circulates in the peripheral blood.[ ] [ ] Mast cells are sparsely distributed in the tissues and have prominent cytoplasmic granules that contain histamine, other chemical mediators, and surface receptors that bind the Fc portion of IgE with high affinity. Mast cells mature within tissues and are often found adjacent to blood vessels and under epithelial surfaces. They are prominent in the GI and respiratory tracts, lymphoid tissues, and skin. Mature mast cells normally do not circulate, are long-lived, and appear to retain some capacity to proliferate. 4

When CD34+ cells from human bone marrow are cultured with stem cell factor (SCF), mast cells are generated.[ ] Human CD34+ cells cultured on fibroblasts also give rise to mast cells that closely resemble human tissue mast cells. This is attributed to the production of SCF by fibroblasts. When factors that support mast cell growth are withdrawn, mast cells undergo programmed cell death (apoptosis).[

5]

The protooncogene c-kit encodes a transmembrane tyrosine kinase receptor for SCF termed Kit (CD117). Kit is significantly expressed on mast cells, hematopoietic stem cells, melanocytes, and germ cell lineages. Mutations in Kit are involved in the hereditary disease piebaldism, which is characterized by loss of pigmentation, whereas over-expression of c-kit messenger ribonucleic acid is reported to be associated with myeloproliferative disorders and myelodysplastic syndromes. Based on the information that the SCF-Kit system plays a role in the development of mast cells, several studies have investigated the role of SCF or Kit in the development of 6

mastocytosis. First, it has been reported that there is increased soluble SCF in the skin of patients with indolent mastocytosis, [ ] although data are not consistent in 7

this regard.[ ] Researchers have also identified activating point mutations of c-kit in the peripheral blood mononuclear cells, bone marrow, and skin lesions of 8 9

patients with mastocytosis. The most common mutation consists of a substitution of valine for aspartic acid (ASP 816 VAL).[ ] [ ] These latter observations suggest that mastocytosis may be in part a result of the presence of “overactive” receptors in some patients with mastocytosis. There is no evidence yet that this

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Box 84-1. Mastocytosis Classification Cutaneous mastocytosis (CM) • Urticaria pigmentosa (UP) • Diffuse cutaneous mastocytosis (DCM) Indolent systemic mastocytosis (ISM) Systemic mastocytosis with associated clonal, hematologic non–mastcell lineage disease (SM-AHNMD) Aggressive systemic mastocytosis (ASM) Mast cell leukemia (MCL) Mast cell sarcoma (MCS) Extracutaneous mastocytoma

9

mutation is passed from generation to generation. Point mutations in Kit appear to be present in most adults with various forms of mastocytosis.[ ] However, the mutation appears absent in many children with CM. Pathologic Effects of Increased Mast Cells Mast cells secrete chemicals that mediate allergic reactions. The pathogenesis of mastocytosis is the result of the increased production of mast cells and their mediators within tissues. Mast cell–derived mediators also circulate through the bloodstream and lymphatic system to produce biologic effects at sites distant from the site of their synthesis ( Table 84-1 ). The effects appear similar regardless of the etiology of the increase in mast cell numbers or the category of disease. The biologic effects of histamine result from the activation of H1 or H2 cell surface receptors and are prominent in mastocytosis. The H1 receptors are blocked by

antihistamines such as chlorpheniramine and are involved in the histamine-induced contraction of bronchial and GI smooth muscle. The H2 receptors play a major role in the increased secretion of gastric acid by parietal cells. They are blocked by drugs such as cimetidine and ranitidine. Histamine enhances vascular permeability by acting on the endothelial cells in the postcapillary venules. The protease tryptase is a major component of the mast cell secretory granule and is secreted and the beta form is released after mast cell stimulation. Histamine and tryptase levels are often elevated in the serum of mastocytosis patients.[ mast cells make prostaglandin D2 and leukotriene C4 . These lipid-derived molecules have a wide spectrum of biologic

11 activities[ ]

10]

Human

(see Table 84-1 ). Mast cells also

produce a variety of proinflammatory and growth factor cytokines, including tumor necrosis factor-α (TNF-α), interleukin-3 (IL-3), and IL-16.[

11]

CLINICAL FEATURES All categories of mastocytosis (see Box 84-1 ) present similar clinical features, although some aspects of disease may predominate in a specific category. The skin, GI tract, lymph nodes, liver, spleen, bone marrow, and skeletal system contribute the most significant management problems. The respiratory tract and endocrine systems are seldom, if ever, TABLE 84-1 -- Clinicopathologic Features of Mastocytosis Associated with Known Mast Cell Mediators I. Systemic Vascular instability

Histamine, LTC4 , LTE4 , PGD2 , PAF, endothelin

Increased vasopermeability

Histamine, LTC4 , LTD4 , PAF

Fibrosis

Transforming growth factor-β

Eosinophilia

IL-5

Lymphocytic infiltrations

IL-16, lymphotaxin

Local anticoagulation

Heparin

Mast cell hyperplasia

IL-3, IL-6, SCF

Cachexia

Tumor necrosis factor-α, IL-6

II. Skin Pruritus

Histamine

Urticaria

Histamine, LTC4 , PAF

III. GI tract Gastric hypersecretion

Histamine

Cramping, abdominal pain

Histamine, LTC4 , PAF

IV. Lungs Bronchoconstriction

Histamine, PGD2 , LTC4 , LTD4 , PAF, endothelin

Secretion of mucus

Histamine, proteases, PGD2 , LTC4

Pulmonary edema

Histamine, LTC4 , PAF

V. Skeletal system Bone remodeling

Tryptase, chymotryptic proteases, IL-6

Osteoporosis

Heparin

LT, Leukotriene; PG, prostaglandin; PAF, platelet-activating factor; IL, interleukin. primarily involved. Patients in every category of mastocytosis sometimes experience flushing or episodic hypotension. Occasionally, hypotension may be provoked by alcohol, aspirin, insect stings, infection, or exposure to iodinated contrast materials. Patients with mastocytosis do not suffer from an increase in bacterial, fungal, or viral infection. Characteristic Patterns of Skin Involvement The most common skin manifestation of mastocytosis in both children ( Figure 84-1 , A and B) and adults ( Figure 84-2 ) is urticaria pigmentosa (UP) (see Box 841 ). It is the most common pattern of skin involvement in CM. UP is also observed in more than 90% of patients with ISM, and is observed in about 50% of patients with SM-AHNMD or those with ASM. UP lesions appear as small yellowish-tan to reddish-brown macules or slightly raised papules ( Figure 84-3 ) and occasionally as raised nodules or plaquelike lesions ( Figure 84-1 , C and D). The palms, soles, face, and scalp tend to remain free of lesions. Rubbing of the lesions usually leads to urtication and erythema over and around the macules, known as Darier's sign. UP is sometimes associated with pruritus that is often exacerbated by changes in temperature, local friction,

1525

ingestion of hot beverages or spicy foods, ethanol, and certain drugs. The diagnosis is confirmed by skin histopathology that shows a pathologic increase in the 12]

number of dermal mast cells.[

Diffuse cutaneous mastocytosis (DCM) is a form of CM and is the result of a diffuse mast cell infiltration in the dermis; there are no discrete lesions ( Figure 84-4 ). It usually occurs before the age of 3 years. The entire cutaneous integument is involved. The skin is normal to yellowish-brown and is thickened. The diagnosis is confirmed by the demonstration of diffuse mast cell infiltrates in the skin ( Figure 84-5 ). 13 14

Young children with UP or DCM may have bullous eruptions with hemorrhage[ ] [ ] ( Figure 84-6 ). Blisters may erupt spontaneously or in association with infection or immunization. Blisters may also occur at birth; thus CM is included in the differential diagnosis of neonatal disorders with blisters. Isolated mastocytoma of the skin is unusual, typically occurring before 6 months of age and cases usually spontaneously

Figure 84-1 Urticaria pigmentosa in childhood. Lesions in A are smaller and more discrete than those seen in B. C, An example of nodular lesions of urticaria pigmentosa with a close-up view (D).

Figure 84-1 Urticaria pigmentosa in childhood. Lesions in A are smaller and more discrete than those seen in B. C, An example of nodular lesions of urticaria pigmentosa with a close-up view (D).

Figure 84-2 Urticaria pigmentosa in an adult with indolent systemic mastocytosis.

Figure 84-3 Close-up of urticaria pigmentosa in an adult with indolent systemic mastocytosis.

Figure 84-4 Diffuse cutaneous mastocytosis in an adult.

Figure 84-5 Biopsy of the skin from a child with diffuse cutaneous mastocytosis showing extensive mast cell infiltration. (Toluidine blue; ×250.)

Figure 84-6 Diffuse cutaneous mastocytosis (confluent urticaria pigmentosa) in a child. A, Extensive diffuse skin involvement. B, Bullous eruption.

Figure 84-7 Colon biopsy showing an increased mast cell number in the mucosa in a patient with indolent systemic mastocytosis. (Toluidine blue; ×400.)

Figure 84-8 A, Paratrabecular focal nodular aggregate of spindle-shaped mast cells surrounds a lymphoid follicle in this bone marrow biopsy specimen. The hematopoietic marrow is normocellular, and the bone trabeculae are slightly thickened. This patient has an indolent form of systemic mast cell disease. (Hematoxylineosin stain; ×60.) B, Higher power demonstrates the spindle shape of the mast cells and the faint granularity of the cytoplasm. (Hematoxylin-eosin stain; ×250.)

Figure 84-8 A, Paratrabecular focal nodular aggregate of spindle-shaped mast cells surrounds a lymphoid follicle in this bone marrow biopsy specimen. The hematopoietic marrow is normocellular, and the bone trabeculae are slightly thickened. This patient has an indolent form of systemic mast cell disease. (Hematoxylineosin stain; ×60.) B, Higher power demonstrates the spindle shape of the mast cells and the faint granularity of the cytoplasm. (Hematoxylin-eosin stain; ×250.)

Figure 84-9 Skeletal scintigraphy showing normal pattern (A) and a diffuse increase (B) in scan intensity in a patient with long-standing indolent systemic mastocytosis.

Box 84-2. Diagnostic Workup I. Routine Studies Complete blood count with differential, platelets, liver function tests, sedimentation rate Examine skin: gross and microscopic Bone marrow biopsy and aspiration Plasma mast cell tryptase II. Additional studies Bone scan and skeletal survey

GI workup: upper GI series, small bowel radiographic study, computed tomography scan, endoscopy Electroencephalography, neuropsychiatric workup

Modified from Metcalfe DD: J Invest Dermatol 96:64S, 1991.

48

using specific antibodies to these proteases, only tryptase-positive, chymase-positive mast cells were identified.[ ] These cells predominate in skin, whereas tryptasepositive, chymase-negative mast cells predominate in alveolar walls and the GI mucosa. The exclusive presence of the former in dermal mastocytosis lesions suggests that mast cell hyperplasia is in part dependent on local factors. Mastocytosis should be suspected in patients without skin lesions if one or more of the following features is present: unexplained ulcer disease or malabsorption, radiographic or technetium 99 bone scan abnormalities, hepatomegaly, splenomegaly, lymphadenopathy, peripheral blood abnormalities, and unexplained flushing or 49] [50]

anaphylaxis. Elevations in the levels of plasma[ B 2 ,[

53]

plasma (total) mast cell tryptase,[

10]

or urinary histamine, or histamine metabolites,[

51]

urine prostaglandin D2 metabolites,[

52]

plasma thromboxane

7

or IL-6[ ] are not definitive diagnostic findings, but are consistent with the diagnosis of mastocytosis. For example, levels 10

of plasma tryptase greater than 20•ng/ml were found in 80% of patients with mastocytosis.[ ] More recently, it has been found that plasma levels of soluble Kit and soluble IL-2 receptor alpha chain (CD25), which are known to be elevated in some patients with hematologic malignancy, are also elevated in patients with mastocytosis, and correlate to severity of

Figure 84-10 Mast cell numbers in the skin of normal subjects (normals), patients with unexplained anaphylaxis or unexplained flushing (UEA/UEF), and patients with mastocytosis from nonlesional (NL) and lesional (L) biopsy sites. Symbols indicate specific categories, which also include patients with urticaria pigmentosa without systemic disease (UP) and patients with diffuse cutaneous mastocytosis (DCM). (From Garriga MM, Friedman MM, Metcalfe DD, et al: J Allergy Clin Immunol 82: 425, 1988.)

(From Garriga MM, Friedman MM, Metcalfe DD, et al: J Allergy Clin Immunol 82: 425, 1988.)

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disease, total tryptase, and bone marrow pathology.[

54]

Reliable tests for these substances may not be generally available except in research laboratories.

In patients suspected of having mastocytosis in the absence of skin lesions, a bone marrow biopsy and aspiration should be performed to confirm the diagnosis and to determine the disease category. Patients with UP or DCM should also undergo this procedure, particularly if peripheral blood abnormalities, hepatomegaly, splenomegaly, or lymphadenopathy is present, to determine whether they have an associated hematologic disorder. Examination of other tissue specimens, such as

those from the lymph nodes, spleen, liver, and GI mucosa, can help define the extent of mast cell involvement. Such studies are performed only when necessary. For example, a GI workup is dictated by symptoms of GI involvement, and lymph nodes are biopsied if lymphoma is suspected. A 24-hour urinary study of 5-hydroxyindoleacetic acid and urinary metanephrines is necessary to help eliminate the possibility of a carcinoid tumor or pheochromocytoma. Patients with mastocytosis do not excrete increased amounts of the former. Idiopathic anaphylaxis and flushing must also be ruled out. Patients 10]

with these disorders do not have histologic evidence of significant mast cell proliferation and should have normal tryptase levels between episodes of anaphylaxis.[

TREATMENT Mast Cell–Mediated Symptoms A principal objective of treatment in all categories of mastocytosis is the control of mast cell mediator–induced signs and symptoms. Most prominent among these are systemic hypotension, gastric hypersecretion, GI cramping, and pruritus. H1 -receptor antagonists, such as hydroxyzine and cetirizine, are instituted to reduce pruritus and flushing. If this is insufficient, the addition of an H2 antagonist, such as ranitidine or cimetidine, may be beneficial. Even with these medications, many patients continue to complain of musculoskeletal pain, headaches, and flushing, in part because of the inability of histamine antagonists to block the effects of high levels of histamine and because of the presence of other mast cell mediators. There may thus be some value in adding a leukotriene-modifying agent. 55]

Disodium cromoglycate (cromolyn sodium) inhibits degranulation of mast cells and has some efficacy in mastocytosis, particularly in the relief of GI complaints.[ Cromolyn sodium does not reduce plasma or urinary histamine levels in patients with mastocytosis. [

56]

Ketotifen, an antihistamine with mast cell stabilizing 57]

properties, is of benefit in relieving the pruritus and whealing associated with mastocytosis, but appears to offer no advantage over hydroxyzine. [ 58

Epinephrine is used to treat episodes of systemic hypotension. [ ] Patients should be taught to administer this medication themselves. If subcutaneous epinephrine is inadequate, intensive therapy for systemic hypotension, as for anaphylaxis, should be instituted. Patients with recurrent episodes of hypotension may be given H1 or H2 antihistamines to reduce the severity of attacks. Episodes of profound hypotension may be spontaneous but have also been observed after insect stings and administration of contrast media. 16 59

Methoxsalen with long-wave ultraviolet radiation (psoralen plus ultraviolet A) relieves pruritus and whealing after 1 to 2 months of treatment.[ ] [ ] Improvement is associated with a transient decrease in the number of dermal mast cells. Relapse of pruritus occurs within 3 to 6 months after discontinuation of therapy. Photochemotherapy should be used only in instances of extensive cutaneous disease unresponsive to other forms of therapy. Some patients report a diminution in the number or intensity of cutaneous lesions after repeated exposure to natural sunlight. Topical steroids, such as 0.05% betamethasone dipropionate ointment applied under plastic wrap occlusion for 8 hours a day over 8 to 12 weeks, may be used to treat UP or DCM. The number of mast cells decreases as lesions resolve. Lesions eventually recur after discontinuation of therapy,[ to improvement in cutaneous lesions for up to 1 year.

60] [61]

although the treatment may lead

GI Symptoms The treatment of GI disease is dictated by the degree of peptic symptoms, diarrhea, and malabsorption. Gastric acid hypersecretion leading to peptic symptoms and ulcerations is controlled with H2 antagonists and proton pump inhibitors. Diarrhea is difficult to manage, and H2 agonists are generally not effective for this symptom. Anticholinergics may give partial relief. In patients with severe malabsorption, oral steroids are effective.[ hypertension in one patient was successfully managed with a portacaval steroid

63 shunt.[ ]

62]

Ascites is also difficult to control. Portal

Another patient with an exudative ascites was treated successfully with systemic

64 therapy.[ ]

Hematologic Abnormalities Patients with mastocytosis and an associated hematologic disorder are managed as dictated by the specific hematologic abnormality.[ that interferon-α2b may help improve mast cell infiltrations,

[66]

survival in MCL, and it has no place in the treatment of indolent forms of mastocytosis associated with poor [69]

68 prognosis.[ ]

another reported no 67 mastocytosis.[ ]

67 benefit.[ ]

65]

Although one report suggests

Chemotherapy has not been shown to produce remission or prolong

One study suggests that splenectomy may improve survival times in patients with

Radiotherapy has been used in the management of refractory bone pain in patients with aggressive disease.

Bone marrow transplantation may be considered for gravely ill patients, although there is as yet little known about long-term prognosis after transplantation.

PROGNOSIS 27 29

The prognosis differs for each category of mastocytosis. The following variables are strongly associated with poor survival[ ] [ ] : constitutional symptoms, anemia, thrombocyto-penia, abnormal liver function tests, lobated mast cell nuclei, a low percentage of fat cells in the bone marrow biopsy, and an associated hematologic disorder. Other variables associated with poor prognosis include male gender, hepatomegaly, splenomegaly, normal bone radiographic findings, absence of UP, and skin and musculoskeletal symptoms. Patients with CM only have the best prognosis. For children with isolated UP, at least 50% of cases are reported to resolve by adulthood.[ evolve into systemic disease. Occasionally, ISM converts to SM-AHNMD.

70]

UP in adulthood may

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Patients with SM-AHNMD have a course that depends largely on the prognosis of the specific hematologic disorder. The mean survival time for patients with MCL is usually less than 6 months. The survival time with ASM is 2 to 4 years with aggressive symptomatic management.

REFERENCES 1. Valent P, Hormy HP, Li CY, et al: Mastocytosis. In Jaffe ES, Harris NL, Stein H, et al, editors: Pathology and genetics of tumors of haematopoietic and lymphoid tissues, Lyon, 2001, IARC Press, p 293. Etiology and Pathogenesis 2. Kirshenbaum AS, Goff JP, Semere T, et al: Demonstration that human mast cells arise from a bipotential progenitor cells population that is CD34+, c-kit+, and expresses aminopeptidase N(CD13), Blood 94:2333, 1999. 3. Rottem M, Okada T, Goff JP, et al: Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+ FcepsilonRI-cell population, Blood 84:2489, 1994. 4. Kirshenbaum AS, Goff JP, Kessler SW, et al: The effect of IL-3 and stem cell factor on the appearance of mast cells and basophils from human CD34+ pluripotent progenitor cells, J Immunol 148:772, 1992. 5. Mekori Y, Oh CK, Metcalfe DD: IL-3 dependent murine mast cells undergo apoptosis upon removal of IL-3: prevention of apoptosis by c-kit ligand, stem cell factor, J Immunol 151:3775, 1993. 6. Longley J, Morganroth GS, Tyrell L, et al: Altered metabolism of mast-cell growth factor (c-kit) ligand in cutaneous mastocytosis, N Engl J Med 328:1302, 1993. 7. Brockow K, Akin C, Huber M, et al: Levels of mast cell growth factor in plasma and in suction skin blister fluids in adults with mastocytosis: correlation with dermal mast cell numbers and mast cell tryptase, J Allergy Clin Immunol 109:82, 2002. 8. Nagata H, Worobec AS, Oh CK, et al: Identification of a point mutation in the catalytic domain of the proto-oncogene c-kit in peripheral blood mononuclear cells of patients with mastocytosis, Proc Natl Acad Sci U S A 92:10560, 1995. 9. Boissan M, Feger F, Guillosson JJ, et al: c-Kit and c-kit mutations in mastocytosis and other hematologic disorders, J Leukoc Biol 67:135, 2000. 10. Brockow K, Akin C, Huber M, et al: Assessment of the extent of cutaneous involvement in children and adults with mastocytosis: relationship to symptomology, tryptase levels, and bone marrow pathology, J Am Acad Dermatol 2003 (in press). 11. Metcalfe DD, Barnam D, Mekori YA: Mast cells, Physiol Rev 77:1033, 1998. Clinical Features 12. Garriga MM, Friedman MM, Metcalfe DD: A survey of the number and distribution of mast cells in the skin of patients with mast cell disorders, J Allergy Clin Immunol 82:425, 1988. 13. Orkin M, Good RA, Clawson CC, et al: Bullous mastocytosis, Arch Dermatol 101:547, 1970.

14. Golitz LE, Weston WL, Lane AT: Bullous mastocytosis: diffuse cutaneous mastocytosis with extensive blisters mimicking scalded skin syndrome or erythema multiforme, Pediatr Dermatol 1:288, 1984. 15. Kudo H, Morinaga S, Shimosato Y, et al: Solitary mast cell tumor of the lung, Cancer 61:2089, 1988. 16. Soter NA: Mastocytosis and the skin, Hematol Oncol Clin North Am 14:517, 2000. 17. Webb TA, Li CY, Yam LT: Systemic mast cell disease: a clinical and hematopathologic study of 26 cases, Cancer 49:927, 1983. 18. Parwaresch MR, Horny HP, Lennert K, et al: Tissue mast cells in health and disease, Pathol Res Pract 179:439, 1985. 19. Cherner JA, Jensen RT, Dubois A, et al: Gastrointestinal dysfunction in systemic mastocytosis: a prospective study, Gastroenterology 95:657, 1988. 20. Ammann RW, Vetter D, Dehyle P, et al: Gastrointestinal involvement in systemic mastocytosis, Gut 17:107, 1976. 21. Clemett AR, Fishbone G, Levine RJ, et al: Gastrointestinal lesions in mastocytosis, Am J Roentg Radium Ther Nucl Med 103:405, 1968. 22. Bank S, Marks IN: Malabsorption in systemic mast cell disease, Gastroenterology 45:535, 1963. 23. Bredfeldt JE, O'Laughlin JC, Durham JB, et al: Malabsorption and gastric hyperacidity in systemic mastocytosis, Am J Gastroenterol 74:133, 1980. 24. Broitman SA, McCray RS, May JC, et al: Mastocytosis and intestinal malabsorption, Am J Med 48:382, 1970. 25. Wesley JR, Vinik AI, O'Dorisio TM, et al: A new syndrome of symptomatic cutaneous mastocytoma producing vasoactive intestinal polypeptide, Gastroenterology 82:963, 1982. 26. O'Dorsio TM, Mekhjian HS: VIPoma syndrome. In Cohen S, Soloway RD, editors: Contemporary issues in gastroenterology, vol 5, New York, 1976, Churchill Livingstone, p 101. 27. Travis WD, Li CY, Bergstralh EJ, et al: Systemic mast cell disease: analysis of 58 cases and literature review, Medicine (Baltimore) 67:345, 1988. 28. Parker RI: Hematologic aspects of systemic mastocytosis, Hematol Oncol Clin North Am 14:557, 2001. 29. Lawrence JB, Friedman BS, Travis D, et al: Hematologic manifestations of systemic mast cell disease: a prospective study of laboratory and morphologic features and their relation to prognosis, Am J Med 91:612, 1991. 30. Horny HP, Parwaresch MR, Lennert K, et al: Bone marrow findings in systemic mastocytosis, Hum Pathol 16:808, 1985. 31. Kettlehut BV, Parker PI, Travis WD, et al: Hematopathology of the bone marrow in pediatric cutaneous mastocytosis: a study of seventeen patients, Am J Clin Pathol 91:558, 1989. 32. Escribano L, Orfao A, Diaz-Augstin B, et al: Indolent systemic mast cell disease in adults: immunophenotypic characterization of bone marrow mast cells and its

diagnostic implications, Blood 91:2731, 1998. 33. Travis WD, Li CY, Hoagland HC, et al: Mast cell leukemia: report of a case and review of the literature, Mayo Clin Proc 61:957, 1986. 34. Peart KM, Ellis HA: Quantitative observations on the iliac bone marrow mast cells in chronic renal failure, J Clin Pathol 28:947, 1975. 35. Frame B, Nixon RK: Bone marrow mast cells in osteoporosis of aging, N Engl J Med 279:626, 1968. 36. Fohlmeister I, Reber T, Fischer R, et al: Bone marrow mast cell reaction in preleukemic myelodysplasia and in aplastic anemia, Virchows Arch [A] 405:503, 1985. 37. Prokocimer M, Polliack A: Increased bone marrow mast cells in preleukemic syndromes, acute leukemia, and lymphoproliferative disorders, Am J Clin Pathol 75:34, 1981. 38. Yoo D, Lessin LS: Bone marrow mast cell content in preleukemic syndrome, Am J Med 73:539, 1982. 39. Chen CC, Andrich MP, Mican JM, et al: A retrospective analysis of bone scan abnormalities in mastocytosis: correlation with disease category and prognosis, J Nucl Med 35:1471, 1994. 40. Travis WD, Li CY: Pathology of the lymph node and spleen in systemic mast cell disease, Mod Pathol 1:4, 1988. 41. Mican JM, DiBisceglie AM, Fong TL, et al: Hepatic involvement in mastocytosis: clinicopathologic correlations in 41 cases, Hepatology 22:1163, 1995. 42. Rogers MP, Bloomingdale K, Murawski BJ, et al: Mixed organic brain syndrome as a manifestation of systemic mastocytosis, Psychosom Med 48:437, 1986. 43. McFarlin KE, Kreusi MJ, Metcalfe DD: A preliminary assessment of behavioral problems in children with mastocytosis, Int J Psychiatry Med 21:281, 1991. Diagnosis 44. Tharp MD, Glass MJ, Seelig LL Jr: Ultrastructural morphometric analysis of lesional skin: mast cells from patients with systemic and nonsystemic mastocytosis, J Am Acad Dermatol 18:298, 1988. 45. Nishioka K, Kobayashi Y, Katayama I, et al: Mast cell numbers in scleroderma, Arch Dermatol 123:205, 1987. 46. Elias J, Boss E, Kaplan AP: Studies of the cellular infiltrate of chronic idiopathic urticaria: prominence of T-lymphocytes, monocytes, and mast cells, J Allergy Clin Immunol 78:914, 1986. 47. Mitchell EB, Crow J, Williams G, et al: Increase in skin mast cells following chronic house dust mite exposure, Br J Dermatol 114:65, 1986. 48. Irani AM, Garriga MM, Metcalfe DD, et al: Mast cells in cutaneous mastocytosis: accumulation of the MCTC type, Clin Exp Allergy 20:53, 1990. 49. Kettlehut BV, Metcalfe DD: Plasma histamine in the evaluation of pediatric mastocytosis, J Pediatr 111:419, 1987.

50. Friedman BS, Steinberg S, Meggs WJ, et al: Analysis of plasma histamine levels in patients with mast cell disorders, Am J Med 87:649, 1989. 51. Keyzer JJ, deMonchy JG, van Doormaal JJ, et al: Improved diagnosis of mastocytosis by measurement of urinary histamine metabolites, N Engl J Med 309:1603, 1983. 52. Roberts LJ II, Sweetman BJ, Lewis RA, et al: Increased production of prostaglandin D2 in patients with systemic mastocytosis, N Engl J Med 303:1400, 1980. 53. Ouwendijk RJ, Zijlstra FJ, Wilson JH, et al: Raised levels of thromboxane B2 in systemic mastocytosis, Eur J Clin Invest 13:227, 1983. 54. Akin C, Schwartz LB, Kitoh T, et al: Soluble stem cell factor receptor (CD117) and IL-2 receptor alpha chain (CD25) levels in the plasma of patients with mastocytosis: relationship to disease severity and bone marrow pathology, Blood 96:1267, 2000. Treatment 55. Horan RF, Sheffer AL, Austen KF: Cromolyn sodium in the management of systemic mastocytosis, J Allergy Clin Immunol 85:852, 1990. 56. Frieri M, Alling DW, Metcalfe DD: Comparison of the therapeutic efficacy of cromolyn sodium with that of combined chlorpheniramine and cimetidine in systemic mastocytosis: results of a double-blind clinical trial, Am J Med 78:9, 1985. 57. Kettelhut BV, Metcalfe DD: A double blind placebo controlled trial of ketotifen versus hydroxyzine in the treatment of pediatric mastocytosis, J Allergy Clin Immunol 83:866, 1989. 58. Turk J, Oates JA, Roberts LJ II: Intervention with epinephrine in hypotension associated with mastocytosis, J Allergy Clin Immunol 71:189, 1983. 59. Czarnetski BM, Rosenbach T, Kolde G, et al: Phototherapy of urticaria pigmentosa: clinical response and changes of cutaneous reactivity, histamine, and chemotactic leukotrienes, Arch Dermatol Res 277:105, 1985. 60. Lavker RM, Schechter NM: Cutaneous mast cell depletion results from topical corticosteroids usage, J Immunol 135:2368, 1985. 61. Barton J, Lavker RM, Schecter NM, et al: Treatment of urticaria pigmentosa with corticosteroids, Arch Dermatol 121:1516, 1985. 62. Friedman BS, Metcalfe DD: Effects of tixocortol pivalate on gastrointestinal diseases in mastocytosis: a preliminary study, Clin Exp Allergy 21:183, 1991. 63. Bonnet P, Smadja C, Szekely AM: Intractable ascites in systemic mastocytosis treated by portal diversion, Dig Dis Sci 32:209, 1987.

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64. Reisberg IR, Oyakawa S: Mastocytosis and malabsorption, myelofibrosis and massive ascites, Am J Gastroenterol 82:54, 1987. 65. Worobeck AS: Treatment of systemic mast cell disorders. Hematol Oncol Clin North Am 14:659, 2000. 66. Kluin-Nelemans HC, Jansen JH, Bruekelman H: Response to interferon alpha-2b in a patient with systemic mastocytosis, N Engl J Med 326:619, 1992. 67. Worobec AS, Krishenbaun AS, Schwartz L: Treatment of three patients with systemic mastocytosis with interferon alpha-2b, Leuk Lymph 18:179, 1995. 68. Friedman B, Darling G, Norton J: Splenectomy in the management of systemic mast cell disease, Surgery 107:94, 1990. 69. Johnstone PA, Mican JM, Metcalfe DD, et al: Radiotherapy of refractory bone pain due to systemic mast cell disease, Am J Oncol 17:328, 1994. 70. Kettelhut BV, Metcalfe DD: Pediatric mastocytosis, Ann Allergy 73:197, 1994.

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Chapter 85 - Urticaria and Angioedema

Allen P. Kaplan

Urticaria is characterized by the appearance of pruritic, erythematous, cutaneous elevations that blanch with pressure, indicating the presence of dilated blood vessels and edema. In its simplest form, it is envisioned to represent the same sort of wheal-and-flare reaction that is observed when histamine is injected into the skin. Biopsy of acute urticarial lesions reveals dilation of small venules and capillaries located in the superficial dermis with widening of the dermal papillae, flattening of the rete pegs, and swelling of collagen fibers. Angioedema is caused by the same or similar pathologic alterations that occur in the deep dermis and subcutaneous tissue. An area involved with angioedema therefore has swelling as the prominent manifestation, and the appearance of the skin itself may be normal. Because angioedema occurs in deeper skin layers, where there are fewer mast cells and sensory nerve endings, the lesions have little or no associated pruritus, and

the swelling may be described as painful or burning. Urticaria may occur on virtually any part of the body, whereas angioedema (in the absence of hives) often involves the face, tongue, extremities, or genitalia. In contrast to other forms of edema, angioedematous swellings do not characteristically occur in dependent areas, are asymmetrically distributed, and are transient. Urticaria and angioedema can occur together; in one series, approximately half of referred patients had both 1

urticaria and angioedema.[ ] This chapter first examines the biochemical mechanisms that lead to the development of urticaria and angioedema and then summarizes the presentation, pathogenesis, and treatment of various clinical entities, as well as the diagnostic studies indicated.

MEDIATOR PATHWAYS The pathogenesis of urticaria and angioedema can involve the release of a diverse array of potential vasoactive mediators that arise from the activation of cells or enzymatic pathways. These are described in detail in Chapters 13 , 14 , 20 , and 21 and are briefly summarized herein as they relate to the skin ( Table 85-1 ). The major noncytotoxic mechanism by which histamine is released requires the combination of an antigen with immunoglobulin E (IgE) antibody bound to basophils or 2

tissue mast cells.[ ] A variety of mediators with vasoactive properties are released. Histamine is released from preformed granules as a consequence of this antigenantibody reaction and is capable of eliciting the classic triple response consisting of vasodilation (erythema), increased vascular permeability (edema), and an axon reflex that increases the extent of the reaction, particularly the erythema. The axon reflex appears to result from the release of substance P from nonadrenergic, 3

noncholinergic type C cutaneous fibers by antidromic conduction. Substance P acts as a potent vasodilator[ ] ; it also stimulates cutaneous mast cells to release 4

histamine and thereby augments the reaction.[ ] In addition, various lipid-derived vasoactive factors are liberated. Of the arachidonic acid metabolites, prostaglandin 2

D2 (PGD2 ) is a vasodilator, and leukotrienes C and D (slow reacting substance of anaphylaxis) can increase vascular permeability [ ] ; these may be synergistic in 5

the skin.[ ] Virtually any stimulus causing cutaneous mast cell degranulation would cause the release of each of these chemicals; however, they have not yet been 2] [6]

rigorously examined, and their role in urticarial reactions has not been defined. Platelet-activating factor (PAF) is also released from mast cells.[ 7 8 metabolite[ ] [ ]

[2]

that can increase vascular permeability when injected directly into skin. not been examined in other types of urticaria or angioedema.

It is a lipid

PAF is released during challenge in patients with cold urticaria, but it has

PAF is 100 to 1000 times more potent than histamine on a molar basis in terms of wheal and flare induction; however, it has a different time course of release, and the quantities released are very small. Although all these factors can contribute to hive formation in the skin, histamine appears to be the major one; therefore, antihistamines have a significant (but incomplete) clinical effect. A second pathway leading to the noncytotoxic release of histamine from basophils or mast cells involves the complement cascade. Three fragments of complement components, termed C3a, C4a, and C5a, act as anaphylatoxins; that is, they interact directly with the cell surface in the absence of antibody to trigger histamine 2

9

release.[ ] On a molar basis, C5a appears to be a more potent permeability factor than C3a or C4a as assessed by intradermal injection.[ ] Because there is particularly 2

rapid inactivation of C3a by the anaphylatoxin inactivator, [ ] only C5a is routinely isolated from serum after complement activation. C5a is then gradually destroyed by the same inhibitor. C5a also functions as a chemotactic factor and can attract neutrophils, eosinophils, or mononuclear cells to a site of inflammation, while C3a 10]

has selective chemotactic activity for eosinophils.[

In virtually all inflammatory reactions (and certainly those in the skin), an increase in vascular permeability leads to activation of the plasma kinin-forming system

once the requisite

1538

TABLE 85-1 -- Mediators of Hives and Swelling Source

Factor

Mast cells (cutaneous)

Histamine Prostaglandin D Leukotrienes C and D Platelet-activating factor or 1-0-alkyl2-acetyl-sn-glyceryl-3phosphorylcholine

Complement system

Anaphylatoxins C3a, C4a, C5a; histamine

Hageman factor–dependent pathway

Bradykinin

Mononuclear cells

Histamine-releasing factors, chemokines

plasma proteins contact surfaces or macromolecular particles that can serve as initiators ( Figure 85-1 ). 11 12

Hageman factor (HF) autoactivates on binding to initiating surfaces (reaction 1; see Figure 85-1 ). [ ] [ ] If as few as 1 in 5000 molecules is active in plasma, this trace of activated Hageman factor (HFa) will digest surface-bound HF to form more HFa, which then initiates coagulation (converts factor XI to factor XIa) and 2

13

fibrinolysis[ ] and converts prekallikrein to kallikrein.[ ] Kallikrein digests a high-molecular-weight (HMW) form of kininogen to yield the vasoactive peptide bradykinin. HMW kininogen accounts for only 15% of the total plasma kininogen, and it not only is a substrate for kallikrein but also has a critical cofactor function 14 15 16

17 18

in contact activation.[ ] [ ] [ ] The substrates of HFa (i.e., prekallikrein and factor XI) each circulate as 1:1 molar complexes with HMW kininogen.[ ] [ ] HMW kininogen binds to initiating surfaces, whereas prekallikrein and factor XI are linked to the surface via this HMW kininogen “bridge.” This appears to place prekallikrein and factor XI in a favorable formation for digestion and activation by HFa.[

19]

Figure 85-1 Diagram of Hageman factor (HF)–dependent pathways of coagulation, fibrinolysis, and kinin generation. Reaction 1 depicts HF autoactivation; reaction 2 describes reciprocal activation of HF and prekallikrein, in which high-molecular-weight (HMW) kininogen functions as a cofactor. HFa, Activated Hageman factor.

TABLE 85-2 -- Testing Procedures for Urticaria and Angioedema Food and drug reactions

Elimination of offending agent, challenge with suspected foods, lamb and rice diet, special diets eliminating natural salicylates and food additives

Inhalant allergens

Skin tests, in vitro histamine release from human basophils, radioallergosorbent test

Collagen vascular diseases

Skin biopsy, CH50 , C4, C3, factor B, immunofluorescence of tissue

Malignancy with angioedema

CH50 , C1q, C4, Cl− INH determinations

Cold urticaria

Ice cube test

Solar urticaria

Exposure to defined wavelengths of light, red cell protoporphyrin, fecal protoporphyrin, and coproporphyrin

Dermographism

Stroking with narrow object (e.g., tongue blade, fingernail)

Pressure urticaria

Application of pressure for defined time and intensity

Vibratory angioedema

Vibration with laboratory vortex for 4 minutes

Aquagenic urticaria

Challenge with tap water at various temperatures

Urticariapigmentosa

Skin biopsy, test for dermographism

Hereditaryangioedema

C4, C2, C1− INH by protein and function

Familial cold urticaria

Challenge by cold exposure, measurement of temperature, white blood cell count, sedimentation rate, and skin biopsy

C3b inactivator deficiency

C3, factor B, C3b inactivator determinations

Idiopathic

Skin biopsy, immunofluorescence (negative), autologous skin test

CH50 , 50% hemolyzing dose for complement; C1− INH, C1 inhibitor.

1542

and the initial report noted a 12% incidence of thyroid immunity (antimicrosomal antibodies) in 140 consecutive patients with chronic hives.[ patients were euthyroid, although 8 of 17 fulfilled criteria for Hashimoto's

79 80 thyroiditis.[ ] [ ]

79]

Most of these

More recent reports confirmed these observations and noted that the

81 24%.[ ]

incidence of either antithyroglobulin or antimicrosomal antibodies or both is At presentation, many patients are already taking levothyroxine for hypothyroidism, and some present with biochemical evidence of either hypothyroidism or hyperthyroidism. The latter abnormality is most commonly associated with leakage of thyroid hormone as an early manifestation of Hashimoto's thyroiditis but may occasionally be due to Graves' disease. Women commonly complain that urticaria exacerbates at the time menses begin, and in some, cyclic urticaria is seen in association with menses. Although 2

immunologic reactions to endogenous hormones have been proposed,[ ] there is little evidence to support such a mechanism. However, hormones clearly have a role in modulating the severity of symptoms. The emotional status of the individual may also affect the course of urticaria and angioedema, as it does allergic rhinitis, atopic dermatitis, or asthma. The resolution of conflicts and the easing of frustrations and daily stresses of living can be of considerable assistance in successful management of urticaria and angioedema. The 2]

notion that psychogenic factors are responsible for chronic urticaria has been abandoned.[

Several forms of urticaria occur mainly in children. Papular urticaria is usually seen in young children, generally on the extremities and other exposed parts. Lesions appear to occur at the site of insect bites. Urticaria pigmentosa typically occurs in childhood and presents a distinctive clinical picture; urtication occurs after stroking of the skin (Darier's sign). Systemic mastocytosis is a more generalized disorder in which there is mast cell infiltration of the skin, skeletal system, liver, spleen, and lymph nodes. Erythema multiforme is a form of the urticaria-angioedema symptom complex in which typical iris or target lesions occur. Mucous membrane involvement is common and some cases may be bullous. Some patients have vasculitis with deposition of immune complexes, and a relationship to herpes simplex

viral infection is suggested.[

82]

PHYSICAL URTICARIA AND ANGIOEDEMA This group of disorders share the common property of being reproducibly induced by environmental factors such as a change in temperature or by direct stimulation of the skin from pressure, stroking, vibration, or light. Cold Urticaria and Related Disorders Cold urticaria is a disorder characterized by the rapid onset of pruritus, erythema, and swelling after exposure to a cold stimulus. The location of the swelling is confined to the portions of the body that have contacted the cold, although the symptoms are often maximal after the exposed area is warmed. Patients typically experience symptoms while outside on cold, windy days, but holding cold objects can cause hand swelling, and eating cold foods may cause lip swelling. Swelling of the tongue and pharynx are less common, and laryngeal edema or abdominal complaints are rarely, if ever, seen. Total body exposure such as occurs while swimming, can cause massive mediator release, resulting in hypotension. Because fatalities by drowning have been reported secondary to cold-induced hypotension, [83]

patients should be warned that swimming in cold water is dangerous.

This disease can begin in any age group and has no obvious gender predilection. If the diagnosis is suspected, a simple test that can confirm the initial impression is to place an ice cube on the patient's forearm for 4 minutes and observe the area for 10 minutes thereafter. If the patient has cold urticaria, the area will become pruritic about 2 minutes after removal of the ice cube, and by 10 minutes a large hive the shape of the ice cube will form ( Figure 85-2 ). Cold urticaria has been reported in association with a variety of diseases

Figure 85-2 Positive ice cube test in a patient with cold urticaria.

Figure 85-3 Histamine release and blood pressure recordings obtained after a challenge for cold urticaria performed by placing one hand in ice water for 4 minutes. Time 0 marks the time at which the hand was removed from the ice bath. (From Kaplan AP, Gray L, Snadt RE, et al: J Allergy Clin Immunol 55:394, 1975.)

(From Kaplan AP, Gray L, Snadt RE, et al: J Allergy Clin Immunol 55:394, 1975.) Challenge studies have documented the release of other mast cell constituents in addition to histamine. These include eosinophilotactic peptides, [ PAF,[

98]

PGD2 ,[

99]

and TNF-α. [

100]

TNF-α and IL-3 are up-regulated on endothelial cells 30 minutes after a cold challenge.[

95] [96]

97]

NCF,[

101]

102 103 104

Although this disease generally responds to antihistamines, cyproheptadine appears to be particularly effective.[ ] [ ] [ ] In my experience, 8 to 16•mg of cyproheptadine daily in divided doses renders the ice cube test negative in most patients and affords considerable objective relief of symptoms when they are exposed to cold. Cyproheptadine does not inhibit histamine release and appears to act as a classic histamine1 (H1 ) receptor antagonist.[

105]

Therefore, any

antihistamine given in sufficient dosage may be effective. Nevertheless, cyproheptadine remains the drug of choice. It is possible that vasoactive factors other than 105

histamine significantly contribute to the urticarial reaction of patients who do not respond well to cyproheptadine[ ] or other antihistamines or whose symptoms are less well correlated with histamine release. Attempts to desensitize patients using ice baths have been reported, but caution must be exercised because highly sensitive patients could have anaphylactoid symptoms. Because this approach is effective, it can be considered for patients who are unresponsive to alternative

therapeutic modalities. Studies of the mechanism by which a temperature change can lead to mediator release have failed to demonstrate the presence of an IgE cryoglobulin but have succeeded in obtaining the cold-dependent release of histamine from skin biopsy samples that were chilled and then warmed.[

106]

However,

1544

chilling and warming basophils did not result in histamine release, even in patients in whom IgE-mediated disease was demonstrable. Therefore, cutaneous mast cells seem to be essential. Although cryoaggregation of an abnormal protein might explain the cold dependence of the reaction, it is also possible that patients have an IgE antibody to a cold-induced skin antigen. Which (if either) of these possibilities is operative is not yet clear. The prognosis and severity of cold urticaria vary considerably. Occasionally the problem resolves spontaneously in a few months. However, it can last for many years, and the course is not predictable. Other cold-dependent syndromes have been reported, but their incidence is unknown. A delayed form of cold urticaria was described in which swelling appeared 9 to 107

18 hours after cold exposure.[ ] Studies of mediator release were unrevealing; the cold sensitivity could not be passively transferred, and biopsy of a lesion revealed edema and a mononuclear cell infiltrate. Family studies suggested a dominant mode of inheritance. A series of four patients was described in which exercise in a cold 108

environment induced hives similar to those seen with cholinergic urticaria[ ] ; however, hive formation did not occur if exercise was performed in a heated environment. This disorder, in which the cold exposure is systemic rather than local, should be suspected if a patient's symptoms suggest either cold urticaria or cholinergic urticaria and standard tests for each disorder are negative. Exercise in a cold room or running on a winter's day lead to generalized urticaria, which confirms the diagnosis. Because of the visual resemblance of the lesions to those typical of cholinergic urticaria, the disorder has been called cold-induced cholinergic urticaria. A study of 13 patients with symptoms suggestive of cold urticaria and cholinergic urticaria revealed 2 who did not have both disorders but had the cold-induced cholinergic type.[

109]

Another related disorder, called systemic cold urticaria, yields severe generalized hive formation on systemic cold challenge 110

occurring over covered or uncovered parts of the body. Symptoms are unrelated to exercise or other activities,[ ] and the results of the ice cube test are negative. Histamine release on cold challenge (with or without exercise, as appropriate) has been seen in cold-induced cholinergic urticaria, as well as in systemic cold urticaria. A treatment regimen of hydroxyzine plus cyproheptadine in high dosage has been used successfully. In addition, a disorder called cold-dependent 110

dermographism has been reported in which prominent hive formation is seen if the skin is scratched and then chilled.[ ] In this disorder the ice cube test and systemic cold challenge yield no hives. Simply scratching the skin yields a weakly positive dermographic response, but dramatic accentuation is seen when the scratched area is chilled. Treatment once again is high-dose antihistamines (e.g., diphenhydramine 200•mg/day or a combination of hydroxyzine 100 to 200•mg/day and cyproheptadine 8 to 16•mg/day). Experimental cromolyn-like drugs, which inhibit mast cell degranulation, are effective in controlling symptoms and in suppressing the positive response to the ice cube test in patients who are poorly responsive to cyproheptadine. Ketotifen, in particular, inhibits histamine release and is effective in a variety of physically induced urticarias. [

111]

Localized cold urticaria, in which only certain areas of the body are affected after cold contact, has been reported after predisposing conditions (e.g., cold injury) or

112

at sites of intracutaneous allergen injections, ragweed immunotherapy, or insect bites. Kurtz and Kaplan[ ] described a patient with cold urticaria confined to the head and face; in this case, there was no identifiable antecedent or associated event. Such cases argue against the presence of a circulating pathogenic factor and speak in favor of a local abnormality of mast cells. Finally, a disorder called localized cold reflex urticaria has been reported in which the ice cube test is positive but hives form in the vicinity of the contact site, not 113 114

where the ice cube is applied. [ ] [ ] The appearance of the hives resembles the punctate lesions of cholinergic urticaria, and there is no confluent hive where the ice cube is applied. A methacholine or acetylcholine skin test for cholinergic urticaria is negative, although the symptoms of one such patient resembled those of cold115]

induced cholinergic urticaria because exercise-induced hives were seen in a cold environment.[ 1. 2. 3. 4. 5. 6. 7. 8.

A summary of the cold-dependent syndromes follows:

Idiopathic cold urticaria Cold urticaria associated with abnormal serum proteins: cold agglutinins, cryoglobulin, cryofibrinogen, Donath-Landsteiner antibody Systemic cold urticaria Cold-induced cholinergic urticaria Cold-dependent dermatographism Delayed cold urticaria Localized cold urticaria Cold reflex urticaria.

Cholinergic Urticaria and Local Heat Urticaria Heat urticaria can occur in a local or a generalized form. The local form is observed when a warm stimulus is in contact with the skin; when suspected, it can be 116

tested for by applying a test tube of warm water at 44° C to the arm for 4 to 5 minutes.[ ] If the patient has the local form of heat urticaria, a hive is seen a few minutes after the test tube is removed. The local form is an extremely rare disorder, and studies have demonstrated histamine and NCF release on warming of the 2

skin, although some heterogeneity in disease pathogenesis may exist.[ ] Mast cell degranulation seems likely, although the results of passive transfer studies have 2

been negative. Association with other forms of physical allergy (e.g., combined cold urticaria and local heat urticaria) is also sometimes seen.[ ] Therapy has been problematic, because administration of antihistamines such as hydroxyzine and cyproheptadine, as well as oral cromolyn, has been ineffective. One patient was successfully desensitized by repeated daily immersion in hot baths, but this approach should be used with caution because systemic reactions are possible. A familial 2

variant of this disorder has been described in which urticaria occurred 1½ to 2 hours after application of a warm stimulus and persisted for 6 to 10 hours.[ ] Sunbathing with pronounced heating of the skin could produce wheals from the temperature effect, but sunlight itself was tolerated. Desensitization of an area could be achieved with repeated challenge every few days. Skin biopsy demonstrated a pronounced inflammatory cell infiltrate in the upper dermis and around hair follicles. The pathogenesis of this form of local heat urticaria is unknown; however, partial control could be achieved with oral administration of antihistamines.

1545

Generalized heat (cholinergic) urticaria is characterized by the onset of small, punctate wheals surrounded by a prominent erythematous flare associated with 117

exercise, hot showers, sweating, and anxiety.[ ] It tends to appear first over the upper thorax and neck but can spread distally to involve virtually the entire body. In some patients, these hives can become confluent and resemble angioedema, whereas other patients may have symptoms characteristic of cholinergic stimulation, such as lacrimation, salivation, and diarrhea. These various stimuli have the common feature of being mediated by cholinergic nerve fibers, which innervate the musculature by way of parasympathetic neurons and innervate the sweat glands by cholinergic fibers that travel with the sympathetic nerves.[ form, expressed only in males, with father to son transmission has been

118]

A rare familial

119 reported.[ ]

The characteristic lesion of cholinergic urticaria can be reproduced by an intradermal injection of 0.01•mg of methacholine in 0.1•ml of saline; a localized hive 2

surrounded by smaller satellite lesions is diagnostic.[ ] These patients therefore demonstrate a hypersensitivity to cholinergic mediators, but they have no evidence of an immunoglobulin-mediated allergy. However, only about one third of patients have a clearly positive skin test; these generally are the most severe cases. Challenge by exercise (e.g., running in a room at 85° F or using a bicycle ergometer for 10 to 15 minutes) is a far more sensitive test. The skin test can be used to confirm the diagnosis but cannot be used as a diagnostic test.[

84] [120]

This disease possibly involves an intrinsic cellular abnormality that leads to mediator release in the 121

presence of cholinergic agents. One study addressing this issue demonstrated an increased number of muscarinic receptors in urticarial sites.[ ] The number of these receptors was further augmented when exercise followed skin patch testing to copper-containing materials. The increased number of acetylcholine-binding sites may be an important key to understanding the pathogenesis of cholinergic urticaria. The importance of copper is unclear, but it may affect ligand-receptor affinity. Evidence that a neurogenic reflex is involved is provided by the observation that placing a patient's hand in warm water with a tourniquet tied proximally does not cause localized hives, whereas removal of the tourniquet can lead to a generalized eruption. Therefore a central perception of the temperature change appears to be followed by an efferent reflex, leading to the urticaria. Such a reflex could also account for the association of hives with anxiety, although it should be emphasized that in these instances the emotional reaction itself may be normal. A photograph of typical cholinergic urticaria is shown in Figure 85-4 ; in this case, the lesions were induced by having the patient run in place for 10 minutes. Studies of mediator release during attacks of cholinergic urticaria demonstrated that, in most cases, elevated plasma histamine levels parallel the onset of pruritus and urticaria[

91]

122] [123] [124]

( Figure 85-5 ). Subsequent studies confirmed the presence of histaminemia in association with cholinergic urticaria [ 122

and reported the

release of eosinophilotactic peptides and NCF.[ ] When patients were challenged while wearing a plastic occlusive suit to produce maximal changes in cutaneous and core body temperature, significant falls in 1-second forced expiratory volume, maximum midexpiratory flow rate, and specific conductance were associated with a rise in residual volume. Four of seven patients also had wheezing detected by auscultation. Therefore, under such conditions, an abnormality in pulmonary function can be detected, reflecting either primary pulmonary involvement or altered pulmonary mechanics secondary to circulating mediators. A clinically significant alteration in pulmonary function is unusual in cholinergic urticaria, and it has no known association with exercise-induced asthma. The drug of choice for the management of cholinergic urticaria is hydroxyzine, and most patients respond to a dose

Figure 85-4 Hives induced in a patient with cholinergic urticaria after running in place for 10 minutes.

Figure 85-5 Time Course of histamine release in three patients with cold-induced cholinergic urticaria who were subjected to a 4° F environment for 15 minutes. (From Kaplan AP, Garofalo J: J Allergy Clin Immunol 68:438, 1981.)

(From Kaplan AP, Garofalo J: J Allergy Clin Immunol 68:438, 1981.) 123

of 100 and 200•mg/24 hours.[ ] Hydroxyzine is considerably more effective than conventional antihistamines, suggesting that it has some additional action; it might, for example, interrupt the neurogenic reflex that leads to the urticaria. Anticholinergic agents given orally are generally ineffective, perhaps because of an 124]

inability to attain a sufficient systemic level. However, injected atropine can reverse the results of the methacholine skin test. [ hydroxyzine, is also effective at 20•mg/day (i.e., twice the usual whom this dose of cetirizine is insufficient.

125 dose).[ ]

Cetirizine, the active metabolite of

Nevertheless, the aforementioned dose of hydroxyzine is effective in many patients for

107] [124]

Patients with combined cold and cholinergic urticaria have been reported, and the time course of the histamine release is characteristic of each disorder.[ Such patients respond well to therapy with a combination of hydroxyzine and cyproheptadine but should be distinguished from patients with cold-induced cholinergic urticaria. [

108]

Exercise-Induced Anaphylaxis The syndrome of exercise-induced anaphylaxis was first described in a series of patients in whom combinations of pruritus, urticaria, angioedema, wheezing, and hypotension occurred as a result of exercise. Symptoms did not occur with each exercise experience, and most of the patients were accomplished athletes. [ The disorder is distinguished from cholinergic urticaria by the following criteria:

126] [127]

1. Although exercise is the precipitating stimulus of each disorder, hot showers, sweating in the absence of exercise, and anxiety do not trigger attacks of 117

exercise-induced anaphylaxis, as they do in cholinergic urticaria.[ ] 2. The hives seen with exercise-induced anaphylaxis are large (10 to 15•mm) rather than the small, punctate lesions that characterize cholinergic urticaria. 3. When patients with exercise-induced anaphylaxis were challenged while wearing an occlusive body suit, no change in pulmonary function was seen, 128]

although histamine release was documented.[ 123

My colleagues and I[ ] described two cases of typical cholinergic urticaria in which lesions became confluent, were associated with prominent elevation of plasma histamine, and were associated with recurrent episodes of hypotension. Therefore, it is clear that some extreme cases of cholinergic urticaria can resemble the exercise-induced anaphylactic syndrome. An important distinguishing feature is that an increase in core body temperature of greater than 0.7° C using hyperthermic blankets or submersion in warmed water causes hives, histamine release, and anaphylactic symptoms in cholinergic urticaria patients with anaphylactic-like 128]

symptoms but not in patients with the exercise-induced anaphylactic syndrome.[ 129

Lewis et al[ ] observed six patients with exercise-induced symptoms. On exercise challenge, three patients developed cholinergic urticaria; two of these individuals had associated angioedema, and one became hypotensive. Challenge in one patient caused giant hives and hypotension (clearly exercise-induced anaphylaxis), whereas challenge in another patient caused periorbital angioedema without hive formation. Therefore, various combinations of exercise-induced hives, angioedema, and hypotension are possible. More recent studies have demonstrated histologic changes consistent with mast cell degranulation on exercise challenge of such

130

patients.[ ] Increased levels of plasma histamine were also documented but did not necessarily correlate with the induction of any particular symptom. There is no general agreement regarding therapy for exercise-induced anaphylactic syndrome, and attempts at prophylaxis using H1 and H2 antagonists have been ineffective. [129]

In contrast, classic cholinergic urticaria is usually responsive to the prophylactic use of hydroxyzine. 126] [127]

Patients with exercise-induced anaphylactic syndrome, in contrast to most other “physical urticarias,” have a high incidence of atopy (half to two thirds)[

as

126 urticaria.[ ]

do other types of physically induced hives, particularly cold Subtypes of exercise-induced anaphylaxis that are food-related have also been described. In one case, exercise-induced anaphylaxis occurred if the exercise was taken between 5 and 24 hours after eating shellfish, whereas exercise alone or eating shellfish 130]

alone did not cause any symptoms.[

In five other reported cases, two patients had symptoms only if exercise occurred within 2 hours after the ingestion of any

131 132 food,[ ] [ ]

131

whereas in three other patients symptoms were precipitated by the specific ingestion of celery within 2 hours of exercise.[ ] These latter patients also had positive skin tests to celery. Therefore, various forms of food-dependent, exercise-induced anaphylaxis are possible, and treatment requires avoidance of specific foods before exercising or avoidance of exercise within certain time intervals after eating. Patients usually continue to exercise but carry an epinephrine kit and 133

diphenhydramine with them. There have been no reported fatalities. Kivity et al[ ] performed skin tests to compound 48/80 (a cutaneous mast cell degranulating agent) and histamine and demonstrated augmented wheal responses to compound 48/80 but not to histamine in subjects with food-induced (skin test–positive), exercise-induced anaphylaxis. Food or exercise alone did not affect the compound 48/80 response, suggesting that increased mast cell releasability was caused by the combination of food plus exercise. Also, in one patient with cholinergic urticaria and associated anaphylaxis, vigorous daily exercise caused desensitization within 1 123

week.[ ] The number of patients with cholinergic urticaria or exercise-induced anaphylactic syndrome that might respond to such treatment is unknown, although it is more likely to be effective in cholinergic urticaria with or without hypotension, because in these cases symptoms recur each time an exercise challenge is done.

1547

Pressure Urticaria (Angioedema) Pressure-induced urticaria differs from most of the aforementioned types of hives or angioedema in that symptoms typically occur 4 to 6 hours after pressure has 134

been applied; it is therefore also designated “delayed pressure urticaria.”[ ] The disorder is clinically heterogeneous because some patients complain of swelling secondary to pressure with normal-appearing skin (i.e., no erythema or superficial infiltrating hive); therefore, the term angioedema is more appropriate. In other patients, the predominant condition is urticaria that may be associated with significant swelling. When urticaria is present, an infiltrative lesion is seen, consistent 135]

with biopsy studies, that is characterized by a perivascular mononuclear cell infiltrate and dermal edema similar to that seen with chronic urticaria.[ prominent infiltration with neutrophils and

135 eosinophils,[ ]

expression of

136 IL-6,[ ]

There is a

and sequential up-regulation of endothelial-leukocyte adhesion molecule-1 137

(ELAM-1) at 6 hours and vascular cell adhesion molecule-1 (VCAM-1) at 24 hours in endothelial cells.[ ] Immediate dermographism is not present, but delayed dermographism is seen and may represent the same disorder. Symptoms occur around tight clothing, the hands may swell with activity such as hammering, foot swelling is common after walking, and buttock swelling may be prominent after sitting for a few hours. Testing can be done using a sling with a 5- to 15-pound weight attached that is placed over the forearm or shoulder for 10 to 20 minutes. Devices that apply gradual pressure (in grams per square millimeter) can also be

136]

used.[

There are few available studies regarding pathogenesis; however, mediators that cause pain rather than pruritus (e.g., kinins) have been considered, because the lesions are typically described as burning or painful. Nevertheless, induced blisters over lesions revealed histamine release after hive formation.[

138]

Antihistamines,

139 corticosteroids.[ ]

however, have little effect on the disorder, and patients with severe disease often must be treated with For chronic treatment, I use the lowest dose, given on alternate days, that allows a significant diminution of symptoms. The usual range required is 15 to 30•mg of prednisone every other day. Although pressure urticaria (angioedema) can occur as an isolated disorder, it is most often seen in association with chronic urticaria, in which case therapy is usually directed toward the chronic urticaria. Immediate pressure urticaria has been described in patients with hypereosinophilic syndrome, characterized by an acute wheal-and-flare reaction within 1 to 2 minutes of applied pressure (e.g., pressing on the back with the thumb). These patients also had dermographism; however, dermatographic patients typically do not 140]

have immediate pressure urticaria and require a stroking motion to produce a hive. [

DERMOGRAPHISM The ability to “write on skin,” termed dermographism, can occur as an isolated disorder that often presents as traumatically induced urticaria. It can be diagnosed by observing the skin after it is stroked with a tongue blade or fingernail. In these patients, the white line that occurs secondary to reflex vasoconstriction is followed by 141 142

pruritus, erythema, and linear swelling, as in a classic wheal-and-flare reaction. This condition is said to be present in 2% to 5% of the population[ ] [ ] ; however, in only a small fraction of these patients is it sufficiently severe to warrant treatment. In approximately 50% of patients, passive transfer studies have suggested an 143

IgE-mediated reaction,[ ] but an antigen has not been identified. Therefore, many patients have an abnormal circulating IgE that confers a particular form of pressure sensitivity to dermal mast cells. Such observations further suggest that histamine is one of the mediators of dermatographism, although demonstration of 2

such release has been difficult because of the localized nature of the reaction. Early studies, however, did suggest that histamine is released into whole blood,[ ] that 115]

induced blisters over lesions contain elevated histamine levels,[

2

that 24-hour urine histamine levels are elevated,[ ] and that the histamine level is increased in the

144

perfusate, as shown by in vivo subcutaneous perfusion studies.[ ] In a single, unusually severe case of IgE-mediated dermographism, elevation of the plasma histamine concentration was documented within 1 minute after stroking of the skin, and the baseline histamine level was abnormal in multiple determinations, suggesting that “leakage” of histamine was ongoing at all times.[

145]

Although the finding is anecdotal because a formal study has not yet been performed, dermographism has been observed as a consequence of drug reactions. [ one case, dermographism could be observed only on challenge with the offending agent, in this instance penicillin.

146]

[147]

The drug of choice for this disease is diphenhydramine, although patients may prefer other antihistamines, depending on their relative efficacies as opposed to adverse side effects. Newer, nonsedating antihistamines are effective when symptoms are mild. The initial objective of therapy is to decrease pruritus so that the stimulation for scratching is diminished. Many patients complain of a sensation of itching or “skin crawling” that is readily relieved by antihistamines. At higher doses, the wheal-and-flare reaction to stroking is also markedly diminished. Dermographism may also be seen in patients with chronic urticaria from a variety of

In

different causes (see later discussion). Severe dermographism is associated with urticaria pigmentosa or systemic mastocytosis, and in systemic disease the wheals may last for many hours. High doses of antihistamines are required to relieve the symptoms. Other vasoactive agents released from cutaneous mast cells can be implicated in dermographism and, in fact, in all forms of physically induced urticaria. An example is the elevation of PGD2 metabolites in the urine of patients with systemic mastocytosis, which may relate to the hypotension associated with this disorder.[

148]

Solar Urticaria Solar urticaria is a rare disorder in which brief exposure to light causes the development of urticaria within 1 to 3 minutes. Typically, pruritus occurs first, at about 30 seconds, followed by erythema and edema confined to the light-exposed area and surrounded by a prominent erythematous zone caused by an axon reflex. The lesions then usually disappear within 1 to 3 hours. If large areas of the body are exposed, systemic symptoms, including hypotension and wheezing, may occur. Although most patients reported have been in the third and fourth decades, this condition can occur in any age group and has no association with other allergic disorders.

1548

Solar urticaria has been classified into six types, depending on the wavelength of light that induces lesions and the ability or inability to passively transfer the disorder with serum.[

115] [149] [150]

Types I and IV can be passively transferred and may therefore be antibody (IgE) mediated; and are associated with wavelengths 151]

of 2800 to 3200 Å and 4000 to 5000 Å, respectively. The antigen has not been identified. The release of histamine[

and chemotactic factors for neutrophils and

152 eosinophils[ ]

after experimental challenge of patients with solar urticaria has been reported, suggesting degranulation of mast cells. Type VI solar urticaria, activated at 4000 Å, is clearly a metabolic disorder in which protoporphyrin IX acts as a photosensitizer; it is synonymous with erythropoietic protoporphyria and is 153

caused by ferrochelatase deficiency.[ ] In contrast to other forms of porphyria, the level of porphyrin excreted in the urine is normal; however, levels of protoporphyrin in red blood cells and levels of protoporphyrin and coproporphyrin in the feces are increased. There is evidence for complement activation when the plasma of patients with protoporphyria is exposed to light of appropriate wavelengths.[ the serum level of

155 protoporphyrin.[ ]

There is generation of C5a chemotactic activity, which is proportional to

Irradiation of the forearms of two patients with this disorder also resulted in in vivo complement activation, as assessed by a

diminution of titers of C3 and C5 and generation of C5a.[ are seen in the dermis of

154]

156 157 patients,[ ] [ ]

156]

Consistent with these observations are the facts that deposition of C3 and accumulation of neutrophils

and that complement fragments can be detected in the serum and suction blister fluid of irradiated skin.[

responds to oral β-carotene, which absorbs light at the same wavelengths as protoporphyrin

156]

The disease

158 IX.[ ]

The mechanism producing light urticaria in types II, III, and V is unknown, but these conditions are induced by inciting wavelengths of 3200 to 4000 Å, 4000 to 5000 Å, and 2800 to 5000 Å, respectively, and cannot be passively transferred with serum. As a simple screen, fluorescent tubes that emit a broad, continuous spectrum can be used to test the patient, and filters can then be used to define the spectrum that causes urticaria. Ordinary window glass 3•mm thick will absorb most

ultraviolet radiation of wavelengths shorter than 3200 Å and will thereby protect patients with type I solar urticaria. Therapy requires administration of antihistamines, avoidance of sunlight, protective garments to cover the skin, and use of topical preparations to absorb or reflect light. No single modality of treatment 159

is uniformly effective.[ ] A 5% solution of para-aminobenzoic acid in ethanol, as in sunscreen lotions, can be helpful in the 2800- to 3200-Å range; however, it is more difficult to screen out the visible spectrum. The most effective agents for this purpose contain titanium oxide, zinc oxide, or both. The efficacy of antihistamine, antimalarial, and corticosteroid agents or psoralen photochemotherapy in these disorders is not clear and must be evaluated for each type. Aquagenic Urticaria Thirteen patients who developed small wheals after contact with water, regardless of its temperature, have been reported. These wheals were distinguishable from 160 161

cold urticaria or cholinergic urticaria. This entity has been termed aquagenic urticaria[ ] [ ] and can be tested for by direct application of a compress of tap water or distilled water to the skin. The diagnosis should be reserved for individuals who have a rare positive test to water in whom tests for all other forms of physical urticaria are negative. Combined cholinergic urticaria and aquagenic urticaria has been reported, and histamine release into the circulation was documented on challenge with water. [

162]

HEREDITARY FORMS OF URTICARIA AND ANGIOEDEMA Familial Cold Urticaria Familial cold urticaria is a rare form of cold intolerance that is inherited as an autosomal dominant trait and histologically is not truly urticaria. After exposure to cold, a systemic reaction occurs consisting of burning papular skin lesions accompanied by fever, chills, arthralgias, myalgias, headache, and leukocytosis. Biopsy of the skin lesions reveals edema and an intense inflammatory infiltrate consisting almost entirely of polymorphonuclear leukocytes, with eosinophils prominent around 163

163

dilated capillaries.[ ] Studies have confirmed a linkage to chromosome 1q44[ ] ; the most common manifestations were rash (100%), fever (92%), arthralgias (96%), and surprisingly, conjunctivitis (84%). The average delay between cold exposure and the onset of symptoms was 2.5 hours, and episodes lasted an average of 163]

12 hours.[

Hereditary Vibratory Angioedema Hereditary vibratory angioedema has been described in a single family in whom it was inherited in an autosomal dominant pattern. It is properly viewed as a 164

physically induced angioedema, because patients complain of intense pruritus and swelling within minutes after vibratory stimuli.[ ] The patients do not have dermographism or pressure urticaria. Lesions can be reproduced by gently stimulating the patient's forearm with a laboratory vortex for 4 minutes. Rapid swelling of 165]

the entire forearm and a portion of the upper arm ensues, and histamine has been shown to be released secondary to such a vibratory stimulus.[

With care, patients 166]

can avoid vibratory stimuli, and their symptoms can otherwise be partially relieved with diphenhydramine. Nonfamilial sporadic cases have also been described.[ [167]

C3b Inactivator Deficiency

168

Three patients deficient in the C3b inactivator (factor I) have been reported.[ ] The complement profile in these patients suggests spontaneous activation of the alternative complement pathway, consisting of depletion of factor B, markedly depressed C3 levels, and a circulating inactive C3 byproduct, without evidence of 168

further degradation to C3c or C3d.[ ] However, intravenous replacement with C3b inactivator leads to elevation of the patient's C3 level. Of particular interest, some patients have histaminuria, and one of the presenting symptoms can be urticaria. This may reflect rapid degradation of C3 with liberation of C3a anaphylatoxin. Family members can have half-normal levels of the C3b inactivator, suggesting an autosomal recessive inheritance.

1549

Urticaria and Amyloidosis 169 170

Familial urticaria has also been seen with amyloidosis, nerve deafness, and limb pain.[ ] [ ] It appears to be inherited as an autosomal dominant condition in which the urticaria has features of cholinergic urticaria, angioedema, or classic hives. The rash is associated with limb pain and seems to be intensified by cold weather. Like familial cold urticaria, it is linked to chromosome 1q44 and is synonymous with Muckle-Wells syndrome. Hereditary Angioedema and Acquired C1− INH Deficiency Hereditary angioedema is an autosomal dominant disorder caused by the absence of C1− INH,[ ] in which patients may have attacks of swelling involving almost any portion of the body. A traumatic episode can initiate an attack. However, such a triggering event may not be evident, and the swelling appears to occur spontaneously. It is not associated with urticaria, and patients with both urticaria and angioedema without a family history invariably have a normal C1− INH concentration. In addition to the family history, the presence of visceral involvement suggests the hereditary disorder. The most severe complication is laryngeal edema, which has been a major cause of mortality in this disease. Patients can also have abdominal attacks lasting 1 to 2 days, consisting of vomiting, severe abdominal pain, and guarding in the absence of fever, leukocytosis, or abdominal rigidity. This can occasionally be difficult to distinguish from an acute abdominal 171

172

condition; however, the attacks are self-limited and are caused by edema of the bowel wall.[ ] Figure 85-6 demonstrates angioedema of the hand in a patient with hereditary angioedema. The ultrastructural lesion seen in tissues of patients with hereditary angioedema consists of gaps in the postcapillary venule endothelial cells, edema, and virtually no cellular infiltrate, [

2]

Figure 85-6 Angioedema of the hand in a patient with hereditary angioedema.

Figure 85-7 Diagram of the pathogenesis of hereditary angioedema, indicating the pathways leading to kinin formation and the steps inhibited by C1− inactivator (C1 − INH). HF, Hageman factor; HFa, activated Hageman factor; HFf, Hageman factor fragment; HMW, high molecular weight.

Figure 85-8 Typical raised lesions in a patient with chronic idiopathic urticaria.

Figure 85-9 Skin biopsy specimen from a patient with chronic idiopathic urticaria (hematoxylin and eosin stain).

Figure 85-10 Diagram of the activation of cutaneous mast cells by immunoglobulin G (IgG) antibody directed to the immunoglobulin E (IgE) receptor.

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119. Onn A, Levo Y, Kivity S: Familial cholinergic urticaria, J Allergy Clin Immunol 98:847, 1996. 120. Commens CA, Greaves CA: Tests to establish the diagnosis in cholinergic urticaria, Br J Dermatol 98:47, 1978. 121. Shelley WB, Shelley CD, Ho AK: Cholinergic urticaria: acetylcholine-receptor dependent immediate-type hypersensitivity reaction to copper, Lancet 1:843, 1983.

122. Soter NA, Wasserman SI, Austen KF, et al: Release of mast-cell mediators and alterations in lung function in patients with cholinergic urticaria, N Engl J Med 302:604, 1980. 123. Kaplan AP, Natbony SF, Tawil AP: Exercise-induced anaphylaxis as a manifestation of cholinergic urticaria, J Allergy Clin Immunol 68:319, 1981. 124. Sigler RW, Levinson AI, Evans R III, et al: Evaluation of a patient with cold and cholinergic urticaria, J Allergy Clin Immunol 63:35, 1979. 125. Zuberier T, Münzberger O, Haustein U, et al: Double blind crossover study of high-dose cetirizine in cholinergic urticaria, Dermatology 193:324, 1996. 126. Sheffer AL, Austen KF: Exercise-induced anaphylaxis, J Allergy Clin Immunol 66:106, 1980. 127. Sheffer AL, Soter NA, McFadden ER Jr, et al: Exercise-induced anaphylaxis: a distinct form of physical allergy, J Allergy Clin Immunol 71:311, 1983. 128. Casale TB, Keahey TM, Kaliner M: Exercise-induced anaphylactic syndromes: insights into diagnostic and pathophysiologic features, JAMA 255:2049, 1986. 129. Lewis J, Lieberman P, Treadwell G, et al: Exercise-induced urticaria, angioedema, and anaphylactoid episodes, J Allergy Clin Immunol 68:432, 1981. 130. Maulitz RM, Pratt DS, Schocket AL: Exercise-induced anaphylactic reaction to shellfish, J Allergy Clin Immunol 63:433, 1979. 131. Kidd JM III, Cohen SH, Sosman AJ, et al: Food dependent exercise-induced anaphylaxis, J Allergy Clin Immunol 71:407, 1983. 132. Novey HS, Fairshter RD, Salness K, et al: Postprandial exercise-induced anaphylaxis, J Allergy Clin Immunol 71:498, 1983. 133. Kivity S, Sneh E, Greif J, et al: The effect of food and exercise on the skin response to compound 48/80 in patients with food-associated exercise-induced urticaria-angioedema, J Allergy Clin Immunol 81:1155, 1987. 134. Estes SA, Yang CW: Delayed pressure urticaria: an investigation of some parameters of lesion induction, J Am Acad Dermatol 5:25, 1981. 135. McEvoy MT, Peterson EA, Black A, et al: Immunohistological comparison of granulated cell proteins in induced immediate urticarial dermographism and delayed pressure urticaria lesions, Br J Dermatol 133:853, 1995. 136. Lawlor F, Bird C, Camp RD, et al: Increased interleukin 6 but reduced interleukin 1 in delayed pressure urticaria, Br J Dermatol 128:520, 1993. 137. Barlow RJ, Ross EL, MacDonald D, et al: Adhesion molecule expression and the inflammatory cell infiltrate in delayed pressure urticaria, Br J Dermatol 131:341, 1994. 138. Kaplan AP, Horakova Z, Katz SI: Assessment of tissue fluid histamine levels in patients with urticaria, J Allergy Clin Immunol 61:350, 1978. 139. Dover JS, Kobza-Black A, Ward AM, et al: Delayed pressure urticaria: clinical features, laboratory investigations, and response to therapy of 44 patients, J Am Acad Dermatol 18:1289, 1988. 140. Parrillo JE, Lawley TJ, Frank MM: Immunologic reactivity in the hypereosinophil syndrome, J Allergy Clin Immunol 64:113, 1979.

141. Mathews KP: Urticaria and angioedema, J Allergy Clin Immunol 72:1, 1983. 142. Soter NA, Wasserman SI: Physical urticaria/angioedema: an experimental model of mast cell activation in humans, J Allergy Clin Immunol 66:358, 1980. 143. Newcomb RW, Nelson H: Dermatographism mediated by IgE, Am J Med 54: 174, 1973. 144. Greaves MW, Sondergaard J: Urticaria pigmentosa and factitious urticaria: direct evidence for release of histamine and other smooth muscle-contracting agents in dermatographic skin, Arch Dermatol 101:418, 1970. 145. Garofalo J, Kaplan AP: Histamine release and therapy of severe dermatographism, J Allergy Clin Immunol 68:103, 1981. 146. Mathews KP: Urticaria and angioedema, J Allergy Clin Immunol 72:1, 1983. 147. Smith JA, Mansfield LE, Fokakis A, et al: Dermatographism caused by IgE mediated penicillin allergy, Ann Allergy 57:30, 1983. 148. Roberts LJ II, Sweetman BJ, Lewis RA: Increased production of prostaglandin D2 in patients with systemic mastocytosis, N Engl J Med 303:1400, 1980. 149. Harber LC, Holloway RM, Sheatley VR, et al: Immunologic and biophysical studies in solar urticaria, J Invest Dermatol 41:439, 1963. 150. Sams WM Jr, Epstein JH, Winkelmann RK: Solar urticaria: investigation of pathogenic mechanisms, Arch Dermatol 99:390, 1969. 151. Hawk JL, Eady RA, Challiner AV: Elevated blood histamine levels and mast cell degranulation in solar urticaria, Br J Clin Pharmacol 9:183, 1980. 152. Soter NA, Wasserman SI, Pathak MA: Solar urticaria: release of mast cell mediators into the circulation after experimental challenge, J Invest Dermatol 72:283, 1979. 153. Bonkowsky HL, Bloomer JR, Ebert PS, et al: Heme synthetase deficiency in human protoporphyria: demonstration of the defect in liver and cultured skin fibroblasts, J Clin Invest 56:1139, 1975. 154. Lim HW, Perez HD, Poh-Fitzpatrick M: Generation of chemotactic activity in serum from patients with erythropoietic protoporphyria and porphyria cutanea tarda, N Engl J Med 304:212, 1981. 155. Gigli I, Schothorst AA, Soter NA, et al: Erythropoietic protoporphyria: photoactivation of the complement system, J Clin Invest 66:517, 1980. 156. Lim HW, Poh-Fitzpatrick MB, Gigli I: Activation of the complement system in patients with porphyrias after irradiation in vivo, J Clin Invest 74:1961, 1984. 157. de la Faille HB, Beerens EG, Van Weelden H, et al: Complement components in blood serum and suction blister fluid in erythropoietic protoporphyria, Br J Dermatol 99:401, 1978. 158. Moshell AN, Bjornson L: Protection in erythropoietic protoporphyria: mechanism of protection by β carotene, J Invest Dermatol 68:157, 1977. 159. Uetsa N, Miyauchi-Hashimoto H, Okamoto T: The clinical and photobiological characteristics of solar urticaria in 40 patients, Br J Dermatol 142:32, 2000.

160. Chalamidas SL, Charles CR: Aquagenic urticaria, Arch Dermatol 104:541, 1971. 161. Tromovitch TA: Urticaria from contact with water, Calif Med 106:400, 1967. 162. Davis RS, Remigio LK, Schocket AL, et al: Evaluation of a patient with both aquagenic and cholinergic urticaria, J Allergy Clin Immunol 68:479, 1981. Hereditary Forms of Urticaria and Angioedema 163. Hoffman HM, Wanderer AA, Broide DH: Familial cold autoinflammatory syndrome: phenotype and genotype of an autosomal dominant periodic fever, J Allergy Immunol 108:615, 2001. 164. Patterson R, Mellies CJ, Blankenship ML, et al: Vibratory angioedema: a hereditary type of physical hypersensitivity, J Allergy Clin Immunol 50:174, 1972. 165. Metzger WJ, Kaplan AP, Beaven MA: Hereditary vibratory angioedema: confirmation of histamine release in a type of physical hypersensitivity, J Allergy Clin Immunol 57:605, 1976. 166. Tong S, Reiman BE, Rauls DO, et al: On familial vibration-induced angioedema, J Allergy Clin Immunol 71:546, 1983. 167. Weiner MH, Metzger J, Simon RA: Occupationally acquired vibratory angioedema with secondary carpal tunnel syndrome, Ann Intern Med 98:44, 1983. 168. Alper CA, Rosen FS, Lachmann PJ: Inactivator of the third component of complement as an inhibitor of the properdin pathway, Proc Natl Acad Sci USA 69:2910, 1972. 169. Muckle TJ, Wells M: Urticaria, deafness, and amyloidosis: a new heredo-familial syndrome, Q J Med 31:235, 1962. 170. Anderson V, Buch NH, Jensen MK, et al: Deafness, urticaria, and amyloidosis: a sporadic case with chromosomal aberration, Am J Med 42:449, 1967. 171. Donaldson VH, Evans RR: A biochemical abnormality in hereditary angioneurotic edema, Am J Med 35:37, 1963. 172. Pearson KD, Buchignani JS, Shimkin PM, et al: Hereditary angioneurotic edema of the gastrointestinal tract, AJR Am J Roentgenol Radium Ther Nucl Med 116:256, 1972. 173. Zuraw BL, Sugimoto S, Curd JG: The value of rocket immunoelectrophoresis for C4 activation in the evaluation of patients with angioedema or C1-inhibitor deficiency, J Allergy Clin Immunol 78:1115, 1986. 174. Klemperer MR, Donaldson VH, Rosen FS: The vasopermeability response in man to purified C1 esterase, J Clin Invest 47:604, 1968 (abstract). 175. Donaldson VH: Mechanisms of activation of C1 esterase in hereditary angioneurotic edema plasma in vitro: the role of Hageman factor, a clot-promoting agent, J Exp Med 127:411, 1968. 176. Curd JG, Yelvington M, Burridge N, et al: Generation of bradykinin during incubation of hereditary angioedema plasma, Mol Immunol 19:1365, 1983.

177. Fields T, Ghebrehiwet B, Kaplan AP: Kinin formation in hereditary angioedema plasma: evidence against kinin derivation from C2 and in support of “spontaneous” formation of bradykinin, J Allergy Clin Immunol 72:54, 1983. 178. Strang CJ, Cholin S, Spragg J, et al: Angioedema induced by a peptide derived from complement component C2, J Exp Med 168:1685, 1988. 179. Carpenter CB, Ruddy S, Shehadeh IH, et al: Complement metabolism in man: hypercatabolism of the fourth (C4) and third (C3) components in patients with renal allograft rejection and hereditary angioedema, J Clin Invest 48:1495, 1969. 180. Schreiber AD, Kaplan AP, Austen KF: Inhibition by CI INH of Hageman factor fragment activation of coagulation, fibrinolysis, and kinin generation, J Clin Invest 52:1402, 1973. 181. Ghebrehiwet B, Silverberg M, Kaplan AP: Activation of the classical pathway of complement by Hageman factor fragment (HFf), J Exp Med 153:665, 1981. 182. Ghebrehiwet B, Randazzo BP, Dunn JT, et al: Mechanism of activation of the classical pathway of complement by Hageman factor fragment, J Clin Invest 71:1450, 1983. 183. Curd JG, Prograis LF Jr, Cochrane CG: Detection of active kallikrein in induced blister fluids of hereditary angioedema patients, J Exp Med 152:742, 1980. 184. Schapira M, Silver LD, Scott CF, et al: Prekallikrein activation and high molecular weight kininogen consumption in hereditary angioedema, N Engl J Med 308:1050, 1983. 185. Zahedi R, Bissler JJ, Davis III AE, et al: Unique C1 inhibitor dysfunction in a kindred without angioedema II: identification of a Ala443 (Val substitution and functional analysis of the recombinant mutant protein, J Clin Invest 95:1299, 1995. 186. Zahedi R, Wisneski J, Davis III AE: Role of the P2 residue of complement 1 inhibitor Ala443 ) in determination of target protease specificity: inhibition of complement and contact proteases, J Immunol 159:985, 1997. 187. Nussberger J, Amstutz Cugno M, Amstutz C, et al: Plasma bradykinin in angioedema, Lancet 351:1693, 1998. 188. Cicardi M, Coppola R, Agostini A: Activation of factor XII and cleavage of high molecular weight kininogen during acute attacks in hereditary angioedema and acquired C1 inhibitor deficiencies, Immunopharmacology 33:361, 1996. 189. Nussberger J, Cugno M, Cicardi M, Agostini A: Local bradykinin generation in hereditary angioedema, J Allergy Immunol 104:1321, 1999. 190. Davis PJ, Davis FB, Charache P: Long-term therapy of hereditary angioedema (HAE): preventive management with fluoxymesterone and oxymetholone in severely affected males and females, Johns Hopkins Med J 135:391, 1974. 191. Frank MM, Sergent JS, Kane MA, et al: Epsilon aminocaproic acid therapy of hereditary angioneurotic edema: a double blind study, N Engl J Med 286:808, 1972.

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192. Sheffer AL, Austen KF, Rosen FS: Tranexamic acid therapy in hereditary angioneurotic edema, N Engl J Med 287:452, 1972. 193. Soter NA, Austen KF, Gigli I: Inhibition by epsilon-aminocaproic acid of the activation of the first component of the complement system, J Immunol 114:928, 1975. 194. Gelfand JA, Sherms RJ, Alling DW, et al: Treatment of hereditary angioedema with danazol: reversal of clinical and biochemical abnormalities, N Engl J Med 295:1444, 1976. 195. Sheffer AL, Fearon DT, Austen KF: Clinical and biochemical effects of stanazolol therapy for hereditary angioedema, J Allergy Clin Immunol 68:181, 1981. 196. Warin AP, Greaves MW, Gatecliff M, et al: Treatment of hereditary angioedema by low-dose attenuated androgens: dissociation of clinical response from levels of C1 esterase inhibitor and C4, Br J Dermatol 103:405, 1980. 197. Sheffer AL, Fearon DT, Austen KF: Hereditary angioedema: a decade of management with stanozolol, J Allergy Clin Immunol 80:855, 1987. 198. Gadek JE, Hosea SW, Gelfand JA, et al: Response of variant hereditary angioedema phenotypes of danazol therapy, J Clin Invest 64:280, 1979. 199. Skriver K, Radziejewska E, Silbermann JA, et al: CpG mutations in the reactive site of human C1 inhibitor, J Biol Chem 264:3066, 1989. 200. Quastel M, Harrison R, Cicardi M, et al: Behavior in vivo of normal and dysfunctional C1 inhibitor in normal subjects and patients with hereditary angioedema, J Clin Invest 71:1041, 1983. 201. Caldwell JR, Ruddy S, Schur P, et al: Acquired C1 inhibitor deficiency in lymphosarcoma, Clin Immunol Immunopathol 1:39, 1972. 202. Hauptmann G, Lang JM, North ML, et al: Acquired C1 inhibitor deficiencies in lymphoproliferative diseases with serum immunoglobulin abnormalities, Blut 32:155, 1976. 203. Schreiber RD, Zweiman B, Atkins P, et al: Acquired angioedema with lymphoproliferative disorder: association of C1 inhibitor deficiency with cellular abnormality, Blood 48:567, 1976. 204. Geha RS, Quinti I, Austen KF, et al: Acquired C1-inhibitor deficiency associated with antiidiotypic antibody to monoclonal immunoglobulin, N Engl J Med 312:534, 1985. 205. Donaldson VH, Hess EV, McAdams PJ: Lupus-erythematosus-like disease in three unrelated women with hereditary angioneurotic edema, Ann Intern Med 86:312, 1977. 206. Alsenz J, Bork K, Loos M: Autoantibody-mediated acquired deficiency of C1 inhibitor, N Engl J Med 316:1360, 1987. 207. Zuraw BL, Curd JG: Demonstration of modified inactive first component of complement (C1) inhibitor in the plasmas of C1 inhibitor deficient patients, J Clin Invest 78:567, 1986.

208. Malbran A, Hammer CH, Frank MM, et al: Acquired angioedema: observations in the mechanism of action of autoantibodies directed against C1 esterase inhibitor, J Allergy Clin Immunol 81:1199, 1988. Chronic Idiopathic Urticaria and Idiopathic Angioedema 209. Atkins PC, Schwartz LB, Adkinson NF, et al: In vivo antigen-induced cutaneous mediator release: simultaneous comparisons of histamine, tryptase, and prostaglandin D2 release and the effect of oral corticosteroid administration, J Allergy Clin Immunol 86:360, 1990. 210. Soter NA, Austen KF, Gigli I: Urticaria and arthralgias as a manifestation of necrotizing angiitis, J Invest Dermatol 63:489, 1974. 211. Soter NA, Mihm MC Jr, Gigli I, et al: Two distinct cellular patterns in cutaneous necrotizing angiitis, J Invest Dermatol 66:334, 1976. 212. Natbony SF, Phillips ME, Elias JM, et al: Histologic studies of chronic idiopathic urticaria, J Allergy Clin Immunol 71:177, 1983. 213. Kern F, Lichtenstein LM: Defective histamine release in chronic urticaria, J Clin Invest 57:1360, 1977. 214. Gratten CE, Walpole D, Francis DM, et al: Flow cytometric analysis of basophil numbers in chronic urticaria: basopenia is related to serum histamine releasing activity, Clin Exp Allergy 27:1417, 1997. 215. Sabroe RA, Francis DM, Barr RM, et al: Anti-FCepsilon AI autoantibodies and basophil histamine releasability in chronic idiopathic urticaria, J Allergy Immunol 102:651, 1998. 216. Phanuphak P, Schocket AL, Arroyave CM, et al: Skin histamine in chronic urticaria, J Allergy Clin Immunol 65:371, 1980. 217. Smith CH, Kepley C, Schwartz L, et al: Mast cell number and phenotype in chronic idiopathic urticaria, J Allergy Clin Immunol 96:360, 1995. 218. Nettis E, Dambra P, Loria MP, et al: Mast cell phenotype in urticaria, Allergy 56:915, 2001. 219. Mekori YA, Giorno RC, Anderson P, et al: Lymphocyte subpopulations in the skin of patients with chronic urticaria, J Allergy Clin Immunol 72:681, 1983. 220. Elias J, Boss E, Kaplan AP: Studies of the cellular infiltrate of chronic idiopathic prominence of T-lymphocytes, monocytes, and mast cells, J Allergy Clin Immunol 78:914, 1986. 221. Peters MS, Schroeter AL, Kephart GM, et al: Localization of eosinophil granule major basic protein in chronic urticaria, J Invest Dermatol 81:39, 1983. 222. Sabroe RA, Poon E, Orchard GE, et al: Cutaneous inflammatory cell infiltrate in chronic idiopathic urticaria: comparison of patients with and without antiFCepsilon RI or anti-IgE autoantibodies, J Allergy Immunol 103:484, 1999. 223. Ying S, Kikuchi Y, Meng Q, et al: Th1/Th2 cytokines and inflammatory cells in skin biopsies from chronic idiopathic urticaria: comparison with the allergeninduced late-phase cutaneous reaction, J Allergy Immunol 109:694, 2002.

224. Gruber BL, Baeza M, Marchese M, et al: Prevalence and functional role of anti-IgE autoantibodies in urticarial syndromes, J Invest Dermatol 90:213, 1988. 225. Hide M, Francis DM, Grattan CE, et al: Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria, N Engl J Med 328:1599, 1993. 226. Tong LJ, Balakrishnan G, Kochan JP, et al: Assessment of autoimmunity in patients with chronic urticaria, J Allergy Immunol 99:461, 1997. 227. Feibiger E, Maurer D, Holub H, et al: Serum IgG autoantibodies directed against the α chain of FCepsilon RI: a selective marker and pathogenic factor for a distinct subset of chronic urticaria patients, J Clin Invest 96:2606, 1995. 228. Ferrer M, Kinet JP, Kaplan AP: Comparative studies of functional and binding assays for IgG anti-FCepsilon RI α (α subunit) in chronic urticaria, J Allergy Clin Immunol 101:672, 1998. 229. Niimi N, Francis DM, Kermeni F, et al: Dermal mast cell activation by auto-antibodies against the high affinity IgE receptor in chronic urticaria, J Invest Dermatol 106:1001, 1996. 230. Ferrer M, Nakazawa K, Kaplan AP: Complement dependence of histamine release in chronic urticaria, J Allergy Clin Immunol 104:169, 1999. 231. Fiebiger E, Hammerschmid F, Stingl G, et al: Anti-FCepsilon RI α autoantibodies in autoimmune-mediated disorders: identification of a structure-function relationship, J Clin Invest 101:243, 1998. 232. Kikuchi Y, Kaplan AP: Mechanisms of autoimmune activation of basophils in chronic urticaria, J Allergy Clin Immunol 107:1056, 2001. 233. Kikuchi Y, Kaplan AP: A role for C5a in augmenting IgG-dependent histamine release from basophils in chronic urticaria, J Allergy Clin Immunol 109:114, 2002. 234. Greaves M. Chronic urticaria, J Allergy Clin Immunol 105:664, 2000. 235. Guo CB, Liu MC, Galli SJ, et al: Identification of IgE-bearing cells in the late-phase response to antigen in the lung as basophils, Am J Respir Dis 10:384, 1994. 236. Charlesworth CN, Hood AF, Soter NA, et al: Cutaneous late-phase response to allergen: mediator release and inflammatory cell infiltration, J Clin Invest 83:1519, 1989. 237. Provost TT, Zone JJ, Synkowski D, et al: Unusual clinical manifestations of systemic lupus erythematosus: I. Urticaria-like lesions: correlations with clinical and serological abnormalities, J Invest Dermatol 75:495, 1980. 238. Braveman FM, Yen A: Demonstration of immune complexes in spontaneous and histamine-induced lesions and normal skin of patients with leucocytoclastic angiitis, J Invest Dermatol 64:105, 1975. 239. Marder RJ, Rent R, Choi EY, et al: C1q deficiency associated with urticaria-like lesions and cutaneous vasculitis, Am J Med 61:560, 1976. 240. McDuffie FC, Sams W Jr, Maldonado JE: Hypocomplementemia with cutaneous vasculitis and arthritis: possible immune complex syndrome, Mayo Clin Proc

48:340, 1973. 241. Gammon WR, Wheeler CE Jr: Urticarial vasculitis, Arch Dermatol 115:76, 1979. 242. Ludivico CL, Myers AR, Maurer K: Hypocomplementemic urticarial vasculitis with glomerulonephritis and pseudotumor cerebri, Arthritis Rheum 22:1024, 1979. 243. Moorthy AV, Pringle D: Urticaria, vasculitis, hypocomplementemia, and immune complex glomerulonephritis, Arch Pathol Lab Med 106:68, 1982. 244. Feig PJ, Soter NA, Yuger HM, et al: Vasculitis with urticaria, hypocomplementemia, and multiple system involvement, JAMA 236:2065, 1976. 245. Agnello V, Koffler D, Eisenberg JW: C1q precipitation in the sera of patients with systemic lupus erythematosus and other hypocomplementemia states: characterization of high and low molecular weight types, J Exp Med 134:228S, 1971. 246. Zeiss CR, Burch FX, Marder RJ: A hypocomplementemic vasculitis urticarial syndrome: report of four new cases and definition of the disease, Am J Med 68:867, 1980. 247. Wisnieski JJ, Naff GB: Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome, Arthritis Rheum 32:1119, 1989. 248. Kaplan AP: Chronic urticaria and angioedema, N Engl J Med 946:275, 2002. 249. Harvey RP, Weg SJ, Schocket AL: A controlled trial of therapy in chronic urticaria, J Allergy Clin Immunol 68:262, 1981. 250. Goldsobel AB, Rohr AS, Siegel SC, et al: Efficacy of doxepin in the treatment of chronic idiopathic urticaria, J Allergy Clin Immunol 78:867, 1986. 251. Sabroe RA, Seed PT, Stut C, et al: Chronic idiopathic urticaria: comparison of the clinical features of patients with and without anti-FCepsilon or anti-IgE autoantibodies, J Am Acad Dermatol 40:443, 1999. 252. Hide M, Francis CM, Grattan CE, et al: The pathogenesis of chronic idiopathic urticaria: new evidence suggests an autoimmune basis and implications for treatment, Clin Exp Allergy 24:624, 1994. 253. Fradin MS, Ellis CN, Goldfarb MT, et al: Oral cyclosporine for severe chronic idiopathic urticaria and angioedema, J Am Acad Dermatol 25:1065, 1991. 254. Kessel A, Golan TD: Low dose cyclosporine A in the treatment of chronic idiopathic urticaria, Allergy 52:312, 1997. 255. Gratten CE, O'Donnell BF, Frances DM, et al: Randomized double-blind study of cyclosporin in chronic idiopathic urticaria, Br J Dermatol 143:365, 1991. 256. Sissons JG, Williams DG, Peters KK, et al: Skin lesions, angioedema, and hypocomplementemia, Lancet 2:1350, 1974. 257. Webb DR, Pearsall HR, Sumida SE: Depression of third component of complement (C3) in chronic angioedema, J Allergy Clin Immunol 55:106, 1975 (abstract).

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Chapter 86 - Atopic Dermatitis

Mark Boguniewicz Donald Y.M. Leung

1] [2]

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease commonly associated with respiratory allergy.[

Recent studies have demonstrated 3]

that AD, like asthma and allergic rhinitis, is associated with the local infiltration of T helper type 2 (Th2) cells that secrete interleukin-4 (IL-4), IL-5, and IL-13.[ 4

More than 50% of patients with AD develop asthma, and approximately 75% develop allergic rhinitis, often as they outgrow AD.[ ] Furthermore, data suggest that 5

the eosinophil is a key effector cell in chronic AD, as in the respiratory allergic diseases, suggesting that AD is truly an allergic disorder.[ ] In addition, patients with AD can react to nonallergic as well as allergic triggers, similarly to patients with asthma and allergic rhinitis. AD can result in significant morbidity, leading to school absenteeism, occupational disability, and emotional stress. Although significant progress has been made in the understanding of AD, its cause is still unknown, and much remains to be learned about the complex interrelationship of genetic, environmental, immunologic, and pharmacologic factors in this disease. With an increased understanding of the complex immune mechanisms involved, clinicians can direct AD therapy from primarily symptomatic relief to more specific immunomodulatory measures.

CLINICAL MANIFESTATIONS Historical Overview 6

In 1892, Besnier[ ] described the prurigo group of diseases, characterized by pruritus and a familial predisposition that included asthma and hay fever along with skin 7

rash. The term atopy, derived from the Greek atopos or “strangeness,” was introduced by Coca and Cooke[ ] in 1923 to describe a familial altered end-organ hypersensitivity to environmental proteins in hay fever and asthma. This definition was subsequently broadened to include the propensity to produce heat-labile 8

9

reaginic antibody, later identified as immunoglobulin E (IgE), to common allergens and to include atopic eczema.[ ] In the 1930s, Hill and Sulzberger[ ] suggested the term atopic dermatitis to describe both the weeping eczema of infancy and childhood and the chronic xerosis and lichenified lesions more typical of older patients. This term also recognized the close relationship among AD, asthma, and allergic rhinitis.

EPIDEMIOLOGY 10

A number of studies suggest an increasing prevalence of AD. Schultz Larsen[ ] in Denmark demonstrated a cumulative incidence rate (up to 7 years) of 12% for twins born between 1975 and 1979, compared with a rate of 3% for twins born between 1960 and 1964. A cross-sectional questionnaire study conducted in 1992 confirmed this increased prevalence.[

11]

In this study of 3000 7-year-olds from Denmark, Germany, and Sweden, the frequency of AD was 15.6%. Similarly,

questionnaire studies of Swedish schoolchildren in 1979 and 1991 showed an increase in the prevalence of AD from 7% in 1979 to 18% in 1991. [ 13]

prevalence of AD in a study of schoolchildren living in northern Norway was found to be 23%. [

12]

The point

In a study from Japan, the authors performed skin examinations

14 AD.[ ]

rather than relying on questionnaires to ascertain the prevalence of childhood and adolescent More than 7000 patients were examined, and AD was documented in 24% of those age 5 to 6 years, 19% of those age 7 to 9 years, 15% of those age 10 to 12 years, 14% of those age 13 to 15 years, and 11% of those age 16 to 18 years. The prevalence of AD in 9- to 12-year-old children was two times higher than in children of similar age examined 20 years earlier, and for 18-yearold adolescents it was five times higher. Recent questionnaire data from the United States in schoolchildren ages 5 to 9 years found the prevalence of AD in this 15]

population to be 17%.[

Of note, studies comparing prevalence of AD between East and West Germany showed that, in contrast to the higher prevalence of asthma 16]

and allergic rhinitis in West Germany, the prevalence of AD is consistently higher in East Germany.[ tests to a panel of allergens) accounted for a significant portion of AD in East Germany.

In this population, “intrinsic” AD (based on negative skin

Increased exposure to pollutants and indoor allergens (especially house dust mites) and a decline in breast-feeding, along with an increased awareness of AD, have 17

18

been suggested as reasons for the increased frequency of AD.[ ] In a prospective study, Zeiger et al[ ] found that restricting the mother's diet during the third trimester of pregnancy and lactation and the child's diet during the first 2 years of life resulted in decreased prevalence of AD in the prophylaxis group compared with a control group at 12 months of age but not at 24 months. Follow-up through 7 years of age showed no difference between the prophylaxis and control groups for AD 19

20

or respiratory allergy. [ ] In a large study of an ethnically and socially diverse group of children in suburban Birmingham, England, Kay and coworkers[ ] found that breast-feeding did not affect the life-time prevalence rate of 20%. A study of prevalence of childhood eczema found a correlation with increased socioeconomic class that did not result from heightened parental awareness.[

21]

1560

22

The effects of genetic and environmental factors on allergic diseases were studied in two Japanese cities with differing climates.[ ] The prevalence of allergic diseases and AD in the city with a temperate climate was significantly higher than in the one with a subtropical climate even after controlling for genetic and environmental factors. In both cities, children from atopic families had a significantly higher risk of contracting respiratory allergies and AD. In a global survey of 23

the prevalence of asthma, allergic rhinoconjunctivitis, and AD, 463,801 children ages 13 to 14 years from 155 centers in 56 countries participated. [ ] The highest prevalences of AD were reported from scattered centers, including sites in Scandinavia and Africa, that were not among centers with the highest prevalences of asthma. On the other hand, the lowest prevalence rates for AD occurred in centers with the lowest prevalence of asthma and allergic rhinoconjunctivitis. Thus the ultimate presentation of an atopic disease may depend on a complex interaction of environmental exposures with end-organ response in a genetically predisposed individual.

Genetics 24]

Although genetic susceptibility to respiratory allergy has been suggested by localization of a locus for atopy on chromosome 11q13,[ been demonstrated in patients with

25 AD.[ ]

linkage to this gene has not

However, linkage of both AD and asthma to polymorphisms within the gene for the β subunit of the high-affinity IgE

receptor on chromosome 11q12–13 has been reported, but remains to be substantiated.[

26]

There has also been particular interest in the role of chromosome 5q31–33 27

28

in AD, because it contains a clustered family of cytokine genes (IL-3, -4, -5, and -13 and GM-CSF) expressed by Th2 cells.[ ] In addition, Chan et al[ ] found that abnormal IL-4 gene expression in AD may be linked to alterations in nuclear protein interactions with IL-4 promoter elements. A study examining linkage between markers at and near the IL-4 gene and AD in 88 Japanese nuclear families found a genotypic association between the T allele of the −590C/T polymorphism of the 29

IL-4 gene and AD.[ ] Since the T allele was reported to be associated with increased IL-4 gene promoter activity compared with the C allele, the data suggest that genetic differences in transcriptional activity of the IL-4 gene may influence predisposition for AD particularly in this population, because of the high frequency of the T allele. In addition, an association of atopy with a gain-of-function mutation in the α subunit of the IL-4 receptor has been reported in a small group of AD patients.[

30]

The authors speculated that the R576 allele may predispose persons to allergic diseases by altering the signaling function of the receptor.

More recently, two studies have shown that a major susceptibility locus for AD maps to chromosome 3q21, as well as loci on chromosomes 1q21, 17q25, and 20p. [31]

These findings are significant in that these loci are linked to psoriasis and may therefore represent genes responsible for dermal inflammation and immune 32]

dysregulation.[

33

Although the mode of transmission remains uncertain, some studies support an autosomal dominant inheritance pattern. Uehara and Kimura[ ] found that 60% of adults with AD had children with AD. The prevalence of AD in children was 81% when both parents had AD, 59% when one parent had AD and the other had respiratory allergy, and 56% when one parent had AD and the other had neither AD nor respiratory allergy. 34

Diepgen and Fartasch[ ] showed that 42% of first-degree relatives of patients with AD also had AD, and 28% had respiratory allergy. In contrast, only 12% of firstdegree relatives of persons with respiratory allergy without skin disease had AD, and 43% had respiratory allergy. A follow-up study from this group investigated the 35

familial aggregation of AD, allergic rhinitis, and allergic asthma in the relatives of 426 patients with AD and 628 subjects with no history of AD.[ ] The odds ratio of familial aggregation for AD was 2.16 if no distinction was made as to the degree of relationship. Further analyses within the members of the family showed a high odds ratio of 3.86 among siblings, whereas the odds ratio between parents and siblings was only 1.90. For AD, the odds ratio differed between mother-sibling pairs (2.66) and father-sibling pairs (1.29), possibly because of environmental events that affect the fetus in utero or the shared physical environment of mother and child. However, the finding that all of the atopic diseases demonstrated a stronger correlation between siblings than between siblings and parents supports the hypothesis that exposure to environmental factors during childhood is responsible for the recently observed increased prevalence of atopic diseases. Atopic Diathesis As discussed previously, AD, along with asthma and allergic rhinitis, is an atopic disease that occurs in individuals with a genetic predisposition to develop an IgE

response to common environmental allergens. Abnormal IgE responses are associated with cellular abnormalities resulting in overproduction of Th2-type cytokines, which also contribute to the eosinophilia seen in these diseases. Early onset of AD has been found to be associated with an increased risk for respiratory allergy. The highest incidence of asthma at a given age has been observed in children with onset of AD before 3 months of age, in those with severe AD and with a family history of asthma.[

36]

The data showing an association of increased risk for asthma, rhinoconjunctivitis, or both with early onset of AD have been confirmed in more recent

37 studies.[ ]

Respiratory allergy occurred in 50% of children who had onset of AD during the first 3 months of life and two or more atopic family members, compared with 12% of children who had onset of AD after 3 months of age and no atopic family members. In a prospective study of children with AD who were observed through 7 years of age, only 14 of 94 children in this cohort had not experienced any signs or symptoms suggestive of asthma or allergic rhinoconjunctivitis.[

38]

In

[39]

addition, children with AD have been shown to have more severe asthma than asthmatic children without AD. This raises the possibility that allergen sensitization through the skin may predispose to more severe and persistent respiratory disease through effects on the systemic allergic response. In support of this hypothesis, murine studies have shown that epicutaneous sensitization with protein antigen can elicit a localized dermatitis, along with elevated serum IgE, airway eosinophilia, and hyperresponsiveness to methacholine. [

40]

In addition, patients with AD react to both allergic and nonspecific triggers, similarly to patients with asthma and allergic rhinitis. Skin hyperreactivity to irritants such as sodium lauryl sulfate (SLS) has been shown in patients with both active and inactive AD, and in patients with allergic respiratory disease even with no skin involvement, compared

1561

41

with nonatopic subjects.[ ] An abnormal intrinsic hyperreactivity of inflammatory cells in atopic individuals may predispose them to a lower threshold of irritant responsiveness. These observations were confirmed and extended in a study showing that the stratum corneum abnormalities in noninvolved AD skin were associated with increased transepidermal water loss even 7 days after application of SLS.[

42]

Furthermore, SLS was shown to induce a similar eosinophilic infiltrate

in patients with allergic rhinitis. Of note, patients with AD do not have a constitutionally impaired stratum corneum barrier,[ transferred through bone marrow

44 transplantation.[ ]

43]

and, in addition, atopy can be

These observations suggest that the cutaneous abnormality in AD results from a complex interaction of resident 45

and infiltrating cells. Furthermore, in a study of bronchial and cutaneous reactivity in asthmatic patients with and without AD,[ ] the authors found a latent predisposition for bronchial asthma in AD patients and implicated circulating activated eosinophils as the common effector cells. Because the ability of eosinophils to reach their target organ depends in part on eosinophil-specific chemotactic factors, increased expression of eotaxin and monocyte chemotactic protein-4 (MCP-4), structurally homologous eosinophil chemoattractants acting through a common CCR3 receptor, has been reported both in the respiratory mucosa of patients with 46] [47]

asthma and rhinitis[

and in AD.[

48]

Natural History 1

AD typically manifests in early childhood, with onset before 5 years of age in approximately 90% of patients.[ ] Patients younger than 2 months of age rarely have any eczematous lesions, possibly because of undeveloped motor skills, which are needed for rubbing and scratching, or because of an immature immune response. In

adults with new-onset dermatitis, especially without a history of childhood eczema, asthma, or allergic rhinitis, other diseases need to be considered ( Box 86-1 ). Although Vickers' 20-year follow-up[ outcomes. Linna et

4 al[ ]

49]

suggested that approximately 84% of children outgrow their AD by adolescence, more recent studies present less optimistic

found that AD had disappeared in only 18% of children who were observed from infancy until 11 to 13 years of age, although it had become 50

less severe in 65%. Kissling and Wuthrich[ ] found that 72% of patients diagnosed during the first 2 years of life continued to have AD 20 years later. In a prospective study from Finland, between 77% and 91% of adolescent patients treated for moderate or severe AD had persistent or frequently relapsing dermatitis as 51]

adults, although only 6% had severe disease.[

In addition, more than half of the adolescents treated for mild dermatitis experienced a relapse of disease as adults.

38

Gustafsson et al[ ] in a prospective study of children with AD observed through age 7 years, found that, although most had milder eczema by 7 years, only approximately one third had no evidence of disease activity. Often, adults whose childhood AD has been in remission for a number of years present with hand dermatitis, especially if daily activities require repeated hand wetting.[

52]

Clinical Features AD has no pathognomonic skin lesions or unique laboratory parameters. Therefore the diagnosis is based on the presence of major and associated clinical features ( Box 86-2 ). Attempts

Box 86-1. Differential Diagnosis of Atopic Dermatitis

Congenital Disorders Netherton's syndrome

Chronic Dermatoses Seborrheic dermatitis Contact dermatitis (allergic or irritant) Nummular eczema Lichen simplex chronicus

Infections and Infestations Scabies Human immunodeficiency virus–associated dermatitis

Malignancy Cutaneous T cell lymphoma (mycosis fungoides/Sézary syndrome)

Immunodeficiencies Wiskott-Aldrich syndrome Severe combined immunodeficiency Hyperimmunoglobulinemia E syndrome

Metabolic Disorders Zinc deficiency Pyridoxine (vitamin B6 ) and niacin deficiency Multiple carboxylase deficiency Phenylketonuria

Proliferative Disorders Letterer-Siwe disease

to standardize signs and symptoms of AD include SCORAD and the Eczema Area and Severity Index (EASI); however, these indexes have been used primarily in 53] [54]

clinical research trials.[

The principal features include severe pruritus, a chronically relapsing course, typical morphology and distribution of the skin lesions, 1

and a history of atopic disease. [ ] The presence of pruritus is critical to the diagnosis of AD, and patients with AD have a reduced threshold for pruritus.[

55]

Acute AD is characterized by intensely pruritic, erythematous papules associated with excoriations, vesiculations, and serous exudate ( Color Plate 19 ). Subacute AD is characterized by erythematous, excoriated, scaling papules, whereas chronic AD is characterized by thickened skin with accentuated markings (lichenification) and fibrotic papules ( Color Plate 20 ). Patients with chronic AD may have all three types of lesions. In addition, patients usually have dry skin. Significant differences can be observed in pH, capacitance, and transepidermal water loss between AD lesions and uninvolved skin in the same patient and or skin of normal 56]

controls.[

During infancy, AD involves primarily the face, the scalp, and the extensor surfaces of the extremities. The diaper area is usually spared; if it is involved, it may be secondarily infected with Candida species, in which case the dermatitis does not spare the inguinal folds. In contrast, infragluteal

1562

Box 86-2. Clinical Features of Atopic Dermatitis

Major Features Pruritus Facial and extensor involvement in infants and children Flexural lichenification in adults Chronic or relapsing dermatitis Personal or family history of atopic disease

Minor Features

Xerosis Cutaneous infections Nonspecific dermatitis of the hands or feet Ichthyosis, palmar hyperlinearity, keratosis pilaris Pityriasis alba Nipple eczema White dermatographism and delayed blanch response Anterior subcapsular cataracts Elevated serum IgE levels Positive immediate-type allergy skin tests

Modified from Hanifin JM, Rajka G: Acta Derm Venereol (Stockh) 2: 44–47, 1980.

involvement is a common distribution in children. In older patients with long-standing disease, the flexural folds of the extremities are the predominant location of lesions. Localization of AD to the eyelids may be an isolated manifestation but should be differentiated from allergic contact dermatitis. 57

The United Kingdom's Working Party proposed a set of revised diagnostic criteria for AD with a sensitivity of 85% and a specificity of 96%.[ ] These included itchy skin plus three or more of the following: history of flexural involvement, history of asthma or hay fever, generalized dry skin, onset of rash before 2 years of age, and flexural dermatitis. Complicating Features Ocular Problems 58

Increased numbers of IgE-bearing Langerhans' cells have been found in the conjunctival epithelium of patients with AD.[ ] These cells can capture aeroallergens and present them to infiltrating T cells, thus contributing to ocular inflammation. Ocular complications associated with AD can result in significant morbidity.

59

Atopic keratoconjunctivitis is always bilateral, and symptoms include itching, burning, tearing, and copious mucoid discharge.[ ] It is frequently associated with eyelid dermatitis and chronic blepharitis and may result in visual impairment from corneal scarring ( Color Plate 21 ). Keratoconus is a conical deformity of the cornea that is believed to result from persistent rubbing of the eyes in patients with AD and allergic rhinitis. Anterior subcapsular cataracts may develop during adolescence or early adult life. Hand Dermatitis

Patients with AD often have nonspecific hand dermatitis. This is frequently irritant in nature and aggravated by repeated wetting, especially in the occupational setting.[

60]

A history of past or present AD at least doubles the effects of irritant exposure and doubles the risk in occupations where hand eczema is a common

61 problem.[ ]

Infections

Patients with AD have an increased susceptibility to infection or colonization with a variety of organisms.[ [63]

[64]

molluscum contagiosum,

and infections with human

65 papillomavirus.[ ]

62]

These include viral infections with herpes simplex virus,

A direct relationship has been demonstrated between interferon-γ (IFN-γ) 66]

concentrations and the cytopathic effect of herpes simplex, as has an inverse relationship between IL-4 and the cytopathic effect of herpes simplex,[ that the T cell–associated cytokine abnormalities seen in AD can enhance viral infections. Superimposed dermatophytosis may cause AD to flare.[ compared with

68 controls.[ ]

67]

suggesting

Patients with AD appear to have a threefold increased incidence of Trichophyton rubrum infections,

Pityrosporum ovale has also been associated with a predominantly head and neck distribution of AD.[

69]

A number of studies have elucidated the importance of Staphylococcus aureus in AD ( Color Plate 22 ). Preferential adherence of this organism in AD may be related to expression of adhesins such as fibronectin and fibrinogen in inflamed skin.[ compared with only 5% of normal

71 subjects.[ ]

70]

S. aureus can be cultured from the skin of more than 90% of patients with AD,

The higher rate of S. aureus colonization in AD lesions compared with lesions from other skin disorders may also be

associated with colonization of the nares, with the hands serving as the vector of transmission.[

72] [73]

Patients without obvious superinfection may have a better 74

response to combined antistaphylococcal and topical corticosteroid therapy than to corticosteroids alone.[ ] Although recurrent staphylococcal pustulosis can be a significant problem in AD, invasive S. aureus infections occur rarely and should raise the possibility of an immunodeficiency such as hyperimmunoglobulinemia E syndrome. [

75]

Differential Diagnosis A number of diseases may be confused with AD (see Box 86-1 ). Scabies can present as a pruritic skin disease. However, distribution in the genital and axillary

areas, the presence of linear lesions, and the results of skin scrapings help distinguish it from AD. An adult who has eczematous dermatitis with no history of childhood eczema and without other atopic features may have contact dermatitis, but, more importantly, cutaneous T cell lymphoma needs to be ruled out. Ideally, biopsies should be sent from three separate sites to increase the yield in identifying abnormal Sézary cells. In addition, an eczematous rash suggestive of AD has been 76]

reported with human immunodeficiency virus.[

A contactant should be considered in patients whose AD does not respond to appropriate therapy. Typical distribution for a suspected contactant may be suggestive. However, allergic contact dermatitis complicating AD may appear as an acute flare of the underlying disease rather than the more typical vesiculobullous eruption. Proper diagnosis depends on confirmation of a suspected allergen with patch testing. Standardized testing with the T.R.U.E. Test is available for 23 of the most common contact allergens (see Chapter 87 )

1563

although more extensive testing may be required in selected cases. Psychological Implications Patients with AD have been characterized as having high levels of anxiety and problems in dealing with anger and hostility.[

77]

Although these emotions do not cause

[78]

AD, they can exacerbate the illness. Patients often respond to stress or frustration with itching and scratching. Stimulation of the central nervous system may intensify cutaneous vasomotor and sweat responses and contribute to the itch-scratch cycle. In some instances, scratching is associated with significant secondary gain or with a strong component of habit. Finally, severe disease can have a significant impact on patients, leading to problems with social interactions and selfesteem.

ROLE OF ALLERGENS Although elevated serum IgE levels can be demonstrated in 80% to 85% of patients with AD[

79]

and a similar number have immediate skin tests or in vitro tests to

80 allergens,[ ]

food and inhalant the relationship between the course of AD and implicated allergens has been difficult to establish. Nevertheless, a number of wellcontrolled studies suggests that various allergens can have an impact on the course of this disease. Foods 81

May[ ] first recognized that patients with AD and positive food allergen skin tests could have negative food challenges to the implicated allergen, distinguishing between symptomatic and asymptomatic hypersensitivity. Thus, triggers for clinical disease cannot be predicted simply by performing allergy testing. However,

double-blinded, placebo-controlled food challenges have demonstrated that food allergens can cause exacerbations in a subset of patients with AD.[

82]

83

Approximately 33% of infants and young children with AD will show clinically relevant reactivity to a food allergen.[ ] Although lesions induced by single positive challenges are usually transient, repeated challenges, more typical of real-life exposure, can result in eczematous lesions. Food-specific T cells have been cloned from lesional skin and peripheral blood of patients with AD.[ in spontaneous basophil histamine

84] [85]

Furthermore, elimination of food allergens results in amelioration of skin disease and a decrease

86 release.[ ]

Aeroallergens The evidence supporting a role for aeroallergens in AD includes the finding of both allergen-specific IgE antibodies[

87]

and allergen-specific T cells.[

88]

Exacerbation

89 Tuft[ ]

of AD can occur with exposure to allergens such as house dust mites, animal danders, and pollens. In the 1940s, demonstrated that introduction of aeroallergens intranasally could exacerbate AD. More recently, 9 of 20 patients with AD who underwent bronchoprovocation with a standardized house dust mite 90

extract in a double-blind, randomized, placebo-controlled fashion developed unequivocal cutaneous lesions after inhalation of dust mite.[ ] All the patients with dust mite–induced dermatitis had a history of asthma, and in eight of these nine patients the skin reaction was preceded by an early bronchial reaction. Therefore the respiratory route may be important in the induction and exacerbation of AD. In addition, studies with patch testing have shown that direct contact with inhalant 91 92

93

allergens can also result in eczematous skin eruptions.[ ] [ ] Using the atopy patch test, Langeveld-Wildschut et al[ ] showed that positive reactions to house dust mite were associated with IgE+ Langerhans' cells in the epidermis of AD patients. In addition, the severity of AD has been correlated with the degree of sensitization to aeroallergens.[ AD

94]

95 96 patients.[ ] [ ]

Most importantly, environmental control measures aimed at reducing dust mite allergen have been shown to result in clinical improvement in These studies suggest that inhalation or contact with aeroallergens may be involved in the pathogenesis of AD.

Microbial Agents 97

98

In addition to their role as infectious agents, both the lipophilic yeast P. ovale (orbiculare) [ ] and the superficial dermatophyte T. rubrum[ ] have been associated with elevated specific IgE levels in patients with AD. Patients with AD predominantly of the head and neck, compared with a group without this distribution and with a group of normal individuals, more often demonstrated positive skin tests, radioallergosorbent tests, and specific histamine release to P. ovale.[ significance of these findings is suggested by clinical improvement of such patients after antifungal

69]

The clinical

67 therapy.[ ]

99

Leung et al[ ] showed that exotoxins secreted by S. aureus are superantigens that can result in persistent inflammation or exacerbations of AD. More than half of the AD patients studied had S. aureus cultured from their skin; the S. aureus organisms secreted primarily enterotoxins A and B and toxic shock syndrome toxin-1. In addition, almost half of the patients had specific IgE antibodies directed against the staphylococcal toxins found on their skin. Basophils from patients with antitoxin IgE released histamine on exposure to the relevant toxin but not in response to toxins to which they had no specific IgE. Other investigators have confirmed these 100] [101]

observations.[

In addition, analysis of the peripheral blood skin homing (cutaneous lymphocyte antigen [CLA]-positive) T cells of superantigen-positive

patients as well as their skin lesions revealed that they had undergone a T cell receptor Vβ expansion consistent with superantigenic stimulation.[ correlation also has been found between the presence of IgE anti-superantigens and severity of

100 AD.[ ]

102] [103]

A

Furthermore, superantigens have been shown to have an

additive effect together with conventional allergens in inducing cutaneous inflammation[

104]

; they augment allergen-specific IgE synthesis[

105]

; and they can induce

106 resistance,[ ]

corticosteroid suggesting that several mechanisms exist by which superantigens could aggravate the severity of AD. Finally, staphylococcal enterotoxin B (SEB) applied to the skin was shown to induce erythema and induration with the infiltrating T cells selectively expanded in response to the specific superantigen.[

107] [108]

Autoantigens 109

Several groups have suggested a role for autoantigens in chronic AD. Valenta et al[ ] reported that the majority of sera from patients with severe AD contain IgE antibodies directed against human proteins. One of these IgE-reactive autoantigens, a 55-kD cytoplasmic protein in skin keratinocytes, has been cloned from a human epithelial complementary

1564

110

DNA (cDNA) expression library and designated Hom s 1. [ ] Although the autoallergens characterized to date have mainly been intracellular proteins, they have been detected in IgE immune complexes of AD sera, suggesting that release of these autoallergens from damaged tissues could trigger IgE- or T cell–mediated responses. In another study, 30% of sera from patients with AD were found to have both IgG and IgE autoantibodies that reacted with an autoantigen termed dense 111

fine speckles 70•kD (DFS70).[ ] These data suggest that skin inflammation in AD, especially in severe cases, could be maintained by endogenous human antigens. Because these autoantigens are primarily nuclear or microsomal in origin, the possibility is raised that damage to the skin by infectious organisms or by scratching could release intracellular antigens that in turn could elicit and perpetuate IgE and T cell responses in AD.

IMMUNOLOGY The finding of elevated serum IgE concentrations and the occurrence of eczematous lesions indistinguishable from AD in patients with primary T cell 112

immunodeficiency disorders suggest an immunologic basis for AD.[ ] In Wiskott-Aldrich syndrome, bone marrow transplantation results in correction of the immunologic defect and resolution of the dermatitis. In addition, nonatopic recipients of bone marrow transplants from atopic donors can develop atopic symptoms 113

and positive skin tests after successful engraftment.[ ] These data suggest that AD results from a bone marrow–derived cell dysfunction rather than a constitutive skin defect. Of note, the concept of intrinsic and extrinsic forms of AD, discussed earlier, has been based on low serum IgE levels and negative allergy skin tests or 114

radioallergosorbent tests (RAST).[ ] A number of cellular and cytokine abnormalities have also been identified, as described in subsequent sections of this chapter. It remains to be seen whether the intrinsic and extrinsic forms of AD represent opposite extremes of the same illness or are, in fact, different diseases with a similar clinical phenotype. Immunoregulatory Dysfunction

A number of immunoregulatory abnormalities have been described in AD ( Box 86-3 ). B cells from patients with AD synthesize high levels of IgE.[ 116] [117]

Lymphocytes from these patients produce increased amounts of IL-4 and express abnormally high levels of IL-4 receptor.[ production of IgE can be inhibited in vitro by the addition of

118 anti-IL-4.[ ]

115]

In addition, the spontaneous

Peripheral blood mononuclear cells (PBMCs) isolated from patients with AD have a 119

decreased capacity to make IFN-γ, which is inversely correlated with serum IgE levels.[ ] Differences that have been noted between the intrinsic and extrinsic forms of AD include the observation that skin-derived T cells from extrinsic AD interacted with B cells to support IgE synthesis, whereas T cells from the intrinsic form of AD did not.[

119]

A number of studies have shown an increased frequency of both circulating[ patients with

119 122 AD.[ ] [ ]

120] [121]

and lesional allergen-specific Th2 cells secreting IL-4, IL-5, and IL-13 in

Furthermore, an increased frequency of circulating skin homing (CLA+) type 2 cytokine-producing cells and decreased frequency of CLA

+ type 1 cytokine-producing cells has been reported in the peripheral blood of patients with AD.[

123]

In addition to acting as an IgE isotype-specific switch, IL-4 also

Box 86-3. Immunoregulatory Abnormalities in Atopic Dermatitis Increased synthesis of IgE Increased levels of specific IgE to multiple allergens, including foods, aeroallergens, microorganisms, and enterotoxins Increased expression of CD23 on B cells and monocytes Increased basophil histamine release Impaired delayed-type hypersensitivity response Decreased CD8 suppressor/cytotoxic T cell number and function Increased secretion of interleukin-4 (IL-4), IL-5 and IL-13 by T helper type 2 (Th2) cells Decreased secretion of interferon-γ by Th1 cells Increased levels of soluble IL-2 receptor Increased levels of monocyte cyclic adenosine monophosphate phosphodiesterase, with increased IL-10 and prostaglandin E2

inhibits the production of IFN-γ and down-regulates the differentiation of Th1 cells.[

124]

IFN-γ production is also inhibited by prostaglandin E2 (PGE2 ) and by IL-

10, both of which are secreted in increased amounts by monocytes from patients with AD.[

125] [126]

In one study, no defect in the capacity of cells from AD patients

127 detected.[ ]

to produce IL-12, an important inducer of IFN-γ, was However, neutralization of IL-10 and IL-4 was able to correct production of IFN-γ. Therefore the activation of Th2-type cells and monocytes may be central to the immune dysregulation in AD. 128

A role for the co-stimulatory molecules CD80/CD86 has been investigated in AD. Using immunohistochemical analysis, Ohki et al[ ] showed predominantly CD86 on Langerhans' cells in both the epidermis and the dermis in AD. They also demonstrated almost complete inhibition of antigen-specific T cell proliferation with an anti-CD86 monoclonal antibody. Studies have also suggested that these accessory molecules differ in their capacity to generate Th1 versus Th2 T cell responses. A recent study found that the expression of CD86 on B cells of AD patients was significantly higher than on B cells from patients with psoriasis or from normal 129

controls, whereas there was no significant difference in CD80 expression among the three subject groups.[ ] Interestingly, total serum IgE from AD patients and normal subjects correlated significantly with CD86 expression on B cells, suggesting a role for CD86+ B cells in IgE synthesis. Purified CD86+ B cells produced significantly more IgE than did CD86-B cells in vitro, and anti-CD86, but not CD80, monoclonal antibody (mAb) significantly decreased IgE production by peripheral blood mononuclear cells stimulated with IL-4 and anti-CD40•mAb. Furthermore, CD86+ B cells had a significantly higher level of IL-4R and CD23 expression than did CD80+ B cells. These data demonstrate the predominant expression of CD86 in AD and suggest a role in IgE synthesis. Immunopathologic Features Routine histologic examination of clinically normal-appearing skin in AD reveals mild epidermal hyperplasia and a sparse,

1565

predominantly lymphocytic infiltrate in the dermis.[

130]

Acute eczematous lesions are characterized by both intercellular edema of the epidermis (spongiosis) and 131

intracellular edema. Of note, a recent study appears to shed light on the mechanism of these changes.[ ] T cell–derived IFN-γ was shown to increase expression of Fas receptor (CD95) on keratinocytes, leading to acantholysis (loss of intercellular cohesion) with subsequent intercellular edema resulting in the characteristic histology. A sparse lymphocytic infiltrate may be observed in the epidermis, whereas a marked perivenular infiltrate consisting of lymphocytes and some monocytes with rare eosinophils, basophils, and neutrophils is seen in the dermis. Mast cells are found in normal numbers in different stages of degranulation. In chronic lichenified lesions, the epidermis has prominent hyperkeratosis with increased numbers of epidermal Langerhans' cells and predominantly monocytes/ macrophages in the dermal infiltrate. Mast cells are usually increased in number but are not degranulated. Immunohistochemical staining of acute and chronic skin lesions in AD shows that the lymphocytes are predominantly CD3, CD4, and CD45RO memory T cells; that 75] [132]

is, they have previously encountered antigen.[

These cells also express CD25 and human leukocyte antigen (HLA)-DR on their surface, indicative of

intralesional activation.[

75]

In addition, almost all of the T cells infiltrating into atopic skin lesions express high levels of the skin lymphocyte homing receptor, CLA, 133]

a ligand for the vascular adhesion molecule, E-selectin.[

Vascular endothelial cells from atopic skin lesions express abnormally high levels of E-selectin, as well as vascular cell adhesion molecule-1 (VCAM-1) and CD54. [134]

Mast cells, monocytes, Langerhans' cells, and keratinocytes are all potential sources of IL-1 and tumor necrosis factor-α (TNF-α), which induce E-selectin, a 135]

molecule critical to the targeting of CLA-expressing T cells to sites of cutaneous inflammation.[

Furthermore, migration of skin-homing T cells into atopic skin

lesions also involves interaction between VCAM-1 and very late antigen-4 as well as CD54 and leukocyte function-associated antigen-1.[ which can be induced by IL-4 and IL-13, is involved in eosinophil and mononuclear cell movement into sites of allergic

136]

In addition, VCAM-1,

137 inflammation.[ ]

In contrast to epidermal Langerhans' cells from normal subjects, Langerhans' cells found in the epidermis and dermis of patients with chronic AD express CD1b, CD36, and HLA-DR surface antigens and are potent activators of autologous resting CD4 T cells.[ infiltrating into the AD skin lesion have surface-bound

139 140 IgE.[ ] [ ]

138]

Furthermore, both Langerhans' cells and macrophages

A distinct population of CD1a inflammatory dendritic epidermal cells in cutaneous lesions in

141 described.[ ]

AD has been These cells are subjected to specific signals leading to the up-regulation of the high-affinity IgE receptor, FcepsilonRI, in AD skin. In addition, while the number of CD1a+ epidermal dendritic cells has been shown to be similar in the inflammatory milieu in lesions from both intrinsic and extrinsic AD, intrinsic AD was characterized by decreased expression of FcepsilonRI, on the CD1a+ epidermal dendritic cells. [

142]

3

Activated eosinophils are present in significantly greater numbers in chronic compared with acute lesions.[ ] These eosinophils undergo cytolysis with release of 143]

granule protein contents into the upper dermis of lesional skin.[ 144]

a lesser extent deeper in the dermis.[

Deposition of eosinophil major basic protein can be detected throughout the upper dermis, and to

Major basic protein deposition is more prominent in involved areas compared with uninvolved skin. It may contribute to the 145]

pathogenesis of AD through its cytotoxic properties and its capacity to induce basophil and mast cell degranulation.[ cationic protein are elevated in AD and correlate with disease

146 severity.[ ]

Furthermore, serum levels of eosinophil

Urinary eosinophil protein X has also been found to correlate with AD disease activity.

[147]

Cytokine Expression 3

Cytokine expression in AD lesions reflects the nature of the underlying inflammation. Hamid, Boguniewicz, and Leung[ ] used in situ hybridization to study IL-4, IL5, and IFN-γ messenger ribonucleic acid (mRNA) expression in acute and chronic skin lesions as well as uninvolved skin of patients with AD. Biopsies from uninvolved atopic skin showed a significant increase in the number of cells expressing IL-4, but not IL-5 or IFN-γ, mRNA. Both acute and chronic lesions had significantly greater numbers of cells that were positive for IL-4 and IL-5 compared with uninvolved or normal skin. Neither acutely involved nor uninvolved atopic skin showed significant numbers of IFN-γ mRNA-expressing cells. In contrast, chronic AD skin lesions, when compared with acute lesions, had significantly fewer IL-4 mRNA-expressing cells and significantly more IL-5 mRNA-expressing cells. T cells comprised the majority of IL-5–expressing cells in both acute and chronic lesions. Activated eosinophils were found in significantly greater numbers in chronic compared with acute lesions. These data suggest that although both acute and chronic lesions in AD are associated with increased IL-4 and IL-5 gene activation, acute skin inflammation is associated with predominantly IL-4 expression,

whereas chronic inflammation is associated with IL-5 expression and eosinophil infiltration. 148

IL-13 expression was also found to be higher in acute AD lesions, compared with chronic AD or psoriatic lesions.[ ] These data suggest that IL-13 may be involved in the pathogenesis of AD and further support the hypothesis that acute inflammation in AD is mediated by Th2-type cytokines. Chronic lesions had increased numbers of IL-12 mRNA– positive cells compared with acute or uninvolved skin. IL-12 is a potent inducer of IFN-γ synthesis, and, consistent with this observation, increased IFN-γ expression has been reported in chronic AD lesions.[

149] [150]

At a clonal level, T cells from AD patients with cow's milk allergy showed

significantly greater production of IL-4, whereas IFN-γ production was greater in the milk-tolerant patients.[ correlated with IL-4 production.

151]

IL-5 and IL-13 cytokine production strongly

Consistent with the observation that the activity of cytokines is dependent on the expression of their receptors, acute AD lesions have been shown to contain a significantly higher number of cells expressing mRNA of the α subunit of the IL-4 receptor (IL-4R-α), compared with chronic AD lesions, uninvolved skin, or 152

normal control skin.[ ] In contrast, chronic AD lesions contained significantly more cells expressing the IL-5R-α mRNA and GM-CSFR-α mRNA than did acute AD lesions, uninvolved skin, or normal control skin. 153]

Differences in cytokine profiles have been suggested between the intrinsic and extrinsic forms of AD.[ 5 and IL-13 in the supernatants from T cells of patients with intrinsic AD.

In this respect, Akdis et al[

119]

showed lower levels of IL-

1566

Of note, neutralization of IL-13 had no effect on the low levels of IgE production by the patients with intrinsic AD. Chemoattractants

The CC family chemokines RANTES, MCP-4, and eotaxin have all been found to be increased in AD skin lesions and probably contribute to the chemotaxis of eosinophils and Th2-type lymphocytes into the skin.[

154] [155]

IL-16, a chemoattractant for CD4+ T cells, is more highly expressed in acute than in chronic AD skin

156 lesions.[ ]

157]

Recent studies suggest a role for cutaneous T cell–attracting chemokine (CTACK/ CCL27) in the preferential attraction of CLA+ T cells to the skin.[ The chemokine receptor CCR3, which is found on eosinophils and Th2-type lymphocytes and can mediate the actions of eotaxin, RANTES, and MCP-4, has been reported to be increased in both lesional and nonlesional skin of AD patients.[ a chemoattractant for the initial influx of inflammatory cells.[ Immunopharmacologic Abnormalities

159]

158]

Leukotriene B4 is also released on exposure of AD skin to allergens and may act as

Leukocytes from patients with AD have genetically determined increased cyclic adenosine monophosphate (cAMP)–phosphodiesterase (PDE) enzyme activity. [ Cellular abnormalities associated with this finding include increased IgE synthesis by B cells, increased IL-4 production by T cells, and increased histamine releasability in basophils.[

161]

160]

162]

The greatest PDE abnormality is seen in AD monocytes that have a unique, highly active isoenzyme.[

Monocytes from patients with AD can modulate T cell dysfunction through the inhibition of IFN-γ production. This is mediated in part by increased monocyte PGE2 125

126

production associated with elevated PDE activity[ ] and by monocyte-associated IL-10.[ ] In addition, enhanced survival or decreased apoptosis of circulating and infiltrating monocytes, in association with increased production of GM-CSF in AD, may play an important role in the establishment of chronic inflammation. [163]

Role of Immunoglobulin E in Cutaneous Inflammation In AD, IgE may play an important role in allergen-induced cell-mediated reactions involving Th2-type cells that are distinct from conventional delayed-type 164]

hypersensitivity reactions mediated by Th1-type cells.[

IgE-dependent biphasic reactions are frequently associated with clinically significant allergic reactions

165 AD.[ ]

and may contribute to the inflammatory process of Immediate-type reactions related to mediator release by mast cells bearing allergen-specific IgE may result in the pruritus and erythema that occur after exposure to relevant allergens. IgE-dependent late phase reactions can then lead to more persistent symptoms. The 166

T cell infiltrate in cutaneous allergen-induced late phase reactions has increased mRNA for IL-3, IL-4, IL-5, and GM-CSF but not for IFN-γ.[ ] These cells are therefore similar to the Th2-type cells found in AD lesions. In addition, the cutaneous late phase reaction is associated with a pattern of adhesion molecule expression similar to that in AD.[

167]

Therefore a sustained IgE-dependent late phase reaction may be part of the chronic inflammatory process in AD.

Furthermore, epidermal Langerhans' cells in AD skin express IgE on their cell surface and are significantly more efficient than IgE-negative Langerhans' cells at 168

169

presenting allergen to T cells.[ ] A study by Jürgens et al[ ] extended these observations by demonstrating that Langerhans' cells from atopic individuals have a much higher level of FcepsilonRI expression. Efficient allergen capture and presentation to Th2 cells in atopic skin may be an important mechanism for sustaining local T cell activation. Skin-Directed Th2-Like Cell Response A number of studies have demonstrated important similarities between the allergic inflammation of asthma and AD. Common features include local infiltration of Th2-type cells in response to allergens, development of specific IgE to allergens, a chronic inflammatory process, and organ-specific hyperreactivity. In both diseases, IL-4– and IL-5–secreting memory Th2-type cells have a central role in the induction of local IgE responses and recruitment of eosinophils.[

3] [170]

The

171

recognition of T cell heterogeneity based on expression of tissue-selective homing receptors[ ] suggests that an individual's propensity for specific allergic disease may be a function of end-organ targeting by their effector T cells. In this respect, T cells migrating to the skin express CLA, whereas most memory/effector T cells isolated from asthmatic airways do not.[

172]

In a study of patients with milk-induced AD, casein-reactive T cells expressed significantly higher levels of CLA than did Candida albicans-reactive T cells from

173

these patients or casein-reactive T cells from patients with milk-induced enterocolitis or eosinophilic gasteroenteritis.[ ] Additional evidence for selective end-organ targeting by T cell subsets in allergic inflammation includes recent data showing that dust mite–specific T cell proliferation in mite-sensitized patients with AD was 174

localized to the CLA-expressing fraction of T cells.[ ] In contrast, T cells isolated from mite-allergic asthmatics that proliferated on exposure to the relevant allergen were CLA negative. Furthermore, CLA-expressing T cells isolated from patients with AD, but not from normal controls, showed evidence of activation (HLA-DR expression) and also spontaneously produced IL-4 but not IFN-γ. This suggests that T cell effector function in AD is closely linked to CLA expression. Immunologic Basis for Chronic Allergic Skin Inflammation The mechanisms responsible for chronic inflammation in AD have not been fully elucidated. However, several interdependent factors are probably involved ( Fig. 861 ). One important factor is likely to be repeated exposure to allergens such as foods, aeroallergens, and microorganisms. This can lead to chronic allergic responses 82 86] [95]

and Th2-type cell expansion. Consistent with this concept, specific allergen avoidance can result in clinical improvement or clearing of AD.[ ] [ clinical improvement after treatment with antistaphylococcal antibiotics may be related to the reduction of S. aureus exotoxin levels on the skin. 175

In addition,

Toxins acting as superantigens can induce CLA expression by stimulating IL-12 production.[ ] In addition, by stimulating epidermal Langerhans' cells and macrophages to secrete IL-1 and TNF-α, staphylococcal exotoxins can induce vascular endothelial E-selectin expression. This in turn would facilitate

1567

Figure 86-1 Immunologic abnormalities in the progression of atopic dermatitis. (From Leung DY: J Allergy Clin Immunol 105:861, 2000.)

(From Leung DY: J Allergy Clin Immunol 105:861, 2000.) migration of CLA+ T cells to the area. Furthermore, toxin-stimulated Langerhans' cells migrating to regional skin-associated lymph nodes act as antigen-presenting cells and could produce IL-12 locally, thus influencing the skin-homing capability of antigen-stimulated T cells. Toxins also stimulate a high number of T cells via the variable domain of the T cell receptor β chain to proliferate and secrete the cytokines implicated in tissue inflammation. By eliciting an IgE response, staphylococcal toxins could exacerbate AD by activating mast cells, basophils, or other FcepsilonRI-bearing cells. In addition, other staphylococcal proteins such as protein A and α-toxin can participate in the induction of local inflammation in AD by releasing TNF-α from epidermal keratinocytes.[ pathways could then lead to persistent cutaneous inflammation.

90] [176]

Such amplifying

Furthermore, cytokines secreted by Th2-type cells after exposure to allergen inhibit IFN-γ production by Th1-type cells; increase IgE synthesis; and promote the 2

177]

migration, differentiation, and proliferation of eosinophils.[ ] Mast cells can also produce IL-4 after allergen stimulation.[ high levels of IL-10 and PGE2 , which antagonize Th1-type

126 cells.[ ]

Monocytes from AD patients express

Therefore, conditions favoring a persistent Th2-type cell response may be established in AD 165

(see Fig. 86-1 ). Monocytes from AD patients also have a lower incidence of spontaneous apoptosis associated with increased production of GM-CSF.[ ] Together with IL-5, GM-CSF contributes to increased survival and infiltration of eosinophils in chronic AD. Finally, allergen-induced inflammation can alter corticosteroid 178]

receptor binding affinity, thus blunting the antiinflammatory effects of corticosteroids. [

The itch-scratch cycle probably contributes to skin inflammation in AD as well. Recent studies demonstrating that keratinocytes are an important source of cytokines have provided new insights into the mechanisms by which scratching could promote inflammation. In this regard, scratching can injure or stimulate keratinocytes, 179

176

leading to the release of cytokines, such as IL-1[ ] and TNF-α,[ ] that are necessary for the induction of adhesion molecules that attract cells into cutaneous sites of inflammation. Both resident and infiltrating cells could then perpetuate the inflammatory process by secreting additional cytokines and mediators. In summary, antigen or superantigen exposure, allergen-induced IgE synthesis and Th2-type cell expansion, mast cell degranulation, and keratinocyte injury may all contribute to chronic AD skin inflammation and possibly to nonspecific cutaneous hyperresponsiveness as well.

MANAGEMENT Conventional Therapy The current understanding of the pathophysiology of AD supports the concept that assessing the role of allergens, infectious agents, irritants, physical environment, and emotional stressors is equal in importance to initiating therapy with

1568

Figure 86-2 Approach to the patient with atopic dermatitis (AD). R/o MRSA, rule out methicillin-resistant Staphylococcus aureus; UV, ultraviolet.

Box 86-4. Selected Topical Corticosteroid Preparations

*

Group 1 Clobetasol propionate (Temovate) 0.05% ointment/cream Betamethasone dipropionate (Diprolene) 0.05% ointment/cream

Group 2 Mometasone furoate (Elocon) 0.1% ointment Halcinonide (Halog) 0.1% cream Fluocinonide (Lidex) 0.05% ointment/cream Desoximetasone (Topicort) 0.25% ointment/cream

Group 3 Fluticasone propionate (Cutivate) 0.005% ointment Halcinonide (Halog) 0.1% ointment Betamethasone valerate (Valisone) 0.1% ointment

Group 4 Mometasone furoate (Elocon) 0.1% cream Triamcinolone acetonide (Kenalog) 0.1% ointment/cream Fluocinolone acetonide (Synalar) 0.025% ointment

Group 5 Fluocinolone acetonide (Synalar) 0.025% cream Hydrocortisone valerate (Westcort) 0.2% ointment

Group 6 Desonide (DesOwen) 0.05% ointment/cream/lotion Alclometasone dipropionate (Aclovate) 0.05% ointment/cream

Group 7 Hydrocortisone (Hytone) 2.5% & 1% ointment/cream

Modified from Stoughton RB: Vasoconstrictor assay-specific applications. In Maibach HI, Surber C, editors: Topical corticosteroids, Basel, Switzerland, 1992, Karger, pp 42–53.

* Representative steroids are listed by group from 1 (superpotent) through 7 (least potent).

must be used cautiously, especially under occlusion, because they may lead to significant atrophic changes and systemic side effects. Topical corticosteroids are available in a variety of bases, including ointments, creams, lotions, solutions, gels, sprays, oil, and even tape (see Box 86-4 ). There is therefore no need to compound these medications. Ointments are most occlusive and as a rule provide better delivery of the medication while preventing evaporative 203

losses. In addition, ointments have been shown to spread more evenly when compared with other formulations such as creams and solutions.[ ] In a humid environment, creams may be better tolerated than ointments, because the increased occlusion can cause itching or even folliculitis. In general, however, creams and lotions, although easier to spread, are less effective and can contribute to skin dryness and irritation. Solutions can be used on the scalp and hirsute areas, although their alcohol content can be quite irritating, especially if used on inflamed or open lesions. In addition to their irritant potential, additives used to formulate the

204]

different bases can cause sensitization. Furthermore, allergic contact dermatitis to the corticosteroid molecule is being recognized with increasing frequency.[ This diagnosis is often difficult to establish on clinical grounds it can present as acute or chronic eczema. Patch testing has been done primarily with tixocortol 205]

pivalate and budesonide.[

Expanded testing has been associated with both false-positive and false-negative reactions.[

206]

Inadequate prescription size often contributes to suboptimally controlled AD, especially when patients have widespread, chronic disease. Approximately 30•g of medication is needed to cover the entire body of an average adult. The fingertip unit (FTU) has been proposed as a measure for applying topical corticosteroids and 207

has been studied in children with AD.[ ] This is the amount of topical medication that extends from the tip to the first joint on the palmar aspect of the index finger. It takes approximately one FTU to cover the hand or groin, 2 FTUs for the face or foot, 3 FTUs for an arm, 6 FTUs for the leg, and 14 FTUs for the trunk. Patients need to be instructed in the proper use of topical corticosteroids. Application of an emollient immediately before or over a topical corticosteroid preparation may decrease the effectiveness of the corticosteroid. Patients often assume that the potency of their prescribed corticosteroid is based solely on the percentage noted after the compound name (e.g., they believe that hydrocortisone 2.5% is more potent than clobetasol 0.05%) and therefore may apply the preparations incorrectly. In addition, patients are often given a high-potency corticosteroid and told to discontinue it after a period of time without being given a lower-potency corticosteroid; this can result in rebound flaring of the AD, similar to what is often seen with oral corticosteroid therapy. A step-care approach with a mid-range or high-potency preparation (although usually not to the face, axillae, or groin), followed by low-potency preparations, may be more successful. Once-daily treatment, which may help with patient adherence to the regimen, has been shown to be effective for fluticasone propionate, a molecule with an increased 208

209

binding affinity for the corticosteroid receptor.[ ] Topical mometasone has been studied in children with AD and is also approved for once-daily use.[ ] Topical corticosteroids usually have been discontinued after the inflammation resolves, while hydration and moisturizers are continued. However, because even normalappearing skin in AD shows evidence of immunologic dysregulation, the use of topical corticosteroids as “maintenance therapy” has been suggested. Van Der Meer 210

et al[ ] showed that, once control of AD with a once-daily regimen was achieved, long-term control could be maintained with twice-weekly applications of fluticasone to areas that appeared to have healed. This approach resulted in delayed relapses of AD compared with placebo therapy. 211

In addition to their antiinflammatory properties, topical corticosteroids can decrease S. aureus colonization in patients with AD.[ ] In a double-blind, randomized 1week trial of desonide compared with a vehicle in children with AD, clinical scores improved and S. aureus density significantly decreased with in desonide group 212

but not in the vehicle group.[ ] Finally, a number of patients with AD may not show a clinical improvement with topical corticosteroids. Reasons for this result may include complication by superinfection or inadequate potency of the preparation used (discussed earlier). In addition, allergen-induced immune activation can alter the T cell response to glucocorticoids by inducing cytokine-dependent abnormalities

1571

in glucocorticoid receptor binding affinity.[

213]

PBMCs from patients with chronic AD have reduced glucocorticoid receptor binding affinity, which can be sustained 214

with the combination of IL-2 and IL-4 in vitro. In addition, corticosteroid unresponsiveness may contribute to treatment failure in some patients.[ ] Endogenous cortisol levels have been found to control the magnitude of cutaneous allergic inflammatory responses, suggesting that impaired response to steroids could contribute

215

216

to chronic AD.[ ] Alternatively, Blotta, DeKruyff, and Umetsu[ ] suggested that chronic corticosteroid therapy can have deleterious, albeit insidious, effects in allergic patients. These results are based on in vitro data that may not recreate the complex milieu in allergic inflammation. A much more frequent reason for failure of corticosteroid therapy is nonadherence to the treatment regimen. Patients or parents often expect a quick and permanent resolution of the AD and become disillusioned by the lack of cure with current therapy. A significant number of patients and caregivers also admit to nonadherence to prescribed topical corticosteroid 217]

therapy due to fear of using this class of medications.[

These findings emphasize the need for both education and alternative therapies.

Systemic corticosteroids, including oral prednisone, should be avoided in the management of a chronic, relapsing disorder such as AD. Often, patients or parents demand immediate improvement of the disease and find systemic corticosteroids more convenient to use than topical therapy. However, the dramatic improvement observed with systemic corticosteroids may be associated with an equally dramatic flaring of AD after discontinuation. If a short course of oral corticosteroids is given, it is usually best to prescribe a tapering dose. Topical skin care, particularly with topical corticosteroids, should be intensified during the taper to suppress rebound flaring of AD. Tar Preparations.

Crude coal tar extracts have antiinflammatory properties that are not as pronounced as those of topical corticosteroids. Nevertheless, in a recent study using the atopy patch test, tar was similar to a topical steroid in its ability to inhibit the influx of a number of proinflammatory cells and in the expression of adhesion molecules in 218

response to epicutaneous allergen challenge.[ ] Tar preparations used with topical corticosteroids in chronic AD may reduce the need for more potent corticosteroid preparations. Tar shampoos (T/Gel, Ionil-T) are often beneficial for scalp involvement. The use of tar preparations on acutely inflamed skin should be avoided because it may result in skin irritation. Other than dryness or irritation, side effects associated with tar products are rare but include photosensitivity reactions and a pustular folliculitis. Wet Dressings.

Wet-wrap dressings reduce pruritus and inflammation by cooling the skin, act as a barrier to trauma associated with scratching, and improve penetration of topical corticosteroids. In one study, children with severe AD showed significant clinical improvement after 1 week of treatment using tubular bandages applied over diluted 219

topical corticosteroids.[ ] Of note, no significant differences were demonstrated among several dilutions of a mid-potency corticosteroid, suggesting that clinical benefit can be achieved with this approach in more severely affected patients even with the use of lower-potency corticosteroids. Although long-term studies with 220

this therapy are lacking, most of the improvement in the latter study occurred during the first week. An alternative approach employs clothing,[ ] using wet pajamas or long underwear, with dry pajamas or a sweatsuit on top. Hands and feet can be covered by wet tube socks under dry tube socks. Alternatively, the face, trunk, or extremities can be covered by wet gauze with dry gauze over it and secured in place with an elastic bandage or pieces of tube socks. Dressings may be removed when they dry, or they may be re-wetted. They are often best tolerated at bedtime. Overuse of wet dressings can result in chilling, maceration of the skin, or, infrequently, secondary infection. Because this approach can be somewhat labor-intensive, it may be best reserved for acute exacerbations of AD along with selective use in areas of resistant dermatitis. Antiinfective Therapy.

Systemic antibiotic therapy may be necessary to treat AD when a secondary infection with S. aureus is present. Semisynthetic penicillins or first- or second-

generation cephalosporins for 7 to 10 days are usually effective. Erythromycin-resistant organisms are fairly common, making macrolides less useful alternatives. 221

Unfortunately, recolonization after a course of antistaphylococcal therapy occurs rapidly.[ ] Maintenance antibiotic therapy, however, should be avoided, because it may result in colonization by methicillin-resistant organisms. The topical antistaphylococcal antibiotic mupirocin (Bactroban), applied three times daily to affected areas for 7 to 10 days, may be effective for treating localized areas of involvement.[

222]

Twice-daily treatment for 5 days with a nasal preparation of mupirocin may

223 AD.[ ]

224]

reduce nasal carriage of S. aureus, which may result in clinical benefit in Although antibacterial cleansers are effective in reducing bacterial skin flora, [ they can cause significant skin irritation. However, a recent double-blind, placebo-controlled study found that daily bathing with an antimicrobial soap containing 1.5% triclocarban resulted in reductions in S. aureus colonization and significantly greater clinical improvement than with the placebo soap.[

225]

226

Patients with disseminated eczema herpeticum, also referred to as Kaposi's varicelliform eruption, usually require treatment with systemic acyclovir.[ ] Recurrent cutaneous herpetic infections can be controlled with daily prophylactic oral acyclovir. Superficial dermatophytosis and P. ovale infections can be treated with topical or, rarely, with systemic antifungal drugs. [

66] [227]

Antipruritic Agents.

Pruritus is the most common and usually the least tolerated symptom of AD. Even partial reduction of pruritus can result in significant improvement in quality of life 228]

for patients with severe AD.[

The participation of histamine in the pruritus of AD has been questioned, and in a dermal microdialysis study looking at mast cell

degranulation, the authors concluded that mediators other than histamine cause pruritus.[

229]

Neuropeptides or cytokines may be important mediators, because 230]

centrally acting agents such as opioid receptor antagonists have been shown to be effective against the itch of AD.[

It is also noteworthy that use of cyclosporin A, 231]

which results in decreased transcription of a number of proinflammatory cytokines, leads to rapid improvement in pruritus for many AD patients. [

Systemic antihistamines and anxiolytics may be most useful through their tranquilizing and sedative effects and can be used primarily in the evening to avoid daytime drowsiness.[

232]

The tricyclic antidepressant doxepin, which has both histamine1 (H1 )- and H2 -receptor binding affinity as well as a long half-life, may be

given as a single 10- to 50-mg dose in the evening. If nocturnal pruritus remains severe, short-term

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use of a sedative to allow adequate rest may be appropriate. Although second-generation antihistamines have been reported to be ineffective in treating the pruritus 233

234 235

associated with AD,[ ] several studies have shown clinical benefit in at least some AD patients.[ ] [ ] Of potential interest, cetirizine, 10•mg twice daily in a placebo-controlled, double-blind study in allergen-specific challenge, reduced the number of both eosinophils and neutrophils in early and late phase reactions, 236

suggesting antiinflammatory as well as antihistamine effects.[ ] In addition, treatment of a large cohort of young AD patients with cetirizine for 18 months reduced the number of children who developed asthma in the subgroups sensitized to house dust mite or pollen, although the primary outcome analysis did not show a

statistical difference between the cetirizine and placebo groups.[ more defined subset of children with AD and allergies.

237]

These findings needs to be confirmed in well-controlled studies, possibly by first studying a

Treatment of AD with topical antihistamines and local anesthetics should be used cautiously because of potential sensitization. A multicenter, double-blind, vehiclecontrolled study of topical 5% doxepin cream resulted in significant reduction of pruritus.[

238]

In this 1-week study, sensitization was not reported, although

rechallenge with the drug after the 7-day course of therapy was not evaluated. Later case reports have documented reactions to topical doxepin.[

239]

Recalcitrant Disease Hospitalization

AD patients who are erythrodermic or who appear toxic may need to be hospitalized. Hospitalization may also be appropriate for patients with severe disseminated disease resistant to first-line therapy. Often, removing the patient from environmental allergens or stressors, together with intense education and assurance of compliance with therapy, results in marked clinical improvement. In this setting, the patient can also undergo appropriately controlled provocative challenges to help identify potential triggering factors. Phototherapy and Photochemotherapy

Ultraviolet (UV) light therapy can be a useful treatment modality for chronic recalcitrant AD. It may exert its therapeutic effect in part by decreasing the expression 240

of activation markers such as HLA-DR, IL-2 receptor, and CD30 on CLA-positive T cells.[ ] Patients who do not experience photoexacerbations of their AD and who are not fair complexioned may benefit from moderate amounts of natural sunlight. However, they should be cautioned not to sunburn and to avoid perspiring, which can induce pruritus. Sunlamp treatment at home usually is not recommended because of the danger of overexposure. Under medical supervision, UVB is effective in the treatment of AD,[

241]

although narrow-band UVB may be a safer alternative.[

242]

The addition of UVA to UVB can increase the therapeutic response.

[243]

244

Alternatively, high-dose UVA1 is a fast-acting and effective phototherapeutic approach in patients with acute exacerbations of AD.[ ] Unlike traditional UVAUVB phototherapy, which appears less effective for acute exacerbations and acts primarily in the epidermis, high-dose UVA1 therapy has been shown to significantly decrease dermal IgE-binding cells, including mast cells and dendritic cells.[

245]

UVA1 may also exert antiinflammatory effects indirectly by down-

regulating proinflammatory cytokines or directly by inducing apoptosis in skin-infiltrating CD4+ T cells. [

246]

Photochemotherapy with oral methoxypsoralen therapy followed by UVA (PUVA) may be indicated in patients with severe AD, especially with failure of topical 247]

therapy in patients with significant corticosteroid side effects.[

Short-term adverse effects may include erythema, pruritus, and pigmentation, whereas long-term

adverse effects include premature skin aging and cutaneous malignancies. [

248]

249]

Topical psoralens combined with UVA may be equally effective.[ 250

251

PUVA therapy in children with severe AD and growth suppression has resulted in accelerated growth.[ ] In a follow-up study,[ ] children with severe AD unresponsive to other therapy were treated with twice-weekly PUVA; significant improvement was observed in 74% after a mean of 9 weeks. In 42%, remission was

sustained 1 year after discontinuation of treatment. However, the long-term risk of cutaneous malignancies has usually precluded treatment of children with PUVA. Immunomodulatory Therapy Because AD is associated with a number of immunoregulatory abnormalities, therapy directed at correction of the immune dysfunction represents a rational alternative for AD unresponsive to conventional therapies. Alternative treatment modalities may be especially useful for patients in whom corticosteroid resistance could be contributing to treatment failure. Interferons

IFN-γ suppresses IgE synthesis[

252]

and inhibits Th2 cell function.[

253]

Treatment with subcutaneous recombinant human interferon-γ (rhIFN-γ) results in reduced

clinical severity and decreased total circulating eosinophil counts in patients with AD.[

254] [255]

Clinical improvement has also been shown to correlate with

reduction in white blood cells, eosinophil, and lymphocyte counts and normalization of the CD4/CD8 ratio among large lymphocytes.[ persistent improvement several months after discontinuation of

257 therapy.[ ]

256]

Some patients may show

Two open, long-term studies showed clinical efficacy in AD patients treated for a 258 259

50••g/m2

minimum of 22 months with rhIFN-γ given daily or every other day.[ ] [ ] These studies demonstrated that patients with AD can be treated on a chronic basis with rhIFN-γ without deterioration of their disease or significant adverse effects. This is noteworthy, because IFN-γ has been shown to have proinflammatory effects in some clinical settings. Importantly, effective dosing with rhIFN-γ is associated with a decrease in eosinophil counts, suggesting that rhIFN-γ acts primarily on the allergic inflammatory response, as opposed to IgE synthesis. Therefore, it is possible that a subset of patients treated with rhIFN-γ would respond to individualized titration of their treatment dose.[

260]

Calcineurin Inhibitors

Calcineurin inhibitors include cyclosporin A, tacrolimus (FK506), and pimecrolimus, an ascomycin derivative. They act primarily on T cells, binding to intracellular 261 262

immunophilins, and this complex in turn inhibits calcineurin, a calcium ion–calmodulin-dependent protein phosphatase involved in signal transduction. [ ] [ ] Activation of calcineurin is necessary for initiation of cytokine gene transcription. In addition, calcineurin inhibitors may also exert an immunosuppressive effect through targeting of a calcineurin-independent activation

1573

pathway for JNK and p38.[

263]

They also affect other cells that contribute to the complex inflammatory milieu in AD, as discussed later. 262]

Cyclosporin A, the first in this class of drugs, binds to cyclophilin. [

Of note, maintenance of chronic inflammation in AD appears to be associated with increased

3

IL-5 gene expression and eosinophil infiltration,[ ] and preliminary in vitro data with mononuclear cells from atopic patients demonstrate suppression of IL-5 264

production by cyclosporin A.[ ] A significant decrease in the number of circulating eosinophils has also been observed with cyclosporin A therapy. These studies provide a rationale for the use of cyclosporin A in AD. The benefit of oral cyclosporin A in severe AD in adults has been demonstrated in both open trials and placebo-controlled studies.[ related quality of life showed significant improvement after treatment with cyclosporin A.

[267]

265] [266]

In addition, health-

Data on oral cyclosporin A in children with AD are limited, with one 268

open study in which cyclosporin A, 5•mg/kg daily for 6 weeks, demonstrated significant improvement.[ ] Discontinuation of treatment resulted in variable relapse. A 1-year study of cyclosporin A in a pediatric population using either intermittent or continuous treatment showed no significant differences between these two approaches with respect to efficacy or safety parameters, and a subset of patients remained in remission after treatment was stopped.[

269]

Short-term oral cyclosporin A therapy can result in increased serum urea, creatinine, and bilirubin concentrations, but these values normalize after treatment is discontinued.[

265] [266]

Because of the concern for progressive or irreversible nephrotoxicity with extended treatment, few patients have been evaluated on 270

maintenance therapy. In one study, [ ] patients with severe AD treated with oral cyclosporin A, 5•mg/kg per day for 6 weeks, were monitored until relapse and then were treated with a second 6-week course. Although this treatment regimen did not result in lasting remission for the majority of patients, a subset of patients 271]

appeared to get extended clinical benefit. In another prospective, open, multicenter study of 100 adults with severe AD, subjects were treated for up to 48 weeks.[ For the first 8 weeks, cyclosporin A was administered at 2.5•mg/kg per day, then adjusted according to clinical response. Cyclosporin A produced rapid and highly significant improvements in all indices of disease activity, including signs and symptoms, body surface area, pruritus, and sleep disturbance. Topically administered cyclosporin A has also been studied in AD. A 3-week double-blind, vehicle-controlled study with both 10% cyclosporin A gel and 10% cyclosporin A ointment failed to produce significant improvement in AD.[

272]

273]

Tacrolimus, another calcineurin inhibitor, has a spectrum of activity similar to that of cyclosporin A, although it is structurally unrelated to cyclosporin A.[ Tacrolimus interacts with FK binding protein-12 (FKBP-12), interfering with both type 1 and type 2 cytokine gene transcription.[

262] [274]

In addition, mast cells,

basophils, eosinophils, keratinocytes, and Langerhans' cells have FKBP and down-regulate their mediator or cytokine expression after treatment with tacrolimus.[ [276]

275]

Treatment with tacrolimus ointment was recently shown to result in decreased FcepsilonRI expression on both Langerhans' cells and inflammatory dendritic 277

epidermal cells, correlating with clinical improvement.[ ] This treatment also resulted in a decrease of the subset of inflammatory dendritic epidermal cells within the pool of CD1a+ epidermal dendritic cells, together with decreased CD36 expression, consistent with decreased local inflammation. 278

Nakagawa et al[ ] showed that treatment with tacrolimus ointment was clinically beneficial in AD, resulting in markedly diminished pruritus within 3 days after initiation of therapy. Biopsy results from days 3 and 7 of treatment revealed markedly diminished T cell and eosinophilic infiltrates. No systemic side effects were noted over the 21 days of treatment. Subsequently, multicenter, blinded, vehicle-controlled 3-week trials with tacrolimus ointment 0.03% and 0.1% in both adults [279]

and children[

280]

with AD showed it to be both safe and effective, with a burning sensation the only significant adverse event. The rapid healing observed with 281]

this topical therapy was suggested to be associated with decreased absorption and contributed to its safety profile.[

Phase 3 studies confirmed these findings,[

282]

and tacrolimus ointment (Protopic) 0.03% for children 2 to 15 years of age and 0.03% and 0.1% for adults has been approved by the U.S. Food and Drug Administration (FDA) for short-term and intermittent long-term use in moderate-to-severe AD. Because AD is a chronic disease, long-term, open studies with tacrolimus ointment applied on up to 100% of body surface area have been performed for up to 12 months in adults and in children, without a suggestion of tachyphylaxis or an increase in skin infections.[ [285]

283] [284]

Indeed, colonization by S. aureus was shown to decrease during long-term therapy with tacrolimus ointment.

In addition, unlike topical corticosteroids, tacrolimus ointment does not cause cutaneous atrophy and has been used for facial and eyelid eczema.[

286]

Recently, the in vitro effects of tacrolimus versus a corticosteroid on expression of CD25 and the co-stimulatory molecules CD80, CD86, and CD40, as well as major histocompatibility complex (MHC) class I and II molecules, were compared and shown to be different.[

275]

In addition, tacrolimus, but not dexamethasone, was

287 proliferation.[ ]

shown to suppress SEB-induced PBMC These studies suggest possible complementary actions of tacrolimus and corticosteroids and a potentially unique role for tacrolimus ointment in steroid-insensitive AD. However, these in vitro observations require in vivo confirmation. Ascomycin compounds binding to macrophilin-12 (FKBP-12) have been developed in oral and topical forms.[

288]

Like tacrolimus, they inhibit cytokine production

289 basophils.[ ]

and have also been shown to inhibit mediator release from mast cells and Topical pimecrolimus (SDZ ASM 981) 1% cream applied twice daily in a randomized, double-blind, placebo-controlled, right-and-left comparison trial in adult patients with moderate AD, was significantly more effective than either a vehicle cream or once-daily treatment over a 21-day period.[

290]

No clinically relevant drug-related adverse effects were noted.[

291]

In a vehicle and betamethasone291

17-valerate 0.1% cream controlled dose-finding study of 260 adults with AD, the 1% concentration of pimecrolimus was found to be most effective.[ ] Although the topical corticosteroid was more effective than the pimecrolimus creams tested in this study, the authors suggested that the efficacy plateau was not reached with the latter during the 3 weeks of treatment. Again, burning or a feeling of warmth was the only adverse event reported more frequently with active drug. Subsequent trials with pimecrolimus 1% cream in children as

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292 293

young as 1 year of age with mild-to-moderate AD also showed both safety and efficacy, [ ] [ ] and the drug is being studied in children as young as 3 months of age with all disease severities. It has been approved by the FDA as Elidel 1% cream for patients as young as 2 years of age who have mild-to-moderate disease. Experimental and Unproven Therapies Allergen Desensitization

A number of uncontrolled trials have suggested that desensitization to specific allergens may improve AD.[ 295 months.[ ]

294]

In a controlled study, patients with AD were treated

with either allergen extract or placebo in a blinded fashion for longer than 24 Some 81% of patients in the active treatment group improved, compared with 40% in the placebo group. However, patients were not treated with the same extract, and standardized allergens were not available. In a more recent double-

blind, controlled trial of desensitization to house dust mite with tyrosine-adsorbed Dermatophagoides pteronyssinus extract, children with AD and immediate 296

hypersensitivity to D. pteronyssinus failed to demonstrate any clinical benefit for desensitization compared with placebo after an 8-month course of treatment. [ ] In a second phase, children to whom D. pteronyssinus extract was initially administered were randomly assigned to continue on active treatment or placebo for an additional 6 months. The clinical scores suggested that extended desensitization may be more effective than placebo, but the numbers were too small to permit confident conclusions. A high placebo effect may have concealed any additional therapeutic effect from active treatment. Further controlled trials with standardized extracts of relevant allergens in AD are needed before this form of therapy can be recommended. Intravenous Gammaglobulin

Because chronic inflammation and T cell activation appear to play a critical role in the pathogenesis of AD, high-dose intravenous immunoglobulin (IVIG) could have immunomodulatory effects in this disease. IVIG could also interact directly with infectious organisms or toxins involved in the pathogenesis of AD. In this respect, IVIG has been shown to contain high concentrations of staphylococcal toxin-specific antibodies that inhibit the in vitro activation of T cells by staphylococcal toxins. [

297]

The mechanism of inhibition by IVIG was shown to be direct blocking of toxin binding to, or presentation by, antigen-presenting cells. In

addition, IVIG has been shown to reduce IL-4 protein expression in AD.[

298]

Treatment of severe refractory AD with IVIG has yielded conflicting results. Studies have not been controlled and have involved small numbers of patients. Kimata [299]

reported dramatic improvement of AD with this treatment modality in a small, uncontrolled study. However, in a study of nine patients with severe AD treated with IVIG (Venoglobulin-I, Alpha Therapeutic Corporation), 2•g/kg monthly for seven infusions, skin disease improved slightly in six patients, while their average 300

daily prednisone dosage did not change significantly.[ ] Mean serum IgE levels did not decrease significantly during IVIG therapy, and in vitro IgE production by PBMCs after IL-4 and anti-CD40 stimulation was not significantly reduced. The authors concluded that IVIG was of no clear clinical benefit in this group of patients. Controlled studies are needed to answer the question of efficacy in a more definitive manner. Traditional Chinese Herbal Therapy

Traditional Chinese herbal therapy (TCHT) in the form of decoctions (boiled and strained extracts) was studied using a standardized formulation in a placebo301

controlled, double-blind, crossover trial involving children with widespread nonexudative AD.[ ] Response to active treatment was superior to placebo with no evidence of systemic toxicity. Patients enrolled in a 1-year study showed significant reduction in their eczema activity scores, although most required continued 302]

therapy.[

Asymptomatic elevation of liver function was noted on one occasion in two children but normalized after discontinuation of the herbal treatments. 303

TCHT was also studied in a double-blind, placebo-controlled crossover study in 40 adults with long-standing refractory AD.[ ] Clinical scores, as well as improvement in itching and sleep, improved significantly during the active treatment phase. No side effects were reported by the patients. Nevertheless, clinical 304]

benefit with TCHT may be short lived, and effectiveness may diminish despite continued treatment.[

A study involving Chinese adult and pediatric patients with

305 decoction.[ ]

recalcitrant AD failed to demonstrate benefit with a standardized Of interest, the authors argued that failure of this formulation was predictable, because practitioners of TCHT would have prescribed a unique herbal preparation for each patient. TCHT used in these studies consists of 10 herbs, some with known pharmacologic properties.[

306]

These may include antimicrobial, sedative, antiinflammatory, and

corticosteroid-like activities. TCHT can inhibit the low-affinity receptor (CD23) for IgE on peripheral blood monocytes in a dose-dependent manner not due to a 307

308

toxic effect.[ ] In addition, down-regulation of CD23 on cutaneous antigen-presenting cells has been demonstrated.[ ] The specific ingredients of TCHT that may be responsible for clinical improvement in AD remain to be elucidated. The possibilities of toxicity associated with long-term use, idiosyncratic reactions, and 309] [310] [311]

adulteration with corticosteroids remain a concern, and TCHT for AD continues to be an investigational therapy.[ Essential Fatty Acids

A number of disturbances in the metabolism of essential fatty acids (EFAs) have been reported in patients with AD.[ [313]

312]

Consequently, clinical trials with either fish 314

oil as a source of n-3 series EFAs or oil extracted from the seeds of Oenothera biennis (evening primrose) as a source of n-6 series EFAs[ ] have been conducted, with conflicting results. A double-blind, placebo-controlled, parallel-group, randomized study that avoided the methodologic and analytic problems of a 315]

number of previous studies found no clinical benefit of either evening primrose oil or fish oil in AD.[ Mycophenolate Mofetil

Mycophenolate mofetil (MMF), a purine biosynthesis inhibitor, has been used as an immunosuppressant in organ transplantation and, more recently, for inflammatory skin disorders.[

316]

Oral MMF, 2•g daily as monotherapy, was first reported to result in clearing of skin lesions in two adults with AD resistant to other

treatment including topical and oral streroids and PUVA.[

317]

Improvement was evident by 2 to 4 weeks after initiation of MMF therapy, and no side effects

1575

were noted. Neither patient experienced relapse during a 12-week follow-up period. An additional 10 consecutive AD patients were treated with 1•g orally twice 318

daily for 4 weeks, then 500•mg twice daily for an additional 4 weeks.[ ] Clinical improvement was demonstrated in the majority of the patients, and six of the seven responders showed lasting remission during 20 weeks of follow-up. The drug was well tolerated, with the exception that one patient developed herpes retinitis. 319

320

Although similar results were reported in another open study,[ ] Hansen et al[ ] reported lack of benefit in five patients with AD treated with 1 to 1.25•g MMF twice daily for up to 12 weeks. Dose-finding and controlled studies need to be carried out. Phosphodiesterase Inhibitors 321]

Because monocytes from AD patients have an abnormal increase in PDE enzyme activity, PDE inhibitors such as Ro 20-1724 decrease IgE synthesis[ release of basophil histamine in PGE2

161 .[ ]

322 vitro.[ ]

and the

Culture of AD monocytes with Ro 20-1724 also resulted in significant reduction of abnormal levels of IL-4, IL-10, and

Patients with AD treated with CP80,633, a potent inhibitor of PDE type 4, applied topically in a blinded, placebo-controlled, paired-lesion study showed

significant clinical improvement with the active drug.[

161]

In addition, a PDE 4 inhibitor, but not PDE 3 or PDE 5 inhibitors, was shown to inhibit SEB-mediated 323]

CLA expression on T cells through its effect on IL-12 production.[ Leukotriene Receptor Antagonists

The role of cysteinyl leukotrienes has not been well defined in AD, although a recent study demonstrated antigen-specific release of leukotriene B4 in vivo in nonlesional skin from patients with AD using skin chambers. [

324]

Preliminary small open trials of leukotriene D4 receptor antagonists in adults with AD suggested

325

326

benefit in some patients with both zafirlukast[ ] and montelukast as adjunctive therapy.[ ] A recent controlled pilot study with montelukast in children with moderate-to-severe AD also showed statistical improvement with active drug, compared with placebo, in the 11 patients completing the protocol, when montelukast 327]

was added to conventional therapy that included a topical corticosteroid.[

SUMMARY AND CONCLUSIONS Although the diagnosis of AD continues to be based on the recognition of characteristic signs and symptoms, significant advances have been made in understanding the immunopathogenesis of this increasingly prevalent disease. These studies have unraveled a multifunctional role for IgE in atopic skin inflammation. Furthermore, Th2-type cells with skin-homing capability, mast cells, Langerhans' cells, keratinocytes, and eosinophils all contribute to the complex inflammatory process in AD. These observations have provided the rationale for development of immunomodulatory and antiinflammatory agents in the treatment of chronic AD. Identification of a specific biochemical or genetic marker could not only improve diagnostic capabilities but also result in more specific strategies for studying the epidemiology and genetics of AD. Undoubtedly, the new insights into mechanisms involved in allergic inflammation will lead to more specific approaches to the therapy and, perhaps eventually, to prevention of this disease.

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239. Shelley WB, Shelley ED, Talanin NY: Self-potentiating allergic contact dermatitis caused by doxepin hydrochloride cream, J Am Acad Dermatol 34:143–144, 1996. 240. Piletta PA, Wirth S, Hommel L, et al: Circulating skin-homing T cells in atopic dermatitis: selective up-regulation of HLA-DR, interleukin-2R, and CD30 and decrease after combined UV-A and UV-B phototherapy, Arch Dermatol 132: 1171–1176, 1996. 241. Jekler J, Larko O: UVB phototherapy for atopic dermatitis, Br J Dermatol 119: 697–705, 1988. 242. George SA, Bilsland DJ, Johnson BE, et al: Narrow band (TL-01) UVB air conditioned phototherapy for chronic severe adult atopic dermatitis, Br J Dermatol 128:49–56, 1993. 243. Midelfart K, Stenvold SE, Voloden G: Combined UVB and UVA phototherapy of atopic eczema, Dermatologica 171:95–98, 1985. 244. Krutmann J, Czech W, Giepgen T, et al: High-dose UVA1 therapy in the treatment of patients with atopic dermatitis, J Am Acad Dermatol 26:225–230, 1992. 245. Krutmann J, Diepgen TL, Luger TA, et al: High-dose UVA1 therapy for atopic dermatitis: results of a multicenter trial, J Am Acad Dermatol 38:589–593, 1998. 246. Morita A, Werfel T, Stege H, et al: Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy, J Exp Med 186:1763–1768, 1997. 247. Morison WL, Parrish JA, Fitzpatrick TB: Oral psoralen photochemotherapy of atopic eczema, Br J Dermatol 98:25–30, 1978. 248. Honig B, Morison WL, Karp D: Photochemotherapy beyond psoriasis, J Am Acad Dermatol 31:775–790, 1994. 249. Yoshiike T, Sindhvananda J, Aikawa Y, et al: Topical psoralen photochemotherapy for atopic dermatitis: evaluation of two therapeutic regimens for inpatients and outpatients, J Dermatol 18:201–205, 1991. 250. Atherton DJ, Carabott F, Glover MT, et al: The role of psoralen chemotherapy (PUVA) in the treatment of severe atopic eczema in adolescents, Br J Dermatol 118:791–795, 1988. 251. Sheehan MP, Atherton DJ, Norris P, et al: Oral psoralen photochemotherapy in severe childhood atopic eczema: an update, Br J Dermatol 129:431–436, 1993. 252. Pene J, Rousset F, Briere F, et al: IgE production by normal human lymphocytes is induced by interleukin 4 and suppressed by interferons gamma and alpha and prostaglandin E2, Proc Natl Acad Sci U S A 85:6880–6884, 1988. 253. Gajewski TF, Fitch FW: Anti-proliferative effect of IFN-γ in immune regulation: I. IFN-γ inhibits the proliferation of Th2 but not Th1 murine helper T lymphocyte clones, J Immunol 140:4245–4252, 1988. 254. Boguniewicz M, Jaffe HS, Izu A, et al: Recombinant gamma interferon in treatment of patients with atopic dermatitis and elevated IgE levels, Am J Med 88:365–370, 1990. 255. Hanifin JM, Schneider LC, Leung DY, et al: Recombinant interferon gamma therapy for atopic dermatitis, J Am Acad Dermatol 28:189–197, 1993.

256. Ellis CN, Stevens SR, Blok BK, et al: Interferon-gamma therapy reduces blood leukocyte levels in patients with atopic dermatitis: correlation with clinical improvement, Clin Immunol 92:49–55, 1999. 257. Reinhold U, Kukel S, Brzoska J, et al: Systemic interferon gamma treatment in atopic dermatitis, J Am Acad Dermatol 29:58–63, 1993. 258. Schneider LC, Baz Z, Zarcone C, et al: Long-term therapy with recombinantinterferon-gamma (rIFN-gamma) for atopic dermatitis, Ann Allergy Asthma Immunol 80:263–268, 1998. 259. Stevens SR, Hanifin JM, Hamilton T, et al: Long-term effectiveness and safety of recombinant human interferon gamma therapy for atopic dermatitis despite unchanged serum IgE levels, Arch Dermatol 134:799–804, 1998. 260. Boguniewicz M, Leung DY: Atopic dermatitis: a question of balance, Arch Dermatol 134:870–871, 1998. 261. Schreiber SL, Crabtree GR: The mechanism of action of cyclosporin A and FK506, Immunol Today 13:136–142, 1992. 262. Liu J, Farmer JD, Lane WS, et al: Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes, Cell 66:807–815, 1991. 263. Matsuda S, Shibasaki F, Takehana K, et al: Two distinct action mechanisms of immunophilin-ligand complexes for the blockade of T-cell activation, EMBO Rep 1:428–434, 2000. 264. Mori A, Suko M, Nishizaki Y, et al: Regulation of interleukin-5 production by peripheral blood mononuclear cells from atopic patients with FK506, cyclosporin A and glucocorticoid, Int Arch Allergy Immunol 104:32–35, 1994. 265. Sowden JM, Berth-Jones J, Ross JS, et al: Double-blind, controlled, crossover study of cyclosporin in adults with severe refractory atopic dermatitis, Lancet 338:137–140, 1991. 266. van Joost T, Heule F, Korstanje M, et al: Cyclosporin in atopic dermatitis: a multicentre placebo-controlled study, Br J Dermatol 130:634–640, 1994. 267. Salek MS, Finlay AY, Luscombe DK, et al: Cyclosporin greatly improves the quality of life of adults with severe atopic dermatitis: a randomized, double-blind, placebo-controlled trial, Br J Dermatol 129:422–430, 1993. 268. Berth-Jones J, Finlay AY, Zaki I, et al: Cyclosporine in severe childhood atopic dermatitis: a multicenter study, J Am Acad Dermatol 34:1016–1021, 1996. 269. Harper JI, Ahmed I, Barclay G, et al: Cyclosporin for severe childhood atopic dermatitis: short course versus continuous therapy, Br J Dermatol 142:52–58, 2000. 270. Granlund H, Erkko P, Sinisalo M, et al: Cyclosporin in atopic dermatitis: time to relapse and effect of intermittent therapy, Br J Dermatol 132:106–112, 1995. 271. Berth-Jones J, Graham-Brown RA, Marks R, et al. Long-term efficacy and safety of cyclosporin in severe adult atopic dermatitis, Br J Dermatol 136:76–81, 1997. 272. Die Rie MA, Meinardi MM, Bos JD: Lack of efficacy of topical cyclosporin A in atopic dermatitis and allergic contact dermatitis, Acta Derm Venereol 71:452– 454, 1991.

273. Kino T, Hatanaka H, Hashimoto M, et al: FK-506, a novel immunosuppressant isolated from a Streptomyces: I. Fermentation, isolation, physico-chemical and biologic characteristics, J Antibiot 40:1249–1255, 1987. 274. Sakuma S, Higashi Y, Sato N, et al: Tacrolimus suppressed the production of cytokines involved in atopic dermatitis by direct stimulation of human PBMC system (comparison with steroids), Int Immunopharmacol 1:1219–1226, 2001. 275. Panhans-Gross A, Novak N, Kraft S, et al: Human epidermal Langerhans' cells are targets for the immunosuppressive macrolide tacrolimus (FK506), J Allergy Clin Immunol 107:345–352, 2001. 276. Wakugawa M, Hayashi K, Nakamura K, et al: Evaluation of mite allergen-induced Th1 and Th2 cytokine secretion of peripheral blood mononuclear cells from atopic dermatitis patients: association between IL-13 and mite-specific IgE levels, J Dermatol Sci 25:116–126, 2001. 277. Wollenberg A, Sharma S, von Bubnoff D, et al: Topical tacrolimus (FK506) leads to profound phenotypic and functional alterations of epidermal antigenpresenting dendritic cells in atopic dermatitis, J Allergy Clin Immunol 107:519–525, 2001. 278. Nakagawa H, Etoh T, Ishibashi Y, et al: Tacrolimus ointment for atopic dermatitis, Lancet 344:883, 1994. 279. Ruzicka T, Bieber T, Schöpf E, et al: A short-term trial of tacrolimus ointment for atopic dermatitis, N Engl J Med 337:816–821, 1997. 280. Boguniewicz M, Fiedler VC, Raimer S, et al. A randomized, vehicle-controlled trial of tacrolimus ointment for the treatment atopic dermatitis in children, J Allergy Clin Immunol 102:637–644, 1998. 281. Bieber T: Topical tacrolimus (FK506): a new milestone in the management of atopic dermatitis, J Allergy Clin Immunol 102:555–557, 1998. 282. Hanifin JM: Tacrolimus ointment: advancing the treatment of atopic dermatitis, J Am Acad Dermatol 44(1 suppl):S1–72, 2001. 283. Reitamo S, Wollenberg A, Schopf E, et al: Safety and efficacy of 1 year of tacrolimus ointment monotherapy in adults with atopic dermatitis, Arch Dermatol 136:999–1006, 2000.

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284. Kang S, Lucky AW, Pariser D, et al: Long-term safety and efficacy of tacrolimus ointment for the treatment of atopic dermatitis in children, J Am Acad Dermatol 44:S58-S64, 2001. 285. Remitz A, Kyllonen H, Granlund H, et al: Tacrolimus ointment reduces staphylococcal colonization of atopic dermatitis lesions, J Allergy Clin Immunol 107:196–197, 2001. 286. Reitamo S, Rissanen J, Remitz A, et al: Tacrolimus ointment does not affect collagen synthesis: results of a single-center randomized trial, J Invest Dermatol

111:396–398, 1998. 287. Hauk PJ, Leung DY: Tacrolimus (FK506): new treatment approach in superantigen-associated diseases like atopic dermatitis? J Allergy Clin Immunol 107: 391– 392, 2001. 288. Griffiths CE: Ascomycin: an advance in the management of atopic dermatitis, Br J Dermatol 144:507–513, 2001. 289. Zuberbier T, Chong SU, Grunow K, et al: The ascomycin macrolactam pimecrolimus (Elidel, SDZ ASM 981) is a potent inhibitor of mediator release from human dermal mast cells and peripheral blood basophils, J Allergy Clin Immunol 108:275–280, 2001. 290. Van Leent EJ, Graber M, Thurston M, et al: Effectiveness of the ascomycin macrolactam SDZ ASM 981 in the topical treatment of atopic dermatitis, Arch Dermatol 134:805–809, 1998. 291. Luger T, Van Leent EJ, Graeber M, et al: SDZ ASM 981: an emerging safe and effective treatment for atopic dermatitis, Br J Dermatol 144:788–794, 2001. 292. Harper J, Green A, Scott G, et al: First experience of topical SDZ ASM 981 in children with atopic dermatitis, Br J Dermatol 144:781–787, 2001. 293. Eichenfield L, Lucky A, Boguniewicz M, et al: Safety and efficacy of pimecrolimus (ASM 981) cream 1% in the treatment of mild to moderate atopic dermatitis in children and adolescents, J Am Acad Dermatol 46:495–504, 2002. 294. Zachariae H, Cramers M, Herlin T, et al: Non-specific immunotherapy and specific hyposensitization in severe atopic dermatitis, Acta Derm Venereol Suppl (Stockh) 114:48–54, 1985. 295. Kaufman HS, Roth HL: Hyposensitization with alum precipitated extracts in atopic dermatitis: a placebo-controlled study, Ann Allergy 32:321–330, 1974. 296. Glover MT, Atherton DJ: A double-blind controlled trial of hyposensitization to Dermatophagoides pteronyssinus in children with atopic eczema, Clin Exp Allergy 22:440–446, 1992. 297. Takei S, Arora YK, Walker SM: Intravenous immunoglobulin contains specific antibodies inhibitory to activation of T cells by staphylococcal toxin superantigens, J Clin Invest 91:602–607, 1993. 298. Jolles S, Hughes J, Rustin M: Intracellular interleukin-4 profiles during high-dose intravenous immunoglobulin treatment of therapy-resistant atopic dermatitis, J Am Acad Dermatol 40:121–123, 1999. 299. Kimata H: High-dose intravenous gamma-globulin treatment for hyperimmunoglobulinemia E syndrome, J Allergy Clin Immunol 95:771–774, 1995. 300. Wakim M, Alazard M, Yajima A, et al: High dose intravenous immunoglobulin in atopic dermatitis and hyper-IgE syndrome, Ann Allergy Asthma Immunol 81:153–158, 1998. 301. Sheehan MP, Atherton DJ: A controlled trial of traditional Chinese medicinal plants in widespread non-exudative atopic eczema, Br J Dermatol 126:179–184, 1992. 302. Sheehan MP, Atherton DJ: One-year follow up of children treated with Chinese medicinal herbs for atopic eczema, Br J Dermatol 130:488–493, 1994.

303. Sheehan MP, Rustin MH, Atherton DJ, et al: Efficacy of traditional Chinese herbal therapy in adult atopic dermatitis, Lancet 340:13–17, 1992. 304. Harper J: Traditional Chinese medicine for eczema, Br Med J 308:489–490, 1994. 305. Fung AY, Look PC, Chong LY, et al: A control trial of traditional Chinese herbal medicine in Chinese patients with recalcitrant atopic dermatitis, Int J Dermatol 38:387–392, 1999. 306. Latchman Y, Whittle B, Rustin M, et al: The efficacy of traditional Chinese herbal therapy in atopic eczema, Int Arch Allergy Immunol 104:222–226, 1994. 307. Latchman Y, Bungy GA, Atherton DJ, et al: Efficacy of traditional Chinese herbal therapy in vitro: a model system for atopic eczema-inhibition of CD23 expression on blood monocytes, Br J Dermatol 132:592–598, 1995. 308. Xu XJ, Banarjee P, Rustin MH, et al: Modulation by Chinese herbal therapy of immune mechanisms in the skin of patients with atopic eczema, Br J Dermatol 136:54–59, 1997. 309. Davis EG, Pollock I, Steel HM: Chinese herbs for eczema, Lancet 336:177, 1990. 310. Ferguson JE, Chalmers RJ, Rowlands DJ: Reversible dilated cardiomyopathy following treatment of atopic eczema with Chinese herbal medicine, Br J Dermatol 136:592–593, 1997. 311. Keane FM, Munn SE, du Vivier AW, et al: Analysis of Chinese herbal creams prescribed for dermatological conditions, Br Med J 318:563–564, 1999. 312. Melnik B, Plewig G: Are disturbances of ω-6-fatty acid metabolism involved in the pathogenesis of atopic dermatitis? Acta Derm Venereol (Stockh) 176: 77–85, 1992. 313. Bjorneboe A, Soyland E, Bjorneboe GE, et al: Effect of dietary supplementation with eicosapentaenoic acid in the treatment of atopic dermatitis, Br J Dermatol 117:463–469, 1987. 314. Sharpe GR, Farr PM: Evening primrose oil and eczema, Lancet 335:667–668, 1990. 315. Berth-Jones J, Graham-Brown RA: Placebo-controlled trial of essential fatty acid supplementation in atopic dermatitis, Lancet 341:1557–1560, 1993. 316. Nousari HC, Sragovich A, Kimyai-Asadi A, et al: Mycophenolate mofetil in autoimmune and inflammatory skin disorders, J Am Acad Dermatol 40:265–268, 1999. 317. Grundmann-Kollmann M, Korting HC, Behrens S, et al: Successful treatment of severe refractory atopic dermatitis with mycophenolate mofetil, Br J Dermatol 141:154–179, 1999. 318. Grundmann-Kollmann M, Podda M, Ochsendorf F, et al: Mycophenolate mofetil is effective in the treatment of atopic dermatitis, Arch Dermatol 137:870–873, 2001. 319. Neuber K, Schwartz I, Itschert G, et al: Treatment of atopic eczema with oral mycophenolate mofetil, Br J Dermatol 143:385–391, 2000.

320. Hansen ER, Buus S, Deleuran M, et al: Treatment of atopic dermatitis with mycophenolate mofetil, Br J Dermatol 143:1324–1326, 2000. 321. Cooper KD, Kang K, Chan SC, et al: Phosphodiesterase inhibition by Ro 20-1724 reduces hyper-IgE synthesis by atopic dermatitis cells in vitro,J Invest Dermatol 84:477–482, 1985. 322. Butler JM, Chan SC, Stevens S, et al: Increased leukocyte histamine release with elevated cyclic AMP-phosphodiesterase activity in atopic dermatitis, J Allergy Clin Immunol 71:490–497, 1983. 323. Santamaria Babi LF, Torres R, Gimenez-Arnau AM, et al: Rolipram inhibits staphylococcal enterotoxin B-mediated induction of the human skin-homing receptor on T lymphocytes, J Invest Dermatol 113:82–86, 1999. 324. Koro O, Furutani K, Hide M, et al: Chemical mediators in atopic dermatitis: involvement of leukotriene B4 released by a type I allergic reaction in the pathogenesis of atopic dermatitis, J Allergy Clin Immunol 103:663–670, 1999. 325. Carucci JA, Washenik K, Weinstein A, et al: The leukotriene antagonist zafirlukast as a therapeutic agent for atopic dermatitis, Arch Dermatol 134: 785–786, 1998. 326. Yanase DJ, David-Bajar K: The leukotriene antagonist montelukast as a therapeutic agent for atopic dermatitis, J Am Acad Dermatol 44:89–93, 2001. 327. Pei AY, Chan HH, Leung TF: Montelukast in the treatment of children with moderate-to-severe atopic dermatitis: a pilot study, Pediatr Allergy Immunol 12:154–158, 2001.

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Chapter 87 - Contact Dermatitis

P. Régine Mydlarski Arnon M. Katz Adam J. Mamelak Daniel N. Sauder

THE PATHOGENESIS OF CONTACT DERMATITIS 1

In 1895, Jadassohn described the first case of contact dermatitis from mercury.[ ] Shortly thereafter, it became apparent that contact dermatitis could result from a primary chemical irritant or a contact sensitizer. Respectively, these disorders became known as irritant contact dermatitis (ICD) and allergic contact dermatitis 2

(ACD). ACD is a prototypic delayed-type hypersensitivity (DTH) reaction mediated by antigen-specific T lymphocytes.[ ] Over the past 20 years, both ACD and ICD have been studied extensively, and major insights into the pathophysiology of these disorders have been gained from experiments on animal models. As early as 1942, contact allergy and DTH reactions were induced in immunologically naive guinea pigs by the passive transfer of lymphocytes from sensitized 3

4

donors.[ ] By 1976, Langerhans' cells (LCs) were observed to be in apposition to lymphocytes, in the dermal lymphatics and in draining lymph nodes (DLNs).[ ] One year later, Fc and C3 receptors, as well as class I and class II major histocompatibility complex (MHC) molecules, were found on LCs. These molecules were previously reported on cells of monocyte/macrophage lineage.[

5] [6]

Furthermore, in vivo murine studies showed that allergen application to LC-deficient skin

7

induced a state of unresponsiveness.[ ] These early experiments suggested that LCs were capable of antigen presentation and were essential for the induction of contact hypersensitivity (CHS).[

8] [9]

Allergic Contact Dermatitis Afferent Phase (Induction Phase, Sensitization, Antigen Recognition)

When a chemically reactive, lipid-soluble, low-molecular-weight molecule termed hapten crosses the stratum corneum, it covalently binds carrier proteins to form a hapten-carrier complex, or immunogen (antigen). The antigen is processed by bone marrow-derived dendritic cells (DCs) known as LCs at the site of epidermal penetration. Once internalized by pinocytosis, the antigens are digested into small peptides that bind to class II MHC molecules. The peptide antigen/MHC complex 10]

is then transported to the LC surface membrane.[

As the major antigen-presenting cells (APCs) in the skin, LCs have long-branching dendritic processes that form a semicontiguous network. After contact with a 11 12 13

chemical sensitizer, the dendrites become rounded, with a reduction in the number of short protrusions.[ ] [ ] [ ] These morphologic changes, in conjunction with modulations of cellular adhesion molecule (CAM) expression, are important for inducing LC movement. The antigen-bearing LCs migrate from the skin, through the efferent lymphatics, and into the regional DLNs. Within 24 hours after hapten application, LCs present antigenic information to naive CD4+ helper T lymphocytes. Clonal proliferation of specific T cells subsequently ensues in the paracortical region of the lymph nodes. Effector and memory T cells are then released into the circulation ( Figure 87-1 ).[

10]

Elicitation Phase (Efferent Phase, Challenge)

When a sensitized individual is re-exposed to a hapten, the hapten-carrier complex is again formed. Now, however, antigen presentation by LCs occurs in the epidermis, dermis, and regional DLNs. Recognition of the peptide antigen/MHC complex by effector and memory T cells results in blast transformation and clonal proliferation of specific T lymphocytes. Activated and resident cells release a series of soluble polypeptide mediators, termed cytokines, leading to a complex

cascade of events (see Epidermal Cytokines and Cytokine Profiles ). An overt inflammatory reaction occurs within 48 hours after re-exposure to the contact allergen ( Figure 87-2 ). [

14]

Irritant Contact Dermatitis When chemicals such as sodium dodecyl sulfate (SDS) or phenol are applied to the skin, an inflammatory response also occurs. In contrast to haptens, these irritants do not activate the immune cascade via the antigen presentation pathway. Primary skin irritation results in keratinocyte (KC) damage, mononuclear cell infiltration, and apposition of lymphocytes to LCs.[

15]

Although the cellular mechanisms of ICD remain unknown, increasing evidence suggests activated KCs act as “signal

transducers” in controlling the host homeostatic responses to exogenous stimuli.[ cytokine reserve, are viewed as key immunoregulators in the skin.

16] [17]

KCs make up 95% of the epidermal cell mass and, by virtue of their wide

Despite the differences in pathogenesis, ACD and ICD are often indistinguishable both clinically and histologically. ACD biopsies show histologic changes as early as 4 to 8 hours after hapten exposure[ uppermost part of the dermis. Later

18]

19]

or sooner.[

In this early period, extravasations of lymphocytes and other mononuclear cells can be observed in the

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Figure 87-1 Afferent (induction) phase of allergic contact dermatitis. Langerhans' cells (LCs) within the epidermis process antigen (hapten-carrier complex). The hapten-LCs migrate through the efferent lymphatics to the draining lymph node, where LCs present the antigen to naive CD4+ T helper lymphocytes. T cell proliferation occurs in the paracortical region of lymph nodes to produce primed (effector and memory) T cells. , keratinocytes (KCs); •, naive T cells; , primed T cells; , hapten. (Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.)

, keratinocytes (KCs); •, naive T cells; , primed T cells; , hapten. (Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.) changes include mononuclear cell migration into the epidermis, spongiosis, and vesiculation. Approximately 24 hours after hapten application, basophils appear, and by day 3, tissue mast cells increase in number. Although the occasional neutrophil may be present in severe ACD, the presence of epidermal or dermal eosinophils is 20] [21]

highly suggestive of this reaction. In ICD, lymphocytes are relatively rare and the bulk of the inflammatory infiltrate consists of neutrophils.[ basophils may be noted in ICD, they constitute 15% of the ACD infiltrate.

[22]

Although

Defective DTH reactions take place when mast cell–deficient mice are treated with a

23]

chemical sensitizer, implying that mast cells are important in ACD development.[ [24] [25] [26] [27] [28] [29]

Moreover, reports on the number of LCs in both reaction types are controversial.

Studies have indicated that LC migration to DLNs occurs late in ICD reactions (i.e., 4 to 5 days after application of sodium lauryl sulfate).

This movement may be induced by KC-derived cytokines. [

30] [31]

T Lymphocyte Immune Regulation The activation of T cells requires two distinct signals: (1) T cell receptor (TCR) and peptide antigen/MHC complex interaction on APCs, and (2) a co-stimulatory, or antigen-nonspecific, adhesion signal ( Figure 87-3 ).[

32]

The first signal is mediated by αβ T cells. CHS responses were abolished in TCRα knockout

Figure 87-2 Efferent (elicitation) phase of ACD. Antigen presentation by Langerhans' cells (LCs) occurs in the epidermis, dermis, and regional lymph nodes. Recognition of the peptide antigen/MHC complex by effector and memory (primed) T cells results in blast transformation and clonal proliferation of specific T lymphocytes. Activated and resident cells release cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ), which undergo a complex cascade of events (see text). , keratinocytes (KCs); , primed T cells; , hapten; CAMs, cellular adhesion molecules; TNF-α, tumor necrosis factor-α. (Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.)

, keratinocytes (KCs); , primed T cells; , hapten; CAMs, cellular adhesion molecules; TNF-α, tumor necrosis factor-α. (Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.) mice (deficient in αβ T cells), confirming the critical effector role of αβ T cells in CHS.[ normal in C57BL/6 mice and enhanced in FVB

33]

In TCRδ knockout mice (deficient in γδ T cells), CHS responses were

34 mice.[ ]

TCRαβ heterodimers interact with their co-receptors, CD4 and CD8, in the development of a CHS response. CD4+ T cells recognize peptides bound to MHC class II

molecules, whereas CD8+ T cells recognize peptides bound to MHC class I molecules. The relative role of CD4+ T helper cells and CD8+ T cytotoxic cells in CHS 35

remains controversial. CHS responses were diminished in CD4 knockout mice but remained normal in MHC class I knockout mice.[ ] These findings supported the traditional view that ACD is mediated by CD4+ T cells. However, CD8 knockout mice showed a decreased CHS response to dinitrofluorobenzene (DNFB) and 36]

oxazolone when compared with wild-type mice, suggesting that both CD4+ and CD8+ T cells play a role in the development of CHS.[

On the basis of the cytokines secreted and the observed immune response, activated CD4+ T helper (Th) lymphocytes have been divided into two subsets, Th1 and Th2 (see Chapter 17 ). Th1 cells produce interleukin-2 (IL-2), interferon-gamma (IFN-γ), tumor necrosis factor-β (TNF-β), IL-12, and macrophage inhibition factor. Th2 cells secrete IL-4, -5, -6, -10, and -13. Some cytokines, including IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and

1583

TNF-α, are produced by both T lymphocyte subsets.[ produce IFN-γ, and T2 cells produce

37]

Similarly, CD8+ T cells have been divided into two categories, termed type 1 (T1) and type 2 (T2). T1 cells

38 39 40 IL-4.[ ] [ ] [ ]

41

Th1 T cells predominate in CHS.[ ] In addition to mediating tumor cytolysis and DTH responses, Th1 cells can inhibit Th2 cell synthesis. These effects are primarily attributed to IFN-γ. IL-2, an early product of Th1 cells, promotes the proliferation of IL-2 receptor–bearing cells and amplifies specific T cell populations. Like IFN-γ, it is one of the most potent enhancers of I-region associated antigen (Ia) expression on LCs. Ia is the murine equivalent of the human leukocyte antigen (HLA)-DR MHC II molecule and is required for antigen presentation. Other functions of Th1 cells include activation of macrophages to kill intracellular parasites and the production of immunoglobulin G2a (IgG2a).[

42] [43]

Th2 cells produce less inflammatory and different immunoglobulin-mediated immune responses. Through their secretion of IL-4 and IL-5 (an autocrine factor for IL42] [43]

2 and a growth factor for eosinophils, respectively), Th2 cells promote B lymphocyte secretion of IgG1 and IgE.[ counterregulate each other. The Th2 cytokine IL-10 inhibits the release of cytokines from Th1 cells ACD

41 reactions.[ ]

[44]

These T cell subsets appear to

and may function as a natural suppressant of both ICD and

The differential production of Th1 and Th2 cytokines mediates, at least in part, the immunologic and inflammatory processes of contact dermatitis.

Epidermal Cytokines The epidermis is composed of KCs, LCs, melanocytes, Merkel cells, and, in mice, dendritic epidermal T cells. In response to injury such as trauma or ultraviolet (UV) radiation, the cytokine production of KCs is key in the skin's immunologic and inflammatory reactions. These cytokines' autocrine, paracrine, and endocrine 17]

influences are effective in converting exogenous stimuli into a host response.[

Stimulated LCs also contribute to the local cytokine milieu in the epidermis, the 14]

efferent lymphatics, and the regional DLNs, even though they represent only 2% to 5% of the epidermal cell mass.[ cytokines are central to understanding both ACD and ICD pathogenesis.

The synthesis and secretion of epidermal

17 45

Epicutaneous hapten application activates both LCs and KCs in vivo.[ ] [ ] Resting KCs secrete low levels of cytokines, whereas activated KCs produce IL-1α, 1β, -3, -6, -7, -8, -10, and -12, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), GM-CSF, TNF-α, transforming growth factor (TGF)-α, TGF-β, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), and interferon-induced protein 10 (IP-10).[

46]

Immature LCs may express macrophage inflammatory protein (MIP)-1α, MIP-2, and IL-1β. As they mature, IL-1β is up-regulated and the expression of 47]

MIPs is decreased. [

Furthermore, cultured LCs synthesize and secrete IL-6 and, after stimulation with phorbol myristate acetate, lipopolysaccharide (LPS), or

48 49 TNF-α.[ ] [ ]

Most of these cytokines and growth factors are not constitutively synthesized in vivo. Rather, they are induced by a variety of “noxious” stimuli. Current contact dermatitis research focuses on the differential expression of these cytokines by allergens and irritants. Cytokine Profiles in Contact Dermatitis In 1990, the phenotypic and functional characteristics of activated LCs in vivo were studied. At different time points, LCs were harvested from the ear skin of naive mice treated with allergens and irritants. Twenty-four hours after hapten application, the epidermal sheets were stained with anti-Ia antibodies. An increase in LC size and class II MHC expression was noted, and these phenotypic changes induced a twofold to fivefold increase in T cell proliferation. Epicutaneous application of a panel of irritants failed to produce these changes. In vivo activation of LCs was therefore central to hapten-specific sensitization.[

50]

Exactly how hapten application induces LC activation still remains unclear. By releasing of a variety of growth factors and cytokines, epidermal cells modulate LC function and participate in immunologic and inflammatory reactions. To characterize the epidermal cytokine profiles in the induction phase of ACD, contact sensitizers such as dinitrochlorobenzene (DNCB) were applied to the skin of mice. Expression of IL-1β, IL-1α, MIP-2, IP-10, and Ia-α messenger RNA (mRNA) was stimulated with hapten exposure. IL-1β mRNA was detected within 15 minutes after allergen application. Cell depletion assays identified epidermal LCs as the major source of this cytokine. Ia-α mRNA expression peaked at 18 hours after antigen exposure.[ molecules were up-regulated within the 24 hours following hapten application.

[45]

51]

This was consistent with the finding that class II MHC

In addition, there was an allergen-specific induction of the two polypeptides, IP-10

and MIP-2. IP-10, produced by KCs in DTH reactions, functions as a mitogenic and chemotactic agent.[

52]

MIP-2, previously reported on LPS-stimulated cells of the 53

monocyte/macrophage lineage, also displays chemotactic activity and elicits a localized inflammatory response in murine models.[ ] Irritant application resulted in nonspecific production of TNF-α and IFN-γ mRNA. These cytokines were both induced by allergens, irritants and tolerogens alike. Clearly, a complex array of signals characterizes the sensitization phase of ACD. Moreover, allergens and irritants appear to differentially regulate epidermal mRNA cytokine expression. The epidermal cytokine profiles of the elicitation and sensitization phases of ACD and ICD were further characterized. When the allergen DNFB was applied to ear skin of naive BALB/c mice, the sensitization and elicitation phases of ACD showed similar patterns of cytokine mRNA expression. IL-1β, IL-6, and IL-10 mRNA were up-regulated from 6 to 24 hours after hapten application but only 24 hours after treatment with the irritant SLS. ACD showed an initial suppression of IL-1α mRNA, followed by an up-regulation at 12 hours after sensitization and 24 hours after challenge. A small up-regulation of IL-1α mRNA was noted 1 hour after treatment with SLS, followed by a 30% to 50% suppression. During the 24-hour observation period in ACD, TNF-α mRNA levels remained the same, but they were up-regulated from 1 to 24 hours after treatment with SLS. GM-CSF mRNA levels increased from 6 to 24 hours after both hapten and irritant exposure. A greater magnitude of cytokine mRNA expression was seen in the elicitation phase compared with the sensitization phase of ACD. Investigators concluded that similar cytokine cascades exist in the sensitization and elicitation phases of ACD. However, different cytokine profiles were induced by allergens and irritants.[

41]

1584

Interleukin-1

IL-1 was the first epidermal cell–derived cytokine to be identified. KCs are the main source of IL-1α in the epidermis, whereas IL-1β is mainly produced by LCs. IL1β is synthesized as a 31-kD precursor molecule that is cleaved by caspase-1, a cysteine protease once known as the IL-1-converting enzyme. Caspase-1–deficient mice failed to develop CHS to both DNFB and oxazolone. Furthermore, when Ac-YVAD-cmk, a caspase-1 inhibitor, was applied to mouse skin before hapten application, LC migration and CHS were inhibited.[

54]

51]

The regulatory role of IL-1 in contact dermatitis is a topic of investigation. IL-1β mRNA is up-regulated within 15 minutes after epicutaneous hapten application.[ Monoclonal anti-murine IL-1β antibodies prevented primary sensitization. In contrast, sensitization was unaffected by the intradermal injection of anti-IL-1α antibodies.[

55]

Systemic administration of recombinant human rhIL-1β caused a net suppression of the ACD response when applied during the sensitization phase.

Administration 48 hours before challenge enhanced ACD, but rh IL-1β application 2 hours before challenge suppressed the ACD response.[ The IL-1 receptor antagonist (IL-1ra) blocks the binding of IL-1 to its receptors while having no inherent agonistic activity.[

57] [58]

Local injection of IL-1ra in mice 59]

significantly impaired the sensitization and elicitation phases of CHS. IL-1ra did not, however, suppress a prototypic ICD reaction.[ the human cutaneous allergic late phase response was inhibited by soluble IL-1 receptors, corroborating these potentiator of the ACD immune response.

60 findings.[ ]

56]

A clinical trial demonstrating

IL-1 therefore appears to function as a

IL-1β–deficient mice, derived by gene targeting in embryonic stem cells, showed defective ACD responses to topically applied trinitrochlorobenzene (TNCB). The responses were reversed by either very-high-dose application of sensitizing antigen or recombinant IL-1β intradermal injection immediately before epicutaneous hapten application.[

61]

Once again, IL-1β appeared to have a role in the host immune response to contact sensitization.

In addition to promoting the secretion of IL-2, IL-6, IL-8, GM-CSF, and other cytokines from neighboring cells, IL-1 has the ability to enhance LC functional 62] [63] [64] [65] [66] [67] [68]

activity when used in conjunction with GM-CSF.[ [69]

The up-regulation of IL-1β gene expression in LCs correlated with their functional 41

activation as potent dendritic APCs. Similarly, epicutaneous hapten application enhanced IL-1β gene expression in LCs.[ ] Intradermal administration of recombinant IL-1β in mice resulted in an increase of Ia-α molecules on the surface of LCs and a decrease of resident LCs in the epidermis, suggesting a possible effect on LC migration in vivo.[

55]

IL-1 is also a potent inducer of CAMs. Intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), endothelial leukocyte adhesion

molecule-1 (ELAM-1), and lymphocyte function–associated antigen-3 (LFA-3) are all induced by IL-1.[ the site of

71 reaction[ ]

70]

These adhesion molecules recruit inflammatory cells to

(see Chapter 9 ).

IL-1β initiates the trafficking of macrophage calcium-type lectin (MGL)+ cells, a subpopulation of dermal macrophages, from the dermis to the regional DLNs.[ Dermal macrophages may also play a role in ACD sensitization.

72]

IL-1α, on the other hand, affects the ACD reaction differently. IL-1α cytokine caused a net suppression of the classic DTH response, possibly by inhibiting the 73 74

development of LCs into potent APCs.[ ] [ ] Although IL-1α and IL-1β are closely related cytokines with many overlapping functions, they have divergent effects on the development of contact dermatitis. Current evidence suggests that IL-1β is central to the induction of cutaneous immune responses. Tumor Necrosis Factor-α

The role of TNF-α in the development of contact dermatitis has remained controversial. Early on, TNF-α was found to maintain cellular viability of murine 75]

epidermal LCs in culture without inducing their functional activation into potent APCs.[

Later, it was observed that the intradermal administration of homologous

recombinant TNF-α caused DC accumulation in DLNs and a reduced frequency of local epidermal LCs.[ migration from the epidermis.

76] [77]

TNF-α was therefore considered a stimulus for LC

However, a suppressive role for TNF-α has also been proposed. When skin is exposed to UVB radiation in vitro, the immunosuppressive photoreceptor transurocanic acid (UCA) is converted to its biologically active cis form. When neutralizing anti-TNF-α antibodies were injected into mice, the immunosuppressive effects of UVB and cis-UCA were reversed. Investigators proposed that cis-UCA, formed by UVB exposure, caused the local release of TNF-α. TNF-α, in turn, suppressed sensitization by altering the function of LCs.[

78] [79] [80]

Furthermore, topical application of TNF-α also impaired the induction of ACD. [

81]

To clarify the role of TNF-α, CHS assays were performed on gene-targeted mutant mice lacking genes for both TNF receptors (p55 and p75). The mice were irradiated with UVB or injected intradermally with cis-UCA before the assay. The knockout mice had a hyporesponsive ACD reaction compare with wild-type mice, reconfirming TNF-α's proinflammatory role. However, after irradiation with UVB or injection of cis-UCA, CHS suppression was noted in both wild-type and gene82

targeted mice.[ ] TNF-α signaling therefore appeared to be only partially involved in UV-induced immunosuppression and did not play a major role in the cis-UCA immunosuppressive mechanism. 83

Using immunohistochemical techniques, investigators found that KC expression of TNF-α in allergic and irritant patch test reactions was unchanged. [ ] However, controversy still existed. Serial human biopsies performed when poison ivy extract was used to induce ACD showed TNF-α and ICAM-1 expression on human KCs 84]

within 4 to 8 hours.[

In 1991, investigators showed total abrogation of both ACD and ICD responses when mice were systemically treated with neutralizing anti-TNF-α monoclonal

41

85

antibodies immediately before challenge.[ ] On the other hand, Bromberg, Chavin, and Kunkel[ ] found that anti-TNF-α antibodies suppressed the ACD response during the sensitization phase but not during the challenge phase. Although the reason for such discrepancies remains unclear, the dose of antibody has been questioned. Other investigators showed that systemic anti-TNF-α antibody administration markedly inhibited the frequency of DCs in DLNs 18 hours after topical allergen and irritant application. Mice exposed to the neutralizing antibody 2 hours before sensitization had a significantly decreased CHS response. When administered 18 hours 86]

after skin sensitization, however, anti-TNF-α antibodies had no apparent effect.[

These results

1585

were consistent with those obtained by Bromberg's group in 1992.[

85]

Most of the biologic activities of TNF are mediated through TNF receptor I (TNF-R1, or p55). LC migration, however, appears to be mediated through TNF receptor II (TNF-R2, or p75). When TNF-R1–deficient mice were exposed to the hapten fluorescein isothiocyanate (FITC), a normal number of FITC+/Ia+ cells (LCs) were found in the DLNs. This suggested that TNF-R1 signaling was not essential for LC migration. However, when anti-TNF-α antibody was present, significantly 87

decreased LC accumulation was observed in the DLNs. This implied that TNF-R2 signaling was responsible for LC migration.[ ] To further characterize these effects, knockout mice lacking p55 or p75 were generated. Although LC migration was normal in p55-deficient mice, migration was markedly depressed in p75deficient mice.[

88]

Once again, a proinflammatory role for TNF-α was suggested in the physiologic state.

TNF-α also has additional roles in chemotaxis and in maintaining the immune response. Similar to IL-1, TNF-α is a potent inducer of CAMs. Specifically, TNF-α increases expression of ICAM-1, VCAM-1, ELAM-1, and LFA-3.[

70] [89]

In addition, TNF-α has been found to promote the degranulation of dermal mast cells in 90]

vitro. These cells subsequently release cytokine and chemokine mediators and thereby contribute to the induction and maintenance of the host immune response. [ Granulocyte-Macrophage Colony-Stimulating Factor 91

GM-CSF is a pleiotropic cytokine that maintains the viability of murine epidermal LCs and augments their immunostimulatory activity.[ ] By themselves, freshly isolated LCs were poor stimulators in a primary mixed epidermal cell–lymphocyte reaction (ECLR). However, in the presence of GM-CSF and IL-1, the LCs were potent stimulators of both the primary and secondary ECLRs.[ the B7 adhesion

92 93 molecule.[ ] [ ]

challenge phase in mice.

GM-CSF induced phenotypic changes on LCs, including increased expression of the Ia antigen and

Moreover, the combination of GM-CSF and TNF-α induced human hematopoietic stem cell differentiation into cells displaying

the morphology, phenotype, and function of LCs.[ [95]

63]

94]

The combination of anti-GM-CSF and anti-IL-3 monoclonal antibodies resulted in partial inhibition of the ACD

Interleukin-6

In addition to regulating B cell differentiation, acute phase protein synthesis, and hematopoiesis, IL-6 may function as a cofactor for thymocyte and T cell proliferation.[

96]

IL-6 plasma levels were elevated after the challenge phase of ACD in mice.[

41]

Immunohistochemical studies showed increased KC-bound IL-6 on

83 sites.[ ]

serial human biopsies from allergic and irritant patch test IL-6 production and lymphocyte proliferation in DLNs peaked 3 days after mice were topically exposed to contact sensitizers, but not after exposure to solvents or to nonsensitizing agents. The investigators consequently suggested that IL-6 responses could be 97

antigen specific.[ ] Furthermore, IL-6 knockout mice had a diminished CHS response after the application of the hapten oxazolone.[ IL-6 remains unclear, it may be associated with CHS induction.

98]

Although the present role of

Interleukin-12

IL-12 was originally called the natural killer cell stimulatory factor for the pleiotropic effects it induces on natural killer cells through IFN-γ secretion, and cytotoxic lymphocyte maturation factor because it enhances the development of Th1-type cell responses. The bioactive form of IL-12 consists of covalently linked p35 and p40 chains. Although it is predominantly secreted by macrophages and B lymphocytes, IL-12 was recently shown to be produced by human KCs.[

99] [100] [101]

Muller,

102 Germann[ ]

Saloga, and showed that the p35 chain of IL-12 was constitutively expressed on human epidermal cells, whereas the p40 chain could be detected only in hapten-treated epidermis. Moreover, neither vehicles nor irritants induced the p40 mRNA chain. When allergen-stimulated LCs were cultured with allogeneic T 102]

lymphocytes, anti-IL-12 monoclonal antibody administration inhibited T cell proliferation by 50%.[

Muller's group similarly examined IL-12 in the lymph nodes and spleens of mice during CHS assays and found that IL-12 p35 mRNA chain was constitutively 103

expressed throughout the experiment.[ ] Induction of the p40 chain was only transiently expressed between 12 and 14 hours after hapten application and was allergen specific. Irritants and solvents did not induce a p40 chain message. Furthermore, studies showed that DCs were the main sources of the p40 chain message. Because IL-12 acts as a proinflammatory cytokine by inducing IFN-γ production, IFN-γ was measured to determine the presence of functional IL-12 protein. Only culture supernatants from allergen-treated mice showed significant amounts of IFN-γ production. The addition of anti-IL-12 antiserum to the supernatant inhibited IFN-γ production by 55%. Anti-IL-12 antiserum was also injected intraperitoneally on days -1, 0, and 1 of sensitization. The antiserum reduced CHS responses by 103]

85%, nearly preventing sensitization.[

Finally, IL-12 was examined with respect to tolerance. Investigators showed that IL-12 treatment prevented tolerance induction in a murine contact allergen system and was actually capable of reversing UV-induced tolerance.[ CHS in vivo.

103]

Overall, the data strongly suggested that IL-12 might be an important factor for the induction of

Interferon-γ 95

Studies by Piguet et al[ ] suggested IFN-γ may also play a role in CHS. The administration of anti-IFN-γ antibodies had a partial suppressive effect on the CHS response. When mice with a targeted gene disruption of the IFN-γ receptor were treated with the haptens oxazolone and TNCB, the development of cutaneous edema

was found to be independent of IFN-γ signaling. Dermal mononuclear infiltrates and epidermal microabscesses, however, were diminished in these mutant mice. Hapten-induced up-regulation of ICAM-1 and MHC class I antigen was also decreased, perhaps accounting for the reduced number of inflammatory cells recruited to 104

the site of reaction.[ ] IFN-γ has not been considered an absolute requirement in ACD development primarily because the IFN-γ–deficient mice are still able to mount a CHS response. Counterregulatory Cytokines Interleukin-10

Previously termed the cytokine synthesis inhibitory factor, IL-10 was originally described as a Th2-derived cytokine that

1586

44] [105] [106]

inhibited the release of cytokines from Th1 cells.[

IL-10 mRNA and protein were detected in murine KC cultures during the induction phase of ACD.[

IL-10 mRNA expression proved to be an allergen-specific event, because irritants and tolerogens could not induce the IL-10 message when tested.[ inducing MHC class II expression on B cells, IL-10 enhanced mast cell proliferation and maintained the viability of cultured B 10 prevented clonal proliferation of antigen-specific T cells by down-regulating MHC class II expression on

110 monocytes.[ ]

107]

108 109 lymphocytes.[ ] [ ]

41]

In addition to

In contrast, IL-

IL-10 not only inhibited IFN-γ 111] [112]

production by Th1 lymphocytes but also halted production of IL-1, IL-6, IL-8, TNF-α, and GM-CSF by T cells, monocytes, and activated macrophages.[ [113]

More recently, the local production of IL-10 was noted to preserve the duration of the ACD response after challenge. Intradermal administration of exogenous 114]

IL-10 before challenge inhibited the ACD elicitation phase.[ responses compared with wild-type C57BL/10 mice.[

115]

Experiments with IL-10 knockout mice have demonstrated enhanced LC migration and CHS

Certainly, an immunosuppressive and tolerogenic role for IL-10 has been demonstrated in vivo. Many

researchers have now suggested a therapeutic role for this cytokine in CHS.[

116] [117]

Transforming Growth Factor-β

TGF-β is an immunosuppressive growth factor that down-regulates IL-1 receptor expression and blocks the activities of IL-1, IL-2, and colony-stimulating factors. [118] [119]

Recombinant human TGF-β (rhTGF-β) prevented up-regulation of Ia antigen expression on murine LCs when treated with IL-1, TNF-α, IL-3, and GMCSF. Systemic administration of rhTGF-β during the elicitation phase completely abrogated the ACD response. Administration of rhTGF-β during the sensitization 120]

phase, however, did not alter LC function or interfere with the final inflammatory reaction. [ caused by the up-regulation of IL-1ra.

[14]

TGF-β–induced immunosuppression could also at least in part be

Though its effects on ICD remain unclear, TGF-β may serve as a therapeutic tool for ACD and other DTH reactions.

Cellular Adhesion Molecules Both ACD and ICD are characterized by a strong cellular infiltrate composed mainly of T lymphocytes and mononuclear cells.[

121]

CAMs are cell surface proteins 71]

involved in the skin's cell-cell and cell-matrix interactions. CAM expression allows for the increased cellular trafficking that occurs at the site of these reactions[ (see Chapter 9 ).

Adhesion molecules include members of the selectin, integrin, and cadherin families and the immunoglobulin gene superfamily. The selectin family is involved in leukocyte–endothelial cell adhesion and is critical in leukocyte trafficking. L-selectin knockout mice had impaired CHS responses, whereas E-selectin and P-selectin 122] [123] [124] [125]

knockouts had normal responses.[

Both LC morphology and CAM expression must change for LCs to migrate. Cultured murine LCs expressed low levels of E-cadherin and had decreased affinity for KCs. E-cadherin may be important in LC migration by potentially modulating KC-LC interactions.[ antigen-4 (VLA-4), suggesting that LC migration and cellular interactions are dependent on this

126]

Activated LCs also up-regulated the expression of very late

127 CAM.[ ]

The presence of surface LFA-3, ICAM-1, ICAM-3, and the B7 molecule on LCs provides secondary nonspecific, adhesion signals for antigen presentation and T cell 128 129 130

131

activation.[ ] [ ] [ ] Specifically, these CAMs enable CD4+ molecules to recognize class II MHC on LCs, and TCRs to recognize processed antigen.[ ] The corresponding ligands of each of these adhesion molecules have also been identified on T cells. ICAM-1 interacts with LFA-1, ICAM-3 with LFA-1, LFA-3 with CD2, and B7 with CD28. Again, without these interactions, MHC-restricted antigen recognition, production of IL-2 by Th1 cells, and clonal proliferation of primed T cells would not occur[

132] [133] [134] [135] [136] [137]

( Figure 87-3 ).

Allergic and irritant-induced skin reactions in gene-targeted CD28-deficient mice were examined. CD28 was required both for optimal induction of ACD and for the complete development of ICD. The expression of B7-1 and B7-2 was induced by allergens in both normal and mutant CD28 gene-deficient mice. Irritants, however, could induce only B7-2.[

138]

Consequently, B7 transgenic mice were used to investigate the progression of ACD. Although CHS kinetics were initially unaffected by the overexpression of B7 139 140

molecules on KCs, the severity and persistence of the ACD response were markedly increased in these animals.[ ] [ ] Increased TNF-α, IL-6, and TNF-β transcripts were detected in the early phase (1 to 14 days) of ACD in the B7-1 transgenic animals. IFN-γ–producing lymphocytes, however, were responsible for the 141]

late phase (14 to 42 days) of the ACD response.[

These studies suggested that overexpression of B7-1 directly or indirectly affects the ACD response. 142 143

ICAM-1 knockout and transgenic mice showed inconsistent results when studied in CHS assays.[ ] [ ] Two reasons were offered for the discrepancy: (1) the absence of proper co-stimulation by ICAM-1+ KCs during CHS, and (2) differences in the efficacy of ICAM-1 for enhancing the LC-mediated afferent phase compared with the KC-mediated efferent phase. [ mice show a similar decrease in CHS

144]

145 response.[ ]

Overall, however, ICAM-1 knockout mice seemed to have depressed CHS responses. LFA-1α chain knockout

Monoclonal anti-CAM antibodies have also been used to examine the ICAM-1–LFA-1 interaction. Anti-LFA-1

Figure 87-3 Langerhans' cell (LC)-T cell interactions require the following cellular adhesion molecules and their corresponding ligands: ICAM-1/LFA-1, ICAM-3/ LFA-1, LFA-3/CD2, B7/CD28, and CD40/CD40L. ICAM, intercellular adhesion molecule; LFA, lymphocyte function–associated antigen; MCHII, major histocompatibility complex II; TCR, T cell receptor. (Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.)

(Modified from Kondo S, Sauder DN: J Am Acad Dermatol 33:786–800, 1995.)

1587

146

monoclonal antibodies inhibited the CHS elicitation phase by 44% after they were locally injected into mice following sensitization with DNCB.[ ] The combination of anti-ICAM-1 and anti-LFA-1 monoclonal antibodies completely inhibited the induction of CHS by FITC. Furthermore, mice treated with anti-ICAM147]

1 or anti-LFA-1 antibodies showed a 79% and 36% reduction, respectively, in FITC-stained Ia+ LC of DLNs.[ are important in cutaneous inflammatory reactions and for LC migration to regional lymph nodes.

These findings implied that ICAM-1 and LFA-1

CD40L is yet another ligand expressed by T cells that contributes to LC–T cell interactions by binding to the CD40 adhesion molecule. CD40L knockout mice had defective dendritic cell migration and exhibited a decreased CHS response. [

148]

Aberrant or exaggerated expression of CAMs in the epidermis or dermal microvasculature may underlie the pathophysiology of contact dermatitis. Future therapies

for contact dermatitis and other related dermatoses may involve modulating CAM expression. The use of monoclonal anti-CAM antibodies may provide a means to alter this abnormal CAM expression.[

14]

Chemokines Chemokines are a group of chemotactic proteins that bind specific receptors expressed on target cells (see Chapter 11 ). Four families of chemokines, all based on a cysteine motif, have been identified: CXC, CC, C, and CX3C. A profound rearrangement in the pattern of chemokine receptors allows DCs to migrate to DLNs and encounter naïve T cells. The kinetics and pattern of chemokine expression in ACD has been documented with IL-8 expression first, followed by monocyte chemoattractant protein (MCP)-1 and RANTES, and finally by CXCR3 receptor antagonist. MCP-1, a CC chemokine, recognizes monocytes, memory T lymphocytes, and natural killer cells. Mice overexpressing MCP-1 in basal KCs showed enhanced CHS responses with increased infiltrating DCs. Chemokines IP-10, IP-9, and Mig differentiate ACD from ICD. IP-10, Mig, and I-TAC (the ligands for CXCR3) selectively attract T lymphocytes. More than 70% of ACD-infiltrating T lymphocytes in the epidermis are CXCR3-positive. I-309 and CCR8 may contribute to the resolution of ACD.[

149]

Chemokine receptors CCR5 and CCR6, expressed on T lymphocytes and immature DCs, and CCR7, expressed on mature DCs, are also important in CHS. Inflammatory stimuli cause LCs to down-regulate CCR5 and CCR6 while up-regulating CCR7, to direct the migration of LCs to regional DLNs. The CHS response 149]

is enhanced in CCR5 and CCR6 knockout mice but abrogated in CCR7 knockout strains.[ Inhibition of Contact Dermatitis

150

Local exposure to UVB radiation inhibited the induction of contact allergy in 40% of individuals treated with DNCB.[ ] Two mechanisms have been proposed to explain this UVB-induced immunosuppression in CHS. First, erythemogenic doses of UVB caused LCs to lose their dendricity, surface markers (most notably MHC class II molecules), and adenosine triphosphatase (ATPase) activity. Furthermore, UVB blocked the ability of LCs to stimulate clonal proliferation of specific T cells. UVB radiation therefore could induce perturbations in LC activity and thereby alter cutaneous antigen presentation. Second, UVB up-regulated the expression of several immunosuppressive soluble mediators, including α-melanocyte-stimulating hormone (α-MSH), prostaglandin E2 , IL-1, TNF-α, IL-10, and TGF-β. Overall, 150] [151] [152] [153] [154] [155]

LCs appear to lose their ability to stimulate Th1 cell proliferation while preserving Th2-type immunity after UVB irradiation.[ Both KCs and dermal mast cells produce TNF-α and IL-10 in response to UV exposure.[ 41 tolerance.[ ]

156]

TNF-α is thought to mediate UVB-induced suppression of CHS, while

157 al[ ]

IL-10 has been implicated in immunologic Alard et reported that mast cell–derived TNF-α was required for UVB-induced CHS suppression. Investigators found that UVB failed to induce tolerance in mast cell–deficient mice, and tolerance occurred if mast cells were triggered to degranulate after ligation 157]

of the IgE receptor.[

These findings imply that mast cells are required for UVB-induced tolerance and may be a major source of IL-10.

At the molecular level, UVB radiation has a number of interesting effects. UVB radiation induced damage in keratinocyte DNA, primarily affecting pyrimidine

bases, with C to T or CC to TT mutations described.[

158]

UVB radiation also altered components of the cell membrane. Changes in the epidermal growth factor 151] [153]

receptor, ICAM-1, and the B7 adhesion molecule were observed and corresponded to suppressed LC function.[ immunosuppression was discussed previously.

The role of urocanic acid in UVB-induced

Corticosteroids can also inhibit contact dermatitis. When topical corticosteroids were co-administered with a contact sensitizer during a patch test, a suppressed ACD 159]

response was noted.[

160] [161]

Epicutaneous application of corticosteroids caused a local decrease in LC numbers in the skin.[

applied to sites with decreased LC density, anergy and eventual tolerance

162 developed.[ ]

When sensitizing compounds were

Corticosteroids may also prevent sensitization by inhibiting LC migration

from the epidermis to regional DLNs. Topical corticosteroids inhibit release of IL-1 and TNF-α, cytokines important in CAM expression and LC migration.[

163] [164]

160] [161] [162]

Finally, in addition to compromising LC function, systemic corticosteroid administration alters T lymphocyte function.[

Other pharmacotherapeutic agents have been less successful in the treatment of contact dermatitis. Antihistamines inhibit mast cells and basophils from releasing histamine, leukotrienes, serotonin, and cytokines, preventing an increase in CAM expression, vascular permeability, and pruritus. Although these cells may be found at ACD and ICD reaction sites, antihistamines have a minimal effect on CHS. [

165]

Cyclosporin A (CsA) impairs LC function without cytotoxic effects on LCs or T

lymphocytes. The administration of CsA and other immunosuppressives such as methotrexate, however, did not modify irritant patch test results.[

166]

The development of specific immunologic tolerance (unresponsiveness) occurs when antigen presentation is inadequate. This can result from application of contact 7

allergens to skin sites deficient in LCs.[ ] With fewer numbers, the allergen bypasses epidermal LCs and reaches the lymphoid system, stimulating the production of suppressor cells. The production of effector T lymphocytes is greatly diminished. The tail skin of mice is relatively deficient in LCs and is an ineffective site for 7

testing CHS. No such area has yet been identified in humans.[ ] Though reports on LC numbers in both ACD and ICD reactions varied, a reduction of LCs appears to interfere

1588

24] [25] [26] [27] [28] [29]

with antigen presentation.[

Systemic and topical corticosteroids, UV radiation, and acquired immune deficiency syndrome (AIDS) may also

interfere with the ability of LCs to function as potent APCs and thereby suppress a CHS response.[

167] [168] [169]

Hyposensitization Hyposensitization is the practice of orally or intravenously administering increasing quantities of an allergen until a maintenance dose or loss of symptoms is reached. [170]

The allergen bypasses epidermal LCs and exerts a direct effect on the production of effector T lymphocytes. Hyposensitization is a short-lived, antigen-specific

response that does not involve suppressor cells.[

171]

In contrast, immunologic tolerance is long-lasting and is mediated by suppressor cells. Hyposensitization may

172]

involve a switch from Th2 to Th1 patterns in T cell regulation.[

However, the true mechanism governing this state of decreased sensitivity remains unclear.

Genetics Genetic factors partly control the susceptibility to development of an ACD reaction. In guinea pigs, the mode of inheritance is autosomal dominant with variable penetrance.[

171] [173]

Studies on the mode of inheritance in humans are limited. European studies have compared the frequency of HLA antigens in patients with

nickel-induced ACD to local controls. The antigens HLA-B7, -B21, -B12, -Bw22, -B35, -B40, -DR4, and -DRw6 were increased in patients exhibiting CHS.[

174]

[175] [176]

However, no two studies found the same HLA antigen to be statistically significantly increased. Because HLA antigen expression is required for the development of CHS, the search for HLA-ACD associations continues. 177 178 179

Others have proposed a TNF-α polymorphism as the basis for UVB-induced immunosuppression of CHS.[ ] [ ] [ ] In humans, a G-to-A polymorphism has been identified at position −308 in the promoter region of the TNFA gene. The TNFA AA genotype was found to be associated with a high susceptibility to experimentally induced ICD and low irritant-threshold individuals.[ valuable marker for determining irritant susceptibility.

180]

Although functional studies are lacking, the TNF-308 polymorphism may also serve as a

CLINICAL ASPECTS Contact dermatitis may be irritant or allergic in nature, and it is often difficult to distinguish between the two clinically. Many allergens have some irritant potential. The end result, dermatitis, is the same for both conditions. Nevertheless, based on historical, clinical, and patch test findings, about 80% of all cases of contact dermatitis are of the irritant type, and only 20% are allergic in etiology. Types of Contact Dermatitis Irritants can cause dermatitis in anyone if they are applied to the skin in sufficient concentration and for a long enough period of time. Allergens, on the other hand, can cause contact dermatitis only in those individuals who are sensitized to the particular substance. More than 85,000 chemicals are estimated to exist in the modern 181]

environment,[

making contact dermatitis a relevant clinical problem. Contact dermatitis is often acquired through work-related circumstances and represents more 182]

than 90% of all occupational skin disease. The cost to society in disability and lost productivity approaches $1 billion per year in the United States.[ diagnosis and treatment is essential for minimizing time off work and preventing persistent states of cutaneous sensitivity.

Early

Predisposing Factors ICD susceptibility is primarily a function of repeated exposure to the irritant and the quality of the skin barrier. Many common substances are capable of producing irritant skin reactions. Solvents, greases, lubricants, and raw foods are just a few. ICD is more prevalent in women who often have repeated exposure to household cleansers, soaps, and detergents. Irritant reactions are also more commonly seen in patients with a history of atopic dermatitis, whose skin barrier function may be

diminished. Skin pigmentation also correlates with susceptibility: very fair skinned individuals are more prone to ICD.[

183] [184]

Predisposing factors to ACD are less clearly understood. Genetics play an important role, as discussed previously. Age may also be important, because ACD is uncommon in infants and children younger than 8 years old. Atopic dermatitis and the impaired cell-mediated immunity these patients possess may actually decrease 185

the incidence of ACD.[ ] Finally, the presence of chronic dermatoses, such as psoriasis, ichthyosis, or other forms of endogenous dermatitis, may affect the skin's barrier function and allow easier absorption of allergens and irritants. Diagnosis Contact dermatitis is diagnosed through a careful history and physical examination. Eruptions characteristically possess sharp, linear edges with geometric outlines; however, the appearance is often quite nonspecific. Important information to elicit includes the duration and progression of the eruption, precipitating and aggravating factors, the temporal relation to the work week, and whether any co-workers are similarly affected. Possible exposures acquired through work, hobbies, or other activities; any previous treatment; and whether an atopic diathesis is present should all be inquired about, in addition to a general medical history. Based on this information, one may ask about specific exposures. For example, review of the Material Safety Data Sheets on occupational exposures or the ingredients in a patient's cosmetic products might be helpful. Clinical Features Clinically, the primary lesions in ACD and ICD are the same. Erythema, edema, and vesiculation may be seen in the acute phase ( Color Plate 23 ). Pruritus is usually very intense in this stage. Within hours to a few days, the vesicles break, leading to exudation, crusting, and increased scaling ( Color Plate 24 ). In more chronic dermatitis, the scaling is more pronounced and the skin is generally thickened in affected areas ( Color Plate 25 ). Lichenification, the thickening of the epidermis with marked accentuation of normal skin creases and markings, results from chronic scratching or rubbing of the lesions. Fissuring, or deep splitting of the skin, may occur, particularly if the palms or soles are involved. Chronic

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lesions may also demonstrate dyspigmentation, especially in darker-skinned individuals. In cases of contact dermatitis, the outlines of the involved area may be suggestive of the cause. For example, a perfectly round plaque on the dorsal wrist could correspond to the back of a watch. A horizontal, linear band might relate to the shape of an adhesive dressing. Finally, in cases of upper eyelid dermatitis, cosmetics should always be suspected. More commonly, however, the outline is nonspecific and the diagnosis is less obvious. The differential diagnosis for contact dermatitis includes other dermatoses in the papulosquamous morphologic group. Endogenous forms of dermatitis, such as atopic, nummular, dyshidrotic, and seborrheic dermatitis, as well as psoriasis,

superficial fungal infections, cutaneous T cell lymphoma, parapsoriasis, lupus erythematosus, and pityriasis rosea may all mimic contact dermatitis in some circumstances. More than half of all cases of contact dermatitis involve the hands, because exposure to exogenous substances is more likely to occur on the hands than anywhere else. ICD is most common over the fingertips, whereas ACD frequently occurs on the dorsal side of the hands, where the skin is thinner and percutaneous absorption of allergens is facilitated. Hand involvement is often related to occupational exposure, and certain vocations are known to possess a greater risk for development of contact dermatitis. Hairdressers, cleaners, bakers, bricklayers, construction workers, and those involved in the rubber, plastic, and metal industries are particularly 186

prone.[ ] A wide variety of irritants and sensitizers have been implicated in hand contact dermatitis, including nickel, potassium dichromate, ethylenediamine, pphenylenediamine (PPDA), latex, food substances, soaps, and detergents. The face is another very common site for contact dermatitis. Facial dermatitis is usually related to the use of cosmetics that contain preservatives, fragrances, pigments, and other sensitizers. Patch Testing Patch testing is the only way to objectively demonstrate that a given allergen can cause allergic skin reactions in a patient, and it is therefore the most useful diagnostic tool in ACD. Patch testing attempts to artificially reproduce the circumstances that created the initial allergic reaction. As such, it has certain inherent limitations. In any case, patch testing has a sensitivity and specificity of approximately 70% in experienced hands.[

187]

Patch testing involves placing small quantities of diluted allergens against the skin for a set period of time. Usually a series of common allergens are placed on small 8-mm aluminum disks (Finn chambers) and taped onto the skin of the upper back for 48 hours. Several such patches can be affixed at the same time, allowing for more than 60 allergens to be tested concurrently. Patients must avoid bathing and strenuous exercise for 2 days. At that time, the patches are removed and the test is read. Areas of erythema with edema covering at least half of the test area are graded as 1+ and indicate a positive test. The additional presence of papules is graded as 2+. Vesicles or bullae indicate a very strong reaction and are graded as 3+. No detectable change in the skin signifies a negative test (grade 0). The patient then returns for a second, late reading, usually about 96 hours after the patches were applied. The timing of the late reading may vary; it can be performed up to 7 days after the initial application of the patches. Late readings significantly increase the sensitivity of the test, allowing weak reactions that may not be present at 48 hours to be detected.[

183]

The area of skin selected for patch testing should be free of any inflammatory lesions before the disks are applied, to prevent false-positive reactions. The so-called “angry back” or “excited skin syndrome” is a cutaneous state of hyperirritability resulting from a single strongly positive patch or a background inflammatory 183

dermatosis present at the time the patches were applied. In this situation, the many positive reactions elucidated are probably an irritant effect.[ ] Other falsepositive results arise from irritant (as opposed to allergic) reactions and from reactions to the tape used to secure the disks. Multiple positive findings within a single test series may also indicate a false-positive result. Retesting at a later date may be required. False-negative results have been attributed to several factors, including (1) use of a negligible allergen concentration, (2) technical errors in patch application or patient care of the patches (e.g., getting the area wet), (3) exclusion of a late reading, and (4) use of a vehicle incompatible with the allergen tested. Patch testing cannot precisely reproduce all of the conditions of the original dermatitis. Local factors, such as sweating, heat, friction, and pressure, may not be reproduced, leading

to a false-negative result. Any use of topical or systemic corticosteroids may also inhibit a positive reaction. Most allergens, including most of the standard patch test allergens, can also act as irritants. Distinguishing between allergic and irritant reactions can be difficult. In general, allergic reactions are more pruritic, last longer, and typically extend beyond the edges of the disk. With ICD, patients report burning symptoms. The reactions tend to be sharply demarcated and are often more pronounced at the edges of the disk, resolving quickly on patch removal. Interpreting a positive patch test must be done carefully. A positive reaction indicates that the patient is allergic to a given allergen. This is different from concluding that the allergen in question is responsible for the dermatitis; such a conclusion can only be made based on the allergen's relevance to the clinical situation. Relevance, in turn, is primarily established through detailed questioning and history. The allergen may then be considered a direct primary cause of the dermatitis or an indirect, aggravating factor. If relevance cannot be established, the allergen must be dismissed as the cause of the dermatitis. Several commercially available standard series, or trays of allergens, formulated by different manufacturers and contact dermatitis societies, aid in the selection of 188

which allergens to test. The North American and European standard series are the two most commonly used sets[ ] ( Table 87-1 ). In addition, specialized trays exist for cosmetics, fragrances, textile dyes, and others. The clinician should be familiar with all available trays, and clinical circumstances should dictate which tray to test. Occasionally, one may suspect a material of uncertain composition or one not present in the available trays. If the material is not known to be irritating and is 183

intended for application on the skin (e.g., a cosmetic), an open or usage test can be performed.[ ] A patient applies the material to the skin of the forearm, without occlusion, twice daily for 1 week. No reaction indicates a negative test. Usage tests must not be performed with nonstandard substances of unknown irritancy (e.g., industrial materials).

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TABLE 87-1 -- Standard Series and Prevalence of Contact Allergy to Various Compounds North American Standard Series

European Standard Series

NACDG Patch Test Allergens

% Allergic(NACDG) [

191]

5-Chloro-2-methyl-4-isothiazolin-3one + 2-methyl 4-isothiazolin-3-one (3:1 in water) 0.01% Bacitracin 20% Balsam of Peru 25%

Balsam of Peru 25%

Balsam of Peru 25%

9.1 10.4

Benzocaine 5%

Black rubber mix 0.6%

Benzocaine 5% Budesonide 0.1%

1.1

Carba mix 3%

5.7

Cobalt chloride 1%

8.0

Ethyleneurea melamine–formaldehyde 5%

5.0

Purified protein derivative (PPD)– black rubber mix 0.6%

Carba mix 3% Cinnamic aldehyde 1% Cobalt chloride (CoCl2 , 6H2 O) 1% Cobalt chloride 2% Colophony 20%

Colophony 20%

Epoxy resin 1%

Epoxy resin 1%

Ethylenediamine dihydrochloride 1%

Ethylenediamine dihydrochloride 1%

Formaldehyde 1%

Formaldehyde (in water) 1%

Formaldehyde 1%

9.2

Fragrance mix 8%

Fragrance mix 8%

Fragrance mix 8%

14.0

Imidazolidinyl urea 2% Lanolin alcohol 30% Mercapto mix 1%

Mercapto mix 2%

Mercaptobenzothiazole 1%

Mercaptobenzothiazole 2%

Neomycin sulfate 20%

Neomycin sulfate 20%

Neomycin 20%

11.6

Nickel sulfate 2.5%

Nickel sulfate (NiSO4 , 6H2 O) 5%

Nickel sulphate 2.5%

14.3

Potassium dichromate 0.25% Paraben mix 15% p-Phenylenediamine 1%

p-Phenylenediamine free base 1%

p-Tertiary butyl phenol formaldehyde resin 1%

p-Tertiary butyl phenol formaldehyde resin (BPF resin)1%

p-Phenylenediamine 1%

6.8

Potassium dichromate 0.5% Primin 0.01% Quaternium-15 2%

Quaternium-15 2%

9.2

Quaternium-15 (cis-1-(3-chloroallyl)3,5,7-triaza-1-azoniaadamanate chloride) 1% Quinoline mix 6%

Thiuram mix 1%

Thiuram mix 1%

Thimerosal 0.1%

10.4

Thiuram mix 1%

6.8

Tixocortol pivalate 1%

2.3

Toluene sulphonamide resin 2% Wool alcohols 30% NACDG, North American Contact Dermatitis Group.

Complications from patch testing are uncommon. The probability of inducing sensitization in the patient is low if standard concentrations of allergens are used.[ Other potential adverse effects include dyspigmentation, secondary infection, skin necrosis, ulceration, and scarring.[

183]

189]

A skin biopsy is not usually performed for diagnosing contact dermatitis. Nevertheless, it may be helpful in unusual or uncertain cases, or for ruling out other inflammatory dermatoses. The typical histologic changes include spongiosis (at times extensive enough to create vesiculation); an early lymphocytic, and later neutrophilic, infiltrate in the epidermis and dermis; and, in more chronic lesions, thickening of the epidermis ( Color Plate 26 and Color Plate 27 ). It is not possible to determine the cause of the dermatitis based on the skin biopsy. Endogenous and exogenous forms of dermatitis have identical histologic features. Other investigations may also be indicated to rule out other forms of skin disease. For example, mycologic tests to exclude tinea might be considered. Treatment The first principle in contact dermatitis treatment is to make an accurate diagnosis. Only then can the causal agent be removed from the patient's environment. In severe

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or persistent cases, this may even necessitate changing occupations. Relatively mild, limited cases of contact dermatitis are treated topically. Cool compresses with aluminum subacetate (Burow's solution) or normal saline are effective in drying out exudative lesions. Chronic, dry lesions do not require compresses. The mainstay of topical treatment consists of corticosteroids of sufficient potency and in an appropriate vehicle. The strength of the agent used depends on the location of the eruption, its severity, response to previous treatment, and the patient's age. Areas of thick skin, such as the palms and soles, generally require more potent agents (e.g., 0.05% betamethasone dipropionate, 0.05% clobetasol propionate, or 0.05% halobetasol propionate). Midpotency agents (e.g., 0.1% betamethasone valerate) can be used for trunk and limb involvement. The least potent agents are indicated for thin skin and for children (e.g., 1% hydrocortisone, 0.2% hydrocortisone valerate, or 0.05% desonide). Ointments are best suited for thicker, drier lesions, whereas creams and lotions should be used on exudative lesions or in intertriginous areas. Lotions or gels work best in hairy regions such as the scalp. Adjuvant treatments including colloidal oatmeal baths, bland emollients, barrier creams, and soap substitutes might also be used. Systemic treatment is reserved for more severe cases or extensive involvement. Systemic corticosteroids (e.g., prednisone 0.5 to 1.0•mg/kg/day) are the agents of choice and should be administered briefly on account of their associated toxicity. Tapering the dose too quickly may result in a rebound flare of the dermatitis. Tapering is usually done over a 2- to 3-week period. Adjuvant systemic treatments may include oral antihistamines to control pruritus and antibiotics to treat secondary infections. Physical therapeutic modalities are also occasionally useful. UV light, UVB monotherapy, or UVA phototherapy in combination with oral or bath psoralens (PUVA therapy) may all be considered. Preventative barrier agents, such as gloves or specialized protective clothing, may be required for industrial exposures.[

190]

Common Allergens The North American Contact Dermatitis Group (NACDG) studied the prevalence of allergic reactions as determined by patch testing between 1994 and 1996. They determined that the 12 most common sensitizers were nickel sulfate, fragrance mix, thimerosal, quaternium-15, neomycin sulfate, formaldehyde, bacitracin, thiuram mix, balsam of Peru, cobalt chloride, PPDA, and carba mix (see Table 87-1 ). The hands were the primary site of involvement in about one third of all cases. 191]

Generalized eruptions and facial involvement were the next most likely affected areas, followed by the arms, legs, and trunk.[ 183

Nickel allergy is one of the most common forms of ACD, with prevalence rates of 10% to 15% reported.[ ] Nickel is ubiquitous in the environment and is found in such diverse objects as jewelry, buttons, zippers, watchbands, and glasses. Nickel allergy is more common in women, and sensitization often occurs with ear piercing. PPDA is a pigment present in many inks and dyes. Exposure commonly occurs through hair coloring. Quaternium-15 is a preservative and formaldehyde releaser present in cosmetics and personal hygiene products. Significant cross-sensitivity to formaldehyde exists in patients who are allergic to quaternium-15. Neomycin sensitization usually occurs in clinical settings, when topical medications containing this aminoglycoside antibiotic are used. Thimerosal (sodium ethylmercurithiosalicylate) is another preservative that is present in cosmetics or topical medications, including eye drops. Cinnamic aldehyde and cinnamic alcohol are closely related agents that are used as fragrances and flavors. They can be present in perfumes, mouthwashes, toothpastes, or other dentifrices. Ethylenediamine is a component in the ethylenediamine class of antihistamines; it is also present in aminophylline and is used as a stabilizer in Kenacomb/Mycolog cream.

Potassium dichromate is important in occupational exposure cases. This sensitizer is present in a wide variety of materials, including chrome-plated metals, tanned leathers, and construction materials such as cement, plaster, glues, and paints. Thiurams are also important in occupational exposures. Found primarily in the rubber industry, they function as accelerators in the vulcanization process. Thiurams may also be present in pesticides and fungicides. 192

193

Increasing reports of contact dermatitis from textiles have now placed the incidence at 1.4% to 5.8%.[ ] Again, women are more commonly affected than men.[ ] Reactions tend to occur in areas where clothing comes in contact with the skin and especially where high moisture is present (e.g., axillae, waistline, neck). However, eruptions may also be generalized or even nummular in morphology. Exposures in garment industry workers may cause hand dermatitis. Most textile-related contact allergens are either dyes used for coloring or resins used in finishing clothing. The textile material itself (e.g., silk, wool, nylon, rubber, fiberglass) much less commonly elicits an irritant contact, allergic contact, or contact urticarial response.[

194]

Textile coloring dyes are classified according to application class (e.g., disperse, acid, basic, direct, vat dyes) and chemical structure (e.g., azo, anthraquinone, azine 195

dyes). The most common textile allergens are disperse azo dyes, particularly disperse blue 124, disperse red 1, and disperse orange 3.[ ] Dye allergy screening can be done with purified protein derivative (PPD) and disperse blue 106. PPD cross-reacts with azo dyes, detecting about 30% of sensitive patients. Disperse blue 106 192

detects about 60% of sensitive patients. If a patient tests positive to these screens, more specialized dye series can then be used.[ ] Disperse dyes are water soluble and are used to color synthetic materials such as polyester and nylon. Allergic patients should be advised to wear natural fibers and to avoid synthetics and synthetic blends. The formaldehyde-containing resins used to make natural fibers such as cotton more resistant to wrinkling and shrinkage are the other major group of textile allergens. The most common allergens are dimethylol dihydroxyethylene urea (DMDHEU) and melamine. Most patients reacting to textile resins will have positive patch test reactions to formaldehyde or formaldehyde releasers (e.g., quaternium-15). However, it is possible to react to formaldehyde resins without reacting to 193

formaldehyde itself.[ ] The best screening agent for this allergy is ethylene urea–melamine formaldehyde. Patients who are allergic to formaldehyde resins should avoid natural fibers that are likely to be treated with formaldehyde resin finishes. Synthetics are less likely to cause a problem in these cases.

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TABLE 87-2 -- Corticosteroid Classes Based on Contact Allergy Cross-Reactivity Class A: Hydrocortisone Type

Class B: Triamcinolone Acetonide Type

Class C: Betamethasone Type

Class D: Hydrocortisone Butyrate Type

Hydrocortisone

Triamcinolone acetonide

Betamethasone

Hydrocortisone butyrate

Hydrocortisone acetate

Halcinonide

Dexamethasone

Hydrocortisone valerate

Methylprednisolone

Fluocinonide

Fluocortolone

Clobetasol propionate

Prednisolone

Fluocinolone acetonide

Desoximetasone

Betamethasone valerate

Prednisone

Desonide

Betamethasone dipropionate

Tixocortol pivalate

Budesonide

Prednicarbate

Amcinonide From Rietschel RL, Fowler JF Jr, editors: Fisher's contact dermatitis, ed 5, Philadelphia, 2001, Lippincott Williams & Wilkins; and Marks JG, Deleo VA: Contact and occupational dermatology, ed 2, St Louis, 1997, Mosby. Most patients who react to topical corticosteroids preparations are sensitive to preservatives and other vehicle components in the preparations. However, corticosteroid molecules and their degradation products have themselves been increasingly recognized for their ability to produce ACD.[ 197 population[ ]

196]

About 3% of a patch test

191 pivalate.[ ]

and 2.3% of the NACDG were sensitive to tixocortol The prevalence of this reaction makes this compound a useful screening agent. For contact allergy testing, topical corticosteroids are grouped into four classes ( Table 87-2 ). Cross-reactivity between groups is infrequent, although it can occur. Furthermore, sensitivity to one agent does not guarantee sensitivity to other agents in the same class. Class C corticosteroids produce the fewest reactions. ACD to topical steroids should be suspected in patients who have steroid-resistant dermatitis or worsening dermatitis after application of topical steroids. Typically, there is a history of a chronically treated atopic or stasis dermatitis. Patch testing can distinguish between patients reacting to a preservative or excipient in the agent and those reacting to the steroid itself. Interpretation of the test is difficult because the allergen has intrinsic antiinflammatory properties. False-negative readings may occur, and late readings (more than 5 days) may be necessary. One may also see an inverse edge effect. That is, with most patch testing there is a higher likelihood of encountering a reaction (irritant or allergic) at the periphery of the patch, where the allergen tends to accumulate. When corticosteroids are used, the allergic reaction is more evident centrally, and a greater antiinflammatory effect is seen at the edge of the disc. The NACDG reported that the prevalence of allergy to fragrance mix (composed of oak moss absolute, cinnamic aldehyde, cinnamic alcohol, α-amyl cinnamic alcohol, geraniol, hydroxycitronellal, isoeugenol, eugenol), a screening mixture, was 11.4% between 1992 and 1994 and increased to 14% between 1994 and 1996. Balsam of Peru sensitivity increased from 5.1% (1985 to 1989) to 7.5% (1992 to 1994) to 10.4% (1994 to 1996). The most common fragrance sensitizers are jasmine 198

absolute, geranium oil burbon, l-citronellol, and spearmint. [ ] Generally, patients who react to balsam of Peru or fragrance mix should avoid all scented products, including perfumes, colognes, and scented toiletry products. Specialized testing is difficult because, with industrial secrecy, it is not always possible to determine all the ingredients in a product. Furthermore, the terms “unscented” and “fragrance free” do not exclude the presence of scented ingredients. These ingredients may be masked by other scents or used for other purposes (e.g., as a preservative). [

199]

Allergic reactions to natural rubber products containing latex have also been increasing in prevalence, especially since the advent of universal precautions in 1987 (see Chapter 82 ). The prevalence of latex allergy is estimated to be about 2%. Risk factors for latex allergy development include multiple surgical or dental procedures (particularly early in life), an atopic diathesis, and occupational exposures. Health care, janitorial, and rubber industry workers are particularly at risk. Latex is derived from the Hevea brasiliensis tree. It is composed of cis-1,4-polyisoprene rubber particle chains; havein, prohavein, and other lutoid proteins; Frey 200

Wyssling particles; and plant cytosol. The latter comprises multiple different carbohydrates, amino acids, nucleotides, and proteins.[ ] Most type I immediatehypersensitivity IgE-mediated reactions to rubber result from sensitivity to natural rubber latex proteins that have not been fully characterized. On the other hand, most type IV DTH reactions to natural rubber that produce ACD are not caused by latex itself. The allergens are the accelerators and antioxidants that are added to

the natural rubber during the vulcanization process. Thiurams (about 72%), carbamates (about 25%), and mercapto compounds (about 3%) are the accelerators that 201

produce sensitivity most of the time.[ ] Patch testing of the thiuram mix (tetraethylthiuram monosulfide, tetramethylthiuram disulfide, dipentaethylenethiuram disulfide), the mercapto mix (mercaptobenzothiazole, N-cyclohexylbenzothiazyl sulfonamide, dibenzothiazyl disulfide, morpholinyldibenzothiazole), and the carba mix (1,3-diphenylguanidine, zinc diethyldithiocarbamate, zinc dibutyldithiocarbamate) can detect these compounds. Patch testing of PPDA may also be useful, because it and a number of its derivatives are used as antioxidants in the rubber manufacturing process. 202

ACD has occurred, however, after exposure to purified latex itself. [ ] The prevalence of this reaction is uncertain. If a DTH reaction is suspected, patch testing of a sample of natural rubber latex can be performed. Caution must be taken when patch testing purified latex, particularly if a possibility for immediate hypersensitivity exists. For patients with known latex allergy, latex and latex-containing products and devices must be avoided. Synthetic rubber compounds such as nitrile, neoprene, silicone, and urethane do not contain latex and are safe for these individuals. Although reactions to gold were once considered rare or irrelevant, increasing numbers have been reported over the

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203

last 5 to 10 years. Of 145 patients reacting to jewelry, 10% reacted to gold when tested with an extended metal series tray.[ ] Gold reactions include lichen planus– like eruptions and persistent “dermal” papular reactions with little or no epidermal component. The papular reaction can be long-lasting, persisting for weeks after the allergen is removed. Stomatitis from dental gold is also common. Patch testing to gold is usually done with 0.5% gold sodium thiosulfate in petrolatum. Late readings should be performed, because gold reactions may be significantly delayed (more than 7 days).[

204]

Plant-derived saps can also cause contact dermatitis. The most frequently encountered plant ACDs are caused by the toxicodendron group of plants, including poison ivy, poison oak, and poison sumac. All produce linear and often streaky eruptions at the sites brushed by the plant ( Color Plate 28 ). Patients often also have crosssensitivity to mango peels and cashew nut shell oil. Photocontact Dermatitis The term photocontact dermatitis describes a form of contact dermatitis that erupts only when the skin is exposed to light. Photocontact dermatitis may be primarily irritant (phototoxic) or allergic (photoallergic) in nature. Clinically, only sun-exposed areas are affected, most prominently the face, the arms, and the V of the upper chest. The skin under the chin, behind the ears, and on the upper eyelids is distinctly spared. Photodistributed eruptions may suggest other disorders, including collagen vascular diseases, metabolic photosensitivities such as the porphyries, and photosensitive drug eruptions. Tars, dyes, psoralens, and drugs such as sulfonamides, tetracyclines, thiazides, and phenothiazines can all produce phototoxic eruptions. Photoallergic reactions are caused by fragrances, sunscreens, and the same drugs that cause phototoxic dermatitis. A photopatch test is used to diagnose photocontact dermatitis. Here, two sets of identical patches are applied to the skin of the upper back and covered with opaque

material. After 48 hours, the covering is removed and the patches are read. One set is then exposed to UVA light from an artificial source, while the other set is shielded. The opaque covering is reapplied to both sets, and the patches are reevaluated at 96 hours. The test is positive when a reaction appears on the irradiated side but is absent on the nonirradiated side. Other Contact Reactions Cutaneous contact reactions are not always dermatitic in morphology. Cheilitis, purpuras, granulomas, dyspigmentations, acneiform eruptions, lichenoid eruptions, and erythema multiforme–like reactions may all occur. Contact urticaria is one of the most common nondermatitic types of contact reaction. Contact urticaria may be caused by either allergic or nonallergic mechanisms, or by a combination of the two. In the nonallergic type, there is no previous sensitization and urticaria results from the nonimmunologic release of vasoactive substances in the skin. Acetic acid, benzoic and sorbic acids (food preservatives), cinnamic acid (a flavoring and fragrance ingredient), balsam of Peru (a fragrance), caterpillar hairs, insect stings, bacitracin, neomycin, and other topical antibiotics can all produce nonallergic contact urticaria in most individuals if the concentration and contact time are sufficient. An “open test” is used to verify nonallergic contact urticaria. A small amount of the test material is placed on the skin of the flexor surface of the forearm for 30 to 45 minutes. Allergic contact urticaria occurs in individuals who have been previously sensitized. IgE is thought to mediate wheal production, although other factors may also play a role. Allergic contact urticaria is more prevalent in patients with a history of atopy. Foods such as milk, eggs, fish, fruits and vegetables, latex, topical antibiotics, metals, animal products (e.g., dog saliva, rat tails), industrial materials (e.g., ammonia, formaldehyde, acrylic acid), and others can produce allergic contact urticaria. Allergic contact urticaria testing may require applying the material to eczematous or scratched skin to get a positive reaction.

PROGNOSIS The prognosis of a patient with contact dermatitis is determined, to a large extent, by the patient's understanding of the causal factors and his or her ability to avoid them. Long-standing disease is a bad prognostic sign, and persistent reactions may never clear. Clearing rates in patients with ACD and ICD range from 30% to 50%. [205] [206]

The multifactorial etiology of many contact dermatitis cases might account for these low clearing rates. This is especially true for cases of hand dermatitis, where both exogenous and endogenous factors may be present and external contactants may exacerbate a preexisting underlying chronic disorder.

CONCLUSIONS Diagnosis of contact dermatitis requires specialized knowledge of the myriad allergens in the modern environment and the chemicals used in various industries and occupations. As new substances are created and used, the list of possible allergens continues to evolve. Several good reference texts are available that list all of the currently known allergens, their usual modes of exposure, cross-sensitizations, and methods of skin testing with them.[ indispensable to any contact dermatitis clinic.

183] [207] [208]

These references are

Although indistinguishable both clinically and histologically, ACD and ICD have different pathogeneses. Transgenic mouse technology has contributed significantly to our understanding of these disorders. Although the major cell types and mediators involved in these reactions have been identified, preliminary understanding of their regulatory mechanisms has led to only limited therapeutic progress. In the years to come, skin-specific transgenic mouse models will advance our understanding

of ACD and ICD reactions. Insights from the laboratory will aid in the development of more effective therapeutic modalities for this common and at times very disabling disease. Agents that block the actions of proinflammatory cytokines or CAMs offer future therapeutic potential. Specific antibodies, soluble receptors, or receptor antagonists may emerge as the new treatments of choice in contact dermatitis.

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1986. 165. Moller H: Allergic contact dermatitis of the mouse ear, Acta Derm Venereol 61: 1–6, 1981. 166. Rietschel RL: Irritant and allergic responses as influenced by triamcinolone in patch test materials, Arch Dermatol 121:68–69, 1985. 167. Bergstresser PR, Toews GB, Gilliam JN, et al: Unusual numbers and distribution of Langerhans cells in skin with unique immunologic properties, J Invest Dermatol 74:312–314, 1980. 168. Furue M, Katz SI: Direct effects of glucocorticosteroids on epidermal Langerhans cells, J Invest Dermatol 92:342–347, 1989. 169. Aberer W, Schuler G, Stingl G, et al: Ultraviolet light depletes surface markers of Langerhans cells, J Invest Dermatol 76:202–210, 1981. Hyposensitization 170. Van Scott EJ, Kalmanson JD: Complete remissions of mycosis fungoides lymphoma induced by topical nitrogen mustard (HN2). Control of delayed hypersensitivity to HN2 by desensitization and by induction of specific immunologic tolerance, Cancer 32:18–30, 1973. 171. Rietschel RL, Fowler JF Jr, editors: Fisher's contact dermatitis, ed 4, Baltimore, 1995, Williams & Wilkins, p 1. 172. Karl S, Ring J: Pro and contra of specific hyposensitization, Eur J Dermatol 9: 325–331, 1999. Genetics 173. Levine B, Ojeda A, Benacerraf B: Studies on artificial antigen: III. The genetic control of the immune response to hapten-poly-L-lysine conjugates in guinea pigs, J Exp Med 118:953–1963. 174. Karvonen J, Silvennoinen-Kassinen S, Ilonen J, et al: HLA antigens in nickel allergy, Ann Clin Res 16:211–212, 1984. 175. Mozzanica N, Rizzolo L, Veneroni G, et al: HLA-A, B, C and DR antigens in nickel contact sensitivity, Br J Dermatol 122:309–313, 1990. 176. Bergstresser PR: Contact allergic dermatitis: old problems and new techniques, Arch Dermatol 125:276–279, 1989. 177. Satoh T, Tokura Y, Takigawa M, et al: Effect of the H-2 and Igh complexes on the susceptibility to ultraviolet B-induced immunosuppression in murine contact sensitivity and contact photosensitivity, Photodermatol Photoimmunol Photomed 7:73–76, 1990. 178. Streilein JW, Bergstresser PR: Genetic basis of ultraviolet-B effects on contact hypersensitivity, Immunogenetics 27:252–258, 1988. 179. Yoshikawa T, Streilein JW: Tumor necrosis factor-alpha and ultraviolet B light have similar effects on contact hypersensitivity in mice, Reg Immunol 3:139– 144, 1990. 180. Allen MH, Wakelin SH, Holloway D, et al: Association of TNFA gene polymorphism at position −308 with susceptibility to irritant contact dermatitis, Immunogenetics 51:201–205, 2000.

Types of Contact Dermatitis 181. Drake LA, Dorner W, Goltz RW, et al: Guidelines of care for contact dermatitis: Committee on Guidelines of Care, J Am Acad Dermatol 32:109–113, 1995. 182. Rietschel RL: Human and economic impact of allergic contact dermatitis and the role of patch testing, J Am Acad Dermatol 33:812–815, 1995. Predisposing Factors 183. Rietschel RL, Fowler JF Jr, editors: Fisher's contact dermatitis, ed 5, Philadelphia, 2001, Lippincott Williams & Wilkins, pp 27–30, 33–50. 184. Wilhelm KP, Maibach HI: Factors predisposing to cutaneous irritation, Dermatol Clin 8:17–22, 1990. 185. Klas PA, Corey G, Storrs FJ, et al: Allergic and irritant patch test reactions and atopic disease, Contact Dermatitis 34:121–124, 1996. Diagnosis 186. Kwangsukstith C, Maibach HI: Effect of age and sex on the induction and elicitation of allergic contact dermatitis, Contact Dermatitis 33:289–298, 1995.

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Patch Testing 187. Nethercott JR, Cooley JE: Getting the most out of patch testing, Curr Opin Dermatol 2:10–17, 1995. 188. Bruynzeel DP, Andersen KE, Camarasa JG, et al: The European standard series: European Environmental and Contact Dermatitis Research Group (EECDRG), Contact Dermatitis 33:145–148, 1995. 189. Fransway AF: Epicutaneous patch testing: current trends and controversial topics, Mayo Clin Proc 64:415–423, 1989. Treatment 190. Mathias CG: Prevention of occupational contact dermatitis, J Am Acad Dermatol 23:742–748, 1990. Common Allergens 191. Marks JG, Belsito DV, Deleo VA, et al: North American Contact Dermatitis Group patch test results for the detection of delayed-type hypersensitivity to topical allergens, J Am Acad Dermatol 38:911–918, 1998. 192. Hatch KL, Maibach HI: Textile dye dermatitis, J Am Acad Dermatol 32: 631–639, 1995.

193. Fowler JF Jr, Skinner SM, Belsito DV: Allergic contact dermatitis from formaldehyde resins in permanent press clothing: an underdiagnosed cause of generalized dermatitis, J Am Acad Dermatol 27:962–968, 1992. 194. Guin JD, Styles A: Protein-contact eczematous reaction to cornstarch in clothing, J Am Acad Dermatol 40:991–994, 1999. 195. Hatch KL, Maibach HI: Textile dye allergic contact dermatitis prevalence, Contact Dermatitis 42:187–195, 2000. 196. Wilkinson SM: Corticosteroid cross-reactions: an alternative view, Contact Dermatitis 42:59–63, 2000. 197. Lutz ME, el Azhary RA, Gibson LE, et al: Contact hypersensitivity to tixocortol pivalate, J Am Acad Dermatol 38:691–695, 1998. 198. Larsen W, Nakayama H, Fischer T, et al: Fragrance contact dermatitis: a worldwide multicenter investigation. Part II, Contact Dermatitis 44:344–346, 2001. 199. Scheinman PL: The foul side of fragrance-free products: what every clinician should know about managing patients with fragrance allergy, J Am Acad Dermatol 41:1020–1024, 1999. 200. Warshaw EM: Latex allergy, J Am Acad Dermatol 39:1–24, 1998. 201. Cohen DE, Scheman A, Stewart L, et al: American Academy of Dermatology's position paper on latex allergy, J Am Acad Dermatol 39:98–106, 1998. 202. Wilkinson SM, Burd R: Latex: a cause of allergic contact eczema in users of natural rubber gloves, J Am Acad Dermatol 39:36–42, 1998. 203. Gawkrodger DJ, Lewis FM, Shah M: Contact sensitivity to nickel and other metals in jewelry reactors, J Am Acad Dermatol 43:31–36, 2000. 204. Bruze M, Andersen KE: Gold—a controversial sensitizer: European Environmental and Contact Dermatitis Research Group, Contact Dermatitis 40:295–299, 1999. Prognosis 205. Hogan DJ, Dannaker CJ, Maibach HI: The prognosis of contact dermatitis, J Am Acad Dermatol 23:300–307, 1990. 206. Nethercott JR, Holness DL: Disease outcome in workers with occupational skin disease, J Am Acad Dermatol 30:569–574, 1994. Conclusion 207. Adams RM: Occupational skin disease, Philadelphia, 1990, WB Saunders. 208. Cronin E: Contact dermatitis, New York, 1980, Churchill Livingstone. 209. Marks JG, Deleo VA: Contact and occupational dermatology, ed 2, St Louis, 1997, Mosby.

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Chapter 88 - Allergic and Immunologic Diseases of the Eye

Neal P. Barney Frank M. Graziano

Many systemic disorders have ocular manifestations. Specific allergic diseases of the eye principally affect the conjunctiva and are well defined and common. Other diseases of the eye with purported immunologic mechanisms are not always recognized and are generally not well defined. This chapter focuses on the five well defined entities that constitute allergic eye disease and other diseases of the eye thought to have an immunologic basis. To put into perspective the discussion of these ocular disorders, the first section of this chapter describes the pertinent anatomy and physiology of the eye. Subsequent sections detail the clinical manifestations, pathophysiology, and treatment of these disorders. Ocular diseases with immunologic implications will be discussed with reference to the principal anatomic structures affected.

ANATOMY AND PHYSIOLOGY OF THE EYE Topographic Anatomy Figure 88-1 demonstrates the ocular anatomy pertinent to the discussion of the allergic and immunologic diseases of the eye. The eye and its adnexa lie within the bony orbit. The pear-shaped orbit has as its medial boundary the nasal cavity and more posteriorly, the ethmoid and sphenoid sinuses. The frontal sinus along with the anterior cranial fossa serves as the superior border. Laterally, the union of the zygomatic and frontal bones forms the temporal portion of the orbit. Beneath the floor of the orbit is the maxillary sinus. The thinnest portion of the bony orbit is found medially. This portion of the orbit is critical because pathologic thinning of the bone may allow the spread of infections, inflammatory diseases, and neoplasms that start in the sinuses through the bone into the orbit. Once in the orbit, these processes primarily affect the extraocular muscles and lacrimal glands. Lacrimal System

The main and accessory lacrimal glands produce the aqueous portion of tears. The main lacrimal gland has two parts. The orbital portion lies in a shallow depression within the frontal bone. The palpebral portion, or portion that overlies the globe itself, may be seen in the superior temporal quadrant by elevating the upper lid off the globe (see Figure 88-1 ). The two parts are separated by a fibrous lateral extension of the levator muscle, but they maintain a connection by a small isthmus of tissue. The ductules conveying the aqueous tears to the ocular surface open in the superior fornix. The fornix is formed where the conjunctiva lining the underside of the upper eyelid is reflected onto the globe (see Figure 88-1 ). The accessory lacrimal glands are located in the fornix. They are histologically identical to the main lacrimal gland. The lacrimal glands are exocrine glands that are surrounded by myofibroblasts. These glands produce their aqueous secretion from acinar cells that line the lumen of the gland. The vascular supply to the main gland is through the lacrimal artery. Neural innervation is complex and may be the major influence on reflex basal and psychogenic gland secretion. However, endocrine and hormonal influences augment secretion and composition of tear fluid. The aqueous portion of tear fluid (also called tear film) is admixed with both mucin and lipid components. The mucin component is derived from goblet cells contained within the conjunctival epithelium and from the epithelial cells themselves. The lipid portion is secreted onto the ocular surface from meibomian glands. The openings of these glands are in the eyelid margins just posterior to the eyelash line. These glands are modified holocrine sweat glands and are contained within the tarsal plate (dense connective tissue that gives form to the eyelid). Inflammatory conditions may alter the volume or composition of any of the three components of tear film. The surface of the eye has a tear volume ranging from 2.6 to 7.4••l. The normal turnover rate of tears on the ocular surface is 12% to 16% per minute. Most commercially available eye drops will dispense (in a single drop) a volume that far exceeds the volume held by the ocular surface. If a therapeutic drop instilled into the eye were to completely displace the tear volume, it would be cleared by tear turnover in 5 to 10 minutes. With each blink, tears are directed nasally across the surface of the eye toward the inferior and superior punctal openings. Passing these small openings, the tears flow horizontally to the nasal lacrimal sac, then downward through the nasolacrimal duct into the nose. Eyelids The eyelids, a composite structure of skin and its appendages, muscle and connective tissue, are lined by conjunctiva (see Figure 88-1 ). The skin begins near the eyelid margin at the mucocutaneous junction. Similar to other facial skin, the epidermis is stratified squamous epithelium about four cell layers thick. The dermis is thin and loosely adherent to the underlying muscular layer (the orbicularis oculi muscle)

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Figure 88-1 Cross-sectional anatomy of the eye. (Illustration by Jim Stahl.)

(Illustration by Jim Stahl.) except in the areas of the eyelid crease, eyelid margins, and medial and lateral angles. Skin appendages include the sweat glands (which tend to be most numerous close to the eyelid margins) and the sebaceous glands (which are found further back from the eyelid margin). Sweat and sebaceous glands empty into the eyelash follicle. The orbicularis oculi muscle surrounds the eye in a sphincterlike pattern and extends from the eyelid margin to the orbital rim. Cranial nerve VII innervates this muscle, which functions in proper closure of the eye. The rigid connective tissue referred to as the tarsus forms the next layer of the eyelid beneath the orbicularis muscle. This gives form to the eyelids and contains the meibomian glands.

Conjunctiva The conjunctiva is a mucous membrane that begins at the eyelid margin, lines the posterior portions of the eyelids (tarsal conjunctiva), and is reflected onto the globe (bulbar conjunctiva). From this reflected area (the fornix), the conjunctiva moves anteriorly, covering the globe up to the limbus, the anatomic junction of the cornea and sclera (see Figure 88-1 ). The conjunctival epithelium varies in its cell type depending on the location, but generally it is a nonkeratinized stratified squamous epithelium. Within the epithelium are the mucin-secreting goblet cells, which discharge their contents onto the ocular surface. Loss of goblet cells compromises the integrity of the tear film. Although Langerhans' cells are found in the epithelium, mast cells are not found in the epithelium of normal conjunctiva. The substantia propria underlies the basement membrane of the epithelium. The superficial portion of the substantia propria contains mast cells, Langerhans' cells, and other resident inflammatory cells. Mast cells are most numerous near the limbus. The cellular composition of the substantia propria may change depending on the age of the individual and is also affected by chronic administration of therapeutic drops, as in glaucoma. The deeper layer of the substantia propria is more fibrous and contains the vasculature and neural innervation to the conjunctiva. This layer serves to attach the conjunctiva to its underlying structures. The bulbar conjunctiva is tightly adherent at the limbus and extends posteriorly to the forniceal portion of the conjunctiva. In this portion, the attachments are quite loose, allowing redundancy of the membrane. This “slack” in the conjunctiva permits free movement of the globe without tension on the inner aspects of the eyelids. The conjunctiva is capable of containing significant amounts of fluid (chemosis) or blood that may have as its source the conjunctival vessels of the orbit. The lymphatic drainage of the conjunctiva is partitioned and tends to be to the preauricular or submandibular nodes.

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Cornea The cornea is the avascular, most anteriorly placed portion of the eye (see Figure 88-1 ). It is composed of stratified squamous epithelium, a stroma, and a single layer of nonreplicating endothelium. The stem cells for the epithelium are contained in the limbal conjunctiva. The stroma is composed of an anterior, densely compact collagen layer (type I collagen) called Bowman's layer. The posterior collagen layer is not as dense and is precisely arrayed to maintain clarity. The cellular elements (keratocytes) are responsible for the elaboration of the collagen and intercellular matrix that forms this stroma. Importantly, under certain conditions, these cells may develop a phenotype similar to that of a fibroblast. This becomes critical in diseases of the eye that threaten sight. The endothelium serves to regulate fluid content of the cornea by an ion pump mechanism. Immunoglobulins and complement components diffuse from the limbus into the cornea. Sclera The optically clear, avascular cornea blends into the white sclera at the limbus. The sclera is the dense connective tissue structure that is responsible for the form and strength of the globe (see Figure 88-1 ). Three anatomic layers of the sclera have been identified: the vascular episclera, the stroma (interwoven collagen bundles

giving a feltlike structure), and the thin lamina fascia which loosely connects the sclera to the outer portion of the choroid. Although the episcleral layer has a fairly rich vascular supply, that of the thick stroma is limited to perforating vessels that bring blood to the middle structures of the eye. The ophthalmic artery (a branch of the internal carotid) gives rise to the ciliary arteries, which supply the sclera. The episcleral vascular plexus has two components: the superficial episcleral plexus and the deep episcleral plexus. It may be possible to distinguish clinical entities of episcleral and true scleral inflammation based on the response of these plexuses (see later discussion of episcleritis and scleritis). These plexuses become intermingled with each other and the conjunctival vasculature, most notably near the limbus. Collagen (type I and type III) forms the majority of the sclera. The water content of the sclera is responsible for its lack of transparency. Fibroblasts are the principal cells found in the sclera; they are found in a relatively quiescent metabolic state. Uvea The uvea is the middle and most vascular structure of the eye (see Figure 88-1 ). It begins anteriorly as the iris and becomes the ciliary body and then the choroid as the vascular tissue sandwiched between the outer sclera and inner retina. Anteriorly, the iris is composed of blood vessels (predominantly in a radial pattern) and connective tissue containing melanocytes. The sphincter muscle is found in the deep stroma, near the pupil margin, just in front of the pigmented epithelial layer of the iris. The dilator muscle lies parallel and just anterior to the posterior pigmented layer. The posterior pigmented layer is a dense, uniform layer of pigmented cells. Interestingly, as this layer becomes contiguous with the same layer of the ciliary body, the cells are nonpigmented. The ciliary body, or middle portion of the uvea, has stromal, muscular, and dual epithelial components. The stromal portion contains capillaries, fibroblasts, and collagen fibrils. The muscular unit controls tension on the zonules of the lens, facilitating accommodation. This unit also plays a role in control of the trabecular meshwork, opening and facilitating aqueous fluid egress from the eye. The epithelium is two layered, with the apical portions of the layers facing and fused to each other. The inner pigmented layer is cuboidal in nature. The nonpigmented layer is the most superficial covering layer of the ciliary body and produces the aqueous humor. The choroid extends posteriorly from the ciliary body throughout the eye (see Figure 88-1 ). This almost entirely vascular structure serves the metabolic needs of the inner aspect of the retina. Retina and Optic Nerve The retina is composed of three layers. The outermost portion contains the photoreceptor layer of rods and cones. The middle cell layer (bipolar layer), receives synaptic connections from the photoreceptors and sends axonal connections to the ganglion cells. The ganglion cell layer sends its axons to the optic disc, where they coalesce as they exit the eye, forming the optic nerve. After a varying course, these axons synapse in the lateral geniculate body of either the same or the contralateral side. A branch of the ophthalmic artery serves the optic nerve; it courses forward and branches within the eye as the retinal artery. Venous return pathways are aligned with the arteries.

ALLERGIC DISEASES OF THE EYE Allergic eye disease is typically divided into five distinct types: allergic conjunctivitis (AC), subdivided into seasonal and perennial allergic conjunctivitis (SAC and PAC, respectively); atopic keratoconjunctivitis (AKC); vernal keratoconjunctivitis (VKC); and giant papillary conjunctivitis (GPC). GPC is considered an iatrogenic form of allergic eye disease. In the discussion that follows, clinical, pathophysiologic, and diagnostic aspects of each ocular process are discussed in detail. These parameters are summarized in Tables 88-1 and 88-2 .

Seasonal and Perennial Allergic Conjunctivitis AC is a bilateral, self-limited conjunctival inflammatory process. It occurs in sensitized individuals (with no gender difference) and is initiated by the binding of allergen to immunoglobulin E (IgE) antibody on resident mast cells. The importance of this process is related more to its frequency than to its severity of symptoms. The two forms of AC are defined by whether the inflammation occurs seasonally (spring, fall) or perennially (year-round). Although the inflammatory symptoms are similar for both entities, SAC (“hay fever conjunctivitis”) is more common. It accounts for the majority of cases of AC and is related to atmospheric pollens (e.g., grass, trees, ragweed) that appear during specific seasons. PAC is often related to animal dander, dust mites, or other allergens that are present in the environment year-round. Both SAC and PAC must be differentiated from the sight-threatening allergic diseases of the eye, namely AKC and VKC.

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TABLE 88-1 -- Allergic Diseases of the Eye Disease

Clinical Parameters

Signs/Symptoms

Differential Diagnosis

Seasonal allergic conjunctivitis (SAC)

Sensitized individuals

Ocular itching

Infective conjunctivitis

Both females and males

Tearing (watery discharge)

Preservative toxicity

Bilateral involvement

Ocular chemosis, redness

Medicamentosa

Seasonal allergens

Often associated with rhinitis

Dry eye

Self-limited

Not sight-threatening

PAC/AKC/VKC

Sensitized individuals

Ocular itching

Infective conjunctivitis

Both females and males

Tearing (watery discharge)

Preservative toxicity

Bilateral involvement

Ocular chemosis, redness

Medicamentosa

Year-round allergens

Often associated with rhinitis

Dry eye

Self-limited

Not sight-threatening

SAC/AKC/VKC

Sensitized individuals

Severe ocular itching

Contact dermatitis

Peak incidence 20–50 years of age

Red, flaking periocular skin

Infective conjunctivitis

Both females and males

Mucoid discharge, photophobia

Blepharitis

Bilateral involvement

Corneal erosions

Pemphigoid

Perennial allergic conjunctivitis (PAC)

Atopic keratoconjunctivitis (AKC)

Vernal keratoconjunctivitis (VKC)

Seasonal/perennial allergens

Scaring of conjunctiva

VKC/SAC/PAC/GPC

Atopic dermatitis

Cataract (anterior subcapsular)

Chronic symptoms

Sight-threatening

Sensitized individuals

Severe ocular itching

Infective conjunctivitis

Peak incidence 3–20 years of age

Severe photophobia

Blepharitis

Males predominate 3:1

Thick, ropey discharge

AKC/SAC/PAC/GPC

Bilateral involvement

Cobblestone papillae

Warm, dry climate

Corneal ulceration and scaring

Seasonal/perennial allergens

Sight-threatening

Chronic symptoms Giant papillary conjunctivitis (GPC)

Sensitization not necessary

Mild ocular itching

Infective conjunctivitis

Both females and males

Mild mucoid discharge

Preservative toxicity

Bilateral involvement

Giant papillae

SAC/PAC/AKC/VKC

Prosthetic exposure

Contact lens intolerance

Occurs any time

Foreign body sensation

Chronic symptoms

Protein buildup on contact lens Not sight-threatening

Clinical Parameters

Prevalence estimates for AC are difficult because allergies in general tend to be considerably underreported. A survey conducted by the American College of Allergy, Asthma, and Immunology (ACAAI) found that 35% of families interviewed experience allergies. Of these, at least 50% reported associated eye symptoms. 1

Most reports agree that AC affects up to 20% of the population. [ ] Importantly, 60% of all allergic rhinitis sufferers have associated AC. The distribution of SAC depends largely on the climate. For example, in the United States SAC induced by grass pollen generally occurs in the Gulf Coast and southwestern areas of the country from March to October, and from May to August in most of the rest of the country (see Chapter 33 ). Conversely, SAC induced by ragweed pollen occurs in most of the country during August through October, but in the southernmost states it can begin as early as July and stretch through November. Tree pollens can become a problem as early as January in the south and March in the north. The dominant symptom reported in AC is ocular itching (see Table 88-1 ). Itching can range from mild to severe. Other symptoms include tearing (watery

discharge), redness, swelling, burning, a sensation of fullness in the eyes or eyelids, an urge to rub the eyes, sensitivity to light, and occasionally blurred vision. As stated previously, AC is often associated with symptoms of allergic rhinitis. Conjunctival hyperemia and chemosis ( Figure 88-2 ) with palpebral edema are typical. Hyperemia is the result of vascular dilatation, and edema (chemosis) occurs because of altered permeability of postcapillary venules. “Allergic shiners” (periorbital darkening), caused by a transient increase of periorbital pigmentation resulting from the decreased venous return in the skin and subcutaneous tissue, are also common. Pathophysiology

It has been understood for some time that antigen cross-linking of IgE antibody bound to the high-affinity IgE receptor (FcepsilonRI) on mast cells induces release of both preformed (granule-associated) mediators (e.g., histamine, tryptase) and newly synthesized mediators (e.g., arachidonic acid metabolites), which have diverse and overlapping biologic effects. Both tissue staining and tear film data have implicated the mast cell and IgE-mediated release of its mediators in the pathophysiology of the ocular allergic

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TABLE 88-2 -- Histopathologic and Laboratory Manifestations of Allergic Ocular Disease Disease

Histopathology

Laboratory Manifestations

Seasonal/perennial allergic conjunctivitis

Mast cell/eosinophil infiltration in conjunctival epithelium and substantia propria

Increased in tears: specific IgE antibody, histamine, tryptase, TNF-α

Mast cell activation Up-regulation of ICAM-1 on epithelial cells Atopic keratoconjunctivitis

Vernal keratoconjunctivitis

Increased mast cells, eosinophils in conjunctival epithelium and substantia propria

Increased specific IgE antibody in tears

Epithelial cell/goblet cell hypertrophy

Depressed cell-mediated immunity

Increased CD4/CD8 ratio in conjunctival epithelium and substantia propria

Increased IgE antibody and eosinophils in blood

Increased collagen

Eosinophils found in conjunctival scrapings

Increased mast cells, eosinophils in conjunctival epithelium and substantia propria

Increased specific IgE and IgG antibody in tears

Eosinophil major basic protein deposition in conjunctiva

Elevated histamine and tryptase in tears

CD4+ clones from conjunctiva found to have helper function for local production of IgE antibody

Reduced serum histaminase activity

Increased collagen

Increased serum levels of nerve growth factor and substance P

Increased ICAM-1 on corneal epithelium Giant papillary conjunctivitis

Giant papillae

No increased histamine in tears

Conjunctival thickening

Increased tryptase in tears

Increased mast cells in epithelium ICAM-1, intercellular adhesion molecule 1; IgE, immunoglobulin E; IgG, immunoglobulin G; TNF-α, tumor necrosis factor-α. inflammatory response. Additionally, a number of clinical studies examining topical antihistamine, mast cell– stabilizing, and dual-acting drugs have demonstrated relief of AC symptoms (see later discussion). Histopathologic and laboratory manifestations of allergic ocular diseases are shown in Table 88-2 . Synthesis of inflammatory mediators varies according to the phenotype and tissue location of the mast cell. Granule-associated neutral proteases (tryptase and chymase) that are unique to mast cells are generally accepted as the most appropriate phenotypic markers to categorize human mast cells into subsets. Mast cells on this basis have been divided into MCT (tryptase) and MCTC (tryptase/ chymase) phenotypes. The phenotype of

Figure 88-2 Allergic conjunctivitis. Arrow indicates area of chemosis in the conjunctiva.

Figure 88-3 Atopic keratoconjunctivitis. Arrows indicate periocular skin lesions of atopic dermatitis.

Figure 88-4 Vernal keratoconjunctivitis. Arrows indicate cobblestone papillae and ropey discharge on the underside (tarsal conjunctiva) of the upper eyelid.

Figure 88-5 Giant papillary conjunctivitis. Arrow indicates numerous papillae 1•mm or greater in size on the underside (tarsal conjunctiva) of the upper eyelid.

TABLE 88-3 -- Grading Scales for Symptom Scores in Conjunctival Provocation Testing Score

Itching

Hyperemia

Chemosis

Lid Swelling

0.0

None

None

None

None

1.0

Intermittent tickling sensation

Mild: dilated blood vessels

Mild: confirmed with slitlamp evaluation

Mild: detectable swelling of lower lid

2.0

Mild continuous ocular itching Moderate: dilated blood vessels

Moderate: raised conjunctiva

Moderate: definite swelling of lower lid

3.0

Severe ocular itching

Severe: numerous and obvious dilated blood vessels

Severe: ballooning of the conjunctiva

Severe: extremely swollen lower lid

4.0

Incapacitating ocular itching

Extremely severe: large, Not applicable numerous dilated blood vessels

Not applicable

Modified from Abelson MB, Chambers WA, Smith LM: Arch Ophthalmol 108:84–88, 1990. In conjunction with the clinical parameters evaluated in the CPT model, many researchers have been able to analyze tear fluid obtained with the CPT procedure to determine mediators and cell types present.[

5] [43] [44]

It has been shown that conjunctival allergen provocation in atopic subjects results in the release in tears of

mediators known to come from mast cells, such as histamine, tryptase, prostaglandins, and leukotrienes C4 and D4 .[

6] [45] [46]

Additionally, it has been shown that

two histamine peaks (at 20 minutes and at 6 hours) occur after allergen provocation. Because only an early tryptase peak is measured, this may indicate either the 47

involvement of basophils in the late phase or, possibly, the presence of refractory mast cells.[ ] Recent techniques facilitating the analysis of cytokines in tears[ could also be combined with the CPT model to better understand both the mechanisms of action of ocular drugs and the pathophysiology of AC.

12]

GENERAL PRINCIPLES OF TREATMENT FOR ALLERGIC EYE DISEASE Treatment of allergic eye disorders should always involve a cooperative effort between the primary care physician, the allergist, and the ophthalmologist. The allergist provides insight into the systemic and local nature of the allergic symptoms and evaluation for systemic therapy, topical therapy, and immunotherapy appropriate to the symptoms. Symptoms that should prompt referral to an ophthalmologist include changes in vision, ocular pain, an irregular or abnormal pupil, corneal opacification, evidence of intraocular inflammation, and an inability to move the eye. This type of approach ensures that diagnostic tests are conducted and appropriate management of allergic and potentially sight-threatening symptoms is instituted. Topical vasoconstrictors, antihistamines, mast cell stabilizers, nonsteroidal antiinflammatory drugs (NSAIDs), and steroids are useful medications to counteract the histamine-induced leakiness and dilation of blood vessels. Some of these topical preparations are available OTC; however, OTC eye drops generally offer only shortterm relief, making the prescription preparation the more beneficial treatment for the long term. There has been significant progress in the number of medications available to treat allergic eye conditions. The classification of the topical ocular drugs is listed in the following sections and summarized in Table 88-4 . 48] [49] [50] [51] [52] [53] [54]

Table 88-5 presents some of the multiple dose–dependent effects of topical ocular allergic drugs on the allergic inflammatory response.[ [55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81]

These effects include inhibition of epithelial cell release of proinflammatory cytokines, inhibition of eosinophil adhesion and chemotaxis, and mast cell–stabilizing properties. Most antihistamines have some stabilizing effects on mast cells, although typically not at pharmacologically relevant doses. The multiple antiallergic effects described previously are not shared by all the drugs in each category, and most of these mechanisms are still under investigation.

TABLE 88-4 -- Topical Drugs Approved for Use in Allergic Eye Disease Classification

Drug (Trade Names)

Vasoconstrictors

Tetrahydrozoline (Visine) Naphazoline (AK-Con, Albalon, Allerest, All-Clear, Antazoline-V, Naphcon, Clear Eyes, Comfort Eye Drops, Degest, Estivin II, Opcon-A, Nafazair-A, Ocu-Zoline, Vasocon, VasoClear)

Histamine1 (H1 ) receptor antagonists

Emedastine (Emadine) Levocabastine (Livostin)

Combination vasoconstrictor and H1

Antazoline (Vasocon-A) Pheniramine (Visine-A, Naphcon-A)

receptor antagonists Combination H1 receptor antagonist and mast cell stabilizers

Olopatadine (Patanol) Ketotifen (Zaditor) Azelastine (Optivar)

Mast cell stabilizers

Cromolyn (Opticrom, Crolom) Lodoxamide (Alomide) Nedocromil (Alocril) Pemirolast (Alamast)

Nonsteroidal anti-inflammatory drug (NSAID)

Ketorolac (Acular)

Corticosteroid

Loteprednol etabonate (Alrex)

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TABLE 88-5 -- Effects of Mast Cell Stabilizers and Other Drugs Drug and Classification

Inhibition of Mediator Release from Human Conjunctival Mast Cells

Inhibitory Effects on Other Cells

References

Antazoline (H1 receptor antagonist)

No effect

Inhibits IL-6, IL-8 release from conjunctival epithelial cells in vitro

[49] [50] [51] [52]

Pheniramine (H1 receptor antagonist)

No effect

Inhibits IL-6, IL-8 release from conjunctival epithelial cells in vitro

[49] [50] [53]

Emedastine (H1 receptor antagonist)

No effect

Inhibits IL-6, IL-8 release from conjunctival epithelial cells in vitro

[49] [50] [53]

Inhibits IL-6, IL-8 release, ICAM-1 expression on conjunctival epithelial cells in vitro

[49] [50] [53]

Conjunctival mast cell TNF-αmediated up-regulation of ICAM-1 on conjunctival epithelial cells in vitro

[49] [50] [52] [53] [54] [55]

Levocabastine (H1 receptor antagonist) No effect

Olopatadine (H1 receptor antagonist,

Histamine, tryptase, PGD2 , TNF α in

mast cell stabilizer)

vitro

Ketotifen (H1 receptor antagonist,

Histamine in vitro

Chemotaxis and activation of eosinophils in vitro

[49] [50] [53] [55] [57] [58]

In vitro data not available

Activation of eosinophils in vitro; eosinophils and neutrophils in tears; ICAM-1 expression in vivo

[49] [50] [51] [58] [59] [60]

Cromolyn (mast cell stabilizer)

Not inhibitory for histamine release in vitro; tryptase in tears

Chemotaxis and activation of eosinophils, neutrophils, monocytes in vitro

[49] [50] [53] [61] [62] [63] [64] [65] [66] [67]

Lodoxamide (mast cell stabilizer)

In vitro data not available; histamine and tryptase in tears

Chemotaxis and activation of eosinophils in vitro; eosinophils, neutrophils, T cells in tears; ICAM-1 expression on conjunctival epithelial cells in vitro

[49] [50] [61] [62] [68] [69] [70] [71] [72] [73]

Not inhibitory for histamine release in vitro

IgE synthesis from B cells; ICAM-1, HLA-DR expression on conjunctival epithelial cells in vitro; activation and survival of eosinophils in vitro

[49] [50] [53] [63] [64] [66] [76] [77] [78] [79]

Not inhibitory for histamine release in vitro

Activation of eosinophils and neutrophils in vitro

[49] [50] [53] [81] [82]

mast cell stabilizer) Azelastine (H1 receptor antagonist, mast cell stabilizer)

Nedocromil (mast cell stabilizer)

Pemirolast (mast cell stabilizer)

[74] [75]

[80]

HLA, Human leukocyte antigen; ICAM-1, intercellular adhesion molecule 1; IgE, immunoglobulin E; IL, interleukin; PGD2, prostaglandin D2; TNF-α, tumor necrosis factor-α. Vasoconstrictors Only two vasoconstrictor preparations are available (tetrahydrozoline and naphazoline), and they are marketed OTC under various trade names (see Table 88-4 ). These topical ocular medications reduce symptoms by constricting conjunctival blood vessels and decreasing hyperemia and chemosis. Discontinuation after prolonged or excessive use of these topical vasoconstrictors may lead to rebound hyperemia of the conjunctiva. Antihistamines Seven topical H1 receptor antagonists are currently approved for ocular use: pheniramine, antazoline, levocabastine, emedastine, azelastine, ketotifen, and olopatadine. Because H1 stimulation is believed to primarily mediate inflammation and itching, vasoconstrictors are sometimes combined with H1 antagonists. Currently pheniramine and the antazoline are available in combination with a vasoconstrictor (see Table 88-4 ). Pheniramine, antazoline, and levocabastine are older, first-generation antihistamines that are less selective H1 antagonists. The second-generation compounds (azelastine, ketotifen, emedastine, and olopatadine) antagonize H1 more selectively, so the adverse effects on the central nervous system are reduced. Some in vitro antiinflammatory effects of these drugs are shown in Table 88-5 . Mast Cell Stabilizers Mast cell stabilizers are defined by their ability to inhibit degranulation of activated mast cells. Some of these drugs also have important antiinflammatory effects on other cells, which contribute to their clinical efficacy (see Table 88-5 ). Mast cell stabilizers are most effective when instituted before or soon after the onset of clinical symptoms. Seven topical mast cell–stabilizing medications are currently approved for ocular use: olopatadine, ketotifen, azelastine, cromolyn, lodoxamide, nedocromil, and pemirolast. Their effects on human conjunctival mast cells are included in Table 88-5 . These compounds were initially characterized as mast cell stabilizers based on studies with rodent

1610

and human mast cells obtained from tissues other than the conjunctiva. Techniques for obtaining functional human conjunctival mast cells have facilitated characterization of topical ocular mast cell stabilizers with a relevant mast cell population.[

82] [83]

The concept of mast cell heterogeneity means that the response to

secretagogues and drugs will vary from species to species and may differ in different tissues of the same species. It follows that these topical mast cell stabilizers vary in their effectiveness in different ocular allergic disease states. For example, cromolyn (the earliest mast cell stabilizer) was developed for the treatment of asthma and is approved for treatment of GPC and VKC. However, clinical studies have found that cromolyn provides minimal benefit over placebo in the treatment of AC.[

84]

Lodoxamide has been very successful in the treatment of VKC.[

85]

Nedocromil has been tested primarily for the treatment of AC and has been

demonstrated to control clinical symptoms (itching, irritation) when compared with placebo.[

78] [79]

Dual-Acting Antihistamine and Mast Cell–Stabilizing Drugs Topical dual-acting ocular medications have both antihistamine and mast cell–stabilizing proprieties that are not correlated with intrinsic H1 receptor antagonism (see Tables 88-4 and 88-5 ). This group of topical ocular medications is currently the most commonly prescribed group for allergic eye disease. The primary dual-acting drugs approved for ocular use are azelastine, olopatadine, and ketotifen. Azelastine is the most recent to receive approval in the United States. Nonsteroidal Antiinflammatory Drugs and Corticosteroids Ketorolac is the only topical NSAID approved for temporary relief of ocular itching due to AC (see Table 88-4 ). Ketorolac treats ocular symptoms in part by its ability to inhibit prostaglandin and thromboxane but not leukotriene synthesis. Corticosteroids are effective for most manifestations of allergy. Only one corticosteroid topical preparation is currently approved for use in ocular allergy— loteprednol etabonate. It is important to understand the adverse effects of topical and systemic corticosteroids on ocular tissue. Topical and systemic steroids can cause cataracts, although the dose and duration of drug administration causing this effect is variable. Additionally, about 4% to 6% of randomly selected individuals respond to topical (and, rarely, systemic) corticosteroids with increased intraocular pressure. Of those individuals with a family history of glaucoma, 20% to 30% 86

respond with elevated intraocular pressure after topical corticosteroid administration.[ ] All patients receiving topical steroids should have a measurement of intraocular pressure 3 months after starting the drug and yearly thereafter. The responsibility for monitoring of the eye for topical steroid applications and complications of this administration should rest with the ophthalmologist. Treatment of Allergic Conjunctivitis AC can be debilitating to some degree and may cause affected individuals to seek any type of help for relief of symptoms. The itching and tearing may be unbearable and sleepless nights frequent. Ocular AC symptoms may be worse than the nasal symptoms of individuals with rhinoconjunctivitis. Furthermore, treatment of the nasal symptoms with topical nasal steroids may help the rhinitis but may not be effective for relieving ocular symptoms. Management of AC is, therefore, primarily aimed at alleviating symptoms. Establishing the cause is the first step in treating AC. The best treatment is avoidance of the specific allergen; however, this is sometimes not possible. Avoidance of scratching or rubbing, application of cool compresses, use of artificial tears, and refrigeration of topical ocular medications are practical interventions to alleviate discomfort. While oral antihistamines may help to relieve eye discomfort, they may also decrease tear production, causing more ocular symptoms.

The treatment of choice for mild to moderate AC is a dual–acting topical ocular medication. The mast cell– stabilizing component of these drugs benefits patients most if treatment is started before the height of symptom onset. Patients usually note rapid onset of relief of itch on drop instillation, because most dual-action medications have high H1 receptor affinity. Drug dosing varies from two to four times per day, and efficacy is judged best by symptom relief. In severe disease, combination therapy is recommended. This therapy may include topical medications (antihistamines, mast cell stabilizers, NSAIDs, or combinations) and oral antihistamines. NSAIDs inhibit cyclooxygenase, resulting in decreased formation of prostaglandins and thromboxanes, but not leukotrienes. Therefore, these compounds are useful in controlling itching and some inflammation, but not the infiltration of inflammatory cells. In extreme cases, use a topical steroid four times a day should be considered. All patients receiving topical steroids should have their intraocular pressure measured with a frequency as described earlier. Immunotherapy performed by an allergist may be beneficial in decreasing the severity of future ocular allergy symptoms. Treatment of Atopic Keratoconjunctivitis AKC is a sight-threatening ocular process. The approach to treatment of this disease is multifaceted and includes environmental controls and use of systemic and topical medications. It is unlikely that an individual with AKC will present to the ophthalmologist without additionally being under the care of an allergist. It is important to review with the patient the removal of environmental allergens in the home, employment, or school setting. The nature of the allergens may be delineated through skin testing. The discussion that follows examines the treatment of ocular symptoms and not the atopic dermatitis that is usually seen in individuals with AKC. The topical application of a vasoconstrictor-antihistamine combination may bring transient relief of ocular symptoms but is unlikely to intervene in the immunopathologic process or its sequelae. The topical administration of steroids eight times per day for 7 to 10 days is clearly beneficial in controlling symptoms and signs. These drugs must be used judiciously, since the chronic nature of the disease may encourage overuse because of the excellent response obtained. The patient must be instructed that steroid use will be transient, advised that there will be careful monitoring for efficacy, and warned of the potential for causing cataracts and glaucoma. Dual-acting

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and mast cell–stabilizing drugs are effective in reducing itching, tearing, and photophobia. Their use is recommended year-round in patients with perennial symptoms. If an exacerbation occurs and the patient is not taking one of these drugs, one should be instituted concurrently with a short burst (7 to 10 days) of topical steroids. Some investigators recommend maximizing the use of systemic antihistamines, since H1 receptors seem most responsible for the symptoms of AKC and newer 19]

antagonists are fairly specific for the H1 receptor.[

Only in rare cases of uncontrolled atopic dermatitis with sight-threatening complications are oral steroids

indicated. The role of immunotherapy is questioned in AKC for the following reasons. First, desensitization to all sensitive allergens is not feasible. Second, some

24]

suggest that, although skin and lung tissues are responsive to hyposensitization, the conjunctiva is not as responsive.[ cyclopeptide that inhibits IL-2, may be useful for its effect on T

Topical or oral cyclosporin A, a specific

87 cells.[ ]

Eyelid and ocular surface abnormalities may require treatment other than that directed toward the underlying pathology of AKC. Trichiasis (misdirected eyelashes), if contributing in any way to cornea compromise, must be corrected surgically. Staphylococcal-associated blepharitis, if present, should receive antibiotic treatment. If, despite adequate control of signs and symptoms of AKC, cornea punctuate staining persists, artificial tears should be used in an effort to avoid the development of corneal epithelial defects. These defects are extremely difficult to heal. Eyelid or ocular surface herpes simplex virus should be treated with antiviral agents (topical or systemic or both, depending on severity). If frequent recurrent episodes of epithelial herpes simplex keratitis occur, prophylaxis with oral acyclovir may be considered in an attempt to reduce recurrences. Treatment of Vernal Keratoconjunctivitis VKC is a sight-threatening ocular process and, as with any atopic condition, avoidance of an offending allergen is of paramount importance. This is often difficult for individuals with VKC due to the large number of allergens to which they react. Seasonal removal of a child from the environment or from the home to reduce allergen exposure usually is not practical. Immunotherapy has the limitations described previously for AKC. For the patient with a significant seasonal exacerbation, a short-term, high-dose pulse of topical steroid therapy under the guidance of an ophthalmologist is warranted. Dosing of the topical steroid eight times daily for 1 week usually brings relief of symptoms. This dosing regimen should be rapidly tapered to maintain patient comfort. As in any chronic ocular inflammatory disease, the risks of prolonged use of steroids are cataracts and glaucoma. Therefore, any limited use of steroids should include additional measures to sustain a decreased 88 89 90

state of inflammation. Cromolyn sodium has been shown effective in VKC.[ ] [ ] [ ] Cromolyn and the other mast cell–stabilizing and dual-acting drugs may need 1 to 2 weeks for effectiveness to begin. Cool compresses may reduce the symptom of ocular itching. Oral medications have a variable role in the treatment of VKC. For the care of severe bilateral disease (with threat to sight) oral steroids may be used, but their use alone in VKC is unusual. Maximizing the use of nonsedating oral antihistamines is often helpful. Aspirin therapy has been shown effective, but usually it requires a 91 92

93

high dose, as much as 2400•mg daily.[ ] [ ] Topical cyclosporin A has shown promise in the treatment of VKC.[ ] The release of IL-2 is diminished with cyclosporin A, thus reducing expansion of certain T cell clones. A corneal shield ulcer is a sight-threatening complication of VKC. Treatment with antibiotic and steroid ointments and occlusive therapy is required. If a plaque forms in the ulcer bed, a superficial keratectomy may be beneficial in promoting epithelial healing.[ [95]

Surgical procedures for treatment of VKC are of historical significance only. Cryoablation of the upper tarsal cobblestones is reported to give short-term improvement. However, scar formation may lead to eyelid and tear film abnormalities. The risk of these adverse permanent changes is probably not warranted, because VKC usually abates 2 to 10 years after onset of symptoms.[

24]

Treatment of Giant Papillary Conjunctivitis Reducing symptoms is the primary aim for management of GPC. A reduction in the wearing time of contacts, from a few hours a day to total abstinence, may be required. One-day use contact lenses may be a consideration for persistent cases of GPC. However, in more serious cases, a more aggressive approach may be

94]

required to prevent ocular tissue damage. OTC “artificial tears” preparations help to wash away environmental allergens and lens debris. Topical mast cell stabilizers have proved effective in the treatment of GPC.[

96] [97] [98] [99]

Dual-acting drugs may be the best therapy for chronic GPC. A patient 100]

with GPC may require continued use of these drugs once contact lens wear is resumed. Steroids have also been approved for the treatment of GPC.[ steroids may be used four times per day for 2 to 4 or lens material.

101 days.[ ]

Topical

A return to contact lens wear can usually be accomplished but may require a change in contact lens style

CONTACT DERMATITIS Contact dermatitis is a delayed inflammatory hypersensitivity reaction resulting from contact with a specific antigen or irritant (see Chapter 87 ). Although it is not an IgE antibody–mediated process, it deserves discussion in this chapter because contact dermatoconjunctivitis is a commonly observed entity in the ocular adnexa. The eye is affected in this disorder not only via direct application of substances containing irritants (often ophthalmic treatments) and specific antigens, but also through eye rubbing after manual contact with an offending irritant or antigen. Because the potential list of substances causing contact dermatitis numbers in the thousands, it is probably one of the most common skin conditions requiring medical attention. It is unclear exactly how many people suffer from the ocular component of this process, but the American Medical Association estimates that the incidence of contact dermatitis, in general, ranges from 1% to 15% of the total population in Western industrialized nations. Symptoms and signs of contact dermatoconjunctivitis can include a sudden rash over the eyelids, tearing, redness, itching, stinging and burning sensations, and a sensation of fullness in the eye or eyelid when swelling is involved. The eyelid may appear thickened, red, and sometimes ulcerated.

1612

If the conjunctiva is involved, vasodilatation, chemosis, watery discharge, and sometimes formation of papillae can be observed. Chronic inflammation may involve occlusion of lacrimal ducts, conjunctival scarring, corneal neovascularization and keratinization; however, sight loss is uncommon. A multitude of irritants and antigens have been implicated in ocular contact dermatitis. Some common substances known to sensitize individuals to ocular contact dermatitis include topical drugs and antibiotics (anesthetics, neomycin, antiviral agents, pilocarpine, timolol); preservatives in ophthalmic solutions (thiomersal, benzalkonium chloride, chlorobutanol, chlorhexidine, EDTA); cosmetics (eye and lip glosses containing waxes, fats, and dyes); perfumes; sunscreens containing paraamino benzoic acid (PABA); fingernail products (containing formaldehyde resins and sulfonamide derivatives); hair products (dyes, permanent solutions); adhesives (false eyelashes); nickel (eyelash curlers and eyeglass frames); irritant plants (poison ivy, sumac, and oak); and latex (gloves). Other irritant substances implicated in contact dermatoconjunctivitis include soaps, detergents, bleach, and solvents. As with allergic ocular processes, the diagnosis of contact dermatoconjunctivitis is predicated on physical examination and a thorough history, including specific questions concerning daily activities, medications, contact lens products, eye drops, cosmetics, occupation, and hobbies, using the above list of culprit irritants and

antigens as a guide. Several tests are used for identification of specific antigens or irritants. Patch testing is the most useful diagnostic tool for evidence of contact dermatoconjunctivitis. Patch testing is discussed in detail in other chapters of this text. The best treatment is avoidance of the offending agent or agents. Substitution of nonirritating products (e.g., contact lens solutions, cosmetics) should be attempted whenever possible. Comfort measures that can be taken include cool compresses four to six times per day, avoidance of hot water and soaps, and application of a mild steroid cream over the affected area. Topical antihistamines and steroid drops may be indicated. Systemic antiallergic agents may be helpful, but could also lead to further discomfort from the dry eyes they may produce.

IMMUNOLOGIC DISEASES OF THE EYE It is beyond the scope of this chapter to approach this broad topic by discussing the ophthalmic manifestations of all immune-mediated diseases. Rather, in this section, diseases localized to the eye for which there is evidence of immune mediation and that have importance to the reader are discussed. The diseases are classified by anatomic location, and treatments are discussed with each entity. The disease states, symptoms and signs, and treatments are summarized in Table 88-6 . Ocular Cicatricial Pemphigoid Systemic pemphigoid may have its most devastating consequences in the eye. The conjunctival component of OCP is characterized by chronic inflammation, scar formation with fornix shortening, and eventual eyelid and eyelash inturning. The changes seen in OCP are sight-threatening (see Table 88-6 ). Patients affected by OCP are usually in the sixth or seventh decade of life. Pemphigoid may begin as early as the third decade of life. There is a moderate to strong 102

female predominance but no racial or geographic predilection.[ ] Presentation is usually as a chronic conjunctivitis with remitting and relapsing symptoms. The patient generally complains of irritation, burning, tearing, and increased mucus production found as strings in the fornix. The initial ocular sign of OCP may be moderate to severe conjunctival redness. The conjunctiva may then become thickened secondary to cellular infiltration and edema fluid, and the epithelium will show areas of patchy mucin or cell loss. Subepithelial fibrosis may progress to cause shrinkage and shortening of the depth of the normal fornix. When this occurs in the superior fornix, the small ductules from the main lacrimal gland may be scarred shut. Eventually, these changes lead to a malposition of the eyelid margin; the eyelashes become inturned and directed toward the conjunctival and corneal surfaces. This contact, along with the contact of the keratinized eyelid margin, leads to a mechanical breakdown of the epithelial surfaces of these structures. Decreased aqueous tear production and a loss of goblet and mucinproducing cells in the epithelium lead to further epithelial changes, and the risk of infection in the cornea is increased. Finally, the conjunctival epithelium may grow beyond the limbus onto the cornea and form a vascularized pannus. The diagnosis of OCP requires a conjunctival biopsy. Light microscopic and immunohistochemical staining of biopsy specimens not only forms the basis for diagnosis but also has given insight into the immunopathophysiology of OCP. The inflammatory cell infiltrate in the substantia propria varies, but both CD4+ and 103

CD8+ T cell numbers are increased by 15- to twentyfold over the number found in controls. [ ] Mast cell and fibroblast numbers are also increased. Along with this cellular infiltration, deposition of collagen is prominent; complement and antibodies are found at the basement membrane zone. The treatment of OCP is systemic and should be a collaboration between medical and ophthalmology physicians. Determination of other sites of involvement must

be performed before any treatment is initiated. Careful attention to symptoms and signs of skin, nose, oral mucosa, esophagus, trachea, gut, anus, and urethral involvement must be sought. The goal of therapy in the ocular tissue should include abolition of all conjunctival redness in the absence of mechanical aggravating factors. Mild early disease may respond to dapsone. Severe ocular disease probably will require oral steroid and cytotoxic therapy. Careful photographic documentation evaluating the extent of redness and scarring is useful to assist in monitoring therapy. Peripheral Ulcerative Keratitis This focal inflammatory reaction may involve to varying degrees any limbal structure. Recall that the lumbus is the anatomic junction of the sclera and cornea (see Figure 88-1 ). Superficially it is covered in part by conjunctiva and in part by peripheral corneal epithelium. The blood vessels supplying the limbus convey to the cornea both cellular elements (chemotactically driven) and serum elements (by diffusion gradients). There are normally higher concentrations of antibody and complement components in the peripheral cornea than in the central cornea. The peripheral infiltrates associated with staphylococcal blepharitis are a good example of non–sight-threatening

1613

TABLE 88-6 -- Immunologic Diseases of the Eye Disease

Clinical Parameters

Signs/Symptoms

Treatment

Ocular cicatricial pemphigoid

Systemic pemphigoid Females predominate Peak incidence 60–70 years of age

Severe conjunctival redness Chronic conjunctivitis Mucous discharge Conjunctival scaring Inturning eyelids and eyelashes Breakdown of corneal and conjunctival epithelium

Mild: dapsone Severe: systemic steroids, immunosuppressive drugs

Sight-threatening

Peripheral ulcerative keratitis

Mild: Both females and males Any age Example-associated with staphylococcal blepharitis Necrotizing (Mooren's ulcer): Both females and males Adults Involvement unilateral (world wide) or bilateral (African males) History of infectious ocular disease History of rheumatic disease

Mild: Pain Photophobia Tearing Redness Peripheral infiltrate on ocular examination Not sight-threatening Necrotizing: Pain Photophobia Tearing Redness Perilimbal ulceration

Mild: Topical steroids and antibiotics Necrotizing: Topical steroids Systemic steroids and other immunosuppressive drugs Surgical intervention

Sight-threatening Episcleritis

Females predominate Peak incidence 40 years of age

Conjunctival redness Diffuse or nodular Minimal pain Not sight-threatening

Cool compress Treat associated blepharitis Artificial tears Topical NSAIDs/steroids

Scleritis

Females predominate Peak incidence 40 years of age, rare in children Anterior: diffuse, nodular, necrotizing Posterior: associated with systemic disease, rarely with ocular infection (40%)

Anterior: Deep, boring pain Diffuse ocular redness Raised nodules Scleral ulceration Necrotizing: Sight-threatening Posterior: Pain with eye movement

Topical steroids ineffective Periocular steroid injection acceptable Oral NSAIDs, systemic steroids, immunosuppressive drugs

Sight-threatening

Uveitis

Associated with systemic and infectious disease (40%) All ages Occurrence: anterior, 70%; intermediate, 20%; posterior, 10%

Anterior: Redness Pain Photophobia Anterior chamber cells and fibrin Pupil miotic Intermediate: Bilateral involvement Mild hazy/blurry vision Posterior: Photophobia Pain

Anterior: Topical steroids Intermediate: Periocular steroids Systemic steroids Posterior: Systemic steroids Immunosuppressive drugs

Sight-threatening NSAIDs, Nonsteroidal antiinflammatory drugs.

1614

peripheral ulcerative keratitis (PUK). Patients with this process can be females or males of any age. Affected individuals present with a few days of unilateral, progressively worsening ocular pain, photophobia, and tearing. Findings include small anterior stromal, oval, yellow-white infiltrates that are separated from the limbus by about 1•mm of clear cornea. Ulceration of the overlying epithelium where these infiltrates occur is often found. Although they are not definitively substantiated, the proposed immune pathophysiologic mechanisms for this process center on the formation of immune complexes, complement activation, and neutrophil recruitment. The location of the ocular infiltrates in PUK represents the area of optimal concentrations of antibody and staphylococcal antigen leading to formation of immune complexes and the immune inflammatory process. Treatment of this type of PUK requires use of antibiotics to reduce the associated staphylococcal blepharitis and the application of mild topical steroids. Mooren's ulcer is an idiopathic vision-threatening form of PUK (see Table 88-6 ). It manifests with pain, redness, photophobia, and tearing. Two types of this process have been identified: unilateral (occurring worldwide) and bilateral (most commonly occurring in males of African descent). This necrotizing process begins at the limbus and progresses circumferentially and centripetally with destruction of the peripheral cornea and conjunctival membrane overlying the sclera. This destructive process may result in perforation. Diagnosis requires exclusion of local infectious diseases such as herpes simplex virus infection, systemic infectious diseases such as syphilis and hepatitis C, local noninfectious diseases such as rosacea blepharitis, and systemic collagen vascular diseases such as rheumatoid arthritis. Although not definitively established, biopsy evidence suggests an immunopathophysiologic process based on immune complex deposition and possibly an 104

autoantibody against a cornea-specific antigen.[ ] Treatment generally requires local temporizing measures (topical steroids) while cultures and systemic evaluation are ongoing. If the diagnosis of Mooren's ulcer is made, conjunctival resection may serve to remove the portion of the membrane that is diseased and perpetuating the inflammatory reaction. Thin areas or small perforations may be stabilized with the application of tissue adhesives such as cyanoacrylate glue. Subsequent long-term

strategies include tissue-addition surgical procedures and immunosuppression. Bilateral disease tends to be more relentless and is perhaps more amenable to immunosuppression.[

105]

Episcleritis and Scleritis These inflammatory disorders of the sclera (see Table 88-6 ) may be differentiated by their clinical presentation, pathophysiology, and course. Episcleritis is a benign inflammatory process of the superficial episcleral and conjunctival vascular plexuses. The inflammation may be diffuse (simple episcleritis) or focal (nodular 106

episcleritis). Episcleritis typically occurs in females, with a peak incidence in the fourth decade of life.[ ] It is characterized by the rapid onset (hours to a few days) of conjunctival redness of varying intensity. Focal or nodular episcleritis is movable over the underlying sclera. There is no true scleral inflammation in the episcleral process. The differential diagnosis of episcleritis includes conjunc-tivitis (e.g., infective, allergic), scleritis, and foreign body. Scleritis is differentiated from episcleritis by the symptom of severe pain in the former condition (often described as deep and boring). Additionally, all redness of episcleral origin will blanch within 20 minutes after application of a single drop of 10% phenylephrine. True scleritis displays residual redness despite phenylephrine administration. Treatment of episcleritis includes patient counseling regarding the benign nature of the inflammation, the possibility of persistence for weeks to months, and the possibility of recurrence in the same or the opposite eye, even with treatment. Cool compresses, treatment of any associated blepharitis, artificial tear preparations, topical NSAIDs, and steroid drops have all been reported to be effective in relieving symptoms and redness. Although steroid drops are effective for relief of symptoms, the potential for development of cataract and glaucoma with prolonged use must be monitored. Scleritis is a sight-threatening true inflammation of the sclera. Although 40% of patients with this process have an associated systemic disease (e.g., rheumatoid 107 108

arthritis), scleritis is discussed here because of the high likelihood that it will be idiopathic.[ ] [ ] The inflammatory process of scleritis may be focused in the anterior or the posterior portion of the eye. Anterior scleritis (the most common form) can be sub-divided into diffuse, nodular, and necrotizing types. Posterior scleritis may involve the retina and optic nerve. Scleritis tends to occur in women more than men, is rare in children, has its peak incidence in the fourth decade of life, and has no racial or geographic predisposition. Infectious causes of scleritis are rare, and when present they follow trauma, herpes zoster, or extension from a severe infectious corneal ulcer. Redness of the eye (usually of all the eye visible between the open lids) and a deep, boring pain that may be so severe as to compromise sleep are the key findings in diffuse anterior disease. Tearing and photophobia are often present but vary widely in their severity; visual acuity is usually good. The patient with anterior nodular scleritis tends to have redness focally in the area of involvement. Redness (diffuse or focal) that surrounds an area of whiteness is typical of necrotizing scleritis. The white central area often appears excavated and may have a bluish tint if the sclera is quite thin. Brown pigment at the base of the excavated white area suggests fullthickness scleral loss with baring of the underlying choroidal layer. Pain with eye movement and a complaint of decreased vision may accompany posterior scleritis. Examination of the inside of the eye may reveal focal or diffuse areas of retinal and choroidal edema secondary to the scleral swelling. Despite the variable causes of scleritis, the pathophysiology of the process tends to be remarkably similar. Granulomatous inflammation and collagen disruption are the main pathologic findings. These pathologic findings may lead to the massive scleral edema or thinning of the sclera if loss of collagen predominates. The prognosis and treatment of scleritis depends greatly on the presenting type. Diffuse anterior scleritis is least likely to recur or to cause long-term loss of vision. Nodular anterior scleritis is intermediate in its likelihood for recurrence and severity, and vision is usually maintained. Necrotizing anterior scleritis tends to be the

most sight-threatening type due to its tissue-destructive nature. The vision decline in posterior scleritis is usually reversible with treatment. Treatment of any form of scleritis with steroid drops is considered ineffective. Regional injection of steroids until

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recently was considered contraindicated. Two case series published in the last seven years have demonstrated the efficacy of this approach, with only increased 109 110

intraocular pressure as a potential significant side effect.[ ] [ ] The mainstay of treatment for all types of scleritis has typically been oral NSAIDs, steroids, or immunosuppressive medications (in that order of approach). Necrotizing scleritis with inflammation poses more of a threat to vision and is most likely to be associated with a systemic disease. The onset of necrotizing scleritis in a patient with a known systemic disease is a sign of a worsening systemic process. Immunosuppressive therapy should be strongly considered for ocular necrotizing scleritis, or for ocular disease associated with active systemic disease. Studies suggest an increased mortality rate in these patients if they do not receive immunosuppressive therapy.[ destructive scleral thinning are objective findings indicating efficacy of therapy.

107]

Reduction of erythema and pain and halting of the

Uveitis Uveitis is a broad term that refers to any intraocular inflammatory process. It is most commonly classified according to the anatomic location of the inflammatory process in the eye (see Table 88-6 ). Anterior uveitis refers to inflammation particularly found in front of the lens. Intermediate uveitis refers to those entities in which the inflammation is centered in the middle portion of the eye (i.e., just posterior to the lens), including the pars plana or posterior portion of the ciliary body and the peripheral retina. Posterior uveitis typically refers to any entity involving (in a focal or diffuse manner) the retina, choroid, or vasculature. Anterior uveitis accounts for about 70%, intermediate uveitis 20%, and posterior uveitis 5% to 10% of all patients with this ocular inflammatory process. An associated systemic disease or infection is found in about 40% of patients with clinical uveitis. The signs and symptoms of uveitis vary with the anatomic location. The most common signs and symptoms in anterior uveitis are the abrupt onset of pain, redness, and light sensitivity or photophobia. These symptoms correspond to inflammation found in the iris and anterior portion of the ciliary body. Redness of the ocular surface is a secondary response to the inflammation in this ocular location. Inflammation in the anterior chamber (see Figure 88-1 ), as seen with a slit-lamp biomicroscope, consists of white blood cells free floating in the aqueous humor. In severe cases, there is a significant fibrinous outpouring within the anterior chamber. This can be so severe as to cause adhesion of the pupil margin to the anterior surface of the normal crystalline lens of the eye. The involved pupil is typically miotic. This is caused by prostaglandin release within the anterior chamber. Inflam-mation in anterior uveitis can drive the formation of cataracts; therefore, lens changes may be found. Patients with intermediate uveitis tends to present with bilateral symptoms of smoky, hazy vision or with the development of diffuse, weblike floaters. Typically, vision is not severely affected by this inflammatory process. Uveitis in this location tends not involve redness of the ocular surface. The anterior chamber may have a cellular infiltrate, but it typically is less than in anterior uveitis, and in general inflammation in the anterior chamber is considered a spillover from the main process posterior to the lens. The pars plana region or posterior aspect of the ciliary body has inflammatory exudates. When inflammation is inactive, there tends to be a

lessening of the height and appearance of the pars plana exudate. Although the greatest concentration of vitreous inflammatory cells is near the pars plana region, there are cells throughout the entire vitreous. This leads to a hazy appearance as the examiner views the posterior retinal structures. Posterior uveitis may manifest with symptoms of redness, pain, light sensitivity, floaters, and, most importantly, profound vision loss. Inflammation in this anatomic location has a large number of different presentations. A posterior uveitis may be limited to a significant vasculitis throughout the retina. A number of infectious entities can also cause retinal inflammation, including cytomegalovirus, herpes simplex, herpes zoster, and toxoplasmosis. Choroidal inflammation in posterior uveitis may be focal or diffuse and may be infectious or noninfectious. Choroiditis may present in a dramatic fashion with numerous white subretinal spots diffusely spread throughout the choroid, or with a single area of granuloma formation. The pathophysiology of uveitis is complex. Approximately 40% of all uveitis is associated with an infectious etiology or systemic disease (e.g., rheumatic diseases). However, 60% of those individuals presenting with uveitis have idiopathic disease. As with other autoimmune disorders, various proposals for the immunopathophysiologic etiology of idiopathic uveitis have been offered. Immune complexes, antinuclear antibodies, and a cell-mediated immune defect have been implicated in a variety of uveitis syndromes. Animal models of intraocular inflammation have also been developed. In these models, immunization of animals with various extracts of ocular tissue from the anterior and posterior portion of the eye have been used. Inflammatory changes in the eye, some consistent with the human manifestations of uveitis, have been demonstrated. An example of the value of this animal model work can be seen with the earliest use of cyclosporine in the treatment of recalcitrant posterior uveitis. Experiments performed in the animal model demonstrated that immunization with a protein isolated from the outer portion of the retina led to significant choroidal and retinal inflammation. Cyclosporin was able to dampen this inflammatory response. [

111] [112]

The diagnosis of uveitis is based on an appropriate clinical history and physical findings. Whereas the presence of intraocular inflammation is often not difficult to determine, finding a systemic process associated with the uveitis is often problematic (60% of uveitis is idiopathic). Historically, individuals with anterior uveitis tend to have recurrence in the same or the opposite eye with similar symptoms and signs. Individuals with intermediate uveitis tend to have a bilateral presentation and to have flareups and recrudescence of inflammation. Importantly, posterior uveitis tends to produce changes in vision. The treatment of uveitis is based on the diagnosis of the ocular process. Infectious processes are treated with antibiotic or antiviral agents. The use of systemic steroids is important for other forms of uveitis. The topical application of steroid drops is effective for anterior uveitis and is usually all that is necessary to bring episodes under control. The penetration of steroid drops is not sufficient to affect that portion of the eye that is most inflamed in intermediate uveitis. Periocular injection of steroids gives excellent relief from

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symptoms and signs of inflammation in this ocular location. Bilateral intermediate uveitis with 20/40 or worse vision in both eyes is generally an indication for the use of oral steroids. Unilateral posterior uveitis of a noninfectious cause may respond well to periocular injection of steroids. Bilateral posterior noninfectious uveitis with 20/40 or worse vision tends to require oral steroids. Posterior uveitis that is bilateral and sight-threatening may respond to oral steroids, but recrudescence tends to occur as the steroids are withdrawn to below a threshold dose. Immunosuppressive agents should be used in posterior uveitis that is vision-threatening and is either unresponsive to oral steroids or requires steroid sparing agents.

SUMMARY The eye is a unique organ because of its varied tissue types, accessibility to direct examination, and observable vascular supply. Although many systemic disorders have ocular manifestations, work on the mechanisms and treatment of specific allergic eye disease is proceeding at a rapid pace. The rapidity of this progress is based on the advantages of working with the eye. Ocular allergy includes clinical disorders that involve different levels of activity of the immune response at the conjunctival interface. In AC, there are minimal changes such as an increase in mast cell activation, minimal presence of migratory inflammatory cells, and early signs of cellular activation. In the more chronic forms, AKC and VKC, there is persistent mast cell, eosinophil, and lymphocyte activation; switching from connective tissue (MCT ) to mucosal-type (MCTC ) mast cells; increased involvement of corneal pathology; and follicular development and fibrosis. The recent development of techniques to isolate and purify conjunctival mast cells should lead to a better understanding of the differences among AC, AKC, and VKC by allowing characterization of cytokine and chemokine profiles, surface receptor expression, and patterns of up-regulation. It will also facilitate examination of cell-to-cell interactions that are critical to the progression of chronic and sight-threatening ocular diseases. Immunologic disorders of the eye are less well defined than ocular allergic processes. The wide variety of immunologic techniques applied to allergic eye disease, however, may now be applied to these disorders. This offers great promise for clinical advances in the diagnosis and treatment of immunologic-based ocular processes.

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104. Sangwan VS, Zafirakis P, Foster CS: Mooren's ulcer: current concepts in management, Indian J Ophthalmol 45:7–17, 1997. 105. Foster CS: Systemic immunosuppressive therapy for progressive bilateral Mooren's ulcer, Ophthalmology 92:1436–1439, 1985. 106. Watson PG, Hayreh SS: Scleritis and episcleritis, Br J Ophthalmol 60: 163–191, 1976. 107. Foster CS, Forstot SL, Wilson LA: Mortality rate in rheumatoid arthritis patients developing necrotizing scleritis or peripheral ulcerative keratitis: effects of systemic immunosuppression. Ophthalmology 91:1253–1263, 1984. 108. Jabs DA, Mudun A, Dunn JP, Marsh MJ: Episcleritis and scleritis: clinical features and treatment results, Am J Ophthalmol 130:469–476, 2000. 109. Croasdale CR, Brightbill FS: Subconjunctival corticosteroid injections for nonnecrotizing anterior scleritis, Arch Ophthalmol 117:966–968, 1999. 110. Tu EY, Culbertson WW, Pflugfelder SC, et al: Therapy of nonnecrotizing anterior scleritis with subconjunctival corticosteroid injection. Ophthalmology 102: 718–724, 1995. 111. Nussenblatt RB, Scher I: Effects of cyclosporine on T-cell subsets in experimental autoimmune uveitis, Invest Ophthalmol Vis Sci 26:10–14, 1985. 112. de Smet MD, Nussenblatt RB: Clinical use of cyclosporine in ocular disease, Int Ophthalmol Clin 33:31–45, 1993.

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1619

Chapter 89 - Adverse Reactions to Foods

Hugh A. Sampson

BACKGROUND AND DEFINITIONS

Historical Background Hippocrates recorded the first report of an adverse food reaction (milk) more than 2000 years ago, and accounts from ancient Rome indicate that the Romans were aware that foods consumed safely by most people could occasionally provoke adverse reactions in others. It was not until 1921 that the first significant advance was made in our understanding of these disorders. Prausnitz and Kustner demonstrated that the substance responsible for Kustner's “allergic” reaction to fish was present 1

in his blood serum and could be transferred to a nonsensitive individual.[ ] After injecting a small amount of Kustner's serum into the skin of his forearm, Prausnitz developed a wheal-and-flare at the passively sensitized site following the ingestion of fish. In 1950, Loveless reported the first blinded, placebo-controlled food trials, 2

and challenged the accuracy of diagnoses based on patients' historical reporting. In one study, eight patients were investigated for milk allergy[ ] and in another, 25 3

4

patients were evaluated for cornstarch sensitivity.[ ] About a decade later, Goldman reported his evaluation of 89 children with suspected milk allergy.[ ] In this study, the diagnosis of food allergy was considered established only when withdrawal of milk from the diet led to complete resolution of symptoms and three successive challenges with milk each duplicated presenting symptoms. In 1976, May published an article on the use of double-blind, placebo-controlled oral food 5

challenges (DBPCFC) to diagnose food allergy,[ ] ushering in the current era of scientific investigation into food allergic disorders. Definitions Any abnormal reaction resulting from the ingestion of a food is considered an adverse food reaction. Such reactions may be the result of food intolerances, adverse 6

physiologic reactions or food hypersensitivities (allergy), or adverse immunologic reactions.[ ] Food intolerances are believed to comprise the majority of adverse reactions to foods and may be due to factors inherent in food ingested, such as toxic contaminants (e.g., histamine in scombroid fish poisoning or toxins secreted by Salmonella, Shigella, and Campylobacter), pharmacologic properties of the food (e.g., caffeine in coffee and tyramine in aged cheeses), characteristics of the host such as metabolic disorders (e.g., lactase deficiency), or idiosyncratic responses of the host. Food aversions may mimic adverse food reactions, but typically cannot be reproduced when the patient ingests the food in a blinded fashion. Food hypersensitivities are most frequent in young children and account for the majority of wellcharacterized food allergic disorders, although a number of non-IgE mediated immune reactions, especially in the gastrointestinal tract, have been delineated.

PREVALENCE OF FOOD HYPERSENSITIVITY Food allergies are most common in the first few years of life. In a prospective study of 480 consecutive newborns followed up through their third birthday, Bock 7

found that about 8% of the cohort was food allergic.[ ] Prospective studies from a number of countries indicate that about 2.5% of newborn infants experience 7] [8] [9] [10]

hypersensitivity reactions to cow's milk in the first year of life.[

IgE-mediated reactions account for about 60% of these milk allergic reactions. Most 8

infants with non–IgE-mediated cow's milk allergy “outgrow” their sensitivity by the third year of life,[ ] but about 25% of infants with IgE-mediated cow's milk allergy retain their sensitivity into the second decade and about 35% develop allergic reactions to other foods.[ 12 children,[ ]

13 Kingdom[ ]

11]

Hypersensitivity reactions to egg occur in about

14 States.[ ]

1.6% of young and to peanut in 0.5% of children in the United and the United Children with atopic disorders tend to have a higher prevalence of food allergy. About 35% of children with moderate to severe atopic dermatitis have IgE-mediated food allergy, many of whom have skin symptoms provoked by ingestion of the food allergen. [ [16]

15]

About 6% of asthmatic children attending a general pulmonary clinic reportedly have food-induced 17

wheezing. Adverse reactions to food additives also have been demonstrated to affect 0.5% to 1% of children. [ ] There is some evidence to suggest that certain food allergies, such as peanut allergy, have been increasing over the past two decades. In a cohort of American children referred for evaluation of atopic dermatitis

18

between 1990 and 1994, allergic reactivity to peanuts nearly doubled compared to a similar referral group evaluated from 1980 to 1984.[ ] Similarly, in a populationbased study of 3-year-olds on the Isle of Wight, sensitization to peanut increased from 1.1% to 3.3% and reactivity to peanut from 0.5% to approximately 1% from 19]

1994 to 1996.[

The prevalence of food hypersensitivity in adults appears to be less common. A survey in the United States indicated that

1620

peanut and tree nut allergy together affect 1.1% of American adults.[ food

20 reactions,[ ]

14]

A survey from the United Kingdom indicated that 1.4% to 1.8% of adults experience adverse

and 0.01% to 0.23% of adults are affected by adverse reactions to food additives.[ 22 reactions.[ ]

21]

Similarly, a study in the Netherlands concluded that about 2%

of the adult Dutch population is affected by adverse food Given the estimated frequency of shellfish allergy (approximately 0.5%)[ to a variety of other foods, it is likely that about 2% of the adult population in the United States is affected by food allergies.

23]

and sensitivities

ANTIGEN HANDLING BY THE GASTROINTESTINAL TRACT Mucosal Barrier The main function of the gastrointestinal tract is to process ingested food into a form that can be absorbed and used for energy and cell growth. This requires that the intestinal immune system be capable of discriminating between harmful and harmless foreign proteins. During this process, both nonimmunologic and immunologic mechanisms operate to block harmful foreign antigens (bacteria, viruses, parasites, food proteins) from entering the interior of the body, thus forming the gastrointestinal “mucosal barrier.” As depicted in Box 89-1 , a variety of immunologic and nonimmunologic factors may destroy or block antigens from entering the body. However, developmental immaturity of these mechanisms in infants reduces the efficiency of the infant mucosal barrier and likely plays a major role in the increased prevalence of gastrointestinal infections and food allergy seen in the first few years of life. Basal acid output is relatively low during the first month of life, [24]

intestinal proteolytic activity is not mature until about 2 years of age,[

antigen binding and transport through mucosal epithelial

26 cells.[ ]

25]

and intestinal microvillous membranes are immature in infants, resulting in altered

In addition, the

Box 89-1. Gastrointestinal Barriers to Ingested Food Antigens Immunologic barriers Block penetration of ingested antigens: Antigen-specific s-IgA in gut lumen Clear antigens penetrating GI barrier: Serum antigen-specific IgA and IgG Reticuloendothelial system Physiologic barriers Breakdown of ingested antigens: Gastric acid and pepsins Pancreatic enzymes Intestinal enzymes Intestinal epithelial cell lysozyme activity Block penetration of ingested antigens: Intestinal mucus coat (glycocalyx) Intestinal microvillus membrane composition Intestinal peristalsis

27

newborn lacks IgA and IgM in exocrine secretions and salivary secretory IgA (s-IgA) is absent at birth and remains low during the early months of life.[ ] The relatively low concentrations of s-IgA in the young infant's intestine and the relatively large quantities of ingested proteins place a tremendous burden on the immature gut-associated immune system. Not surprisingly, the early introduction of numerous food antigens has been shown to stimulate excessive production of IgE 28 29

antibodies[ ] [ ] or other adverse immune responses in genetically predisposed infants. A prospective study of more than 1200 unselected infants demonstrated a direct linear relationship between the number of solid foods introduced into the diet by 4 months of age and the subsequent development of atopic dermatitis, with a

threefold increase in recurrent eczema at 10 years of age in infants who had received 4 or more solid foods before 4 months of age.[

30]

The gut-associated lymphoid tissue (GALT) must mount a rapid and potent response against potentially harmful foreign substances and pathogenic organisms, but must remain unresponsive to enormous quantities of nutrient antigens and about 1014 commensal organisms forming the normal gut flora. GALT comprises four distinct lymphoid compartments: Peyer's patches (PPs) and the appendix (aggregates of lymphoid follicles throughout the intestinal mucosa), lamina propria 31

lymphocytes and plasma cells, intraepithelial lymphocytes interdigitated between enterocytes, and mesenteric lymph nodes.[ ] PPs are located primarily in the distal small intestine, where specialized epithelial cells, M cells, overlie PPs. M cells express a number of receptors (lectinlike molecules), which serve to sample antigens (foreign proteins) from the gut lumen, especially large particulate antigens and a number of bacteria and viruses. Antigens can pass through the M cell intact where they are taken up by macrophages underlying the dome formed by the M cells. Macrophages then carry the antigens to resident precursor T cells and B cells within the PPs, where an active immune response can be generated. Although antibodies of all immunoglobulin classes can be produced following oral administration of antigen, IgM+ B cells primarily are switched to IgA-B cell precursors, exit PPs through intestinal lymphatics, and “home” to the lymphoid compartments of the gastrointestinal tract and mammary glands. s-IgA antibodies, a dimeric form of IgA that is found in intestinal secretions, do not activate complement or bind to Fc receptors, and therefore do not induce inflammatory responses. s-IgA antibodies are directed against bacterial or viral surface molecules that can prevent their binding to the epithelium or agglutinate bacteria and viruses, resulting in a complex that becomes trapped in the mucous barrier and passes out in the stool.[

32]

Recent studies indicate that B cells in the 33]

lamina propria can undergo isotype switching in the absence of T cells and produce IgA, which is directed mainly against commensal flora in the gut.[ clear invading organisms through phagocytic cells that express Fcα receptors,

[34]

IgA can

or by transporting antigen–s-IgA immune complexes across the epithelium in

35

association with the polymeric Ig receptor in the distal ileum.[ ] Complexes then travel via the portal vein to the liver sinusoids where they are taken up by Kuppfer cells, which destroy the microorganism and release free s-IgA. Bile duct epithelial cells express receptors on their basal surface, which actively take up free s-IgA and transport it into the bile duct for recirculation in the gut lumen.

1621

Antigen Penetration of the Gastrointestinal Barrier Despite the evolution of this elegant multitiered barrier system, about 2% of ingested food antigens are absorbed and transported throughout the body in an “immunologically” intact form, even through the mature gut.[

36]

Prausnitz and Kustner were the first to demonstrate how rapidly food antigens were absorbed and

1

transported to mast cells in the skin.[ ] A few years later, Freeman reported similar findings in the nose when he injected serum from an egg allergic patient into the middle turbinate on one side of his nose. [ after ingesting an egg.

37]

Nasal stuffiness and pruritus that lasted for hours developed on the passively sensitized side of the patient's nose shortly

In a classic series of experiments, Walzer and colleagues used sera from egg and fish allergic patients to passively sensitize volunteers.[

38] [39] [40] [41]

Volunteers

39 40

were given intradermal injections of 0.05•ml of patient and control sera. Approximately 24 hours later, they were fed fish or eggs.[ ] [ ] A wheal-and-flare reaction developed at the sensitized site within several minutes to 1 hour after ingestion of the relevant antigen in more than 90% of subjects, but no reaction occurred at the control site. Using a similar sensitizing protocol and introducing food in specific locations along the gastrointestinal tract, it was demonstrated that food antigens 41

were most readily absorbed from the small intestine, colon, and rectum and somewhat more slowly from the esophagus and stomach.[ ] Several factors were shown to decrease antigen absorption, including increased stomach acidity and the presence of other food in the gut, whereas increased absorption resulted from decreased stomach acidity and the ingestion of alcohol.[

42]

The rapidity with which food allergic reactions can occur following allergen ingestion has perplexed many investigators. However, recent studies evaluating the regulatory role of intestinal epithelial cells (IECs) in animal models provide a probable explanation for this phenomenon. Studies in sensitized rats demonstrated that intestinal antigen transport proceeds in two phases.[

43]

In the first phase, transepithelial transport occurs via endosomes, is antigen-specific and mast cell-

independent, and occurs 10 times faster in sensitized rats than in nonsensitized control subjects.[

44]

Antigen-specific IgE antibodies bound to the mucosal surface of

45 entry.[ ]

IECs via FcepsilonRII were shown to account for this accelerated allergen In the second phase, paracellular transport predominates. Allergenic proteins entering during the accelerated first phase activate local mast cells, which release cytokines capable of “loosening” intercellular tight junctions. Consequently, the sensitized host has larger amounts of allergen cross the “gastrointestinal barrier” much more rapidly than in the nonsensitized host. The appearance of the gastrointestinal tract surface following the ingestion of a food allergen was first investigated by passively sensitizing rectal, colonic, and ileal mucosa in normal volunteers and patients with ileostomies and colostomies. Ingestion of the responsible food allergen initially provoked pallor at the sensitized site followed rapidly by edema, hyperemia, marked mucus secretion, and occasionally petechiae and bleeding.[

46]

Subsequent studies were carried out in food allergic

patients by instilling small amounts of food allergen in the stomach under endoscopic observation, first with rigid endoscopes[

47]

and more recently with flexible

48 biopsy.[ ]

endoscopes followed by The physical findings described in passively sensitized patients were confirmed, and histochemical staining of biopsy samples demonstrated degranulation of mast cells following food challenge. Oral Tolerance Induction 39 40

As noted earlier, intact food antigens penetrate the gastrointestinal tract and enter the circulation in both normal children and adults.[ ] [ ] However, these intact proteins do not normally cause clinical symptoms because most individuals develop tolerance to ingested antigens. In mucosal tissues, soluble antigens, such as foods and inhaled antigens, are usually poor immunogens, inducing a state of unresponsiveness known as oral tolerance. Oral tolerance is defined as the specific immunologic unresponsiveness to antigens induced by their prior feeding. Unresponsiveness of T cells to ingested food proteins may be the result of three different mechanisms: T cell deletion, T cell anergy, or induction of regulatory T cells. Clonal deletion of antigen-specific T cells by apoptosis has been demonstrated in experimental animal models fed very large (probably nonphysiologic) doses of antigen.[

49]

Even though deletion may contribute to oral tolerance to food proteins, it is not believed to play a significant role in humans. Some recent studies suggest 50

that T cell anergy may be one of the major mechanisms by which oral tolerance is induced in humans. [ ] T cell anergy is characterized by reduced proliferation following stimulation with antigen and functional antigen-presenting cells (APCs). The presentation of antigen in the presence of appropriate co-stimulation by APCs leads to priming and the development of an active immune response. However, when antigen is presented to T cells in the absence of co-stimulatory factors, effector

31

function fails to develop and tolerance ensues. In the gastrointestinal tract, IECs are believed to play a major role in tolerance induction to food antigens.[ ] IECs act as nonprofessional APCs, which present antigen to T cells via MHC class II molecules but lack the appropriate co-stimulatory molecules to prime T cells efficiently. In addition, dendritic cells residing within the noninflammatory environment of PPs express interleukin (IL)-10 and IL-4, which favor generation of tolerance. Lastly, the induction of regulatory T cells can mediate bystander suppression to fed antigen by the production of inhibitory cytokines, such as T cell growth factor (TGF)-β, 49

IL-4, and IL-10. T cells designated as Th3 and Tr-1 cells are potent sources of TGF-β. [ ] These cells are generated in mucosal lymphoid tissue in response to lowdose antigen and mediate “bystander tolerance” within the gastrointestinal tract by inhibiting activation of surrounding lymphocytes. In a similar way, cognate interaction between tolerized T cells and APCs may alter APCs to promote tolerance in subsequently encountered naive T cells. The Notch-Serrate receptor-ligand 51]

pair has been suggested as the possible effector of this cognate tolerance induction.[

It also appears that the gut flora plays a significant role in the induction of oral tolerance in that animals raised in a germ-free environment from birth fail to develop 52

normal tolerance.[ ] The generation of regulatory T cells appears to require a threshold level of inflammatory response to bacterial challenge and it has been suggested that a lack of appropriate early inflammatory challenge may prevent the normal generation of TGF-β–producing lymphocytes, leading to specific impairment of oral tolerance to low-dose antigen exposure.[

53]

Several prospective studies have indicated that exclusive breast-feeding may promote the development of oral tolerance

1622

54 55

and prevent some food allergy and atopic dermatitis[ ] [ ] The protective effect of breast-feeding may be due to several factors: decreased exposure to foreign proteins, breast milk s-IgA that provides passive protection against foreign proteins and pathogens, and soluble factors in breast milk, which may induce earlier maturation of the gut barrier and the infant's immune response. The resistance of s-IgA to proteolytic digestion and the decreased proteolytic activity in the infant gut allows s-IgA antibodies to reach sites in the infant's intestine where foreign antigens and microorganisms may be encountered. Low levels of s-IgA in maternal breast milk have been associated with increased prevalence of cow milk allergy.[ may stimulate lymphocytes to mature and produce IgA.[ antigens have been demonstrated in breast

58 59 milk[ ] [ ]

55]

56]

In addition to the passive protection provided by s-IgA, soluble factors in human milk 57]

Although prolonged breast-feeding has been reported to prevent the development of asthma,[

and shown to sensitize and provoke symptoms in young

food

60 infants.[ ]

Normal Immune Response to Ingested Antigens 61]

Low concentrations of detectable serum IgG, IgM, and IgA food-specific antibodies are commonly found in normal individuals.[

In general, the younger an infant

62 response.[ ]

when a food antigen is introduced into the diet, the more pronounced the antibody Following the introduction of cow's milk, serum milk protein-specific IgG antibodies increase over the first month, achieving peak antibody levels after several months, and then generally decline, even though cow's milk proteins continue to be ingested. [

63]

Individuals with various inflammatory gastrointestinal disorders (e.g., celiac disease, food allergy, inflammatory bowel disease)

64

frequently have high levels of food-specific IgG and IgM antibodies. However, these antibodies do not indicate that the patient cannot tolerate these foods.[ ] The increased levels of food-specific antibodies (not IgE) appear to be secondary to increased gastrointestinal permeability to food antigens and simply reflect dietary intake. Several studies have demonstrated increased lymphocyte proliferation or IL-2 production following food antigen stimulation in vitro in patients with food allergy, 65

66 67

celiac disease, and inflammatory bowel disease.[ ] However, in vitro T cell responses also are commonly found in normal individuals as well.[ ] [ ] Although not diagnostic, stimulation of T cells from food allergic patients in vitro demonstrates the presence of increased numbers of Th2-type cells (IL-4, IL-5, and IL-13 positive).

FOOD ALLERGENS Sensitization to food allergens may occur in the gastrointestinal tract, considered “traditional” or class 1 food allergy, or as a consequence of an allergic sensitization to inhalant allergens, considered class 2 food allergy. [

68]

The major food allergens that have been identified in class 1 allergy are water-soluble glycoproteins, which 69

have molecular weights ranging from 10 to 70•kD and are stable to treatment with heat, acid, and proteases.[ ] However, there are no obvious physicochemical properties common to the class 2 food allergens. The majority of these generally plant-derived proteins are highly heat-labile and difficult to extract, often making standardized extracts for diagnostic purposes unsatisfactory. A limited number of the class 1 and 2 food allergens have been identified, cloned, sequenced, and expressed as recombinant proteins ( Table 89-1 ). Many of the plant-related allergens are homologous to pathogen-related proteins (PRs), which are expressed by the plant in response to infections or other stress factors, or comprise seed storage proteins, profilins, peroxidases, or protease inhibitors common to many plants ( Table 89-2 ).[

68]

The variety of animal-related allergens appears to be more limited in number and cross-reactivity.

Cow's milk generally represents the first foreign proteins introduced into an infant's diet. It is the most common food allergy in young children (if both IgE- and non– 70] [71]

IgE-mediated hypersensitivities are considered) and has been implicated in a variety of hypersensitivity reactions. [ TABLE 89-1 -- Major Class 1 Food Allergens Protein Fraction

Protein (Approximate %)

M.W. (kD)

Caseins

76–86

19–24

••αs1 -Casein

53–70

27

••α s2 -Casein

45–50

23

••β-Casein

25–35

24

Nomenclature

Cow's Milk

Bos d 8

Cow's milk

••κ-Casein

8–15

19

Whey

14–24

••β-Lactoglobulin

7–12

36

Bos d 5

••α-Lactalbumin

2–5

14.44

Bos d 4

Serum albumin

0.7–1.3

69

Bos d 6

Ovalbumin

54

45

Gal d 1

Ovomucoid

11

28

Gal d 2

12–13

77.7

Gal d 3

Vicilin

—

63.5

Ara h 1

Conglutin

—

17/19

Ara h 2

Glycinin

—

64

Ara h 3

Glycinin G1 acidic chain

—

40

Profilin

—

13

Gly m 3

—

12.3

Gad c 1

—

36

Pen a 1

Chicken Egg White

Ovotransferrin Peanut

Soybean

Fish Parvalbumin Shrimp Tropomyosin

Lipid Transfer Proteins (pathogen-related proteins group 14) Apple

—

9

Mal d 3

Apricot

—

9

Pru ar 3

Peach

—

10

Pru p 3

Plum

—

9

Pru d 1

Corn

—

9

Zea m 14

M.W., Molecular weight.

1623

TABLE 89-2 -- Class 2 Food Allergens (Cross-reactive and Associated with Oral Allergy Syndrome) Protein

Nomenclature

Protein Fraction

M.W. (kD)

Latex–Fruit Cross-reactivity Pathogen-related protein 2 group Latex Avocado Banana Chestnut Fig Kiwi

Hev b 2

1,3 gluconase

34/36

Chitinase Endochitinase

5 32

Pathogen-related protein 3 group Latex Avocado Chestnut

Hev b 6.02 Pers a 1

Pathogen-related protein 5 Apple

Mal d 2

Thaumatinhomologue

31

Cherry

Pru av 2

Thaumatin

23.3

Birch Bet v 1 Homologues (pathogen-related proteins 10) Apple

Mal d 1

Bet v 1 homologue

Cherry

Pru av 1

Bet v 1 homologue

Apricot

Pru ar 1

Bet v 1 homologue

Pear

Pyr c 1

Bet v 1 homologue

Carrot

Dau c 1

Bet v 1 homologue

Celery

Api g 1

Bet v 1 homologue

Parsley

pcPR 1 and 2

Bet v 1 homologue

Hazelnut

Cor a 1

Bet v 1 homologue

18

16

17

Birch Bet v 2 Homologues (celery-mugwort-spice syndrome) Latex

Hev b 8

Profilin

Celery

Api g 4

Profilin

Potato

14

Profilin

Cherry

Pru av 4

Profilin

15

Pear

Pyr c 4

Profilin

14

Peanut

Ara h 5

Profilin

15

Soybean

Gly m 3

Profilin

14

M.W., Molecular weight. 72

contains at least 20 protein components, which may lead to antibody production in humans.[ ] The milk protein fractions are subdivided into casein and whey proteins. The caseins are generally found in micellar complexes, which give milk its “milky” appearance, and constitute from 76% to 86% of the protein in cow's milk.[

73]

The casein fraction is precipitated from skim milk by acid at pH 4.6 and is comprised of four basic caseins (αs1 , αs2 , β, and κ comprising 32%, 10%, 28%,

and 10% of the total milk protein, respectively). The noncasein fraction, or whey, consists of β-lactoglobulin, α-lactalbumin, bovine immunoglobulins, bovine serum albumin, and minute quantities of various proteins (e.g., lactoferrin, transferrin, lipases, esterases). Extensive heating destroys several of the whey proteins (bovine serum albumin, bovine γ-globulin, and α-lactalbumin). However, routine pasteurization is not sufficient to denature these proteins, but may increase the allergenicity 72]

of some milk proteins, such as β-lactoglobulin.[

Sequential (linear) allergenic (IgE) epitopes have been mapped on the caseins, β-lactoglobulin and α-lactalbumin.

[74] [75] [76]

4

Goldman et al[ ] were among the first to investigate the relative allergenicity of milk proteins. Of 50 milk allergic children with positive skin tests, 30 of 50 were 4

positive to two or more of the milk proteins: caseins, 34; α-lactalbumin, 22; β-lactoglobulin, 20; bovine serum albumin, 21.[ ] Of 45 children challenged with 77]

purified milk proteins, 62% reacted to β-lactoglobulin, 60% to casein, 53% to α-lactalbumin, and 52% to bovine serum albumin.[

It is likely that these proteins 78]

contained small amounts of other milk proteins and more recent studies with more highly purified proteins suggest that the casein proteins are more allergenic.[ [79]

Immunoblotting techniques also have shown cross-reactivity among milk proteins in cows, goats, and sheep, due to the high degree of homology between these proteins. Oral challenge studies in cow's milk allergic children indicated that at least 90% of cow's milk allergic children will react to goat's milk.[ about 10% of milk allergic children will react to beef, with a slightly higher number reacting to rare Chicken egg is the most common IgE-mediated food allergy in children.[

82]

80]

Interestingly,

81 beef.[ ]

The yolk is considered less allergenic than the white, although IgE antibodies to chicken

gamma globulin, apovitellenin I, and contaminating egg white proteins can be demonstrated.[ ovomucoid, ovalbumin, and ovotransferrin have been identified as the major allergens.[

83]

71] [84]

The egg white contains 23 different glycoproteins; however

Although ovalbumin comprises the majority of the protein in egg 85

white, ovomucoid has been shown to be the dominant allergen when highly purified egg white proteins are used.[ ] Ovomucoid is comprised of 186 amino acids arranged in three tandem domains, a set tertiary structure, and six sequential (linear or continuous) IgE binding sites. Blinded oral food challenges with ovomucoid86

depleted egg white demonstrated that ovomucoid is responsible for clinical reactivity in the vast majority of egg allergic children.[ ] In addition, it was shown that about half of children allergic to eggs may be able to ingest small amounts of egg protein in extensively heated (baked) products, such as breads, cakes, and cookies. These children appear to lack IgE antibodies to sequential (continuous) epitopes and because the prolonged high temperature destroys the tertiary structure (discontinuous or conformational epitopes) of the egg white proteins, the children fail to react.[ Peanuts are the most common food allergy beyond the age of 4 years.[

87]

71] [85]

Peanut proteins are traditionally classified as albumins (water-soluble) and globulins (saline-

solution soluble), the latter of which is further subdivided into arachin and conarachin fractions.[

71]

Thirty-two protein bands have been identified on SDS-PAGE 88]

(sodium dodecyl sulfate–polyacrylamide gel electrophoresis); however, three with molecular weights of 63.5•kD (Ara h 1),[ 90 91 3)[ ] [ ]

17•kD (Ara h 2),[

89]

and 64•kD (Ara h

92 identified[ ]

have been identified as major allergens. Ara h 4 to 7 have been ; Ara h 5 is a profilin, whereas Ara h 4 appears to be an isoform of Ara h 3, and Ara h 6 and 7 appear to be isoforms of Ara h 2. A wide variety of peanut products including flour and reprocessed peanuts have been shown to retain their allergenicity.[

allergenicity.

93]

However, refined peanut oil was found to be safe in 60 peanut allergic individuals, whereas pressed (or extruded) oils retained some of their

[94]

Ara h 1 (64.5•kD) belongs to the vicilin family of seed storage proteins. Using peanut-allergic patient sera and overlapping decapeptides representing the entire primary amino acid sequence of Ara h 1, 23 different IgE-binding sequential

1624

95]

epitopes were identified, 4 of which were recognized by more than 80% of peanut-allergic patient sera.[

Mutational analysis of the immunodominant epitopes 96

revealed that a single amino acid substitution, especially in the center of each epitope, could dramatically reduce IgE binding. [ ] Expression of these mutated recombinant proteins may prove valuable in the development of safer vaccines for immunotherapy. Ara h 2 (17.5•kD) is a member of the conglutin family of storage

97

proteins, which has been fully sequenced and a full-length cDNA clone isolated.[ ] Ten IgE-binding continuous epitopes have been identified, although three are clearly immunodominant, and mutational analysis of these major allergenic epitopes demonstrated that single amino acid substitutions drastically reduced IgE binding.[

97]

T cell epitope mapping with peanut-specific T cell lines demonstrated four immunodominant regions on the Ara h 2 molecule, three of which mapped to 98

90 91

different locations than the immunodominant IgE-binding epitopes.[ ] Ara h 3 (64•kD) is a member of the glycinin family of storage of proteins.[ ] [ ] Glycinins represent a class of seed storage proteins that are first synthesized as precursor proteins of approximately 60•kD and then are proteolytically cleaved into 40- and 20kD proteins. A full-length cDNA of the 64-kD protein has been isolated and four IgE-binding sequential epitopes have been identified.[

99]

Soybeans are a second member of the legume family that provokes a significant number of hypersensitivity reactions, predominantly in infants and young children. Because soybeans provide an inexpensive source of high-quality protein, soybean protein is used in many commercial foods. 100

Approximately 10% of the seed proteins are water-soluble albumins and the remainder are salt-soluble globulins.[ ] Four major protein fractions have been separated by ultracentrifugation: 2S (contained in whey fraction), 7S (50% β-conglycinin), 11S (glycinin), and 15S (aggregated glycinin). A number of soy proteins 101]

have been isolated and characterized: a 34-kD thiol protease-like protein (Gly m Bd 30K), a 13-kD profilin (Gly m 3),[ 103]

40-kD glycinin G1 acidic chain,[

binding epitopes on peanut Ara h up to 8•ml of soy

106 oil.[ ]

and a 22-kD G2 glycinin protein.[

105 3.[ ]

104]

a 70-kD α-subunit of β-conglycinin,[

102]

a

Interestingly, the allergenic epitopes on glycinin G1 acidic chain are homologous to IgE-

Similar to highly refined peanut oil, refined soy oil did not provoke allergic reactions to soy in eight patients after ingesting

Using an enzyme-linked immunosorbent assay (ELISA)-inhibition assay, however, European investigators found detectable soy protein in

107 products.[ ]

soy fat Six of seven soy lecithin preparations contained 1.03 to 27.2•mg/g of soy protein, four of five margarines contained 0.1 to 0.29•mg/g, and three of eight soy oil preparations contained 0.11 to 3.3•mg/g of soy protein. 14

Tree nut allergies affect about 0.6% of the American population.[ ] In a national registry of peanut and nut allergic individuals, walnuts were the nut provoking the most allergic reactions (34%), followed by cashews (20%), almonds (15%), pecan (9%), pistachio (7%), and hazelnut, Brazil nut, pine nut, and macadamia nut, all 108

less than 5%.[ ] Skin testing reveals extensive cross-reactivity among tree nuts. Although individuals allergic to one nut can clearly tolerate other nuts, too few patients have been systematically challenged to a variety of nuts to determine the extent of clinical cross-reactivity. In one study, 14 children underwent 19 109

DBPCFCs to nuts; one patient reacted to five nuts, one to two nuts, and the remaining 12 children to one nut each.[ ] Overall, there were seven reactions to walnuts, six to cashews, three to pecans, two to pistachios, and one to filbert. Patients allergic to nuts do not necessarily need to avoid peanuts (a legume), and vice versa. However, recent surveys suggest that about 35% to 50% of peanut allergic patients are also reactive to at least one tree nut.[

110] [111]

112 113 114

Fish are one of the most common causes of food allergic reactions in adults, and a common cause in children as well. [ ] [ ] [ ] Edible fish are found predominantly in the Osteichthyes class, in which there are hundreds of species. The major allergen in cod, Gad c1, is a parvalbumin that has been isolated from the myogen fraction of the white meat. A similar protein, Sal s1, has been isolated from salmon. It is heat-stable and resistant to proteolytic digestion, has a molecular weight of 12•kD and an isoelectric point of 4.75, and is composed of 113 amino acids.[ be arranged in three domains, two of which bind calcium.[ that is similar to Gad c1 and up to 29 protein

116]

117 fractions.[ ]

115]

The three-dimensional structure of Gad c1 has been defined and shown to

Using SDS-PAGE and immunoblot analyses, 10 common fish species were shown to have a protein

Eleven patients with histories of fish allergy had multiple positive skin tests to various fish; 7 of 11

reacted to only one fish species in DBPCFCs, one reacted to two fish, two reacted to three fish, and one patient did not react to any fish during blinded challenges. Unlike many other food allergens, the fish protein fractions responsible for clinical symptoms in some patients appear to be more susceptible to manipulation (e.g., heating, lyophilization) than other foods, because reactions occurred during open feedings of the fish in approximately 20% of those with negative DBPCFCs using 118

lyophilized fish.[ ] Furthermore, most patients allergic to fresh cooked salmon or tuna could ingest canned salmon or tuna without difficulty, indicating that preparation led to destruction of the major allergens. Nevertheless, allergic reactions following exposure to airborne allergen emitted during cooking are not uncommon.[

119]

120

Shellfish allergens are considered a major cause of food allergic reactions in adults, affecting up to 0.5% of the U.S. adult population.[ ] This group consists of a wide variety of mollusks (snails, mussels, oysters, scallops, clams, squid, and octopus) and crustacea (lobsters, crabs, prawns, and shrimp). Shrimp allergens have been most extensively studied. Eighteen precipitating antigens have been detected by crossed immunoelectrophoresis (CRIE); seven appeared to be allergens as determined by CRIE using a pool of sera from six shrimp allergic subjects.[ the major allergen in

122 123 shrimp.[ ] [ ]

121]

Tropomyosin, a protein found both in muscle and elsewhere, has been identified as

Considerable cross-reactivity among crustacea has been demonstrated by skin test and radioallergosorbent test (RAST)

124 analyses.[ ]

125

Invertebrate tropomyosins are highly homologous and tend to be allergenic[ ] : those from crustaceans (e.g., shrimp, crab, crawfish, and lobster), arachnids (house dust mites), insects (cockroaches), and mollusks (squid, snails), whereas vertebrate tropomyosin tends not to be allergenic. Wheat (spelt) and other cereal grains share a number of homologous proteins and are not infrequently implicated in food allergic reactions in children. One study suggested that the globulin and glutenin fractions were the major allergenic fractions in IgE-mediated reactions, gliadins in celiac disease, and albumins in Baker's asthma.[

126]

Sera from 20 pediatric patients with asthma, eczema, or both revealed extensive cross-reactivity between wheat, rye, and barley by RAST analyses.

1625

In a second study of pediatric patients, 70 of 225 children were found to have at least one positive prick skin test to wheat, rye, oat, barley, rice, or corn: 28 of 70 to 127

one grain, 16 of 70 to two grains, 12 of 70 to three grains, 6 of 70 to four grains, 6 of 70 to five grains, and 2 of 70 to six grains.[ ] However, only 15 patients had at least one positive DBPCFC to a cereal grain; two children reacted to two grains and two reacted to three grains. SDS-PAGE and immunoblot analyses with patient sera revealed 27 to 31 protein bands in the range of 7.8 to 66.5•kD for each of the six cereal grains studied. Nonspecific binding to lectin fractions was noted with each grain, and extensive immunologic cross-reactivity was noted among the cereals, as was seen with skin testing. In addition, homologies to proteins in grass pollens account for a large number of clinically irrelevant positive skin tests to wheat and other cereal grains. Recent studies have suggested that the water-insoluble gliadin fraction may be important in clinical reactivity to wheat, but further studies are warranted.[

128] [129] [130]

Pathogenesis-related proteins (PRs) have been shown to comprise a large number of class 2 allergenic proteins (see Table 89-2 ) found in various vegetables and 131 132

fruits.[ ] [ ] These proteins are induced when pathogens, wounding, or certain environmental stresses, such as drought or heat, stress the plant. PRs have been classified into 14 families, although six PR families account for the majority of cross-reactivity among plant proteins. Allergens homologous to the PR-2 β-1, 3glucanase proteins are responsible for “latex-fruit” cross-reactivity seen between latex (Hev b2) and avocado, banana, chestnut, fig, and kiwi. Two families of

chitinases that are similar to the latex allergen, Hev b6.02, have been identified as allergens in a number of vegetables: PR-3 type proteins are found in chestnut and avocado (Pers a1), and PR-4 type proteins are wound-induced proteins found in tomato and potato (Win 1 and Win 2 proteins). PR-5 type thaumatin-like proteins have been identified as cross-reacting proteins found in apples (Mal d2) and cherry (Pru av2). The PR-10 type proteins are homologous to the major birch pollen allergen, Bet v1, and account for cross-reactivity between birch pollen and fruits of the Rosaceae species, such as apple (Mal d1), cherry (Pru av1), apricot (Pru ar1), and pear (Pyr c1), or vegetables of the Apiaceae species, such as carrot (Dau c1), celery (Api g1), and parsley (pcPR 1 and 2), and hazelnut (Cor a1). The lipid transfer proteins (LTPs), or PR-14 type proteins, form a family of 9-kD proteins distributed widely throughout the plant kingdom. LTPs have been identified as major allergenic proteins in Prunoideae, such as peach (Pru p1 in peach skin and Pru p3 in the fruit), apple (Mal d3), apricot, plum, and cherries. Gly m1, a major allergen in soybean, is an LTP. Profilin is an actin-binding protein that was first identified in birch pollen (Bet v2) and is now recognized as an allergenic protein in a number of fruits and 131

vegetables.[ ] Profilins are responsible for the celery-mugwort-spice syndrome and is responsible for oral allergy syndrome (OAS) to apple, pear, carrot, celery (Api g4), and potato in birch pollen allergic patients. Profilins have also been identified in tomato, peanut (Ara h5), and soybean (Gly m3), but whether these proteins cause allergic reactions remains to be established.

PATHOPHYSIOLOGIC MECHANISMS In the susceptible host, a failure to develop or a breakdown in oral tolerance may result in hypersensitivity responses to ingested food antigens. Traditionally, the Gell 133

and Coombs classification[ ] has been used to provide a framework for discussing hypersensitivity reactions, but generally food allergic disorders involve more than one of the classic mechanisms described. IgE-Mediated Reactions The best characterized food allergic reactions involve IgE-mediated responses. A failure to develop or a breakdown in oral tolerance results in excessive production of food-specific IgE antibodies. These antibodies bind high-affinity FcepsilonRI on mast cells and basophils as well as low-affinity FcepsilonRII (CD23) on macrophages, monocytes, lymphocytes, eosinophils, and platelets. When food allergens penetrate mucosal barriers and reach IgE antibodies bound to mast cells or basophils, mediators are released that induce vasodilatation, smooth muscle contraction, and mucus secretion, which result in symptoms of immediate hypersensitivity. The activated mast cells also may release a variety of cytokines, which may induce the IgE-mediated late-phase response. During the initial 4 to 8 hours, primarily neutrophils and eosinophils invade the site of response. These infiltrating cells are activated and release a variety of mediators including platelet activating factor, peroxidases, eosinophil major basic protein, and eosinophil cationic protein. In the subsequent 24 to 48 hours, lymphocytes and monocytes infiltrate the area and establish a more chronic inflammatory picture. With repeated ingestion of a food allergen, mononuclear cells are stimulated to secrete “histamine134]

releasing factor” (HRF), a cytokine that interacts with IgE molecules bound to the surface of basophils (and perhaps mast cells) and increases their releasability.[ The “spontaneous” generation of HRF by activated mononuclear cells in vitro has been associated with increased bronchial hyperreactivity in patients with asthma [135]

134]

and increased cutaneous irritability in children with atopic dermatitis.[

A variety of symptoms have been associated with IgE-mediated allergic reactions: generalized (shock), cutaneous (urticaria, angioedema, and a pruritic morbilliform rash), oral and gastrointestinal (lip, tongue, and palatal pruritus and swelling, laryngeal edema, vomiting, and diarrhea), upper and lower respiratory (ocular pruritus and tearing, nasal congestion, and wheezing). An increase in plasma histamine has been associated with the development of these symptoms following blinded food

challenges.[

136]

It is not known why foods provoke different constellations of symptoms in different individuals. In IgE-mediated gastrointestinal reactions,

endoscopic observation has revealed local vasodilation, edema, mucus secretion, and petechial hemorrhaging.[ and PGF2 have been observed following adverse food reactions leading to diarrhea.[ activation of other cell types through IgE-mediated mechanisms.[

138]

137]

Increased stool and serum prostaglandin (PG) E2

Atopic dermatitis and chronic airway hyperreactivity (asthma) involve

140]

Non–IgE-Mediated Food Hypersensitivity Type II antigen-antibody dependent cytotoxic reactions occur when specific antibody binds to a surface tissue antigen or hapten associated with a cell and induces complement activation. Complement activation products promote the generation of various inflammatory mediators that lead to subsequent tissue damage. A few reports have implicated an antibody-dependent

1626

140

thrombocytopenia secondary to the ingestion of milk.[ ] However, little evidence supports any significant role for type II hypersensitivity in food allergic disorders. Type III antigen-antibody complex-mediated hypersensitivity has been implicated in patients with a variety of complaints and elevated serum food antigen-antibody complexes. However, food antigen-antibody complexes have been demonstrated in the sera of normal individuals, as well as patients with suspected food hypersensitivity. The complexes formed by the interaction of IgG, IgA, or IgM antibodies to β-lactoglobulin are found 1 to 3 hours after ingesting milk in normal children and adults.[

141]

Although IgE-food antigen complexes are more commonly found in patients with food hypersensitivity, there is little support for food 142

antigen-immune complex mediated disease.[ ] Type IV cell-mediated hypersensitivity has been implicated in food allergic disorders in which the onset of clinical symptoms occurs several hours after the ingestion of a suspected food allergen. Ingestion of the sensitizing antigen may provoke mucosal lesions. Several investigators have found increased lymphocyte proliferation to food antigens in food allergic individuals, but increased proliferation is found in many asymptomatic 67

subjects as well.[ ] Cell-mediated hypersensitivity reactions are likely to contribute to a number of gastrointestinal disorders, such as allergic eosinophilic esophagitis and gastroenteritis, and celiac disease. In several adverse food reactions, pathogenic factors are not well defined, but are believed to involve immune mechanisms. Both antigen-antibody complex and cellmediated reactions are presumed to be responsible (at least in part) for these pathogenic states, as depicted in Box 89-2 .

CLINICAL MANIFESTATIONS OF FOOD HYPERSENSITIVITY Categorizing food hypersensitivity disorders into those pri-marily involving IgE-mediated reactions, those not involving IgE-mediated mechanisms, and those that may involve both IgE- and non–IgE-mediated mechanisms is most useful from a clinical and diagnostic standpoint, as depicted in Box 89-2 .

Gastrointestinal Food Hypersensitivity Reactions IgE-Mediated Hypersensitivity

IgE-mediated gastrointestinal food hypersensitivity has been studied extensively in rodent models, which has elucidated a number of physiologic changes provoked 143

by the ingestion of a food allergen.[ ] There is a sharp increase in gastric acid secretion in the stomach, delayed gastric emptying, and mast cell degranulation, with an increase in intraluminal histamine and serum rat mast cell protein II (RMCP II, similar to tryptase in humans). In the intestine, Na+ , Cl− , and water absorption decrease sharply while aboral contractility markedly increases, leading to diarrhea. Histologically, intestinal mast cells appear degranulated, and although significant disruption of the basement membrane and underlying collagenous matrix is apparent by ultrastructural examination, the mucosal architecture appears largely unchanged following a reaction. [

144]

Cromolyn sodium, H1 antihistamines, and H2 antihistamines failed to block the allergic response. [

The effect of repeated daily ingestion of allergen has been investigated using a similar model in rats sensitized with Box 89-2. Food Hypersensitivity Disorders IgE-mediated: Cutaneous: urticaria, angioedema, morbiliform rashes and flushing Gastrointestinal: oral allergy syndrome, gastrointestinal anaphylaxis Respiratory: acute rhinoconjunctivitis, bronchospasm (wheezing) Generalized: systemic anaphylaxis with or without shock Mixed IgE- and cell-mediated: Cutaneous: atopic dermatitis Gastrointestinal: allergic eosinophilic esophagitis; allergic eosinophilic gastroenteritis Respiratory: asthma Cell-mediated: Cutaneous: contact dermatitis, dermatitis herpetiformis Gastrointestinal: food protein–induced enterocolitis; food protein–

145]

induced proctocolitis; food protein–induced enteropathy syndromes, celiac disease Respiratory: food-induced pulmonary hemosiderosis (Heiner's syndrome) Unclassified: cow's milk–induced anemia; arthritis; migraine

146

ovalbumin.[ ] Following the first feeding of ovalbumin in sensitized rats, serum RMCP II levels increased to approximately 16 times normal on the first day and ranged between 2 and 5 times normal on the subsequent 4 days. When the rats were rested for 9 days and then re-fed ovalbumin, serum RMCP II levels again increased, but only to five times normal concentrations. In the sensitized rats, the initial allergen challenge led to a marked mast cell activation (release of RMCP II and probably other mast cell mediators) followed by a period of partial desensitization. Subsequent exposures to allergen, even after short periods of avoidance, provoked lesser (but significant) amounts of mast cell mediator release. Similar effects are seen in humans, which accounts for the need to have an extended period of complete food allergen avoidance in order to elicit a distinctive response during the oral food challenge. The phenomenon of partial desensitization due to frequent allergen exposure also explains why food allergic patients with primarily mild cutaneous symptoms who are placed on a strict elimination diets frequently experience more severe generalized symptoms if the food allergen is accidentally ingested after prolonged avoidance. Human studies of IgE-mediated food hypersensitivity initially focused on roentgenologic changes associated with the ingestion of food allergens. In one of the first reports, a wheat sensitive patient was studied following a deliberate feeding of wheat. Hypertonicity in the transverse and pelvic colon and hypotonicity in the cecum and ascending colon were noted.[

147]

In a later study, four patients were evaluated after administering barium mixtures containing specific allergens. Gastric 148]

retention, hypermotility of the intestine, and colonic spasm were observed.[ were followed fluoroscopically to compare the outcome of barium

In a third study, 12 food allergic children underwent a single-blind food challenge and

1627

149

sulfate meals with and without food allergen.[ ] Again, the most prominent findings included gastric hypotonia and retention of the allergen test meal, prominent pylorospasm, and increased or decreased peristaltic activity of the intestines. The rigid gastroscope was used by several investigators in the late 1930s to observe allergic reactions in the stomach. In one study, six patients with gastrointestinal 47

food allergy, four patients with asthma symptoms exacerbated by food ingestion, and three control subjects were evaluated.[ ] A small amount of food allergen was placed on the stomach mucosa through the gastroscope and the mucosa was revisualized 30 minutes later. The gastric mucosa of patients with gastrointestinal allergy was markedly hyperemic and edematous with patches of thick gray mucous and scattered petechiae, as noted earlier by Walzer and colleagues using passively

sensitized intestinal mucosal sites.[

42]

Only mild hyperemia of the gastric mucosa was noted in patients with wheezing provoked by food ingestion. More recent 137

studies of intragastral provocation under endoscopic control (IPEC) have confirmed these earlier observations.[ ] In addition, increased numbers of intestinal mast cells were observed before challenge in food allergic patients compared with normal control subjects, and significant decreases in stainable mast cells and tissue histamine content following a positive food challenge. 68

OAS is elicited by a variety of plant proteins, especially pathogenesis-related proteins,[ ] cross-reacting with airborne allergens. Symptoms are provoked almost exclusively in the oropharynx and rarely involve other target organs. It is estimated that OAS affects up to 50% to 70% of adults suffering from pollen allergy, 150 151

especially to birch, ragweed, and mugwort pollens.[ ] [ ] Local IgE-mediated mast cell activation provokes the rapid onset of pruritus, tingling and angioedema of the lips, tongue, palate and throat, and occasionally a sensation of pruritus in the ears or tightness in the throat. Symptoms are generally short-lived and are most commonly associated with the ingestion of various fresh fruits and vegetables. [

152] [153]

Ragweed allergic patients may experience OAS following contact with

153 154 bananas.[ ] [ ]

various fresh melons (e.g., watermelon, cantaloupe, honeydew) and Symptoms may be more prominent following the ragweed season, corresponding to their seasonal increase in ragweed-specific IgE levels. Birch pollen allergic patients may develop symptoms following the ingestion of raw potatoes, carrots, celery, apples, pears, hazelnuts and kiwi.[ [68]

various “pathogenesis-related proteins.” 89-2

157 ).[ ]

153] [155] [156]

Cross-reactivity between birch pollen and various fruits and vegetables is due to homology among

For example, Mal d 1, the major apple allergen, is 63% homologous to the major birch pollen allergen, Bet v 1 (see Table 158]

Other birch pollen–related proteins have been identified in hazelnut (Cor a 1),[

celery (Api g 1),[

159]

and pears (Pyr c 1).[

160]

Similarly, the birch

161 potato.[ ]

Oral symptoms also have been described among several pollen profilin, Bet v 2, cross-reacts with profilins found in pear (Pyr c 4), celery (Api g 4), and fruits in the Prunoideae subfamily and appear to be due to a homologous 9-kD protein found in these fruits: peach (Pru p 1), cherry, apricot, and plum, and Brazil nuts (Ber e 1).[

162] [163]

OAS patients generally can ingest these foods in the cooked form without difficulty.

Diagnosis is based on a suggestive history and positive “prick + prick” skin tests with the implicated fresh fruits or vegetables in patients with allergic rhinitis. Skin tests with commercial extracts are often negative because the responsible allergen is often destroyed in the manufacturing process. [ that successful treatment of pollen-induced rhinitis with immunotherapy will also eliminate oral allergy

153]

Several recent studies suggest

164 165 symptoms.[ ] [ ]

Gastrointestinal anaphylaxis is a form of IgE-mediated gastrointestinal hypersensitivity, which often accompanies allergic manifestations in other target organs and 69

results in a variety of symptoms. [ ] Symptoms generally develop within minutes to 2 hours of consuming the responsible food allergen and consist of nausea, abdominal pain, cramps, vomiting, and diarrhea. In food allergic children with atopic dermatitis, frequent ingestion of a food allergen appears to induce partial desensitization of gastrointestinal mast cells resulting in less pronounced symptoms, such as occasional minor complaints of poor appetite and periodic abdominal 166 167]

pain. However, carbohydrate absorption studies, a measure of gut wall integrity, demonstrate malabsorption in such patients.[ ] [ symptoms is seen in young infants with frequent vomiting, leading to a loss of consistent vomiting immediately following feeding.

A similar diminution of

Diagnosis is established by clinical history, determination of food-specific IgE antibodies (prick skin tests or RAST), complete elimination of the suspected food allergen for up to 2 weeks with resolution of symptoms, and oral food challenges. DBPCFCs provoke typical symptoms if the allergen has been strictly eliminated from the patient's diet for 10 to 14 days.

Mixed IgE and Non–IgE-Mediated Disorders Mixed IgE- and non–IgE-mediated disorders may involve both IgE- and cell-mediated mechanisms. A murine model of allergic eosinophilic gastroenteritis was established by exposing mice to enteric-coated antigen. Mice developed an extensive Th2-associated eosinophilic inflammatory response involving the esophagus, 168]

stomach, small intestine, and Peyer's patches, and developed gastric dysmotility, gastromegaly, and cachexia.[ allergens developed a picture consistent with allergic eosinophilic

Similarly, mice sensitized with respiratory

169 esophagitis.[ ]

Allergic eosinophilic esophagitis, gastritis, and gastroenteritis are characterized by infiltration of the esophagus, stomach and intestinal walls with eosinophils, basal 170

zone hyperplasia, papillary elongation, absence of vasculitis, and peripheral eosinophilia in about 50% of patients.[ ] The eosinophilic infiltrate may involve the mucosal, muscular, and serosal layers of the stomach or small intestine, and clinical symptoms correlate with the extent of eosinophil infiltration of the bowel wall. [171] [172] [173] [174]

Eosinophilic infiltration of the muscular layer leads to thickening and rigidity, provoking symptoms of obstruction, whereas infiltration of the serosal area results in ascites containing eosinophils. Peripheral blood T cells from these patients were shown to secrete excessive amounts of IL-4 and IL-5 compared with normal controls subjects,[

175]

and recently increased numbers of lymphocytes expressing IL-5 and TNF-α were demonstrated in biopsy specimens of

patients with allergic eosinophilic esophagitis.[

176]

Allergic eosinophilic esophagitis (AEE) is seen most frequently during infancy through adolescence, and typically presents with chronic gastroesophageal reflux disease (GERD), intermittent emesis, food refusal, abdominal pain, dysphagia, irritability, sleep disturbance, and failure to respond to conventional reflux medication. [

177]

One study of children

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178

younger than 1 year of age with GER found that 40% had cow's milk–induced reflux.[ ] Allergic eosinophilic gastritis also is more common between infancy and adolescence, and presents with postprandial vomiting, abdominal pain, anorexia, early satiety, hematemesis, failure to thrive, and gastric outlet obstruction. Allergic eosinophilic gastroenteritis (AGE) may occur at any age and may present with symptoms similar to esophagitis or gastritis. Weight loss or failure to thrive is a hallmark of this disorder. Up to 50% of patients with these allergic eosinophilic disorders are atopic, and food-induced IgE-mediated reactions have been implicated in a minority of patients. Adults with food-induced symptoms often have the mucosal form of AEG with IgE-staining cells in jejunal tissue, elevated IgE in duodenal fluids, atopic disease, elevated serum IgE concentrations, positive prick skin tests to a variety of foods and inhalants, peripheral blood eosinophilia, iron deficiency anemia, and hypoalbuminemia. Generalized edema secondary to hypoalbuminemia may occur in young infants with marked protein-losing enteropathy, often in the presence of minimal gastrointestinal symptoms such as occasional vomiting and diarrhea.[ stenosis in infants with outlet obstruction and postprandial projectile responsible food allergens (frequently multiple foods).[

181]

180 emesis.[ ]

179]

Rarely, allergic eosinophilic gastroenteritis may present as pyloric

Resolution of symptoms may require 3 to 8 weeks following the elimination of

Diagnosis is dependent on the gastrointestinal biopsy demonstrating a characteristic eosinophilic infiltration, typically 15 or more eosinophils per 40× high-power 70 173 177

field. Eosinophilic infiltrates may be patchy so it is recommended that up to eight sites be biopsied to rule out eosinophilic gastroenteritis.[ ] [ ] [ ] Patients with the mucosal form frequently exhibit food allergies and often have atopic symptoms, elevated serum IgE concentrations, positive skin tests and RASTs to a variety of allergens, and peripheral eosinophilia. Laboratory studies consistent with this diagnosis include peripheral eosinophilia, Charcot-Leyden crystals in the stools, positive prick skin tests to foods, anemia, hypoalbuminemia, and abnormal D-xylose tests. Elimination of the responsible food allergen from the diet for up to 8 weeks may be necessary to bring about resolution of symptoms and up to 12 weeks to bring about normalization of intestinal histology. This often requires the use of an amino acid–derived formula or an oligoantigenic diet. To identify the responsible foods, challenges are required that consist of reintroducing the suspect food allergen (often for 3 to 5 days) and demonstrating recurrence of symptoms and eosinophilic infiltrate on biopsy. Treatment of this disorder is often unsatisfactory. If a food allergen is identified to be provoking symptoms, it should be strictly avoided. An alternative approach is to use oral corticosteroids, which generally bring about rapid symptomatic relief. However, reexacerbations of symptoms are frequent when steroids are discontinued. Prednisone is initiated at 1•mg/kg per day (up to 100•mg) and then tapered. If exacerbations recur, a regimen of low-dose prednisone or every other day prednisone may be successful in suppressing symptoms. Infantile colic is an ill-defined syndrome of paroxysmal fussiness characterized by inconsolable, “agonized” crying, drawing up of the legs, abdominal distention, and 182

excessive gas. It generally develops in the first 2 to 4 weeks of life and persists through the third to fourth month of life.[ ] A variety of psychosocial and dietary factors have been implicated in the etiology of infantile colic, but in a disorder that has no standardized diagnostic criteria, consists of subjective symptoms, and resolves in a relatively short time, it is difficult to establish an etiology for this ailment. However, double-blind crossover trials in bottle- and breast-fed infants suggest that IgE-mediated hypersensitivity may be a pathogenic factor in some infants.[ colicky infants.

183] [184]

Allergic mechanisms probably account for only 10% to 15% of

Diagnosis of food-induced colic can be established by the implementation of several brief trials of hypoallergenic formula. Symptoms should resolve when the child is placed on the hypoallergenic formula and recur when the regular formula or breast-feeding resumes. A food allergic etiology is confirmed when two placebocontrolled, crossover blinded trials of the suspected allergen results in symptoms during the allergen challenges and resolution during the placebo challenges. In infants with food allergen–induced colic, symptoms are generally short-lived so periodic rechallenges every 3 to 4 months should be carried out. Non–IgE-Mediated Hypersensitivity A number of gastrointestinal disorders are believed to be secondary to cell-mediated hypersensitivities (see Box 89-2 ). Food protein-induced enterocolitis syndrome is a disorder most commonly seen in young infants between 1 week and 3 months of age who present with protracted 185 186

vomiting and diarrhea, which not infrequently results in dehydration.[ ] [ ] Vomiting generally occurs 1 to 3 hours following feeding, and continued exposure may result in bloody diarrhea, anemia, abdominal distention, and failure to thrive. Symptoms are most commonly provoked by cow's milk or soy protein–based formulas, but rarely result from food proteins passed in maternal breast milk. A similar enterocolitis syndrome has been reported in older infants and children as a 186

77 186]

result of egg, wheat, rice, oat, peanut, nuts, chicken, turkey, and fish sensitivity. [ ] Hypotension occurs in about 15% of cases following allergen ingestion.[ ] [ In adults, shellfish (e.g., shrimp, crab, lobster) sensitivity may provoke a similar syndrome, with severe nausea, abdominal cramps, and protracted vomiting. Stools

often contain occult blood, neutrophils, eosinophils, and Charcot-Leyden crystals. Prick skin tests to the suspected foods are negative. Jejunal biopsies classically reveal flattened villi, edema, and increased numbers of lymphocytes, eosinophils, and mast cells. Although the immunopathogenic mechanism of this syndrome remains to be elucidated, some studies suggest that food antigen–induced secretion of tumor necrosis factor alpha (TNF-α) from local mononuclear cells may be 187] [188]

responsible for the secretory diarrhea and hypotension.[

Diagnosis can be established when elimination of the responsible allergen leads to resolution of symptoms within 72 hours and oral challenge provokes symptoms. [188] [189]

However, secondary disaccharidase deficiency may persist longer and may result in ongoing diarrhea for up to 2 weeks. Oral food challenges consist of administering 0.3 to 0.6•g/kg body weight of the suspected protein allergen. Vomiting generally develops within 1 to 4 hours of administering the challenge food. Diarrhea or loose stools often develop after 4 to 8 hours. In conjunction with a positive food challenge, the peripheral blood absolute neutrophil count increases at least 3,500 cells/mm3 within 4 to 6 hours of developing symptoms, and neutrophils

1629

and eosinophils may be found in the stools. About 15% of food antigen challenges lead to profuse vomiting, dehydration, and hypotension, so they must be performed under medical supervision. Food protein-induced colitis, like the food-induced enterocolitis syndrome, generally presents in the first few months of life. Although often secondary to cow's milk 174 190 191 192 193 194 195

or soy protein hypersensitivity, the majority of such cases today are in breast-fed infants.[ ] [ ] [ ] [ ] [ ] [ ] [ ] These infants generally do not appear ill, often have normally formed stools, and typically are discovered because of the presence of blood (gross or occult) in their stools. Blood loss is usually minor but occasionally can produce anemia. Lesions are confined to the distal large bowel and consist of mucosal edema with infiltration of eosinophils in the epithelium and lamina propria. In severe lesions with crypt destruction, neutrophils also are prominent. Diagnosis can be established when elimination of the responsible allergen leads to resolution of hematochezia, generally with dramatic improvement within 72 hours 170 195

of appropriate food allergen elimination, but with complete clearing and resolution of mucosal lesions taking up to 1 month. [ ] [ ] Reintroduction of the allergen leads to recurrence of symptoms within several hours to days. Sigmoidoscopy findings are variable but range from areas of patchy mucosal injection to severe friability with small aphthoid ulcerations and bleeding. Colonic biopsy reveals a prominent eosinophilic infiltrate in the surface and crypt epithelia and the lamina propria.[ seen.

192]

Cow's milk and soy protein–induced proctocolitis usually resolve within 6 months to 2 years of allergen avoidance, but occasional refractory cases are

Dietary protein-induced enteropathy (excluding celiac disease) generally presents in the first several months of life with diarrhea (mild to moderate steatorrhea in 196 197

about 80%) and poor weight gain.[ ] [ ] Symptoms include protracted diarrhea, vomiting in up to two thirds of patients, failure to thrive, and malabsorption, demonstrated by the presence of reducing substances in the stools, increased fecal fat, and abnormal D-xylose absorption. Cow's milk hypersensitivity is the most frequent cause of this syndrome, but it also has been associated with sensitivity to soy, egg, wheat, rice, chicken, and fish.

Diagnosis requires the identification and exclusion of the responsible allergen from the diet, which brings about a resolution of symptoms within several days to weeks. On endoscopy, a patchy villous atrophy is evident and biopsy reveals a prominent mononuclear round cell infiltrate and a small number of eosinophils, not 196 197 198

unlike celiac disease, but generally much less extensive. [ ] [ ] [ ] Colitis-like features are usually absent, but anemia occurs in about 40% of affected infants and protein loss occurs in the majority. Complete resolution of the intestinal lesions may require 6 to 18 months of allergen avoidance. Unlike celiac disease, loss of clinical reactivity frequently occurs, but the natural history of this disorder has not been well studied. Celiac disease is a more extensive enteropathy leading to malabsorption. Total villous atrophy and extensive cellular infiltrate are associated with sensitivity to gliadin, the alcohol-soluble portion of gluten found in wheat, rye, and barley, and possibly oat. Celiac disease represents an interplay between the environment and 199

genetics, with a strong association with HLA-DQ2 [α1*0501, β1*0201], which is present in more than 90% of patients with celiac disease.[ ] The incidence of celiac disease is reported as 1:250 in the United States (Celiac Disease Foundation). The recent striking increase in celiac disease in Sweden compared to genetically 200

201

similar Denmark,[ ] and the variation in prevalence associated with changes in patterns of gluten feeding in Sweden[ ] strongly implicate environmental factors (i. e., feeding practices) in the etiology of this disorder. The intestinal inflammation in celiac disease is precipitated by exposure to gliadin. Gluten-specific T cells are found in biopsies of patients with celiac disease, and without exception they respond to gluten-derived peptides bound to the disease-associated HLA-DQ2 or HLADQ8 molecules.[

202]

Several gluten-derived peptides have been characterized, which are the result of selective conversion of specific glutamine residues in gluten 203]

peptides into glutamic acid by tissue transglutaminase [tTGase]. This deamidation creates epitopes that bind efficiently to DQ2 and are recognized by T cells.[ addition, most patients develop IgA antibodies against gliadin and tTGase.[ epitopes on the transglutaminase

205 molecule,[ ]

204]

In

Even though virtually all celiac disease patients possess autoantibodies to distinct

these antibodies do not appear to be responsible for the pathology.

Initial symptoms often include diarrhea or frank steatorrhea, abdominal distention and flatulence, weight loss, and occasionally nausea and vomiting. Oral ulcers and other extraintestinal symptoms secondary to malabsorption are not uncommon. Villous atrophy of the small bowel is a characteristic feature of celiac patients ingesting gluten. IgA antibodies to gluten are present in more than 80% of adults and children with untreated celiac disease.[ increased IgG antibodies to a variety of foods, presumably the result of increased food antigen absorption.

206]

In addition, patients generally have

Diagnosis is dependent on demonstrating biopsy evidence of villous atrophy and inflammatory infiltrate, resolution of biopsy findings after 6 to 12 weeks of gluten 207

elimination, and recurrence of biopsy changes following gluten challenge.[ ] Revised diagnostic criteria have eliminated the requirement for gluten challenge, instead placing a greater focus on serologic studies. Quantitation of IgA antigliadin antibodies may be used for screening with IgA anti-tTGase antibodies in patients older than 2 years of age. Once the diagnosis of celiac disease is established, lifelong elimination of gluten-containing foods is necessary to control symptoms and to avoid the increased risk of malignancy. Cutaneous Food Hypersensitivity Reactions The skin is a frequent target organ in IgE- and non–IgE-mediated food hypersensitivity reactions (see Box 89-2 ). Ingestion of food allergens may lead to the rapid onset of cutaneous symptoms or aggravate more chronic conditions. IgE-Mediated Food Hypersensitivity

Urticaria and angioedema on an acute basis are said to be among the most common symptoms of food allergic reactions. However, the prevalence of these reactions is unknown. Because the onset of symptoms follows within minutes of ingesting the responsible allergen, the cause-and-effect nature of the reaction is often obvious to the patient. Consequently, most individuals with these reactions do not seek medical assistance nor do they necessarily relate them to their physicians. The foods most commonly incriminated in adults are fish, shellfish, nuts, and peanuts, and in children are eggs,

1630

milk, peanuts, and nuts. Acute urticaria secondary to contact with foods also is believed to be common, but again the true prevalence is unknown. Foods most often incriminated include raw meats, fish, vegetables, and fruits.[

208] [209] [210]

The significance of food hypersensitivity in chronic urticaria and angioedema (symptoms lasting more than 6 weeks) is subject to controversy. However, most evidence suggests that food allergy is rarely responsible for chronic symptoms. In one series, food allergy was implicated in only 1.4% of 554 adult patients with 211

212]

chronic urticaria,[ ] whereas a more recent series suggested that food allergy is frequently responsible for chronic urticaria.[ necessary to resolve this discrepancy.

Further controlled trials are

Diagnosis is based on the demonstration of food-specific IgE antibodies (skin test or RAST), resolution of skin symptoms with complete elimination of the putative food from the diet, and development of symptoms following challenge. Mixed IgE- and Non–IgE-mediated Food Hypersensitivity

Atopic dermatitis is one form of eczema that generally begins in early infancy and is characterized by typical distribution, extreme pruritus, chronically relapsing 213

course, and association with asthma and allergic rhinitis.[ ] The role of allergen-specific IgE antibodies in the pathogenesis of atopic dermatitis involves a number of cell types. Langerhans' cells, “professional” APCs in the skin, are increased in lesions of atopic dermatitis and display allergen-specific IgE antibodies on their surface.[ [215]

214]

The high-affinity receptors for IgE on Langerhans' cells, through bound antigen-specific IgE antibodies, play a unique role as “nontraditional” receptors.

These IgE-bearing Langerhans' cells are up to 1,000-times more efficient than non–IgE-bearing APCs at presenting allergen to T cells (primarily Th2 cells) and

activating T cell proliferation.[

216]

Infiltrating T lymphocytes in acute eczematous lesions express predominantly Th2 cytokines, IL-4, IL-5, and IL-13, whereas T

cells in chronic lesions express predominantly IL-5 and IL-13.[

217] [218] [219]

This is in contrast to classic “delayed” cell-mediated responses, such as the tuberculin

response, wherein cells express primarily mRNA for IFN-γ and IL-2, but not IL-4 and IL-5.[ allergy have been shown to induce sharp increases in plasma histamine 222 products.[ ]

136 concentrations,[ ]

220]

Oral food challenges in children with atopic dermatitis and food

activation of plasma eosinophils,[

221]

and elaboration of eosinophil

Milk-specific, cutaneous lymphocyte antigen-bearing T cells capable of homing to the skin have been identified in the circulation of children with IgE-

mediated milk-induced skin symptoms, cells not present in patients with milk-induced gastrointestinal disease. [

223]

In addition, food antigen–specific T cells have

been cloned from active skin lesions and normal skin of patients with atopic dermatitis (AD).[

224]

Peripheral blood mononuclear cells from food-allergic children 134

with atopic dermatitis elaborate an IgE-dependent HRF that primes basophils and possibly other IgE-bearing cells, and has been correlated with disease activity.[ ] The generation of HRF was associated with increased spontaneous basophil histamine release in vitro, increased basophil releasibility in vitro, and increased cutaneous hyperirritability (state of increased reactivity to a variety of minor nonspecific stimuli, such as detergents, heat, and cold). Spontaneous generation of HRF decreased to background levels over a 6- to 9-month period when the food allergens were identified and removed from the diet. In addition, spontaneous basophil histamine release returned to background levels, basophil releasability normalized, and cutaneous hyperirritability diminished. Using density gradient centrifugation, circulating eosinophils changed within 24 hours from a prechallenge normodense profile to a postchallenge hypodense profile in three patients studied to date, suggesting acute release of eosinophil-priming cytokines such as IL-5 or GM-CSF (unpublished). Biopsy specimens obtained at the site of a challenge-induced morbilliform lesion 8 to 14 hours after a positive challenge revealed eosinophil infiltration and deposition of major basic protein. Recently a murine model of foodinduced atopic dermatitis-like skin lesions was described, which should facilitate dissecting immunopathogenic mechanisms in this disorder. [

225]

In a recent study, 35% to 40% of children presenting to a university-based dermatologist with moderate to severe atopic dermatitis were found to be food allergic 15

226

following allergy evaluation and DBPCFCs.[ ] An earlier study demonstrated a direct correlation between disease severity and the likelihood of food allergy.[ ] In a follow-up study of 34 children with atopic dermatitis, 17 children with food allergy placed on an appropriate allergen elimination diet experienced marked, significant improvement in their eczematous rash over the 4-year follow-up period compared with food nonallergic children and food allergic children not adhering to an allergen elimination diet.[

227]

In a prospective, blinded, randomized, controlled trial of egg elimination in young children with atopic dermatitis and positive

228 al[ ]

RASTs for egg, Lever et demonstrated a significant decrease in both the area of affected skin and symptom scores in children avoiding egg compared with control subjects. In our series of nearly 500 children with atopic dermatitis and food allergy, approximately one third of symptomatic food hypersensitivities are “outgrown” in 2 to 3 years (Sampson et al; unpublished). The probability of developing tolerance appeared to depend on the food antigen responsible (e.g., development of tolerance to soy was common whereas development of tolerance to peanut was rare) and the stringency of allergen elimination. Results of prick skin tests often remained unchanged, but concentrations of allergen-specific IgE decreased significantly. The pathogenic role of food allergy in adults with atopic dermatitis remains to be elucidated. 229

230 231

232

Diagnosis is based on demonstration of food-specific IgE antibodies[ ] or allergen-specific patch tests,[ ] [ ] elimination diets, and oral food challenges.[ ] At the time of first evaluation, skin symptoms provoked by DBPCFCs generally consist of a markedly pruritic, erythematous, morbilliform rash, which develops in predilection sites for atopic dermatitis. Urticarial lesions are rarely seen. Interestingly, urticaria is frequently seen in follow-up challenges conducted 1 to 2 years later in patients who had adhered to an appropriate allergen elimination diet and had experienced clearing of their eczema, but who remained food sensitive. Although history had not suggested other food-induced complaints, food challenges provoked intestinal symptoms in nearly one half of patients (nausea, abdominal cramping, and vomiting and diarrhea), upper respiratory symptoms in about one third, especially laryngeal edema (sensation of itching and tightness in the throat, persistent throat clearing with dry hacking cough, and hoarseness), and wheezing in about 10% of positive challenges. If absorption studies are performed, such as lactuloserhamnose or

1631

lactulose-mannitol, most patients will be found to have malabsorption even though gastrointestinal complaints are minimal.[

167]

Non–IgE-mediated Food Hypersensitivity

Food-induced contact dermatitis is seen frequently among food handlers, especially among those who handle raw fish, shellfish, meats, and eggs.[ Patch tests can be used to confirm the diagnosis.

210] [233] [234]

Dermatitis herpetiformis (DH) is a chronic blistering skin disorder associated with a gluten-sensitive enteropathy. It is characterized by a chronic, intensely pruritic 235 236

papulovesicular rash symmetrically distributed over the extensor surfaces and buttocks.[ ] [ ] The histology of the intestinal lesion is virtually identical to that seen in celiac disease, although villous atrophy and inflammatory infiltrate are generally milder, and T cell lines isolated from intestinal biopsy specimens of patient with DH produce significantly more IL-4 than T cell lines isolated from patient with celiac disease. [

237]

Like patients with celiac disease, virtually all patients with 238]

DH have circulating IgA antibodies against tTGase, the quantity of which appears to correlate with the extent of the jejunal mucosal lesions.[

Diagnosis of DH depends on the presence of the characteristic skin lesions and demonstration of IgA deposition at the dermal-epidermal junction of the skin. Although many patients have minimal or no gastrointestinal complaints, biopsy of the small bowel generally reveals intestinal involvement. Elimination of gluten from the diet generally leads to resolution of skin symptoms and normalization of intestinal findings over several months. Administration of sulfones, the mainstay of therapy, leads to rapid resolution of skin symptoms but has virtually no effect on intestinal symptoms. Respiratory Food Hypersensitivity Reactions IgE-Mediated and Mixed IgE- and Non–IgE-mediated Food Hypersensitivity

Acute respiratory symptoms secondary to food allergy represent pure IgE-mediated reactions, whereas chronic respiratory symptoms represent a mix of IgEmediated symptoms (see Box 89-2 ). Both upper and lower respiratory reactions have been provoked by DBPCFC in some children, with spirometric measurements demonstrating significant decreases in forced vital capacity, forced expiratory volume in 1 second (FEV1 ), and maximal mid-expiratory flow (MMEF) during positive food challenges.[

239]

Rhinoconjunctivitis alone is infrequently a manifestation of food allergy, although it is commonly seen with other allergic symptoms. In a survey of 323 patients with 240

chronic rhinitis attending an allergy clinic, only two patients [0.6%] had nasal symptoms reproduced during blinded food challenges.[ ] Within minutes to 2 hours of ingestion, food allergens may induce typical signs and symptoms of rhinoconjunctivitis, including periocular erythema and pruritus, and tearing; and nasal congestion, pruritus, sneezing, and rhinorrhea. During DBPCFCs, nasal lavage fluid histamine was found to increase up to tenfold with the onset of nasal symptoms in some children.[

241]

Despite the notion that milk ingestion frequently leads to nasal congestion in young infants, only 0.08% to 0.2% of infants in three

epidemiologic surveys were found to develop nasal symptoms following a milk challenge.[

7] [8] [10]

Children with atopic disorders and food allergy frequently

experience nasal symptoms during oral food challenges. Of 480 children referred to Bock for evaluation of adverse food reactions,[

242]

about 16% experienced

respiratory symptoms (sneezing, rhinorrhea, nasal obstruction, wheezing, cough, ocular signs) during DBPCFCs, but only 2% were confined to the respiratory tract alone. Approximately 25% of 112 patients with histories of adverse food reactions occurring after 10 years of age were found to develop respiratory symptoms 243]

following oral challenge, with the majority being nasal symptoms secondary to fruit or vegetable sensitivities.[

In our studies of children with atopic dermatitis, nasal symptoms typically develop within 15 to 90 minutes of initiating the DBPCFC and last about 0.5 to 2 hours. Nasal and periocular pruritus are commonly followed by prolonged bursts of sneezing and copious rhinorrhea. Nasal fluid histamine and eosinophil cationic protein were found to increase significantly in children experiencing nasal symptoms. [

241]

Asthma alone is also an infrequent manifestation of food allergy. Although ingestion of food allergens are rarely the main aggravating factor in chronic asthma, there is some evidence to suggest that food antigens can provoke bronchial hyperreactivity.[ induced respiratory reactions were demonstrated in about 6% to 8.5% of

16] [244]

In surveys of children with asthma attending pulmonary clinics, food-

16 245 246 children.[ ] [ ] [ ]

Bock found that about 25% of 279 children referred for evaluation 247

with histories of food-induced wheezing and asthma actually experienced wheezing as one of their symptoms during DBPCFC.[ ] Similarly, a study of 88 children with atopic dermatitis and asthma revealed acute bronchospasm in 15% of patients (dyspnea, cough, wheezing) during DBPCFCs, with 8% demonstrating greater 248]

than a 20% decrease in FEV1 . [

In a study of 26 asthmatic patients with food allergies, 12 developed mild, acute bronchospasm (cough, wheezing) during

DBPCFCs, and 7 of the 12 (58%) had a significant increase in airway reactivity as demonstrated by a greater than twofold decrease in their postchallenge methacholine inhalation challenge PD20 FEV1 (provocation dose eliciting 20% decrease in FEV1 ).[

239]

Asthmatic reactions secondary to airborne food allergens 119]

have been reported in cases in which susceptible individuals are exposed to vapors or steam emitted from cooking food, such as fish,[ and garbanzo beans.

mollusks, crustacea, eggs,

Diagnosis of food-induced respiratory disease is based on patient history, evidence of food-specific IgE such as positive skin tests or RAST to food antigens, and oral food challenges. DBPCFCs following strict elimination of suspected food allergens are usually the only way to confirm the diagnosis of food-induced wheezing. Because many factors can exacerbate wheezing, elimination diets alone are not generally useful. Whether prechallenge and postchallenge studies of airway hyperreactivity should be performed remains to be established. Non–IgE-Mediated Food Hypersensitivity

Food-induced pulmonary hemosiderosis (Heiner's syndrome) is a syndrome of recurrent episodes of pneumonia associated with pulmonary infiltrates, hemosiderosis, 249

gastrointestinal blood loss, iron-deficiency anemia, and failure to thrive first reported in infants by Heiner.[ ] Hemosiderin-laden macrophages may be found in morning aspirates of the stomach or seen in biopsy specimens of the lung. This rare syndrome is most often associated with a non–IgE-mediated

1632

250]

hypersensitivity to cow's milk, but reactivity to egg and pork have also been reported.[

Although peripheral blood eosinophilia and multiple serum precipitins to

cow's milk are a relatively constant feature, the immunologic mechanisms responsible for this disorder are not known.[

251] [252] [253]

Both antigen-antibody

254

complexes and lymphocyte-mediated hypersensitivity responses to milk and less frequently other foods, such as buckwheat,[ ] are postulated in the immunopathogenesis of this disorder. However, this theory is based primarily on the presence of elevated serum levels of milk-specific IgG antibodies and the in vitro proliferative response of patient lymphocytes to milk antigen. Diagnosis is based on the elimination of the precipitating allergen and subsequent resolution of symptoms. The presence of characteristic laboratory data including precipitating antibodies to cow's milk (or the responsible antigen) is also considered necessary to make the diagnosis. Food-Induced Generalized Anaphylaxis 255 256

Food allergies are the single leading cause of generalized anaphylaxis seen in hospital emergency departments,[ ] [ ] accounting for about one third of cases seen. In addition to the cutaneous, respiratory, and gastrointestinal symptoms noted earlier, patients may develop cardiovascular symptoms including hypotension, vascular collapse, and cardiac dysrhythmias, presumably due to massive mast cell mediator release. However, most food-induced anaphylactic reactions are not associated 257 258

with major increases in serum β-tryptase.[ ] [ ] In a series of 12 fatal or near-fatal anaphylactic reactions, all patients experienced severe respiratory compromise, 10 of 12 experienced nausea and vomiting, and only 7 of 12 patients (or one of six fatal reactions) experienced cutaneous symptoms. About one third of patients 257

developed a biphasic reaction and one fourth experienced prolonged symptoms (typically 2 to 3 days).[ ] Factors that appear to be associated with severe reactions include the presence of asthma, a history of previous severe reactions, denial of symptoms, and failure to initiate therapy expeditiously. In a recent survey of 32 food259

induced anaphylactic deaths,[ ] a number of factors about these severe reactions were highlighted: anaphylactic reactions to foods affect both sexes equally, a large majority of the victims were adolescents or young adults, and virtually all these individuals were known to have food allergy with a prior history of some type of reaction to the food culprit that caused the fatal reaction. For 24 of 25 (96%) subjects for whom data were available, all but one was known to have asthma. Very few (3 of 32, or 10%) of the group had epinephrine available for use at the time of their reaction, and about 10% of subjects who received epinephrine in a timely fashion did not survive. Peanuts or tree nuts are responsible for the vast majority (94%) of the fatalities in the United States. Food-associated exercise-induced anaphylaxis is an unusual form of anaphylaxis that occurs only when the patient exercises within 2 to 4 hours of ingesting a food, 260 261 262 263 264

but in the absence of exercise, the patient can ingest the food without any apparent reaction.[ ] [ ] [ ] [ ] [ ] Patients generally have asthma and other atopic disorders, positive prick skin tests to the food that provokes their symptoms, and occasionally a history of reacting to the food when they were younger. This disorder appears to be more common in female than male patients, and is most prevalent in the late teens to mid-thirties. The exact mechanisms involved in this disorder is unknown, and several foods have been implicated including wheat, shellfish, fruit, milk, celery, and fish. Diagnosis is based on an unequivocal history of an isolated ingestion followed by the rapid onset (within 1 to 2 hours) of classic IgE-mediated symptoms and the demonstration of food-specific IgE antibodies by prick skin test or RAST. Lacking this evidence, a physician-supervised food challenge is generally warranted to ensure that the suspected food is truly responsible for the anaphylactic reaction. Such challenges should be done in a hospital setting by a physician experienced in the treatment of anaphylactic reactions. Other Food-Induced Hypersensitivity Reactions

Box 89-2 lists other symptoms ascribed to food allergy. However, convincing proof that any of these disorders is due to immunologic mechanisms is lacking. It should be noted that subjects experiencing a classic allergic reaction to a food may become irritable or lethargic following an IgE-mediated reaction. 265]

Ingestion of pasteurized, whole cow's milk by infants, especially those younger than 6 months of age, frequently leads to occult gastrointestinal blood loss,[

and

266 anemia.[ ]

occasionally to iron deficiency Substitution of infant formula (including cow's milk-derived formulas that have been subjected to more extensive heating) for whole cow's milk generally normalizes fecal blood loss within 3 days. Considerable interest has resurfaced on the possible role of food allergy in inflammatory bowel disease (Crohn's disease and ulcerative colitis). Although considerable circumstantial evidence makes such hypotheses attractive, convincing proof remains to be established. Given the presence of food antigen-immune complexes in many individuals following meals and the presence of mast cells in joint synovium, several workers have suggested that arthritis may be due to food hypersensitivity. However, exacerbation of arthritis has been established in only two cases associated with ingestion of a 267]

specific food by DBPCFCs.[

There are several disorders in which symptoms have been associated with reactivity to ingested foods, but in which an associated immune response is unclear. Some investigators have claimed that certain neurologic disorders are due to adverse food reactions and suggested that oligoantigenic diets may be useful in the treatment of migraines and epilepsy. One study found that 15% of 80 adult patients with frequent migraine headaches had clearing of their headache while on a specific food elimination diet, and they had exacerbation of their symptoms during double-blind challenge with a single food.[

268]

DIAGNOSING ADVERSE FOOD REACTIONS As with most medical disorders, the diagnostic approach to adverse food reactions begins with the medical history and physical examination. Based on information derived from these initial steps, various laboratory studies may be ordered ( Box 89-3 ).[

229]

The value of the medical history is largely dependent upon the patient's recollection of symptoms and the examiner's ability to differentiate between disorders provoked by food hypersensitivity and other etiologies ( Box 89-4 ). In some

1633

Box 89-3. Methods for Evaluating Food Allergic Reactions Medical history Diet diary Elimination diet Prick skin testing (PST) Radioallergosorbent tests (RAST) Basophil histamine release assays (BHR) Intestinal mast cell histamine release (IMCHR) Intragastral provocation under endoscopy (IPEC) Double-blind placebo-controlled food challenge (DBPCFC) Intestinal biopsy following allergen elimination and feeding

cases, it may be useful in diagnosing food allergy (e.g., acute events such as systemic anaphylaxis following isolated ingestion of shrimp). However, in several series, 269 270 271

less than 50% of reported food allergic reactions could be verified by DBPCFC. [ ] [ ] [ ] Information required to establish that a food allergic reaction occurred and to construct an appropriate blinded challenge at a later date include the following: (1) the food presumed to have provoked the reaction, (2) the quantity of the suspected food ingested, (3) the length of time between ingestion and development of symptoms, (4) whether similar symptoms developed on other occasions when the food was eaten, (5) whether other factors (e.g., exercise) are necessary to provoke a reaction, and (6) how long since the last reaction to the food occurred. Although any food may cause an allergic reaction, a few foods account for about 90% of reactions: adults—peanuts, nuts, fish, and shellfish; young children—egg, milk, peanuts, soy, and wheat (fish in Scandinavian countries). In chronic disorders (e.g., atopic dermatitis, asthma, chronic urticaria), history is often an unreliable indicator of the offending allergen. Diet diaries are frequently used as an adjunct to history. Patients are instructed to keep a chronological record of all foods ingested over a specified period of time, including items just placed in the mouth, such as chewing gum. Any symptoms experienced by the patient are also recorded. The diary is then reviewed to determine whether there are any relationships between foods ingested and symptoms experienced. Occasionally, this method detects an unrecognized association between a food and a patient's symptoms. As opposed to the medical history, it collects information on a prospective basis and is not as dependent on a patient's memory. Diagnostic elimination diets are frequently used both in diagnosis and management of adverse food reactions. Once certain foods are suspected of provoking allergic

disorders, they are completely omitted from the diet. The success of these diets depends on the identification of the correct allergens, the ability of the patient to maintain a diet completely free of all forms of the offending allergen, and the assumption that other factors do not provoke similar symptoms during the period of study. These conditions are rarely met. In a young infant reacting to cow's milk formula, resolution of symptoms following substitution with a soy formula or casein hydrolysate (Alimentum, Nutramigen) or elemental Box 89-4. Differential Diagnosis of Adverse Food Reactions Gastrointestinal disorders (vomiting and/or diarrhea) Structural abnormalities Hiatal hernia Pyloric stenosis Hirschsprung's disease Tracheoesophageal fistula Enzyme deficiencies (primary versus secondary) Disaccharidase deficiency (lactase, sucrase-isomaltase, glucosegalactose) Galactosemia Phenylketonuria Malignancy Other Pancreatic insufficiency (cystic fibrosis, Schwachman-Diamond syndrome) Gallbladder disease Peptic ulcer disease Contaminants and additives Flavorings and preservatives

Sodium metabisulfite Monosodium glutamate Nitrites/nitrates Dyes Tartrazine, ? other azo dyes Toxins Bacterial (Clostridium botulinum, Staphylococcus aureus) Fungal (aflatoxins, trichothecenes, ergot) Seafood associated Scombroid poisoning (tuna, mackerel) Ciguatera poisoning (grouper, snapper, barracuda) Saxitoxin (shellfish) Infectious organisms Bacteria (Salmonella, Shigella, Escherichia coli, Yersinia, Campylobacter) Parasites (Giardia, Trichinella) Virus (hepatitis, rotavirus, enterovirus) Mold antigens (?) Accidental contaminants Heavy metals (mercury, copper) Pesticides Antibiotics (penicillin)

Pharmacologic agents Caffeine (coffee, soft drinks) Theobromine (chocolate, tea) Histamine (fish, sauerkraut) Tryptamine (tomato, plum) Serotonin (banana, tomato) Phenylethylamine (chocolate) Tyramine (cheese, pickled herring) Glycosidal alkaloid solanine (potatoes) Alcohol Psychological reactions Food aversions Food phobias

Modified from Sampson HA: J Allergy Clin Immunol 78:212–219, 1986.

1634

formula (Neocate, EleCare) is highly suggestive of cow's milk or other food allergy, respectively, but also could be due to lactose intolerance. Although avoidance of suspected food allergens is recommended before blinded challenges, elimination diets alone are rarely diagnostic of food allergy, especially in chronic disorders such as atopic dermatitis or asthma.

Prick skin tests are highly reproducible [

272]

and frequently used to screen patients with suspected IgE-mediated food allergies. The criteria for interpreting prick skin

269 May[ ]

tests established by Bock and have proven useful to many investigators. Glycerinated food extracts (1:10 or 1:20) and appropriate positive (histamine) and negative (saline) controls are applied by the prick or puncture technique. Any food allergens eliciting a wheal at least 3•mm greater than the negative control is 273

considered positive; all others are considered negative. Recently Hill et al[ ] suggested that prick skin tests inducing mean wheal diameters greater than 8•mm are diagnostic of milk, egg, and peanut allergies. Acceptance of such criteria will require universal standardization of skin test reagents and testing procedures. Until then, positive skin tests indicate the possibility that the patient has symptomatic reactivity to the specific food (overall positive predictive accuracy is less than 50%), whereas negative skin tests essentially confirm the absence of IgE-mediated reactions (negative predictive accuracy is greater than 95%), if good quality food 242 270 274 275 276 277

extracts are used.[ ] [ ] [ ] [ ] [ ] [ ] Therefore the prick skin test may be considered an excellent means of excluding IgE-mediated food allergies but is only “suggestive” of the presence of clinical food allergies. There are some exceptions to this general statement: (1) IgE-mediated sensitivity to several fruits and vegetables (e.g., apples, oranges, bananas, pears, melons, potatoes, carrots, celery) are frequently not detected with commercially prepared reagents, presumably because of the lability of the responsible allergen [

153]

; (2) children younger than 1 year of age may have IgE-mediated food allergy in the absence of positive skin 278

tests, and infants younger than 2 years may have smaller wheals, presumably due to lack of skin reactivity[ ] ; and conversely, (3) a positive skin test to a food that when ingested in the absence of other foods provokes a serious systemic anaphylactic reaction may be considered diagnostic. 274

Intradermal skin testing is more sensitive than the prick skin test but is much less specific when compared to the DBPCFC.[ ] In Bock's study, no patient with a positive intradermal skin test to a food and a concomitant negative prick skin test had a positive DBPCFC. In addition, intradermal skin testing increases the risk of inducing a systemic reaction compared to prick skin testing, and therefore is not recommended. RASTs and similar in vitro assays (including ELISAs) for identifying food-specific IgE antibodies are also frequently used to screen for IgE-mediated food allergies. Although generally considered slightly less sensitive than skin tests, one study comparing Phadebas RAST with DBPCFCs found prick skin tests and RASTs to have 270

similar sensitivity and specificity to food challenge outcome when a Phadebas score of 3 or greater was considered positive.[ ] More recently, the use of a quantitative measurement of food-specific IgE antibodies (CAP System FEIA; Pharmacia-Upjohn Diagnostics) has been shown to be more predictive of symptomatic 279 280

IgE-mediated food allergy ( Table 89-3 ).[ ] [ ] Food-specific IgE levels exceeding the “diagnostic values” indicate that the patient is greater than 95% likely to experience an allergic reaction if he or she ingests the specific food. In addition, the IgE levels can be monitored and if they decrease to less than 2•kUA /L for egg or less than 7•kUA /L for milk, the patient should be rechallenged to determine whether he or she has “outgrown” the food allergy. Basophil histamine release (BHR) assays have generally been reserved for use in research settings, but the recently developed semiautomated methods that use small amounts of whole blood are being promoted for screening multiple food allergens. The use of whole blood in these assays should circumvent the problem of high 134]

spontaneous basophil histamine release seen in food allergic individuals continuing to ingest the responsible allergen.[

One such method was employed in a study 281

comparing BHR to prick skin tests, RASTs, food antigen-induced intestinal mast cell histamine release, and food challenges in suspected food allergic children.[ ] As demonstrated in earlier studies, the food allergen-induced BHR correlated most closely with RAST results. In the few children challenged in this study, the BHR did not appear to be any more predictive of clinical sensitivity than the prick skin test or RAST, but further studies are warranted.

TABLE 89-3 -- Food-Specific IgE Concentrations Predictive of Clinical Reactivity

Decision Point (kUA /L)

Allergen Egg (Infants ≤ 2 yr)

*

Sensitivity

7

Specificity

61

PPV 95

2 Milk (Infants < 1 yr)

†

15

NPV 98

38

95 57

94

5

95

53

95

Peanut

14

57

100

100

36

Fish

20

25

100

100

89

Soybean

30

44

94

73

82

Wheat

26

61

92

74

87

∼15

—

—

∼95

—

Tree nuts

‡

From Sampson HA: J Allergy Clin Immunol 107:891–896, 2001. PPV, Positive predictive value; NPV, negative predictive value. * From Boyano MT et al: Clin Exp Allergy 31:1464–1469, 2001. † From Garcia-Ara et al: J Allergy Clin Immunol 107:185–190, 2001. ‡ Tentative values.

1635

IPEC was first used for the diagnosis of food allergy more than 50 years ago.[

47]

In a recent study, small quantities of food extract (1:10 solution of food in normal 137

saline) were applied to the mucosa of the stomach, and the site was observed and scored.[ ] IPEC in 30 patients with food allergy previously documented by doubleblind challenge provoked reactions on the gastric mucosa in all patients. Tissue histamine and stainable mast cells in biopsies of the site were decreased compared to prechallenge samples. Prick skin tests and RASTs were positive in only about one half of these patients. Although the sensitivity of this test appears superior to skin tests, RASTs, and BHR, especially in patients with gastrointestinal allergy, the specificity of IPEC in skin test positive, nonreactive patients has not been evaluated. Furthermore, patients often experience systemic symptoms, suggesting this procedure may be no safer than oral challenges.

DBPCFC has been labeled the “gold standard” for the diagnosis of food allergies.[

229]

Many investigators have used DBPCFCs successfully in children and adults to

6 282 283 284 complaints.[ ] [ ] [ ] [ ]

examine a variety of food related The selection of foods to be tested in DBPCFCs is generally based on history and skin test (RAST) results. Foods unlikely to provoke food allergic reactions may be screened by open or single-blind challenges. However, for research purposes, positive reactions by these methods should be confirmed by DBPCFC, except perhaps in very young infants. Before undertaking DBPCFC, several factors need to be taken into consideration ( Box 89-5 ). Suspect foods should be eliminated for 7 to 14 days before the challenge, longer in some non–IgE-mediated gastrointestinal disorders. Antihistamines should be discontinued long enough to establish a normal histamine skin test, and other medications should be minimized to levels sufficient to prevent breakthrough of acute symptoms. In some asthmatic patients, short bursts of corticosteroids may be necessary to ensure adequate pulmonary reserve for testing (FEV1 > 70% predicted).

Box 89-5. Methods for Conducting DBPCFCs Eliminate suspected foods for 7 to 14 days before challenge. Discontinue antihistamines (positive histamine skin test); minimize other medications. Administer challenge to patient on an empty stomach. Administer an equal number of food and placebo challenges; randomized by noninterested party (dietitian). Use lyophilized food, which may be blinded in liquid or capsules. Administer 10•g over 1 hour; first dose is ≤ 250•mg. Use a standardized scoring system. Observe for 2 to 4 hours (IgE-mediated) or up to 4 days depending on type of reaction being studied. Have appropriate equipment available to treat systemic anaphylaxis. All negative challenges must be confirmed by an open feeding under

observation.

285

The food challenge is administered in the fasting state, starting with a dose unlikely to provoke symptoms (5 to 250•mg of lyophilized food). [ ] The dose is then doubled every 15 to 60 minutes, depending on the type of reaction suspected. Once the patient has tolerated 10•g of lyophilized food blinded in capsules or liquid, clinical reactivity is generally ruled out. If the blinded challenge is negative, however, it must be confirmed by an open feeding under observation to rule out the rare false-negative challenge. To control for a variety of potential confounding factors, an equal number of food antigen and placebo challenges are necessary; the order of administration should 286

be randomized by a noninterested third party (e.g., dietitian).[ ] A standardized scoring system should be used for all challenges. The length of observation is dependent upon the type of reaction suspected, for example, generally up to 2 hours for IgE-mediated reactions, up to 4 to 8 hours for milk-induced enterocolitis, 3 to 4 days for allergic eosinophilic gastroenteritis, and so on. Results of blinded challenges for objective signs and symptoms are rarely equivocal, but can be made more objective by monitoring a variety of laboratory parameters, such as plasma histamine, pulmonary function tests, and nasal airway resistance. As noted earlier, 257] [287]

however, serum β-tryptase is rarely shown to increase following food allergic reactions. [

DBPCFCs are the best means of controlling for the variability of chronic disorders (e.g., chronic urticaria, atopic dermatitis), any potential temporal effects, and acute exacerbations secondary to reducing or discontinuing medications. Other precipitating factors are controlled or (at least) neutralized, and psychogenic factors and patient or observer bias are eliminated. Although the diagnostic accuracy is excellent, rare false-negative challenges may occur when a patient receives insufficient material during the challenge or lyophilization alters the relevant allergenic epitopes (e.g., fish). Overall, the DBPCFC has proven to be the most accurate means of diagnosing food allergy at the present time. In non–IgE-mediated food allergies, such as dietary protein-induced enterocolitis, allergen challenges may require up to 0.3 to 0.6•g of food per kilogram of body 186 288

weight given in one or two doses.[ ] [ ] In other non–IgE-mediated disorders, such as allergic eosinophilic esophagitis or gastroenteritis, the patient may require several feedings over a 1- to 3-day period to elicit symptoms. In most IgE-mediated disorders, challenges to more foods often may be conducted every 1 to 2 days, whereas with non–IgE-mediated disorders, challenges to new foods often need to be at least 3 to 5 days apart. Oral food challenges should be conducted in a clinic or hospital setting, especially if an IgE-mediated reaction or dietary protein-induced enterocolitis is suspected, 282 289

and only when trained personnel and equipment for treating systemic anaphylaxis are immediately available.[ ] [ ] Patients with histories of life-threatening anaphylaxis should be challenged only when the causative antigen cannot be conclusively determined by history and laboratory testing, or the patient is believed to have “outgrown” the sensitivity. The evaluation of many “delayed” reactions (e.g., most IgE-negative gastrointestinal allergies) can be conducted safely on an outpatient basis. When patients' symptoms are largely subjective, three crossover trials with reactions developing only during the allergen challenge are necessary to conclude that a cause-and-effect relationship exists. Other studies may be conducted to exclude other disorders. As discussed earlier, non–IgE-mediated gastrointestinal

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hypersensitivity disorders generally require laboratory and endoscopic studies (with or without oral food challenges) to arrive at the correct diagnosis. In addition, several other disorders must be excluded (see Box 89-4 ). Practical Approach to Diagnosing Food Allergy The diagnosis of food allergy remains a clinical exercise dependent upon a careful history, selective skin tests, or

Figure 89-1 Evaluation of adverse food reactions. *Up to 2 wks for IgE-mediated reactions; up to 8 wks for non–IgE-mediated food hypersensitivity

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303. Fahy JV, Fleming E, Wong HH, et al: The effect of an anti-IgE monoclonal antibody on the early- and late-phase responses to allergen inhalation in asthmatic subjects, Am J Respir Crit Care Med 155:1828–1834, 1997. 304. Boulet LP, Chapman KR, Cote J, et al: Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response, Am J Respir Crit Care Med 155:1835–1840, 1997. 305. Casale TB, Bernstein IL, Busse W, et al: Use of anti-IgE humanized monoclonal antibody in ragweed-induced allergic rhinitis, J Allergy Clin Immunol 100: 100–110, 1997. 306. MacGlashan-DW, Bochner BS, Adelman DC, et al: Down-regulation of Fc(epsilon)RI expression on human basophils during in vivo treatment of atopic patients with anti-IgE antibody, J Immunol 158:1438–1445, 1997. 307. Li XM, Zhang TF, Huang CK, et al: Food allergy herbal formula-1 (FAHF-1) blocks peanut-induced anaphylaxis in a murine model, J Allergy Clin Immunol 108:639–646, 2001. 308. Sampson HA: Immunological approaches to the treatment of food allergy, Pediatr Allergy Immunol 12(suppl 14):91–96, 2001. Natural History of Food Hypersensitivity 309. Host A: Cow's milk protein allergy and intolerance in infancy, Pediatr Allergy Immunol 5(suppl 5):5–36, 1994. 310. Bock SA: The natural history of food sensitivity, J Allergy Clin Immunol 69:173–177, 1982. 311. Hill DJ, Firer MA, Ball G, Hosking CS: Recovery from milk allergy in early childhood: antibody study, J Pediatr 114:761–766, 1989. 312. Burks AW, James JM, Hiegel A, et al: Atopic dermatitis and food hypersensitivity reactions, J Pediatr 132(1):132–136, 1998. 313. Hill DJ, Firer MA, Shelton MJ, Hosking CS: Manifestations of milk allergy in infancy: clinical and immunological findings, J Pediatr 109: 270–276, 1986. Prophylaxis of Food Hypersensitivity 314. Grulee CG, Sanford HN: The influence of breast feeding and artificial feeding in infantile eczema, J Pediatr 9:223–225, 1936. 315. Zeiger R, Heller S: The development and prediction of atopy in high-risk children: follow-up at seven years in a prospective randomized study of combined maternal and infant food allergen avoidance, J Allergy Clin Immunol 95: 1179–1190, 1995. 316. Hanson DG: Ontogeny of orally induced tolerance to soluble proteins in mice; priming and tolerance in newborn, J Immunol 127:1518–1524, 1981. 317. Kajosaari M, Saarinen UM: Prophylaxis of atopic disease by six months; total solid food elimination, Arch Paediatr Scand 72:411–414, 1983. 318. Peanut allergy. London, Department of Health, 1998.

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Chapter 90 - Adverse Reactions to Food and Drug Additives

Robert K. Bush Steve L. Taylor Susan L. Hefle

Many substances are added to foods and pharmaceutical products for a wide variety of technical functions. The Food and Drug Administration (FDA) periodically publishes a small volume commonly known by the acronym EAFUS (Everything Added to Food in the United States), which lists 2922 substances allowed for 1

addition to foods in the United States.[ ] Similarly, pharmaceutical products contain an enormous number of additives or “inactive ingredients,” many of which can 2

be found in the U.S. Pharmacopeia. [ ] Approximately 773 chemical agents are approved for use in drug products by the U.S. FDA.[

3]

Food additives serve many functional purposes ranging from coloring and flavoring to nutrient and antimicrobial. A list of the major functional categories and examples within each category is provided in Box 90-1 . Many food ingredients serve more than one technical function; for example, sugar can serve as a sweetener, a bulking agent, and a preservative. With a few exceptions such as sugar, the intake of specific food additives is rather small. Although many additives may be used in any given food product, the additives are typically minor ingredients of the composite food. Additives in pharmaceuticals also serve numerous functions. Among the major functions of drug additives are coloring, flavoring, emulsifier, thickener, binding agent, and preservative ( Box 90-2 ). In contrast to foods, the inert ingredients of pharmaceutical products often comprise the majority of such products. The active pharmaceutical ingredients are frequently present as a small fraction of the total mass, although there are exceptions.

LABELING OF FOOD AND DRUG ADDITIVES

In the United States, food labels provide an ingredient statement that identifies virtually all of the intentional ingredients in the food product. Some standardized foods with well known and defined compositions are not required to have an ingredient statement. The ingredient statement lists all of the ingredients in the composite food product in descending order of predominance. A few groups of ingredients are allowed to be declared collectively without a listing of all of the components; examples include spices, natural flavors, and artificial flavors. Because numerous individual flavors are often added to a food product in extremely small quantities, the individual listing of these ingredients would add substantially to the length of the ingredient statement. Processing aids are also not required to be listed on ingredient statements. A processing aid is a substance that is used during processing but that serves no functional purpose in the finished product, such as boiler water additives used in the generation of steam or enzymes used in small quantities but inactivated during the process. Recent guidelines from the FDA indicate that any processing aid that is capable of eliciting an allergic reaction in a sensitive consumer should be declared on the ingredient statement. Labeling regulations differ in other parts of the world. In most countries, the guidelines for listing ingredients are less stringent than in the United States. The added ingredients in over-the-counter pharmaceutical products are declared in an ingredient statement on the package or in an insert. For prescription drugs, information on the added ingredients is provided in the package insert.

CLINICAL AND DIAGNOSTIC APPROACHES TO ADVERSE REACTIONS TO FOOD AND DRUG ADDITIVES Prevalence of Adverse Reactions to Food and Drug Additives Many food and drug additives have been reported to cause adverse reactions ranging from lethargy to severe asthma and anaphylaxis. Many of the food and drug additives listed in Boxes 90-1 and 90-2 have been reported to cause adverse reactions. However, many of these adverse reactions have not been verified by appropriate diagnostic challenge procedures. Several food and drug additives have been extensively studied, including synthetic colorants, sulfites, monosodium glutamate (MSG), aspartame, and benzoates. The prevalence of food allergies and food additive-induced sensitivities has been assessed in several large studies. In a Dutch study that started with a survey of 1483 Dutch adults and proceeded through clinical challenge trials, only three individuals were identified with food additive 4

sensitivities, [ ] amounting to 0.2% of the population. In a large Danish study of food additive-induced sensitivities that started with a survey of 4274 Danish school children and proceeded through clinical trials, an intolerance to food additives confirmed by double-blind challenge occurred in 2% of the children selected from the 5 6

7

survey on basis of atopic history but only 0.13% of the entire surveyed population.[ ] [ ] Previously, Young et al[ ] had evaluated the prevalence of sensitivities to food additives among a British population using a combination of a survey questionnaire given to 18,582 individuals and a series of mixed additive challenges conducted at home with self-reporting of symptoms. The researchers

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Box 90-1. Common Categories of Food Additives

Category

Example

Starches and complex carbohydrates

Corn starch, modified starch

Preservatives (antimicrobials)

Potassium sorbate, sodium benzoate

Preservatives (antioxidants)

Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT)

Preservatives (anti-browning)

Potassium metabisulfite, sulfur dioxide

Nutrients

Vitamin A, ferrous sulfate

Flavors

Ethyl vanillin, cinnamic aldehyde

Anticaking agents

Sodium aluminosilicate

Emulsifying agents

Lecithin

Sequestrants

Citric acid

Stabilizers and gums

Tragacanth gum, xanthan gum

Acidulents

Phosphoric acid, hydrochloric acid

Flavor enhancers

Monosodium glutamate

Colors

Tartrazine, annatto

Enzymes

Papain

Leavening agents

Sodium bicarbonate

7

estimated the prevalence of adverse reactions to food additives as 0.01% to 0.23%.[ ] Thus, food additive-induced sensitivities seem to occur rarely in the overall population. The frequency of adverse reactions to drug additives is also unknown but is less common than food additive reactions. Many agents causing adverse reactions in drugs exist as single case reports. Cellulose derivatives (carboxy methylcellulose) have caused three cases of anaphylactic shock when used in a corticosteroid

8

solution. [ ] Emulsifying agents such as gums (arabic and tragacanth) have caused urticaria when administered in antihistamines and corticosteroid tablets.[ Individuals have developed anaphylaxis to gelatin in vaccines, which has been confirmed by the presence of positive prick skin tests to the vaccine and the

8]

8 9

demonstration of specific IgE-antibodies to gelatin in the patient's serum.[ ] [ ] Potentially, egg albumin in erythromycin tablets could cause problems for the eggallergic individual, although this has not been reported. Sulfites used as antioxidants in drugs are well recognized as causing asthma and other adverse reactions as discussed later. Ethylenediamine is associated primarily with contact dermatitis, and sensitization occurs through cutaneous exposure. Ethylenediamine is also a 8

component of aminophylline and has produced urticaria, exfoliative dermatitis, and anaphylaxis in sensitized individuals.[ ] Likewise, thermosal, a preservative, 8

often acts as a contact sensitizer, but has caused anaphylaxis when administered in intravenous heparin solutions.[ ] Overall, the role of excipients in drugs as a cause of adverse reactions remains unknown.

Box 90-2. Common Categories of Drug Additives

Category

Example

Encapsulation agents

Carboxymethyl cellulose, gelatin

Emulsifying agents/solvents

Dextran, gums, egg albumin, polyoxyethylated castor oil

Synthetic sweetness

Saccharin, aspartame

Vehicles

Oils, alcohols, propylene glycol

Stabilizing agents/antioxidants

Ethylenediamine, sulfites

Dyes

Tartrazine, sunset yellow, ponceau red, xanthene dyes

Preservatives

Benzoates, parabens, thimerosal, cholorobutamel

Adjuvants

Aluminum hydroxide, zinc oxide

Food and pharmaceutical additives have been most frequently linked to chronic disorders such as asthma, chronic urticaria, and atopic dermatitis. Because these illnesses are chronic in many patients and tend to flare episodically, the establishment of a causative role for food and/or pharmaceutical additives in these conditions can be difficult. Recommended diagnostic approaches for patients with urticaria or asthma are discussed in subsequent paragraphs.

Additive Challenges in Patients with Urticaria Challenge studies are used to confirm an adverse reaction to a food or drug additive based on medical history. The initial history should ascertain the nature and severity of the symptoms and the time interval between exposure and the occurrence of urticaria. Further information should include whether symptoms are reproduced upon subsequent exposure to the food or drug additive in question. Also, an approximation of the amount consumed should be obtained as well as any association with extenuating circumstances such as exercise or consumption of alcohol. Patient Selection

The need for a diagnostic food or drug additive challenge depends on the circumstances. When an acute episode has occurred, the process becomes somewhat easier because a cause and effect relationship can be established. For the clinician, a diagnostic challenge for acute urticaria may be helpful in recommending appropriate avoidance procedures. For the researcher, a diagnostic challenge may be useful in establishing the role of a new agent in provoking urticaria and angioedema. In the case of chronic urticaria and angioedema, diagnostic challenge procedures become somewhat more difficult because the episodes of urticaria and angioedema occur sporadically over time, which makes false-positive results more likely. Epidemiologists may wish to challenge all available patients with chronic urticaria to determine the prevalence of food additive reactions. The clinician may wish to evaluate

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only those patients with histories suggestive of food additive-induced urticaria or evaluate those whose urticaria improves on an additive-free diet. Attention should be directed at the activity of the urticaria at the time of the study. If the urticaria is active, then patients are more likely to react in a positive manner to the challenge. If the urticarial reaction is quiescent, the patient is more likely to have a negative challenge. Procedures

Before initiating a challenge procedure, consideration must be given to the severity of the likely reaction. If a patient has experienced anaphylaxis, all necessary equipment to resuscitate the patient must be available. Likewise, trained personnel should be available and the procedure conducted in the morning when assistance is more likely to be at hand. The use of medications, particularly antihistamines, which can influence the result of the study, needs to be assessed. A decision whether to continue or withhold antihistamines needs to be made. Withholding antihistamines immediately before or within 24 hours of the challenge is likely to lead to false-positive reactions, whereas continuation of antihistamines may block the reaction and lead to false-negative reactions. The longer the interval between the last dose of antihistamine and the challenge, the more likely the subject is to experience breakthrough urticaria. This is particularly accentuated if the placebo is given first and in closer proximity

to the protective effects of the last antihistamine dose.[

10]

If it is decided to discontinue antihistamines, H1 antihistamines of short duration of activity should be

withheld for a minimum of 48 hours, hydroxyzine needs to be withheld for a minimum of 72 hours, and second-generation antihistamines should be withheld for up to 1 week. Oral corticosteroids can be continued and usually are not thought to affect the reaction. Alternatively, it may be appropriate to continue antihistamines at the lowest effective dose to stabilize the underlying chronic urticaria at a tolerable level. The dose of antihistamine chosen should provide improvement in patient comfort without completely abolishing the urticaria. This dose of antihistamine should not be sufficient to affect the results of the additive challenge (i.e., produce a false-negative result). Thus, it is important to establish a stable baseline before a food or drug additive challenge. Furthermore, it is imperative to establish the fact that the patient responds consistently to the challenge. This may require several blinded challenges with the active agent and placebo controls. A sample format for conducting a challenge procedure is shown in Figure 90-1 . It is recommended that the procedure be conducted in the morning after an overnight fast and the food or drug additive agent in question should be withheld at least 48 hours before the challenge. The initial challenge procedure may consist of a single blind challenge using a placebo control. If there is no objective finding such as appearance of urticaria on two occasions, then the presumptive diagnosis is not correct. If a positive response is observed on single-blind challenge, either on one or both occasions, then doubleblind challenges may be necessary for confirmation. If only subjective symptoms such as pruritus occur, it may be necessary to repeat the challenge with three active and three placebo challenges. If a patient has objective findings such as urticaria and angioedema, a double-blind, placebo-controlled challenge in which a positive response occurs (urticaria) with the active food or drug additive and a negative response occurs with the placebo, then a presumptive diagnosis of an adverse reaction to the additive can be made. However, because of the evanescent nature of urticaria, it may be appropriate to repeat the challenge with either additional active agent or placebo controls to ensure that the reaction is consistent and reproducible. If an equivocal reaction occurs with the placebo, a third challenge may be necessary with either active or placebo control. Dosages.

The maximal amounts of food additives used for placebo-controlled trials are listed in Box 90-3 . Where a provoking dose has been reported in the literature, it is usually recommended that the starting dose be approximately one log lower than the lowest dose known to cause a reaction. If the provoking dose is not known, it may be calculated based on estimates of the quantity ingested obtained from the patient's history. The doses are usually administered in opaque capsules; however, it may be appropriate to consider liquid or solution challenges if this more closely mimics the natural exposure. Once the starting dose is obtained, incremental twofold increasing doses are typically administered at times varying from 30 to 60 minutes. Controls.

Many reports of food additive-associated urticarial reactions include a surprising number of studies done without appropriate placebo controls. One also needs to consider the time interval between the last dose of antihistamine and the administration of a placebo in the challenge sequence. If the placebo is administered closer

to the last dose of an antihistamine, it may lead to false-positive responses when the protective effect of the antihistamine is lost during the course challenge. The use of multiple placebos randomly interspersed during the challenge enhances the design of the study and eliminates the bias of the loss of the protective effect of the antihistamine. It is also important to blind both subjects and observers to avoid unspoken signals of concern and apprehension that may lead to positive responses. When conducting double-blind, placebo-controlled trials over the course of 2 separate days, the placebo day must take place over the same length of time as the day in which the active challenge is given because urticaria may vary over the time of the challenge. Criteria for Positive Reactions.

11 12

In the case of urticaria, a scoring system based on the “Rule of Nines” may be used.[ ] [ ] In this system, the body is divided into areas of 9% which are then scored on a scale from 0 to 4. Zero equals no urticaria; 1 equals 25% of the areas involved; 2 equals 50% of the areas involved; 3 equals 75% of the areas involved; 4 equals confluence of urticaria in the areas involved. A score of 9 or a 30% increase from the baseline urticaria is considered a positive challenge. [

12]

If the blinded challenge procedures are negative, a final open feeding without reaction after negative challenge is considered appropriate to ensure the correctness of the diagnosis. Additive Challenges in Asthmatic Patients Procedures

As with urticaria, an initial history describing the symptoms and the interval between exposure and onset of symptoms is important in establishing the role of food or drug additives in asthma. Furthermore, reproducibility of symptoms

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Figure 90-1 Food/drug additive challenge information form. (From Bock SA, Sampson HA, Atkins FM, et al: J Allergy Clin Immunol 82:986, 1988.)

(From Bock SA, Sampson HA, Atkins FM, et al: J Allergy Clin Immunol 82:986, 1988.)

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Box 90-3. Suggested Maximum Doses for Additives Used in Challenge Protocols

Tartrazine/FD&C dyes

50•mg

Sulfites

200•mg

Monosodium glutamate

2.5–5•g

Aspartame

150•mg

Parabens/benzoates

100•mg

BHA/BHT

100•mg

Nitrates/nitrites

100•mg

From Simon RA: J Nutr 130:1063S, 2000.

when the patient is exposed to the offending agent, as well as the amount consumed, should be obtained. Before performing challenges, a general physical examination, pulmonary function tests, and, where applicable, skin tests or and radioallergosorbent tests (RASTs) may be helpful in determining whether an IgE mechanism is involved in the adverse reaction to the food or drug additive. Challenges may be used to confirm a diagnosis when the history is equivocal or only suggestive. Single-blind challenges can be used as a screening approach and if negative on two occasions, excludes the possibility that the food or drug additive is the culprit. Double-blind, placebo-controlled challenges may be necessary when the results of a single-blind challenge are equivocal or when conducting research protocols. Patient Selection

As in the case of urticaria, unstable asthma can lead to false-positive challenge results. Similarly, using medications that can block the reaction can lead to falsenegative studies. Asthma must be stable at the time of the challenge. For adults, an forced expiratory volume in 1 second (FEV1 ) of greater than 1.5•L or greater than 70% of the predicted or prior best FEV1 is required.[

12]

Occasionally, patients may need a course of oral or inhaled corticosteroids before the challenge to obtain

stability. For safety, proper precautions must be in place. Venous access may be necessary if there is a risk of anaphylaxis. Equipment and trained personnel for resuscitation and initiation of endotracheal intubation and mechanical ventilation should be available. Procedures should be conducted in the morning when assistance is most readily available. On the day of challenge, patients need to withhold inhaled or oral β2 -agonists. Inhaled salmeterol needs to be withheld for a minimum of 24 hours. Similarly, inhaled anticholinergics should be avoided on the day of the procedure. First-generation antihistamines should be withheld for 24 to 72 hours and chromones (cromolyn and nedocromil) for 48 hours. Second-generation antihistamines may need to be discontinued for 1 week. Patients can continue to receive theophylline, which usually does not interfere with the challenge. The effect of leukotriene modifiers has not been studied. Likewise, continuing or increasing doses of inhaled or oral corticosteroids may be necessary to maintain stable lung function because unstable pulmonary function can lead to false-positive results. Protocol Design

For initial screening or to establish the dosages for a double-blind procedure, a single-blind protocol conducted in an open fashion can be used. Because so few asthmatic patients are likely to react to an additive challenge (except for sulfites), a single-blind challenge simplifies the process. More importantly, a single-blind challenge can individualize doses, which is not possible when a double-blind protocol is used. During each step of an active challenge, the dose is incrementally increased twofold. If a patient has a 10% to 15% decrease in FEV1 with a dose of the agent, one can give a dose half-way between the usual incremental increase to lessen the risk of a severe reaction. This would not be possible with a double-blind protocol.

Dosages.

Food or drug additive challenges are usually conducted using opaque capsules but a liquid or solution challenge can be used if it more closely mimics the natural exposure. The maximum dosages recommended for several food and drug additives are listed in Box 90-3 . Protocols for sulfite challenges are listed in Boxes 90-4 and 90-5 . As in the case of

Box 90-4. Capsule and Neutral-Solution Metabisulfite Challenge

*

Preparing the patient and collecting preliminary data • Withhold short-acting aerosol sympathomimetics and cromolyn/ nedocromil sodium for 8 hours and short-acting antihistamines for 24 to 48 hours before pulmonary function testing. • Measure pulmonary function: Forced expiratory volume in 1 second (FEV1 ) must be greater than or equal to 70% of predicted normal value and greater than or equal to 1.5•L in adults. (Test contraindicated in patients with an FEV1 below those levels. Standards for children have not been defined.)

Performing the single-blind challenge • Administer placebo (powdered sucrose) in capsule form. Measure FEV1 . • Administer capsules containing 1, 5, 25, 50, 100, and 200•mg of potassium metabisulfite at 30-minute intervals. Measure FEV1 30 minutes after administering each dose and if the patient becomes symptomatic. • If no response, administer 1, 10, and 25•mg of potassium metabisulfite in water-sucrose solution at 30-minute intervals. Measure FEV1 30 minutes after each dose and if symptoms occur. Positive response is indicated by a decrease in FEV1 of 20% or more.

Performing the double-blind challenge • Perform challenge and placebo procedures on separate days, in random order. • Placebo day: Administer only sucrose in capsules and solution. Measure FEV1 30 minutes after each dose and if patient becomes symptomatic. • Challenge day: Same protocol as single-blind challenge day.

From Bush RK: J Respir Dis 8:23, 1987. Used with permission.

102 124

* Protocol used in the University of Wisconsin prevalence study.[ ] [ ] Perform this test only where the capability for managing severe asthmatic reactions exists.

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Box 90-5. Acid-Solution Metabisulfite Challenge

*

Preparing the patient and collecting preliminary data • Withhold aerosol sympathomimetics and cromolyn sodium for 8 hours and antihistamines for 24 to 48 hours before pulmonary function testing. • Measure pulmonary function: Forced expiratory volume in 1 second (FEV1 ) must be greater than or equal to 70% of predicted normal value and greater than or equal to 1.5•L in adults. (Test contraindicated in patients with an FEV1 below those levels. Standards for children

have not been defined.)

Performing the bisulfite challenge • Dissolve 0.1•mg of potassium metabisulfite in 20•ml of a sulfite-free lemonade crystal solution. Have the patient swish the solution around for 10 to 15 seconds, then swallow. • Measure FEV1 10 minutes after the first dose. Then, administer 0.5, †

1, 5, 10, 15, 25, 50, 75, and 100 per 20•ml of the solution at 10minute intervals. Measure FEV1 10 minutes after each incremental increase in dose. Positive response is signified by a decrease in FEV1 of 20% or more.

From Bush RK: J Respir Dis 8:23, 1987. Used with permission.

* Protocol investigated by the Bronchoprovocation Committee-American 124

Academy of Allergy, Asthma and Immunology.[ ] Perform this test only where the capability for managing severe asthmatic reactions exists. † Doses in excess of 100•mg are likely to produce nonspecific bronchial reactions in asthmatic patients due to the high levels of free SO2 that are generated.

challenges for urticaria, it is recommended that the starting dose be one log lower than the lowest dose known to cause a reaction. If the provoking dose is unknown, an estimate may be obtained based on the history of the person's exposure. Controls.

If a single-blind challenge is positive, the challenge should be repeated in a double-blind, placebo-controlled manner. The initial dose in the double-blind protocol can be based on the patient's previously established provoking dose or a onefold to twofold lower dose. It should be accompanied by at least two placebo challenges. The challenges with placebo should take place over the same time course as the length of the active challenge to exclude the possibility that decreases in pulmonary function are caused by the withholding of bronchodilator therapy.

Criteria for Positive Reactions.

A stable baseline pulmonary function is necessary to initiate the challenge. Three consistent FEV1 maneuvers with values within 5% of each other are necessary. Patients with unstable asthma may develop bronchoconstriction when performing forceful expiratory maneuvers. Therefore, several efforts may be necessary to establish a stable baseline. Pulmonary function is measured before the challenge and before the next scheduled dose or sooner if symptoms develop. A 20% or greater decrease in the FEV1 from the baseline is considered a positive response. For most clinical applications, the FEV1 is chosen as the gold standard for measurement of pulmonary function. This is because the FEV1 , when properly performed, is highly reproducible. Some investigators have used measurements of airway resistance or specific airway conductance for challenges in asthmatic patients. These measurements are technically more difficult and subject to more variability than the FEV1 ; therefore, they are more difficult to interpret and require larger changes in the magnitude of response to be considered positive. In patients who have failed to respond in a double-blind, placebo-controlled challenge, open feeding without reaction is necessary to verify the negative response.

FOOD AND DRUG ADDITIVES KNOWN OR SUSPECTED TO CAUSE ADVERSE REACTIONS Of the thousands of food and drug additives, relatively few have been identified as causing significant adverse reactions. Often, appropriate methods to substantiate the relationship between the additive and the reported adverse reaction have not been conducted. The following section describes in detail some of the most commonly implicated food and drug additives in adverse reactions. Synthetic Food Colorants (Dyes) Numerous synthetic colorants are allowed for use in foods.[

13]

These synthetic colorants fall into two general structural categories: the azo dyes and the non-azo 14]

dyes. Adverse reactions have been reported to only a few of these colorants, primarily tartrazine, also known as FD&C Yellow #5.[ Tartrazine

A number of studies have been conducted on the role of tartrazine in various food sensitivity reactions but especially in asthma and chronic urticaria. Although many of these studies have purported to show a causative role for tartrazine in asthma, in chronic urticaria, and, occasionally, in other food sensitivities, a cause-and-effect 14 15

14 15

role for tartrazine in these illnesses has not been established. [ ] [ ] Stevenson et al[ ] [ ] reviewed the literature on tartrazine sensitivity and concluded that tartrazine-induced asthma does not occur even among aspirin-sensitive asthmatics who were thought to be at higher risk for tartrazine sensitivity. Previously, they 14

14 15

had concluded that tartrazine was a rare cause of chronic urticaria.[ ] Stevenson et al[ ] [ ] argued that several methodologic flaws in earlier studies were responsible for the positive associations in these studies between tartrazine and either asthma or urticaria.

16

Tartrazine has been frequently linked to chronic urticaria. Lockey [ ] was the first to associate tartrazine with urticaria by identifying three patients who had experienced urticaria following the ingestion of yellow color-coated drugs. He “confirmed” the reactions by conducting nonblinded, uncontrolled trials with dilute solutions of tartrazine. Since then, many more studies have been conducted on the role of tartrazine in urticaria. Several methodologic flaws make interpretation of the results of clinical trials of the role of tartrazine in chronic urticaria difficult. Many of the positive reactions observed with high frequency in some of these studies are likely false-positive reactions resulting from the order effect of always administering the placebo first and the high likelihood of breakthrough urticaria from the withdrawal of antihistamines. Many of the clinical trials were conducted with single-blind or open challenges, which increases the difficulty in interpretation of the results, especially if medications are withheld. Several of the studies were conducted with a double-blind challenge design, but even these studies could have been

1651

compromised if the patients always received placebo before tartrazine. However, the percentage of positive responders was lower in the double-blind challenge trials. In virtually all of the numerous studies on the role of tartrazine in chronic urticaria, antihistamines were withheld or no information was provided on medication status during challenge, which raises the question of breakthrough urticaria. Several studies had other complicating factors. A mixture of colors was used in several of the studies so that the positive reactions cannot be attributed to any one substance. 14

Stevenson et al[ ] conducted the first double-blind, placebo-controlled challenge trial of tartrazine in chronic urticaria in which antihistamines were not withheld. Their patients had a history suggestive of possible tartrazine sensitivity. Only 1 of 24 patients developed urticaria on single-blind challenge, and this reaction was confirmed on double-blind challenge. A comparatively large challenge dose of 50•mg of tartrazine was used to provoke this response, whereas earlier studies by other investigators had used lower amounts of tartrazine ranging from 0.1 to 25•mg. This same group has more recently indicated that 0 of 65 patients with chronic urticaria reacted to double-blind challenges with 50•mg of tartrazine.[ abstract, antihistamines were likely not withheld.

17]

Even though the status of antihistamine therapy in these 65 patients was not reported in this

18

The study by Murdoch et al[ ] illustrates further the challenges involved in evaluating the role of tartrazine in chronic urticaria. They identified three patients who reacted to a panel of azo dyes (tartrazine, sunset yellow, amaranth, and carmoisine); two of the patients reacted to tartrazine alone in a double-blind, placebo18

controlled, in-hospital challenge. Murdoch et al[ ] also documented the release of histamine and its metabolites into the plasma and urine during these urticarial flares, adding some credibility to a diagnosis of tartrazine-induced urticaria. However, medications were withheld from one of these patients during challenge, and no information is presented on the medication status of the other patient. Perhaps these positive reactions were simply breakthrough urticaria. Furthermore, one of these 19

patients required large provoking doses of tartrazine, either 50 or 150•mg. Murdoch et al[ ] subsequently demonstrated that even normal subjects release histamine into plasma and urine when subjected to such large and unrealistic challenge doses with tartrazine. In summary, despite numerous reports that tartrazine can provoke chronic urticaria, convincing evidence is lacking.

In 1958, Speer[

20]

stated in a textbook on childhood asthma that tartrazine caused asthma in some children. He presented no data to support this assertion. Later, 21 22

Sampter and Beers[ ] [ ] attempted to link aspirin intolerance to tartrazine-induced asthma. However, their studies were largely observational, and inferences on a cause-and-effect relationship cannot be drawn from these observations. Numerous studies have since been conducted on tartrazine-induced asthma. Similarly to the situation with chronic urticaria, the interpretation of the many studies of the role of tartrazine in causation of asthma is difficult. Again, methodologic flaws occurred in many of these studies. In several studies of tartrazine-induced asthma, medications were withheld, leading to concerns that the withdrawal of 15

medications was a significant contributing factor in the development of asthma.[ ] When critical medications were continued during challenge, the frequency of positive asthmatic responses to tartrazine was almost zero in most of the studies. Only a few studies have identified more than one case of tartrazine-induced asthma 23 24

25

while medications were continued and double-blind challenges were used.[ ] [ ] Weber et al[ ] demonstrated that the continued use of bronchodilators during challenge of aspirin-sensitive asthmatic patients was critical to the avoidance of false-positive responses. 15

The definitive study by Stevenson et al[ ] involved 43 aspirin-tolerant and 194 aspirin-sensitive asthmatic patients. Beta agonists, cromolyn, and antihistamines were withheld but theophylline, bronchodilators, and inhaled and systemic corticosteroids were continued during challenge. The studies were single-blind, but all positive reactions were confirmed by double-blind challenges. None of their patients reacted to tartrazine at doses up to 50•mg. This evidence suggests that tartrazine does not provoke asthma. 4 14

Several cases of angioedema related to tartrazine have been reported, [ ] [ ] but there were only a small number of patients and the status of medications before challenge was not reported. There is no compelling evidence for a role of tartrazine in angioedema. Tartrazine has been linked to atopic dermatitis in only a few studies.[ [28]

6] [26] [27] [28] [29]

However, mixtures of food colors were used in several of these studies,[

6] [26]

so positive results cannot necessarily be attributed to tartrazine. Also, the medication status of patients involved in these studies was not revealed. Devlin and 27

David[ ] performed a well controlled study using multiple double-blind, placebo-controlled challenges with tartrazine (50•mg) in 12 children with atopic dermatitis and a history suggestive of possible tartrazine provocation. In only one of these patients did the multiple tartrazine challenges correspond consistently to the highest symptom score. It seems probable that this single patient is indeed sensitive to tartrazine, although the challenge dose is high compared with typical dietary 29

exposures. Worm et al[ ] assessed the release of sulfidoleukotrienes from peripheral leukocytes in nine patients with atopic dermatitis exacerbated by oral challenge with food additives, nine patients with atopic dermatitis not exacerbated by oral challenge with food additives, and 10 control subjects. Tartrazine elicited increased sulfidoleukotriene release in 0/10 control subjects, 1/9 atopic dermatitis patients with negative oral challenges to the food additive mixture, and 3/9 atopic dermatitis patients with positive oral challenges to the food additive mixture. 30]

Michaelsson et al[

demonstrated that tartrazine (1 to 10•mg) provoked purpura in five of seven patients. Four additional cases of tartrazine-induced purpura have

been noted in the medical literature: two by Thune and Granholt[

31]

and one each by Criep[

32]

33]

and by Kubba and Champion.[ 7

The overall prevalence of tartrazine sensitivity in the population cannot be estimated from current information. Young et al[ ] estimated the prevalence of sensitivity to tartrazine, amaranth, sunset yellow, and carmoisine to be 0% to 0.12% of their large survey population, but their challenge proce-dures left much to be desired,

including the use of self-reporting of symptoms and use of mixed additives for challenges. However, this study would seem to indicate that the prevalence of tartrazine sensitivity is quite low. Sunset Yellow

Sunset yellow, also known as FD&C Yellow #6, has been much less commonly linked to food sensitivities than tartrazine. Sunset yellow has been implicated in several

1652

isolated cases of gastrointestinal illness confirmed by blinded challenges.[

34] [35]

Like tartrazine, sunset yellow has also been implicated in urticaria and angioedema. 17

However, these studies are subject to many of the same methodologic flaws previously discussed regarding tartrazine. Simon et al[ ] failed to identify a single reactor to sunset yellow among a total of 65 patients with chronic urticaria who were subjected to double-blind, placebo-controlled food challenge (DBPCFC) under 28

conditions in which antihistamines were likely not withheld. Worm et al[ ] evaluated the role of a group of food additives in the possible provocation of atopic dermatitis and indicated that 6 of 15 patients experienced worsening of their atopic dermatitis on double-blind challenge with an additive mixture, but sunset yellow 36

was only 1 of 22 common food additives included in the challenge trial. Sunset yellow has also been implicated in asthma.[ ] However, the asthmatic reactions were not confirmed clinically with actual measurements of lung function and the status of medications in these children was not revealed. Sunset yellow has been implicated in one case of purpura[ appears to be very

30]

and a single case of orofacial granulomatosis.[

37]

As noted earlier, the prevalence of sunset yellow sensitivity in the population

7 low.[ ]

Other Synthetic Colors

Several other synthetic food colors have occasionally been implicated in urticaria, angioedema, asthma, and atopic dermatitis. These synthetic colors include amaranth (FD&C Red #2), erythrosine (FD&C Red #3), brilliant blue (FD&C Blue #1), ponceau 4R, carmoisine, quinoline yellow, patent blue, azorubin, new coccine, indigo carmine (FD&C Blue #2), brilliant black BN, and fast green (FD&C Green #3). The studies are plagued by the same methodologic flaws previously discussed for tartrazine. Thus, there is no compelling evidence for the involvement of these colors in urticaria, angioedema, asthma, or atopic dermatitis. New coccine has been implicated in three cases of purpura,[

30]

but this finding appears to be an isolated report. A single case of leukocytoclastic vasculitis has been

ascribed to ponceau red 4R and confirmed by a placebo-controlled oral challenge with 50•mg of the dye.[ to ingestion of carmoisine. [

37]

38]

A case of orofacial granulomatosis has also been linked

7

Young et al[ ] estimated the prevalence of sensitivities to green S, quinoline yellow, and indigo carmine was 0 to 0.11%.

Natural Food Colorants Many natural colorants are allowed for use in foods. They include annatto, carmine, carotene, turmeric, paprika, beet extract, and grape skin extract. These types of

colorants are not used to any extent in pharmaceutical applications. Several studies have reported positive reactions following challenges with mixtures of natural 6 39 40

41 42

colors[ ] [ ] [ ] or mixtures of natural and synthetic colors.[ ] [ ] The natural colorants involved in these challenges were annatto, betanin, curcumin, turmeric, βcarotene, canthaxanthin, and beet extract. The adverse reactions were asthma, urticaria, atopic dermatitis, colic, and vomiting. No one color can be identified as the causative factor when challenges are conducted with mixtures. Annatto

Annatto is obtained as an extract from the seeds of the fruit of the Central and South American tree, Bixa orellana. Bixin, the principal pigment in annatto, is a carotenoid. The extracts are red in color but annatto is often used to impart an orange or deep-yellow color to the finished food. 43

Nish et al[ ] reported a possible IgE-mediated allergic reaction to annatto extract. The patient experienced angioedema, urticaria, and severe hypotension within 20 minutes of ingesting a breakfast cereal containing annatto. The patient had a strongly positive skin test to annatto extract, and an IgE-binding protein was identified through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with immunoblotting. Because annatto extract is derived from a seed, the presence of proteins in the extracts is likely. IgE-mediated allergies to annatto proteins are possible, although this is the only reported case in the medical literature. 7

Young et al[ ] estimated the prevalence of annatto sensitivity at 0.01% to 0.07%. Carmine

Carmine and cochineal extract are derived from dried female insects of the species, Dactylopius coccus Costa, which lives as a parasite on the prickly pear cactus. An aqueous-alcoholic extract of the dried insects is made and concentrated, by removal of the alcohol, to obtain the color additive, cochineal extract. The coloring principle of cochineal extract is carminic acid. Carmine is the aluminum or calcium-aluminum lake of the coloring principles, primarily carminic acid, obtained by aqueous extraction of cochineal. Carmine and cochineal extract have a red color. 44 45

46

Carmine is widely used in cosmetics, but only a few cases of dermatologic reactions have been attributed to it.[ ] [ ] Park[ ] reported a case of severe anaphylactic shock possibly linked to the cutaneous use of carmine. A soldier involved in a casualty simulation drill was smeared with a make-up stick to simulate burns. An immediate anaphylactic response ensued characterized by severe hypotension and tachycardia. Unfortunately, no follow-up was done on this patient to confirm the role of carmine in this case. Carmine-associated occupational asthma has been reported[

47] [48]

and the affected individuals also reacted to ingestion or oral challenges with carmine. In one of

47 studies,[ ]

these two individuals were subjected to oral challenge with carmine; one responded with asthma and gastrointestinal upset after challenge with 1•ml of cochineal extract diluted in 100•ml of water. The other experienced asthma after drinking Campari, a beverage that contains carmine. Numerous cases of anaphylaxis 45 47 48 49 50 51 52 53

45

have been reported from the ingestion of carmine-containing foods or beverages.[ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] Kagi et al[ ] described a single individual with anaphylaxis characterized by rhinitis, asthma, urticaria, and multiple gastrointestinal complaints after ingestion of Campari-orange. This individ-ual had positive skin prick tests to the Campari beverage, carmine, and carmine-containing cosmetics, indicating a possible IgE-mediated reaction. Another case of probable IgE-mediated

allergy to carmine has been described in an individual who reacted after ingestion of a carmine-containing yogurt with urticaria, angioedema, and asthma. [ [49]

49]

A

[50]

histamine-release assay using the patient's basophils was also positive, another indication of an IgE-mediated reaction. Wuthrich et al reported five cases of anaphylaxis associated with the ingestion of Campari and established the likelihood of an IgE-mediated mechanism through demonstrations of positive skin prick tests and RASTs. Baldwin et al[

51]

described the case of

1653

a patient who experienced severe anaphylaxis after the ingestion of a carmine-colored popsicle. In addition to a positive skin prick test with carmine, these investigators demonstrated a positive passive transfer reaction in this patient's husband, which further establishes an IgE-mediated mechanism.[

51]

This same group

52 foods.[ ]

of investigators reported two further cases of carmine-induced anaphylaxis associated with the likely ingestion of carmine-containing Both patients had positive skin prick tests to carmine. Because carmine is obtained from an extract of insect bodies, it is likely to contain proteins and could elicit IgE-mediated 53]

reactions in rare cases such as these. Several IgE-binding proteins have been identified in carmine using sera from carmine-sensitive patients.[ Sulfites

Sulfites or sulfiting agents used as food or drug additives include sulfur dioxide (SO2 ), and inorganic sulfite salts, sodium and potassium metabisulfite (Na2 S2 O5 , K2 S2 O5 ), sodium and potassium bisulfite (NaHSO3 , KHSO3 ), and sodium sulfite (Na2 SO3 ). Sulfites have been used for centuries as food additives but they can also occur naturally in many foods, particularly fermented beverages such as wines.[

54]

Uses in Foods

Sulfites are added to many different types of foods for technical purposes ( Box 90-6 ). These include inhibition of enzymatic browning, inhibition of nonenzymatic browning, antimicrobial effects, dough conditioning actions, antioxidant effects, bleaching applications, and a host of processing aids.

Box 90-6. Technical Attributes of Sulfites in Foods

Technical Attribute

Examples of Specific Food Applications

Inhibition of enzymatic browning

Fresh fruits and vegetables Salads

*

*

Guacamole

*

Shrimp (black spot formation) Pre-peeled raw potatoes Inhibition of nonenzymatic browning

Dehydrated potatoes Other dehydrated vegetables Dried fruits

Antimicrobial actions

Wines Corn wet milling to make cornstarch, corn syrup

Dough conditioning

Frozen pie crust Frozen pizza crust

Antioxidant action

No major U.S. applications

Bleaching effect

Maraschino cherries Hominy

From Taylor SL, Bush RK, Nordlee JA: Sulfites. In Metcalfe DD, Sampson HA, Simon RA, editors: Food allergy. Adverse reactions to foods and food additives, Boston, 1996, Blackwell Scientific, p 348. Used with permission. * No longer allowed by U.S. Food and Drug Administration.

In many products, sulfites serve more than one purpose. For example, in wines, the function is to prevent bacterial growth and acetic acid formation and to prevent browning.[

54]

Because of the wide variety of applications of sulfites as food additives, a wide range of use levels and residual sulfite concentrations can be found in foods ( Box 907 ). These residual concentrations can range from an undetectable level (less than 10•ppm) to greater than 2000•ppm (mg of SO2 equivalents per kg of food). Some of the more highly sulfited foods pose the greatest hazard to sulfite-sensitive individuals. Sulfites are extremely reactive in food systems. A dynamic equilibrium exists between free sulfites and many bound forms of sulfites. The fate of sulfites added to 54]

foods is variable and depends on the nature of each individual food.[ 55]

acid, bisulfite ion, or sulfite ion.[

Sulfites readily dissolve in water and depending on the pH of the medium can exist as sulfuric

At acidic pHs (less than pH 4.0), sulfur dioxide can be released as a gas from food or solutions containing sulfites. 54

Sulfites react with a variety of food constituents including aldehydes, ketones, reducing sugars, proteins, amino acids, and other constituents.[ ] Some of these reactions are readily reversible whereas others, for example, that between acetaldehyde and sulfites to form acetaldehyde hydroxysulfonate, are virtually irreversible. [56]

Before its use was eliminated by the FDA in 1986, high concentrations of sulfite (500 to 1000•ppm) were recommended to prevent enzymatic browning in lettuce. Because lettuce is comprised mostly of cellulose and water, there was little chance for sulfite to react with other food components. Consequently, most sulfite 57

added to lettuce exists as free inorganic sulfite.[ ] Lettuce is unique in this regard because most other foods contain substances that readily react with sulfites. In most foods, bound forms of sulfite predominate. The presence of free sulfite in lettuce may explain why sulfite-sensitive individuals reacted so vigorously to sulfited lettuce in salad bars before its use was banned. Uses in Drugs 3 58

Sulfites are added to a number of pharmaceutical products.[ ] [ ] Box 90-8 contains a partial list of drugs that may be used by asthmatic patients that contain sulfites. Because of concerns over sulfite-induced asthma, sulfites have been removed from many drugs, especially those intended for asthmatic patients. Sulfites have two primary uses as drug ingredients: their antioxidant properties and the prevention of nonenzymatic browning. The nonenzymatic browning reactions involve reducing sugars that interact with amino acids or amines. These reactions can occur in enteral feeding solutions and dextrose solutions that can be prevented by addition of sulfites. Epinephrine can undergo similar reactions that would diminish its potency; therefore, sulfites are routinely added to epinephrine. The level of use of sulfites in pharmaceutical products varies from 0.1% to 1.0% although a few products may contain higher levels. In a few cases, paradoxical 59 60

bronchoconstriction has occurred where sulfiting agents have been used to stabilize bronchodilator medications.[ ] [ ] However, epinephrine has not been reported to have any paradoxical effects, even though it contains sulfites; therefore, epinephrine should never be denied or avoided by a sulfite-sensitive asthmatic patient, 58] [61]

because it may prove lifesaving.[

Aside

1654

Box 90-7. Estimated Total SO2 Level as Consumed for Some Sulfited Foods *

≥100•ppm Dried fruit (excluding dark raisins and prunes) Lemon juice (nonfrozen) Lime juice (nonfrozen) Wine Molasses Sauerkraut juice Grape juice (white, white sparkling, pink sparkling, red sparkling) Pickled cocktail onions

50 to 99.9•ppm Dried potatoes Wine vinegar Gravies, sauces Fruit topping

Maraschino cherries

10.1 to 49.9•ppm Pectin Shrimp (fresh) Corn syrup Sauerkraut Pickled peppers Pickles/relishes Corn starch Hominy Frozen potatoes Maple syrup Imported jams and jellies Fresh mushrooms

≤10•ppm Malt vinegar Canned potatoes Beer Dry soup mix Soft drinks

Instant tea Pizza dough (frozen) Pie dough Sugar (especially beet sugar) Gelatin Coconut Fresh fruit salad Domestic jams and jellies Crackers Cookies Grapes High fructose corn syrup

* From Taylor SL, Bush RK, Nordlee JA: Sulfites. In Metcalfe DD, Sampson HA, Simon RA, editors: Food allergy. Adverse reactions to foods and food additives, Boston, 1996, Blackwell Scientific, p 349. Used with permission.

Box 90-8. Some Antiasthmatic Preparations Containing Sulfites

Epinephrine

Adrenaline, Parke-Davis Ana-Kit, Hollister-Stier Epi-Pen, Dey

Isoetharine HCl

Isoetharine HCl, Roxane

Isoproterenol

Isoproterenol parenteral solution (injectable), Abbott (injectable)

Hydrocortisone

Hydrocortone phosphate (injectable) Merck

Dexamethasone

Decadron LA (injectable), Merck Decadron phosphate (injectable), Merck Dexone (injectable), Keene

Modified from Taylor SL, Bush RK, Nordlee JA: Sulfites. In Metcalfe DD, Sampson HA, Simon RA, editors: Food allergy. Adverse reactions to foods and food additives, Boston, 1996, Blackwell Scientific, p 347. Used with permission.

from epinephrine, sulfite-sensitive individuals should be alerted to the possible presence of sulfites in medications and seek alternative formulations. Clinical Manifestations of Adverse Reactions to Sulfites 55

A host of adverse reactions and symptoms have been attributed to sulfiting agents.[ ] Many of these responses have not been documented by appropriate diagnostic challenges. For normal individuals, exposure to sulfites appears to pose little risk. Toxicity studies in normal individuals showed that ingestion of up to 400•mg of 62

63

sulfite daily for 25 days did not have any ill effect.[ ] Adverse reactions suggestive of a hypersensitivity response have been observed. Epstein [ ] described a patient who developed contact sensitivity through exposure to sulfiting agents used in a restaurant. The delayed hypersensitivity was confirmed by patch testing. 64]

Belchi-Hernandez[

described an individual with sulfite-induced urticaria provoked by ingestion of sulfite in foods and beverages. The role of sulfites in the

reaction was confirmed by a double-blind, placebo-controlled challenge. Several other cases of angioedema and urticaria have been reported.[ Wüthrich[

68]

65] [66] [67] [68] [69] [70]

conducted single-blind, placebo-controlled challenges with sodium bisulfite in 245 patients with suspected sulfite sensitivity. Seventeen patients 69]

developed urticaria or angioedema in response to these challenges. Wüthrich et al[

reported a case of acute intermittent urticaria with an associated vasculitis due 70

to sulfites based on a placebo-controlled, single-blind challenge. Confirmatory double-blind challenges were not performed. Simon,[ ] using a rigorous blinded, placebo-controlled trial and objectives criteria for positive reactions was unable to demonstrate positive reactions to encapsulated metabisulfite (200•mg maximum dose) in 75 patients with chronic urticaria, anaphylaxis, or both in patients with a history suggestive of sulfite sensitivity. Whereas some individuals may develop urticaria or anaphylactoid reactions as adverse reactions to sulfites, the frequency with which these reactions occur, at best, is extremely rare and requires confirmation by rigorous double-blinded challenges.

1655

Anaphylactic-like (anaphylactoid) events have been described in several individuals, although appropriate confirmatory testing was not performed in many cases. 71]

Prenner and Stevens[

described a nonasthmatic individual who developed urticaria and angioedema after eating sulfited foods in a restaurant. A non–placebo-

controlled, single-blind challenge with sodium metabisulfite reproduced some of the patients symptoms. Sokol and Hydick[

72]

identified an individual with sulfite-

induced anaphylaxis. This individual had a positive skin test to sulfite, and histamine was released from the patient's basophils on incubation with sulfites.[

72]

In

73 al[ ]

addition, multiple single-blind, placebo-controlled oral challenges reproduced the patient's symptoms. Yang et described three patients with systemic anaphylaxis symptoms that were confirmed by sulfite challenges. These individuals had positive skin tests to sulfites and two of the three had positive PrausnitzKustner transfer tests. 74

Systemic adverse reactions have been attributed to intravenous and inhalation administration of pharmaceuticals containing sulfiting agents.[ ] A patient who experienced flushing and urticaria from the intravenous use of metoclopramide, which contained a sulfiting agent, underwent placebo-controlled oral challenge with 74

75

sodium metabisulfite and developed flushing and a decline in pulmonary function. [ ] Schwartz[ ] described two nonasthmatic individuals who developed abdominal distress and hypertension, which was confirmed by oral challenge with potassium sulfite with a negative placebo-controlled challenge. Sulfiting agents do not appear to play a role in patients with idiopathic anaphylaxis.[

76] [77]

Similarly, sulfites do not appear to enhance the likelihood of anaphylaxis

[77]

in patients with systemic mastocytosis. Overall, the risk of adverse reactions to sulfiting agents for nonatopic, nonasthmatic individuals appear to be low. Properly performed placebo-controlled double-blind challenges need to be undertaken in suspected adverse reactions to confirm whether sulfite sensitivity is responsible. Although sulfiting agents play a limited and somewhat controversial role in the production of nonasthmatic adverse reactions, the role in the production of acute 78

bronchospasm and severe asthma is better established. Kochen[ ] suggested that ingestion of sulfited food can cause asthma. He described a child who experienced cough and wheeze when exposed to dehydrated sulfited fruits packaged in sealed plastic bags. No challenge studies were done to confirm the observation, however. Subsequently, Freedman[

79] [80]

demonstrated that sulfite solutions containing 100•ppm of sodium metabisulfite could induce changes in pulmonary function in

individuals with histories of wheezing with ingestion of sulfited orange drinks. 81

82

Recognition of sulfite sensitivity in asthma was enhanced by the reports of Stevenson and Simon[ ] and Baker et al.[ ] Studies by both groups of investigators showed that exposure to potassium metabisulfite in controlled challenges were capable of causing significant bronchoconstriction and intravenous administration 82]

could lead to respiratory arrest.[

Subsequently, a number of investigators have documented the fact that exposure to sulfiting agents through ingestion of sulfited 73 83 84]

beverages or foods and medications can indeed lead to severe bronchoconstriction. Furthermore, the potential for fatal reactions to sulfite exposure is real.[ ] [ ] [ In many instances, the individuals who possibly succumbed from an adverse reaction to sulfite had not been properly diagnosed. Nonetheless, the bulk of evidence indicates that severe bronchoconstriction, hypotension with loss of consciousness, and death can occur in some subjects, particularly those with steroid-dependent asthma. Prevalence of Adverse Reactions to Sulfites

The prevalence of adverse reactions to sulfiting agents is not precisely known. Presently, only estimates of prevalence can be made as a result of the nature of the population studied and the challenge methods employed. Simon et al[

85]

conducted potassium metabisulfite capsule and solution challenges on a group of 61 adult 86]

asthmatic patients. Five of the 61 (8.2%) had a 25% or greater decrease in FEV1 upon challenge. Buckley et al[

challenged 134 adult asthmatic patients using a

single-blind protocol with potassium metabisulfite capsules, 4.6% of the individuals were thought to have sulfite sensitivity. In these studies, the population consisted of a large portion of steroid-dependent asthmatic patients, in whom sulfite sensitivity was more frequent than had been found in patients with milder asthma.[

59]

In

87 al[ ]

the largest prevalence study reported to date, Bush et conducted capsule and neutral solution sulfite challenges in 203 adult asthmatics. None were selected for history of sulfite sensitivity. Of these, 120 patients were not receiving oral or inhaled corticosteroids, whereas 83 were steroid-dependent. In the nonsteroiddependent group, only one showed a 20% or greater decline in FEV1 after single-blind and confirmatory double-blind challenge. The response rate in the steroiddependent asthma group was higher and was estimated to be approximately 8.4%. The prevalence of sulfite sensitivity in the asthmatic population as a whole was less than 3.9%. Steroid-dependent asthmatic patients appeared to be most at risk.[

87]

88

89

Limited studies have been conducted in children. Sulfite-sensitive asthma has been reported in a 2-year-old child.[ ] Towns and Mellis[ ] evaluated 29 children ages 5½ to 14 years. All had moderate to severe asthma. Challenges were performed with placebo on one day and sequential administration of sodium metabisulfite capsules or solutions performed on a second day. Using a 20% decline in peak expiratory flow rate as a positive response, 66% of these children were thought to be sulfite sensitive. However, there was no significant improvement in patients' asthma as the result of avoidance diets.[

89]

In contrast, only 4 of 56 asthmatic children

(7%) responded to single-blind challenges with sulfiting capsules and only 2 of 56 (3.5%) reacted to oral metabisulfite solution at 50 to 100•mg.[

90] [91]

The

92 children.[ ]

prevalence of sulfite sensitivity may increase with age in severely asthmatic This study used inhalation of metabisulfite for the challenge, and thus may reflect heightened bronchial responsiveness to inhaled SO2 and may not necessarily indicate an increased risk of an asthma attack from the ingestion of sulfiting agents in foods. Whether sulfite sensitivity is really more frequent in children and is age dependent remains to be established. Differences in challenge procedures such as capsule versus acidic beverage solutions may account for some of the differences. Nonetheless, the overall presence of sulfiting sensitivity, particularly in adult asthmatic patients, is small but significant. Steroid dependency appears to pose an

additional risk, particularly in adults. Mechanisms of Sensitivity to Sulfites

The mechanisms of sulfiting agent sensitivity are not known; the number of possible explanations have been hypothesized.

1656

Asthmatic patients respond with significant bronchoconstriction upon inhalation of less than 1.0•ppm of sulfur dioxide.[ itself and bisulfite (HSO3 − ) but not sulfite (SO3 ) causes bronchoconstriction to develop in asthmatic patients.[

94]

93]

Furthermore, inhalation of sulfur dioxide

Field et al[

95]

challenged 15 individuals with

doubling concentrations of sulfur dioxide gas or a metabisulfite solution. All subjects reacted to the metabisulfite solution, and 14 of the 15 reacted to inhaled sulfur dioxide with a 20% or greater decrease in FEV1 . It was their conclusion that the mechanism of sulfite sensitivity involves mechanisms other than that of generated SO2 alone.[ response.[

95]

Some asthmatic patients respond either to oral or inhalation challenge with sulfite, but inhalation is more apt to produce a bronchoconstrictive

96]

97

Delohery et al[ ] observed that 10 sulfite sensitive asthmatics reacted to an acidic metabisulfite solution when administered as a mouthwash or swallowed, but not when it was instilled through a nasogastric tube. Furthermore, these subjects did not respond when they held their breath while swallowing the solution. It was hypothesized that some individuals respond to these forms of challenge due to the inhalation of sulfur dioxide during the swallowing process.[

97]

Even though exposure to inhaled sulfur dioxide is one plausible explanation for sulfite-induced asthma, other mechanisms have been suggested including cholinergic reflex mechanisms, IgE mechanisms, and deficiencies in tissue levels of sulfite oxidase.[

98]

Diagnosis

The diagnosis of sulfite sensitivity cannot be established by the patient's history alone. A positive sulfite challenge does not always correlate with the patient's history. [87]

Therefore the diagnosis of sulfite sensitivity should only be made in individuals who demonstrate an objective response upon appropriate challenge. 74 99

Skin testing by prick and intradermal methods has identified some individuals with positive challenge responses.[ ] [ ] However, other individuals with equally severe bronchospasm or other reactions have had negative skin tests. The general approaches to diagnostic challenges in asthmatic patients have been outlined previously in this chapter. However, no standardized procedure has yet been established for sulfiting agent challenges. Patients may be challenged with capsules, neutral solutions, or acidic solutions of metabisulfite. Examples of protocols that have been previously reported in the literature are listed in Boxes 90-4 and 90-5 .

[100]

Currently, a capsule challenge may be preferable because most sulfite exposure is likely to occur to bound forms of sulfites in foods rather than in free form such as sulfite in lettuce. Solutions may be considered for patients who have reacted to beverages such as sulfited wine. When conducting challenges in single-blind fashion, positive results should always be confirmed by double-blind procedure. It has been noted that there is an order effect of challenge. For example, if a patient receives placebo on the first day and experiences no reaction, the individual may experience a reaction on the subsequent challenge day regardless of whether placebo or active challenge with sulfite is administered. To overcome this possibility, the order of administration of active and placebo challenges should be randomized and a third challenge day with either active or placebo capsules should be considered. Treatment 55 101 102

Sulfite sensitive individuals should avoid sulfite-treated foods and drugs[ ] [ ] [ ] that have been shown to trigger the response. Because individuals may vary in their sensitivity to sulfited foods, it might be necessary to perform challenges with foods containing sulfites to determine which ones are tolerated. Clearly, all sulfitesensitive asthmatic patients should be instructed to avoid the more highly sulfited foods, that is, those foods having in excess of 100•ppm of SO2 equivalents (see Box 90-7 ). Those with lower thresholds may be better advised to avoid all sulfited foods from their diets, although adherence to such diets can be extremely difficult. Packaged foods containing more than 10•ppm residual SO2 equivalents must declare the presence of sulfites on the label. Sulfite-sensitive consumers should be able to avoid sulfited foods by carefully reading labels. However, they need to be aware that the terms sulfur dioxide, sodium or potassium bisulfite, sodium or potassium metabisulfite, and sodium sulfite carry the same meaning as sulfites or sulfiting agents. No evidence exists to suggest that sulfite-sensitive individuals need to be concerned about foods having less than 10•ppm residual sulfite equivalents or sulfur dioxide equivalents. Whereas avoidance of prepared and packaged foods is relatively straightforward, avoidance of sulfites in restaurant foods is more difficult. The FDA ban on sulfites from fresh fruits and vegetables in restaurants has significantly reduced the risk of severe problems; however, other unlabeled sulfited foods remain in restaurants. The major contributing problem is sulfited potatoes. Therefore, sulfite-sensitive individuals should be instructed to avoid all potato products in restaurants except baked potatoes with skins intact. Challenge studies with sulfited foods have been conducted in sulfite-sensitive asthmatic patients. Based on the suspected mechanisms of sulfite-induced asthma, clinical challenges with acidic solutions of sulfite in lemon juice or other vehicles supports the prediction that these would be more hazardous than other forms of sulfited foods.[

61] [97]

Furthermore, it has been conclusively demonstrated that sulfited lettuce (banned under current FDA regulations) can trigger asthmatic reactions 101 103

101

in sulfite-sensitive individuals.[ ] [ ] Other foods in which sulfites may exist primarily in the bound form, such as shrimp, [ ] are less likely to induce responses. To establish an appropriate avoidance diet as discussed earlier, patients may need to be challenged with individual foods to determine which ones they can tolerate. [101]

Where possible, sulfite-sensitive individuals should avoid pharmaceutical agents that contain sulfites. Some bronchodilator solutions, subcutaneous lidocaine, intravenous corticosteroids and others may pose a risk for sensitive subjects. Many pharmaceutical companies have undertaken efforts to eliminate sulfiting agents in their products. A partial list of medications that do contain sulfites but that may be used in asthmatic patients is given in Box 90-8 . However, package inserts should always be consulted for the most current information. When patients experience bronchoconstriction as a consequence of sulfite exposure, inhaled bronchodilator medications that do not contain sulfites are the treatment of choice. If a patient exhibits hypotension or other evidence of a severe systemic reaction, injectable epinephrine is appropriate, even though the epinephrine

solution may contain sulfite as a preservative. Self-administration of automatically dispensed epinephrine, 0.3•ml, 1:1000 solution (0.3•mg), available as

1657

Epi-PEN, (Dey; Napa, California) can be lifesaving. Similar devices are available for children; these deliver 0.30•ml of the 1:2000 solution of epinephrine (Epi-Pen, Jr). 104]

Whereas a few studies have been conducted with agents that may block the response to sulfite, including cromolyn sodium, atropine, doxepin, and vitamin B12 ,[ [105]

these treatments have been conducted only in limited number of individuals. Therefore, they remain investigational and cannot be recommended as standard therapy. Strict avoidance remains the fundamental approach to sulfite sensitivity. Regulatory Restrictions

Because of reports of life-threatening reactions to sulfiting agents, the FDA moved in 1986 to regulate certain uses of sulfites. The FDA banned the use of sulfites from fresh fruits and vegetables other than potatoes. The ban affected lettuce, cut fruits, guacamole, mushrooms, and many other applications including the once commonly used practice of sulfiting fresh fruits and vegetables in salad bars. Furthermore, the declaration of sulfites on the label when sulfite residues exceed 10• ppm is required on packaged foods and alcoholic beverages. The food industry has moved to replace sulfites in some products and the search for effective alternatives continues. Furthermore, the levels of sulfite use have been reduced for other products. Whereas the actions of the various governmental agencies have helped protect sulfite-sensitive individuals from hazards associated with sulfited foods, there has been no action to limit the use of sulfites in drugs. Pharmaceutical corporations, however, have made efforts to eliminate sulfites in many products used in the treatment of asthmatic patients. Because regulations are only as good as the enforcement policies, sulfite-sensitive individuals and their physicians need to be alert to avoid inadvertent exposure. Monosodium Glutamate MSG is a popular flavor enhancer that is added to many foods but that also occurs naturally. MSG is the sodium salt of one of the most common amino acids in the human body and occurs naturally in virtually all foods. 106 107

Reports of MSG sensitivity have existed for many years.[ ] [ ] The classic example of MSG sensitivity is the MSG symptom complex, formerly called the “Chinese restaurant syndrome,” a mild, subjective, and transient syndrome characterized by (1) a burning sensation of the back of the neck, forearms, and chest; (2) facial pressure or tightness; (3) chest pain; (4) headaches; (5) nausea; (6) upper body tingling and weakness; (7) palpitations; (8) numbness in the back of the neck, arms, and back; (9) bronchospasm (in asthmatic patients only); (10) drowsiness.[

106]

Numerous human challenges have been conducted to confirm the existence of

the MSG symptom complex. Most subjects experienced no symptoms or no differences existed in the frequency of reactions to MSG versus placebos. A few 106 107

individuals may react with such mild, subjective symptoms when exposed to doses of MSG in excess of 3•g.[ ] [ ] Although it is not impossible to ingest 3•g of MSG in a single meal, it is not a common occurrence. Furthermore, symptoms have only occurred in the absence of food. In a study of 130 individuals with selfreported reactions to MSG who participated in a double-blind, placebo-controlled challenge protocol that used up to 5•g of MSG given without food, symptoms were more common (38.5%) with MSG compared to placebo (13.1%).[ when MSG was given with

108 109 food.[ ] [ ]

108]

However, on rechallenge, the results were inconsistent.[

108]

The responses were not observed

Thus the MSG symptom complex has not been firmly established and, if it occurs at all, it occurs only at very high levels

of human exposure, such as 2.5 to 3•g in the absence of food.[ MSG ingestion has also been linked to asthma.[

109] [110]

106]

Twenty-nine cases of MSG-induced asthma have been reported in the medical literature. [

110]

However,

111 DBPCFC.[ ]

there is only one report of MSG-induced asthma that was confirmed in a The remainder of the cases are anecdotal reports or reports of single-blind challenges performed in patients with a history of unstable asthma and conducted with placebos administered first in the challenge sequence. Some positive reactions 109 112

have occurred after a latency period of 6 to 12 hours after the challenge[ ] [ ] ; these are more likely to be false-positive responses. Any positive responses have tended to occur following challenge with large doses of MSG of 2•g or more. In a study of 100 asthmatic patients (30 with a history of asthma attacks after eating MSG-containing foods and 70 with a negative history of MSG-induced asthma) undergoing challenge with up to 2.5•g of MSG, compared to placebo, no significant response as measured by a decrease in FEV1 was observed.[

113]

Thus, even in history-positive patients, the existence of MSG-induced asthma has not been

114]

conclusively established.[

Other reactions have also been attributed to MSG ingestion. However, most of these reports were either anecdotal or were conducted with very poor experimental 115

designs. Kenney,[ ] in a rigorous double-blind, placebo-controlled study, failed to confirm MSG as a cause of severe symptoms in six subjects reporting histories of severe reactions attributed to MSG. MSG does not appear to be a significant factor as a cause of chronic urticaria. In 65 patients with idiopathic, chronic urticaria, two subjects had a positive response on a single-blind challenge with doses up to 2.5•g of MSG, but neither responded in a double-blind, placebo-controlled challenge.[

116]

Van Bever et al[

been ascribed to

37 118 MSG,[ ] [ ]

117]

reported two cases of MSG-induced atopic dermatitis confirmed by DBPCFC. Several cases of orofacial granulomatosis have

but these were basically anecdotal reports with no confirmation by challenge.

Aspartame Aspartame is a nonnutritive sweetener that has enjoyed considerable popularity in food and beverage applications. Numerous anecdotal reports of adverse reactions to aspartame have been made, such as headaches and various neuropsychiatric symptoms including seizures.[ emerged from these

3 120 complaints.[ ] [ ]

3] [119]

However, no clear symptom complex ever

Furthermore, careful evaluation of individuals with self-reported aspartame sensitivity including the use of single-blind and 121

122

double-blind challenges failed to identify a single aspartame reactor out of 61 individuals evaluated.[ ] Kulczycki[ ] identified two cases of aspartame-induced urticaria using DBPCFC. However, in a randomized, double-blind, placebo-controlled crossover study, aspartame was no more likely than placebo to elicit urticaria 123]

or angioedema.[ [116]

In 65 patients with chronic urticaria, none responded to doses of aspartame up to 150•mg in a double-blind, placebo-controlled challenge study.

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Protein Hydrolysates Protein hydrolysates are typically processed from the protein fractions of soybeans, wheat, corn, milk, peanuts, yeast, or gelatin. Soybean, wheat, and peanuts in particular are common allergenic foods and the allergens reside in the protein fraction. If the proteins are completely hydrolyzed to a mixture of amino acids, the allergens are destroyed and the protein hydrolysates may be safe to eat even for individuals with allergies to the source food. However, protein hydrolysates can vary in the degree of hydrolysis from minimally hydrolyzed (5% to 10% of peptide bonds broken) to virtually completely hydrolyzed. The protein hydrolysates with a high degree of hydrolysis are often used in foods as flavor enhancers because they contain large quantities of MSG. The protein hydrolysates with lesser degrees of hydrolysis are used for a wide variety of purposes including texture improvement, water binding, emulsification, and flavor enhancement. On labels in the United States, the source of the protein hydrolysate must be identified unless the hydrolysate is part of the flavoring formulation. The hydrolysates made from yeast are typically called yeast autolysate or autolyzed yeast. Casein and whey hydrolysates are often used in hypoallergenic infant formulas. However, IgE-mediated allergic reactions have occurred to casein hydrolysate formulas in highly sensitized infants.[ [128] [129]

Haddad et al[

130]

124] [125] [126] [127] 131]

and Schwartz et al[

An infant formula based on partially hydrolyzed whey protein caused much more frequent reactions.[

124]

produced protein fragments by partial digestion of b-lactoglobulin with pepsin or pepsin and trypsin that were able

to bind to IgE from patients with cows' milk allergy. Commercial hydrolysates also contain IgE-binding peptide fragments.[

132]

Hydrolysates from other sources may also elicit allergic reactions on occasion. A commercial hydrolyzed soybean protein and an acid-hydrolyzed soy sauce made from hydrolyzed soy protein were able to bind serum IgE from soybean-allergic individuals.[ sold as a flavor enhancer was unable to bind to IgE from peanut-allergic been

134 patients.[ ]

133]

In contrast, an extensively hydrolyzed peanut protein hydrolysate

A case of contact urticaria from protein hydrolysates in a hair conditioner has

135 reported.[ ]

Benzoates/Parabens Sodium benzoate and benzoic acid are widely used antimicrobial preservatives in foods. The methyl, ethyl, n-propyl, n-butyl, and n-heptyl esters of parahydroxybenzoic acid (the butyl ester is not used in foods), also known collectively as the parabens, are used on a much more limited basis as preservatives in foods. But, the parabens are extensively used as preservatives in pharmaceuticals and cosmetics. p-Hydroxybenzoic acid is also known as salicylic acid, which is structurally related to aspirin, a well-known cause of sensitivity disorders. Numerous cases of adverse reactions to benzoates and parabens have been recorded. Contact dermatitis is a well-recognized reaction to parabens in sunscreens, eye 136 137

138

drops, and shampoos.[ ] [ ] Several case reports involve rather serious anaphylactic reactions that are worthy of note. Michils et al[ ] reported the case of a teenager who had experienced several food-associated reactions in which sodium benzoate seemed to be the common factor. One of these episodes involved flush,

139

angioedema, dyspnea, and severe hypotension. An oral challenge with 20•mg of sodium benzoate produced itching and urticaria. Nagel et al[ ] reported a case of bronchospasm and pruritus following intravenous administration of a hydrocortisone preparation containing methyl- and n-propyl-paraben. The patient was able to tolerate a hydrocortisone preparation without the parabens. Direct and passive transfer skin tests were positive, suggesting immediate hypersensitivity as the mechanism. 140

Benzoates and parabens have been implicated as causative factors in some cases of chronic urticaria and angioedema.[ ] Sodium benzoate and, to a lesser extent, the parabens were included in many of the clinical trials involving tartrazine. These studies are plagued by the methodologic flaws described earlier for tartrazine. Many of these studies reported a rather high prevalence of benzoate/paraben sensitivity among patients with chronic urticaria, but antihistamines were either withheld prior to and during challenges or no information was provided on the medication status of the patients involved in the study. In one study in which antihistamines were continued, a much lower prevalence of reactions to p-hydroxybenzoic acid (5%) and sodium benzoate (11%) were found during the clinical evaluation of 111 patients with chronic urticaria.[

141]

Benzoic acid has been identified as a cause of mild, perioral contact urticaria in children who smeared food around their faces.

[142]

Van Bever et al[

117]

found that sodium benzoate (100•mg) exacerbated atopic dermatitis following DBPCFC in three of six patients challenged. Sodium benzoate and 30] [33]

p-hydroxybenzoic acid has also been identified as a possible cause of purpura in a small number of patients.[

Sodium benzoate and the parabens have not been

140 Jacobsen,[ ]

thoroughly evaluated for their role in asthma. As pointed out by most of the studies that have been conducted lacked proper placebo controls or blinding techniques. Thus, any role for benzoate or parabens in asthma remains unproven. 7

Young et al[ ] estimated the prevalence of adverse reactions to sodium benzoate at 0.01% to 0.11%, but they conducted challenges with mixtures of aspirin and benzoate. Because aspirin is a well-established sensitizing substance, the majority of their reactions were likely due to aspirin and not to benzoate. A single case of 143]

perennial rhinitis induced by benzoates has been reported in which the response was confirmed on repeated, double-blind challenge.[ Sorbate/Sorbic Acid

Sorbic acid and its potassium salt are widely used antimicrobial preservatives in foods. They are used especially for the prevention of mold growth on food products. Sorbates have been infrequently implicated in adverse reactions. Many of the studies on sorbate have the same methodologic flaws as described for tartrazine. 144

145

Volonakis et al[ ] encountered no responders among 226 patients with chronic urticaria who were challenged with 50 to 200•mg of sorbic acid. Grater[ ] reported a patient with dermatitis who had a positive patch test to several food colors and to sorbic acid. Sorbic acid can also cause contact urticaria in the perioral region especially in children who smear sorbate-containing foods around their face. [

142] [146]

Sorbic acid has caused contact dermatitis on rare occasion.[

BHA/BHT Butylated hydroxyanisole (BHA) and butylated hydroxytoluene are popular antioxidants used in a wide variety of food products.

147]

1659

Several studies on patients with chronic urticaria have included BHA and BHT as test materials, but these studies are plagued by the same methodologic flaws as described with tartrazine. Hannuksela and Lahti[

148]

identified no patients with BHA/BHT sensitivity while conducting DBPCFCs on 44 patients with chronic 116]

urticaria and 91 patients with atopic dermatitis. In the 65 patients tested by Simon[ none reacted. Weber et al

[25]

with chronic urticaria undergoing challenge with up to 100•mg of BHA/BHT,

identified 2 of 45 asthmatic patients with BHA/BHT sensitivity through DBPCFC.

Nitrates/Nitrites Sodium nitrate and sodium nitrite are used as curing agents in meat products. Only a very few adverse reactions have been attributed to nitrate or nitrite, and most of these reports are compromised by the previously described methodologic flaws. Volonakis et al[

144]

were unable to identify any responders among a group of 226 149]

patients with chronic urticaria who were challenged with 100•mg of nitrate/nitrite. Moneret-Vautrin et al[ patients with sodium nitrite using DBPCFC.

116 Simon[ ]

reported on the provocation of chronic urticaria in five

was unable to identify any reactors to nitrates or nitrites in 65 patients with chronic urticaria. 150

A case of anaphylaxis in a 22-year-old man that occurred after ingestion of nitrates and nitrites was recently reported.[ ] The reaction was confirmed by a doubleblind, placebo-controlled challenge. Another patient with chronic generalized pruritus was found to respond to a 10-mg dose of sodium nitrate on a double-blind challenge, a repeat challenge was not performed. [

151]

Further studies are needed to confirm the role of nitrates and nitrites in these generalized reactions.

Flavors Numerous different flavoring substances are used in foods and pharmaceuticals. Many flavoring formulations contain hundreds of different chemical compounds. With foods, natural and artificial flavors typically appear as the final ingredient on the list of ingredients on the food label, because the flavors are the least prevalent components of the formulated food product. 152] [153]

Few reports of allergic reactions to flavors exist. IgE-mediated allergic reactions from flavoring ingredients in typical food products are even more rare.[ 154

Flavorings do not usually contain allergenic proteins but they can on rare occasions. Gern et al[ ] reported the cases of four milk-allergic patients who had reactions to foods labeled as nondairy or “pareve” (containing neither meat nor milk products); two cases involved consumption of hot dogs and two cases involved consumption of bologna. In all of these situations, traces of milk protein were identified in the food product. The milk protein emanated from hydrolyzed sodium 155

caseinate, a milk protein, that made up part of the natural flavoring in the product. St. Vincent and Watson[ ] documented the cases of two patients in whom allergic reactions developed after ingesting an unsuspected source of cow's milk protein found in the flavoring of dill pickle-flavored potato chips. The ingredient label listed “dill pickle seasoning” but did not identify the presence of milk or milk products. McKenna and Klontz[

156]

described the case of an individual who

suffered a severe, life-threatening systemic allergic reaction from ingestion of a soup mix. The soup was found to contain peanut flour as a component of the natural flavoring. Although the only examples in the literature involve milk and peanuts, the possibility exists that similar reactions can occur to soybeans, eggs, seafood, and 152]

other allergenic materials used occasionally in the formulation of certain flavorings.[ low, even in the few flavorings in which allergenic materials are

However, the level of allergen resulting from flavors would be extremely

152 used.[ ]

Flavoring substances can also cause contact sensitization in the oral cavity on rare occasions. Most of these episodes involve products that are in prolonged contact with the oral cavity such as chewing gum, toothpaste, hard candy, cigarettes, or denture products.[ sensitization reactions. Balsam of Peru is probably the most notorious of these agents,[

157] [158]

153]

Few of the components of flavorings can elicit contact

but it is not a very common flavoring ingredient. Other known 153

offending substances include anethole, anise oil, cinnamon and cinnamic compounds, eugenol, menthol, peppermint oil, and spearmint oil.[ ] Even these substances rarely cause these reactions unless they are used in products that have prolonged contact with the oral cavity, especially when the substances are present at comparatively higher concentrations. Lecithin Lecithin generally refers to a complex, naturally occurring mixture of phosphatides. Lecithin is used as an emulsifier in both food and pharmaceutical applications. The primary sources of lecithin are soybeans and eggs. Whereas lecithin is primarily a mixture of various phospholipids, it can contain soy protein and soy allergen 159] [160] [161] [162]

residues.[

Despite the widespread use of lecithin as a food ingredient, allergic reactions to soy lecithin have been described on only a few occasions.

[162] [163]

The low levels of soy protein in lecithin coupled with the low level of use of soy lecithin in many food applications probably limits exposure to a level that is below the threshold for elicitation of an allergic reaction for most soy-allergic individuals. Papain 164

Papain is a proteolytic enzyme that is used occasionally in the processing of foods. One of its primary uses is as a meat tenderizer. Mansfield and Bowers[ ] reported the case of a patient who experienced an IgE-mediated allergic reaction to papain in a meat tenderizer. The reaction was systemic, requiring epinephrine for treatment. In further studies on a group of 500 unselected allergy patients, 5 of 475 with seasonal pollen allergies had positive skin tests and DBPCFCs to papain, whereas none of 25 patients without pollen allergies had similar responses.[

165]

Gums Many different types of gums are used in foods and pharmaceuticals. The major gums are guar, tragacanth, xanthan, carrageenan, acacia, locust bean, and alginate. Several of these gums are legumes, including guar, tragacanth, locust bean, and acacia; some other members of the legume family such as peanut are intensely allergenic. The gums are primarily composed of complex polysaccharides, although residues of proteins can occur in the gums, on occasion.

1660

Gums are a well known cause of occupational asthma, particularly guar gum.[ of allergic reactions to gum tragacanth have been 169 enema.[ ]

167 168 reported.[ ] [ ]

166]

Allergic reactions from the ingestion of gums are much less frequent. Several cases

Carrageenan has been implicated in a case of anaphylaxis resulting from its use in a barium

A single case of allergic sensitization to gum Arabic (acacia gum) has been described, but the nature of the allergen, protein residues versus carbohydrate 170

171]

epitopes, was uncertain. [ ] Several patients were described as experiencing flares of chronic urticaria after taking thyroid tablets containing acacia gum[ patients and 9 of 14 others had IgE antibodies against acacia extract, although challenges were not conducted. Allergic sensitization to carob bean gum was

; these

172]

confirmed by challenge in an infant.[ Lactose

Lactose is the disaccharide that occurs naturally in cow's milk. Commercial lactose can contain residues of proteins, although the level of proteins in lactose is unknown. Lactose is a common ingredient in many pharmaceutical applications and is a common food ingredient. No confirmed cases of allergic reactions of milkallergic individuals to lactose have been described, but a case of urticaria attributed to lactose may be associated with contaminating milk proteins,[ is not proven.

173]

although that

Gelatin Gelatin is most commonly derived from beef or pork. Kosher gelatin is made from fish skins from several species of fish including cod. Gelatin is usually considered as a protein with rather low allergenic potential. The major sources of gelatin, beef and pork, are rarely allergenic. However, kosher gelatin is obtained from a frequently allergenic source, fish. The allergens in fish are localized in the edible muscle tissue. The gelatin is obtained from the fish skins, although remnants of muscle tissue are likely to adhere to the skin. The fate of any fish muscle allergens in the gelatin-making process is unknown. However, the gelatin-making process involves rather significant modifications of protein structure. Beef and pork gelatin is often used as a vaccine expander, and allergic reactions to injected pharmaceutical preparations containing gelatin have been reported.[ [174] [175] [176]

9]

Presumably, most of these reactions were associated with beef and pork gelatin. Both IgE-mediated and cell-mediated allergic reactions have been

177 reported.[ ]

178

Gelatin (presumably beef and pork) contained in a hemostatic sponge used in a surgical procedure was also implicated in an allergic reaction.[ ] Allergic reactions to gelatin used as a food ingredient are rarely reported. In a study of 26 children with allergic reactions to vaccines, seven had allergic reactions to gelatin-containing foods including two who had reactions before vaccination and five who had reactions after vaccination.[ Cross-reactions occur between beef and pork gelatin and gelatins derived from other mammalian species.[ individuals with known gelatin

180 allergies.[ ]

180]

179]

However, IgE to fish gelatin is rarely observed in

In a study of 10 fish-allergic individuals, three patients had specific IgE to fish gelatin, suggesting that fish gelatin 181]

might be an allergen in subjects with fish allergy. [

However, allergic reactions to fish gelatin contained in foods has not been documented.

Inulin Inulin is a fructan polysaccharide found in many different types of plants including chicory, artichoke, salsify, and Jerusalem artichoke. Inulin is frequently used as an intradermally injected pharmaceutical for tests of renal function. No allergic reactions have been reported to inulin as a pharmaceutical agent. Increasingly, inulin is also used as a food ingredient because of its ability to stimulate the growth of beneficial intestinal bacteria, particularly the bifidobacteria. A single case of 182

anaphylaxis to inulin has been reported in an individual who reacted to ingestion of salsify, artichokes, and several inulin-containing foods.[ ] In this patient, both intradermal challenge and DBPCFC were positive. Strong skin prick tests were noted in this individual with inulin, and extracts of salsify, artichoke, and inulincontaining foods. Wheat Starch Starch is a very frequently used food ingredient. Starch is most frequently derived from corn, a rarely allergenic food source. However, starch can occasionally be 183]

derived from wheat. Wheat starch contains trace residues of wheat proteins,[ unique proteins that may not be allergenic.

183 allergenic,[ ]

but allergic reactions to wheat starch remain undocumented. Starch granules contain

although commercial wheat starch might also contain residues of proteins from other wheat fractions that are

Edible Oils Edible oils are frequently derived from commonly allergenic foods such as peanut, soybean, and sunflower seed, although they may also come from less commonly allergic foods such as corn, olive, or safflower seed. However, the allergenic activity of foods resides in the protein fraction and highly purified oils do not typically 184

185

186

contain any detectable protein. Thus, peanut oil,[ ] soybean oil,[ ] and sunflower seed oil[ ] can be safely ingested by individuals allergic to the source product from which the oil is derived. However, less well-refined and cold-pressed oils can contain protein residuals and may be hazardous for consumption by allergic individuals.[

187] [188] [189]

Gourmet oils made from allergenic sources such as sesame seeds or tree nuts may also contain protein residues and are likely to be

hazardous to consumers with allergies to the source food.[

190] [191]

192]

The allergenicity of edible oils has been extensively reviewed.[

Other Drug Additives Benzalkonium chloride is a preservative used in albuterol and metaproterenol nebulizer solutions and in beclomethasone and ipratropium nebulizer solutions in other 3 10

countries.[ ] [ ] It has been reported to cause paradoxical bronchoconstriction in 60% of asthmatics. The reaction is frequently accompanied by cough and a burning sensation, and occasionally by facial flushing and pruritus. It can be blocked by prior use of b2 agonists, cromolyn, and partially by H1 -histamine receptor antagonists. The mechanism may involve non-IgE–mediated mast cell mediator release. Reduction on the concentration of commercial nebulizer solutions has 10]

reduced the likelihood of reactions.[

Other potential sources that contain benzalkonium include nasal saline, nasal corticosteroids, and nasal decongestant solutions.

1661

3

Benzyl alcohol is a common preservative in many injectable drugs and solutions and is a rare cause of contact dermatitis or angioedema.[ ] Toxicity to high doses has occurred in newborns resulting in mortality from intraventricular hemorrhage.[

3]

Propylene glycol is used as a drug solubilizer in a number of topical, oral, and injectable medications. Toxicity is the main concern with high doses in infants, leading to lactic acidosis, hyperosmolality, and seizures. Contact dermatitis has been reported; 4.5% of 487 with contact dermatitis were reported to react to propylene glycol. [3]

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Chapter 91 - Adverse Reactions to Vaccines

John M. Kelso

PUTTING ADVERSE REACTIONS TO VACCINESIN PERSPECTIVE Success of Vaccines 1

The Centers for Disease Control and Prevention (CDC) compiled a list of the greatest public health achievements of the century just past.[ ] At the top of the list is vaccination. Table 91-1 summarizes the impact of vaccines on dramatically reducing or even eliminating the morbidity from infectious diseases. These are remarkable accomplishments, and it is against this background of enormous benefit that adverse reactions to vaccines must be evaluated. Rarity of Serious Reactions In the overall picture, the rarity of serious adverse reactions to vaccines should also be considered. A total of 213,947,411 doses of all vaccines were distributed in the United States in 2000 (CDC; personal communication). The Vaccine Adverse Event Reporting System (VAERS) received 13,368 reports involving the administration of 22,396 immunizations (some reports involve reactions after the administration of more than one vaccine at a single visit). Thus, there is one report generated for every 10,000 doses administered. Even given the limitations of this passive reporting system, serious adverse reactions to vaccines overall are rare. Evolution of an Immunization Program When an infectious disease causes widespread morbidity and mortality, the adverse events caused by a vaccine developed to combat the disease may be more readily accepted. Once the vaccine leads to a dramatic decline in the disease, however, vaccine side effects become relatively more apparent and less acceptable. A 2

theoretical evolution of an immunization program has been described[ ] ( Figure 91-1 ). Before vaccine availability, incidence of the disease is high (stage 1). As a program of vaccination against the disease is begun, incidence of disease decreases while vaccine use increases (stage 2). Some rate of adverse events attributable to the vaccine will, of course, reflect its usage. The number of vaccine adverse events is initially small in relation to the number of disease cases but, as the natural disease is largely eliminated by widespread vaccine usage, this same number of vaccine adverse events becomes large by comparison to disease cases (stage 3). Attention to these vaccine adverse events may lead to a loss of confidence in the vaccine and lower vaccination rates with resultant resurgence in disease. If confidence is restored, vaccination rates increase (stage 4). Eventually, high vaccination rates may lead to the eradication of the disease and vaccination may be stopped, eliminating vaccine adverse events (stage 5). Consequences of Not Vaccinating The risk of adverse events after vaccination must be put in the context of the consequences of not vaccinating vulnerable populations as well. Many U.S. states allow exemptions from childhood immunizations based on philosophical objection. Based on reports to the CDC surveillance system, children who were not vaccinated

3

4

against measles were 35 times more likely to contract the disease than those vaccinated.[ ] A rubella outbreak occurred in Nebraska in 1999.[ ] All 83 cases were either unvaccinated or had an unknown vaccination status. At least one infant with congenital rubella syndrome was born to one of the cases. A polio outbreak 5

occurred in the Dominican Republic in the year 2000.[ ] All 19 cases were either unvaccinated or inadequately vaccinated. Military recruits in the United States previously received routine vaccination against adenovirus. When the sole manufacturer of the vaccine ceased production, the incidence of adenoviral respiratory illness escalated and led to two deaths.[

6]

Monitoring for Vaccine Adverse Events 2

Since 1990, VAERS has been in place in the United States.[ ] Established by the CDC and FDA, VAERS is a passive surveillance system; that is, it relies on reporting by health care providers and parents or patients. A report to VAERS is actually a report of a suspected association between vaccine administration and an 7

adverse event. VAERS receives more than 10,000 reports annually, exceeding the number of reported cases of vaccine preventable diseases themselves.[ ] Whereas VAERS provides important data for evaluation of causality between a vaccine and a subsequent event, more thorough epidemiologic evaluation requires that the number of subjects vaccinated without the subsequent event be known, and that the number not vaccinated with and without the subsequent event also be known. To meet this need, CDC has established the Vaccine Safety Datalink (VSD), which monitors the immunization and medical records of more than half a million children 8

enrolled in four large staff model HMOs.[ ] These postlicensure surveillance systems are particularly important given that prelicensure

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TABLE 91-1 -- Decrease in Reports of Vaccine-Preventable Diseases in the United States Peak Year Year

*

Year 2000

CasesReported

CasesReported

†

% Decrease

Diphtheria

1921

206,939

2

99.9

Haemophilus influenzae type b

1984

20,000

1212

93.9

Measles

1941

894,134

81

99.9

Mumps

1968

152,209

323

99.7

Pertussis

1934

265,269

6755

97.4

Poliomyelitis

1952

21,269

0

100.0

Rubella

1969

57,686

152

99.7

Tetanus

1948

601

26

95.6

* Data from MMWR 45:RR-12, 1996. † Data from MMWR 49:51/52, 2001.

studies may not include enough subjects to detect rare adverse events.[

9]

RECENT CHANGES IN U.S. VACCINE RECOMMENDATIONS RELATED TO ADVERSE REACTIONS Thimerosal 10

Because mercury is neurotoxic, in 1997 the FDA undertook a review of mercury-containing vaccines.[ ] Thimerosal (50% mercury by weight) has been used for decades as an additive to several vaccines as a bactericidal agent. It was determined that infants receiving recommended vaccines in the first few months of life might be receiving an amount of mercury, in the form of thimerosal, exceeding federal guidelines on permissible exposure. Although no harmful effects of the exposure to mercury in vaccines had been reported, recommendations were made to reduce or eliminate the thimerosal content of vaccines. Substantial progress has been made in 11]

reducing the amount of mercury a child might be exposed to through vaccination.[ Oral Polio Vaccine

When polio struck more than 21,000 people each year in the United States, a few cases caused by the oral polio vaccine itself may have seemed less consequential. However, with the eradication of natural disease in the Western Hemisphere, the eight or nine vaccine-associated polio cases per year became all of the cases. Thus, beginning in the year 2000, the routine use of OPV was discontinued in favor of an all IPV schedule.[

12]

Rotavirus Vaccine In 1998, an oral vaccine against rotavirus was introduced and recommended for routine vaccination of infants. However, in the first several months of its widespread use, numerous reports of intussusception were reported through the VAERS.[

13]

After careful review of the cases of intussusception, all of which occurred within 1 to

2 weeks after vaccination, the recommendation for the use of the vaccine and the vaccine itself were withdrawn.[

IGE-MEDIATED REACTIONS TO VACCINES

14]

IgE-Mediated Reactions to Vaccine Constituents Other than the Immunizing Agent Gelatin

Gelatin is added to many vaccines as a stabilizer ( Box 91-1 ). The first case report of an allergic reaction to the gelatin

Figure 91-1 Evolution of an immunization program. (Modified from Chen RT, Rastogi SC, Mullen JR, et al: Vaccine 12:542, 1994.)

(Modified from Chen RT, Rastogi SC, Mullen JR, et al: Vaccine 12:542, 1994.)

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Box 91-1. Gelatin Content of Vaccines

Vaccine

Gelatin Content

DTaP (Acel-Immune, Lederle)

15••g per 0.5•ml dose

*

DTaP (Tripedia, Aventis Pasteur)

28••g per 0.5•ml dose

*

Influenza (Fluzone, Aventis Pasteur)

250••g per 0.5•ml dose

†

Japanese Encephalitis (JE-VAX, Aventis Pasteur)

500••g per 1.0•ml dose

†

Measles, Mumps, Rubella (Attenuvax, BiavaxII, MeruvaxII, MMRII, MRVAXII, Mumpsvax, Merck)

14,500••g per 0.5•ml dose

†

Rabies (RabAvert, Chiron)

12,000••g per 1.0•ml dose

†

Varicella (Varivax, Merck)

12,500••g per 0.5•ml dose

†

Yellow Fever (YF-VAX, Aventis Pasteur)

7500••g per 0.5•ml dose

*

* Vaccine manufacturers, personal communication. † Package inserts.

component of a vaccine described a young woman who suffered a classic anaphylactic reaction after receiving an MMR immunization; it was also reported that she 15

developed a swollen tongue and itchy throat within minutes of eating gelatin in any form.[ ] Laboratory evaluation revealed that the only component of the vaccine to which she made IgE antibody was gelatin. Subsequent reports from Japan evaluated 26 children who had allergic reactions to measles, mumps, or rubella vaccines containing gelatin, and found 24 of the 26 made IgE antibody to gelatin, whereas none of 26 control children who had been vaccinated uneventfully made such antibodies.[

16]

Similar reports involving measles-mumps-rubella (MMR) vaccine followed from other countries,[

vaccines, including

18 19 varicella[ ] [ ]

[20]

and Japanese encephalitis,

17]

and reports regarding other gelatin-containing

were also rendered. Vaccine manufacturers in Japan removed gelatin or changed to a less 21]

allergenic gelatin in vaccines, with a resultant decline in reported cases of allergic reactions after immunizations.[

A history of allergy to the ingestion of gelatin

should be sought before the administration of any gelatin-containing vaccine. A negative history, however, may not exclude an allergic reaction to gelatin injected 16

with the vaccine.[ ] Most persons with IgE antibody to gelatin and immediate-type allergic reactions to gelatin-containing vaccines attributable to those antibodies have not reported allergic reactions to the ingestion of gelatin prior to vaccination, although some may do so afterward. Presumably the different route of exposure, injection versus ingestion, accounts for this fact.[ 16 Japan[ ]

than in other

17 23 countries.[ ] [ ]

22]

The incidence of gelatin allergy among vaccine recipients suffering anaphylactic reactions has been higher in

A recent report found a strong association between gelatin allergy and HLA-DR9, an HLA type unique to Asians, suggesting

a genetic susceptibility to gelatin allergy. [

24]

This type of HLA analysis might shed light on the risk for allergic reactions to other vaccines.

Egg

Concern has existed over the administration of vaccines “grown in eggs” to egg-allergic recipients. A report in 1983 described two egg-allergic children who suffered allergic reactions to measles vaccine,[

25]

seeming to support the need for caution. However, a subsequent review of the reported cases of anaphylaxis to measles or

MMR vaccine found no other cases in egg-allergic recipients.[

15]

Furthermore, hundreds of egg-allergic children had been administered the vaccines without adverse 26

reaction. Measles and mumps vaccines are grown in chick embryo fibroblast cultures (as opposed to “eggs”) and contain negligible or no egg protein.[ ] Follow-up studies in which measles or MMR vaccine was administered in the normal manner to egg-allergic children without adverse reaction have confirmed the safety of this approach.[

27] [28]

The safety of administration of other vaccines possibly containing egg protein has also been investigated. Influenza vaccine is grown in the allantoic fluid of embryonated chicken eggs.[

29] [30] [31]

It contains measurable quantities of egg proteins. [

26] [32]

The amount varies broadly from 0.2 to 42••g/ml. Egg-allergic

[33]

32

patients have been administered the vaccine without reaction, either by several graded doses or a two-dose protocol. [ ] In the former study, the egg content of the vaccine was not stated; in the latter, the egg content was less than or equal to 1.2••g/ml. The safety of administering any vaccine with larger amounts of egg 34 35

protein to egg-allergic persons is not known. Negative prick and intradermal skin tests to the vaccine are reassuring.[ ] [ ] Positive skin tests to influenza vaccine, however, are difficult to interpret. Up to one third of patients receiving influenza vaccine uneventfully may show an increase in IgE antibodies to egg protein after 36

37

vaccination.[ ] Furthermore, patients who tolerate egg ingestion may have positive intradermal skin tests to influenza vaccine, yet receive it without reaction.[ ] Thus, it seems that if the egg protein content of a particular lot of influenza vaccine can be determined to be less than or equal to 1.2••g/ml, then the vaccine can be safely administered to egg-allergic persons by a two-dose protocol (and perhaps even as a single dose). If the egg content of the vaccine cannot be determined, it seems appropriate to perform prick (full strength) and intradermal (1:100) skin tests with the vaccine. If the skin tests are positive, a more cautious (several dose) protocol for vaccine administration seems prudent. 26]

Yellow fever vaccine is cultured in chicken embryos and contains egg protein.[

38] [39] [40]

Anaphylactic reactions in egg-allergic persons have been reported.[

Additional cases have been reported in persons whose egg-allergic status is not known. [

41]

Persons presenting for yellow fever vaccination are routinely asked if 42

they are allergic to eggs. However, a patient has been reported who was allergic to a heat-labile egg protein.[ ] She ate cooked eggs without difficulty and denied egg allergy when asked before receiving her yellow fever vaccine. She nonetheless developed a urticarial reaction to the vaccination. Subsequent skin tests were positive for raw egg and vaccine. The vaccine, which is not heated during manufacturing, would still contain egg proteins that would otherwise be destroyed by heat. Thus the clinical history may not identify all persons allergic to egg proteins present in yellow fever or other vaccines.

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Latex

Natural rubber latex is used in some medication vial stoppers. There is a single case report of a latex allergic patient who suffered an anaphylactic reaction after 43

administration of hepatitis B vaccine drawn through a rubber stopper. [ ] She had no reaction to a subsequent dose administered without puncturing the stopper. A search of the VAERS database through 1998 revealed 14 reports of immediate-type allergic reactions to various vaccines in patients also reported to be latex allergic. Some of these may possibly have been due to latex allergy (Vitale Pool MD, CDC, personal communication). Storage of liquid in a vial where the liquid is in contact 44

with a natural rubber latex stopper can cause the release of latex allergens into the solution.[ ] This could, in theory, subsequently cause a reaction when administered to a latex-allergic patient. However, most vaccine vial stoppers are made of synthetic rubber (as opposed to natural rubber latex) and pose no risk to latex-allergic persons (Larry Pickering M.D., CDC; personal communication). IgE-Mediated Reactions to Specific Vaccines Diphtheria

Many recipients of diphtheria vaccine generate IgE antibody to the vaccine[

45] [46] [47]

; however, most go on to receive subsequent doses uneventfully.[ 47]

reactions to subsequent doses may be more frequent in those who have made antidiphtheria IgE,[

46]

Local

but generalized reactions have been reported only rarely.[

48] [49]

A Danish vaccine, Di-Te-Pol, contains diphtheria toxoid, tetanus toxoid, and inactivated polio virus. A 16-month-old child developed generalized urticaria after his 50

third dose of the vaccine.[ ] Basophil histamine release was demonstrated to Di-Te-Pol and diphtheria vaccine but not to the other two constituents. Thus, it appears that this generalized IgE-mediated reaction was to diphtheria toxoid. Another interesting case involved a generalized urticarial reaction after administration of a conjugate Haemophilus influenzae type b (Hib) vaccine in which the 51

conjugate protein CRM 197 is a mutant diphtheria protein.[ ] Subsequent skin testing revealed positive reactions to the vaccine in question but not to other Hib vaccines using different conjugate proteins. This case also demonstrates the importance of determining which vaccine component is responsible for a reaction. The child in this report may safely receive other Hib vaccines with different conjugate proteins, yet he may react to the heptavalent pneumococcal conjugate vaccine because it contains the same CRM 197 conjugate protein to which he is allergic. Hepatitis B 48]

A 1994 Institute of Medicine report described as many as 18 cases of anaphylaxis after hepatitis B vaccine (gleaned from the VAERS database).[

No IgE antibody

testing is described; however, the nature and timing of the reactions were convincing enough for the report to conclude that “the evidence establishes a causal relationship between hepatitis B vaccine and anaphylaxis.” CDC estimates the risk to be 1:600,000 vaccine doses.[ protein but no adverse reactions have been attributed to this component per

49]

Hepatitis B vaccines contain up to 5% yeast

52 se.[ ]

Haemophilus influenzae Type B 48] [53]

Reports of reactions consistent with anaphylaxis have been rare after Hib vaccination.[

Only one report has demonstrated IgE antibody to an Hib vaccine

component, which was not the Hib polysaccharide, but rather the protein in a conjugate vaccine (see earlier section on diphtheria).[

51]

Influenza

There are a number of vague references to anaphylaxis to influenza vaccine in the medical literature, but no reports of specific cases, despite administration of more than 60 million doses per year in the United States. The use of this vaccine in egg-allergic individuals is described earlier under “Egg.” Japanese Encephalitis

Japanese encephalitis vaccine can cause typical immediate-type allergic reactions, consisting of urticaria with or without wheezing occurring 5 to 60 minutes after 20] [54] [55]

vaccination.[

Most of these patients have anti-gelatin IgE, which is not found in those immunized uneventfully. These allergic reactions are presumably 15] [16] [17] [18]

due to gelatin allergy, as has been described with other gelatin-containing vaccines.[

Japanese encephalitis vaccine has also been particularly prone to cause an unusual, late-onset urticaria and angioedema reaction.[

56]

Reports from several countries

have placed the incidence of such reactions as high as 1% of recipients. The median interval between vaccination and these reactions is 2 to 3 days.[

56] [57] [58]

56 58 59

Although the reactions are usually confined to the skin, some cases have involved hypotension or respiratory distress.[ ] [ ] [ ] One death occurred 60 hours after Japanese encephalitis vaccination (and 12 hours after plague vaccination); however, the cause of death and relationship, if any, to vaccination could not be determined.[

57]

57] [60]

Two studies have suggested that a prior history of urticaria, asthma, rhinitis, or eczema increases the risk of these late-onset reactions.[

Measles-Mumps-Rubella

There are many reports of anaphylactic reactions to MMR vaccine. [

15] [16] [17] [23] [25] [61] [62] [63] [64] [65] [66] [67] [68]

distributed each year, making the incidence of such reactions on the order of 2 to 10 per as a stabilizer has been determined to be the likely cause in 27% to 92% of reactions to MMR vaccine and egg allergy.

17 23 million.[ ] [ ]

16 17 23 cases.[ ] [ ] [ ]

There are, however, tens of millions of doses

As described earlier, allergy to gelatin added to the vaccine

As detailed earlier, there is no relationship between anaphylactic

Pertussis 69] [70]

Natural infection with pertussis causes the production of anti-pertussis IgE in many recipients.[

Immunization with either whole-cell or acellular pertussis 71] [72]

vaccine results in the production of anti-pertussis IgE in about one third of the recipients after the primary series and in as many as two thirds after a booster.[ The production of these antibodies appears unrelated to atopic status (i.e., having asthma, allergic rhinitis, eczema, or food allergy[ Although the presence of the IgE antibodies may predispose to local reactions to subsequent doses of the vaccine,[

72]

72] [73]

71]

) or alum content.[

they have not generally predisposed to systemic

72 73 reactions.[ ] [ ]

Immunization to pertussis vaccine has also been examined for the possibility that it would enhance IgE production to allergens.[ children nor adults did pertussis vaccine increase the level of IgE antibody to common aeroallergens.

74] [75]

In neither

1669

Rabies

A few cases consistent with immediate type allergic reactions to human diploid cell rabies vaccine have been reported but without any confirmatory laboratory data 76]

regarding IgE antibody.[

Interestingly, many cases consistent with serum sickness have been reported[

76] [77] [78] [79] [80]

and many of them have been associated

[78] [79]

80

with IgE antibody to a vaccine constituent, even though such reactions are generally thought to be IgG immune-complex related. [ ] The timing of the reactions is from 2 to 21 days after vaccine administration. The symptoms have included those typical of serum sickness such as arthralgia, fever, and malaise, and 81 82

urticaria has been a prominent feature. The rabies virus used in the vaccine is inactivated with beta propiolactone (BPA).[ ] [ ] However the cell culture medium in which the virus is grown contains human albumin among other ingredients, and the BPA added to inactivate the virus also alters the albumin. This BPA-altered albumin has been shown to stimulate the production of specific IgE antibody in vaccine recipients who go on to report the serum sickness–like reaction to subsequent doses.[

78] [79]

How IgE antibody could result in this clinical picture is not adequately explained.

Tetanus

As with diphtheria toxoid, many recipients of tetanus vaccine generate specific IgE antibodies to the toxoid.[ antibodies may be related to the aluminum adjuvant.

[84]

45] [46] [47] [83] [84] [85]

The production of these IgE

Most persons go on to receive subsequent doses without systemic reaction.[

46] [84] [85]

A small number of 48]

reports of apparent anaphylaxis, including fatalities, have been reported after tetanus toxoid administration but few have included assessment for IgE antibody.[ a single case report, a 5-year-old is described with generalized urticaria and angioedema after his fourth dose of response to tetanus toxoid. He subsequently received a dose of the vaccine uneventfully by graded dosing. Varicella

86 DPT.[ ]

In

Subsequent skin testing revealed a positive

A recent report described the first 3 years of postlicensure safety surveillance for varicella vaccine.[ of three per million doses distributed. Reports from

18 Japan[ ]

and the United

19 States[ ]

87]

Among the reports were 30 consistent with anaphylaxis, a rate

have implicated gelatin allergy as the cause of some of these reactions.

Yellow Fever 38 39 40

Before 1999, the only reports of anaphylaxis to yellow fever vaccine dated from the 1940s and described only three patients.[ ] [ ] [ ] The 1999 report reviewed VAERS data over a 7-year period and identified 40 cases of probable or possible anaphylaxis to yellow fever vaccine and estimated the rate of such reactions at 7 per 41

million doses.[ ] Clearly other anaphylactic reactions to yellow fever vaccine occurred in the 50 years between reports of such reactions. This underscores the importance of vaccine safety surveillance systems such as VAERS to establish the occurrence and frequency of rare reactions. It also emphasizes the importance of persons administering vaccines to report to VAERS. The possible association between reactions to yellow fever vaccine and egg allergy is discussed previously. The vaccine also contains gelatin, which has been proven responsible for many allergic reactions to other vaccines.[

15] [16] [17] [18] [19] [20]

Suggested Approach to a Suspected IgE-Mediated Reaction to a Vaccine A suggested approach to a suspected IgE-mediated reaction to a vaccine is outlined in Figure 91-2 . The first step in the evaluation is to determine if the nature and timing of the reaction are consistent with anaphylaxis. Although many schemes have been used to classify anaphylactic reactions, that proposed in Note 1 to Figure 41

91-2 seems reasonable and has been used in the evaluation of vaccine reactions. [ ] Pertinent, too, is a history of similar reactions to the same or other vaccines, or to vaccine constituents. The various elements that make up a vaccine are clearly labeled in manufacturer package inserts. If the reaction occurred with the first dose of a vaccine, the chance that the immunizing agent itself is the allergen is greatly diminished. One should also inquire about allergic reactions to food, because influenza and yellow fever vaccines contain egg protein.[ (see Box 91-1 ).

26] [29] [30] [31] [88]

Yellow fever vaccine may also contain chicken proteins,[

26] [88]

and many vaccines contain gelatin

Once a history has been obtained of a vaccine reaction occurring shortly after administration that is consistent with mast cell degranulation, it is appropriate to determine whether future doses of the suspect vaccine, or other vaccines with common components, are required for the particular patient. If a patient's reaction occurred with a vaccine in which only one dose is normally given, or to the final dose in a series, one could argue that the patient can simply be labeled “allergic” to the vaccine in question without further testing. However, given the potential for cross-reaction with common components in other vaccines and with foods, a more thorough evaluation, even if no further doses are required, may be appropriate. Many vaccines are given as a series, because some recipients require several doses to achieve a “protective” response. However, some recipients may generate an adequate response to fewer than the usual number of doses. In this circumstance, it may be reasonable to determine the level of immune response in terms of antibody level achieved in a particular patient by the doses already received. Protective levels of specific antibody to the immunizing agent have been determined for many vaccines and some are routinely available in commercial reference laboratories. If a patient can be determined to have already mounted a sufficient antibody response to be considered protected, then consideration can be given to not giving further doses of the vaccine. One caveat here is that the level of protective antibody may not persist as long in persons vaccinated with fewer than the usual number of doses. Antibody levels may need to be checked again at some interval, particularly if the patient, because of travel or other reasons, may have an increased chance of exposure to the infectious agent to which he or she was immunized.

If the patient must receive further doses of a vaccine, either because a test for specific antibody is not available or because the patient demonstrated subprotective levels of specific antibody, skin testing with the vaccine should proceed. The vaccine should first be tested by the prick method. Full-strength vaccine can be used, unless the history of reaction was truly life-threatening, in which case beginning even the prick test with dilute vaccine is appropriate. If the full-strength prick test is negative, with appropriate positive and negative controls, an intradermal test with the vaccine diluted 1:100 should be performed, again with appropriate controls. If the

1670

Figure 91-2 Suggested approach to suspected immediate-type allergic reactions to vaccines.

TABLE 91-2 -- Suggested Intervals between Administration of Immunoglobulin Preparations for Various Indications and Vaccines Containing Live Virus

Indication

Dose (including mg IgG/kg)

Time Interval (months) Before Live Virus Vaccination

Tetanus (TIG) prophylaxis

250 units (10•mg IgG/kg) IM

3

••Contact prophylaxis

0.02•ml/kg (3.3•mg IgG/kg) IM

3

••International travel

0.06•ml/kg (10•mg IgG/kg) IM

3

Hepatitis B prophylaxis (HBIG)

0.06•ml/kg (10•mg IgG/kg) IM

3

Rabies immune globulin (HRIG)

20•IU/kg (22•mg IgG/kg) IM

4

Hepatitis A (IG) prophylaxis:

Varicella prophylaxis (VZIG)

125 units/10•kg (20–40•mg IgG/kg) IM (maximum 625 units)

5

••Standard (i.e., nonimmunocompromised contact)

0.25•ml/kg (40•mg IgG/kg) IM

5

••Immunocompromised contact

0.50•ml/kg (80•mg IgG/kg) IM

6

••Red blood cells (RBCs), washed

10•ml/kg (negligible IgG/kg) IV

0

••RBCs, adenine-saline added

10•ml/kg (10•mg IgG/kg) IV

3

••Packed RBCs (Hct 65%)

10•ml/kg (60•mg IgG/kg) IV

6

••Whole blood cells (Hct 35%–50%)

10•ml/kg (80–100•mg IgG/kg) IV

6

••Plasma/platelet products

10•ml/kg (160•mg IgG/kg) IV

7

Replacement therapy for immune deficiencies

300–400•mg/kg IV (as IgIV)

8

••Immune thrombocytopenic purpura

400•mg/kg IV (as IgIV)

8

••Immune thrombocytopenic purpura

1000•mg/kg IV (as IgIV)

10

••Kawasaki disease

2•g/kg IV (as IgIV)

11

Measles prophylaxis (IG)

Blood transfusion

Treatment of

Modified from MMWR 45:RR-12, 1996. level of antibodies to these agents provides information on the ability to make antibodies to protein antigens. Until the introduction of the heptavalent pneumococcal protein-conjugate vaccine, most people had not been vaccinated against pneumococcus. Measuring levels of antipneumococcal antibodies before and after immunization with the 23-valent pneumococcal vaccine provides information on the ability to make antibody to polysaccharide antigens. [

155]

In certain clinical circumstances, such as in patients already on intravenous immunoglobulin (IVIG) antibody replacement therapy, the measurement of antibodies to vaccines is not helpful, because these antibodies are abundant in the IVIG donor pool and cannot be distinguished from recipient antibodies. A novel approach has been developed for use in this situation: vaccination with a “neoantigen,” that is, an antigen to which neither the donors to the IVIG pool nor the recipient would have been exposed.[

156] [157]

Immunoglobulin Preparations

49 89

IG preparations interfere with the desired immune response to live viral vaccines,[ ] [ ] presumably because the IG preparations contain antibodies to the immunizing virus, or because of other immunomodulation. IG preparations given within 2 weeks after live virus vaccination could prevent adequate immunization. When an IG preparation is given first, the suggested interval until vaccination is listed in Table 91-2 .[

49]

SUMMARY The dramatic reduction or elimination of many diseases by vaccination is perhaps the greatest public health achievement in history. Whereas mild, self-limited adverse reactions to vaccines are common, serious complications or long-term sequelae are rare. In virtually all cases, the risk of an adverse outcome from the disease itself is far greater than the risk from vaccination. When a serious adverse event possibly related to vaccination occurs, it should be investigated to determine the cause and to make recommendations to the individual patient regarding subsequent immunizations. Adverse reactions may or may not be IgE-mediated, and may be due to the immunizing agent itself or to some other vaccine constituent. Serious reactions should also be reported even if the provider is not certain of the relationship of the reaction to vaccination. Public health agencies and vaccine manufacturers need these reports to quantify rare adverse vaccine reactions and to further improve vaccine safety.

REFERENCES Putting Adverse Reactions to Vaccines in Perspective 1. Centers for Disease Control and Prevention: Ten great public health achievements—United States, 1900–1999, MMWR 48:241, 1999. 2. Chen RT, Rastogi SC, Mullen JR, et al: The Vaccine Adverse Event Reporting System (VAERS), Vaccine 12:542, 1994. 3. Salmon DA, Haber M, Gangarosa EJ, et al: Health consequences of religious and philosophical exemptions from immunization laws: individual and societal risk of measles, JAMA 282:47, 1999.

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4. Danovaro-Holliday MC, LeBaron CW, Allensworth C, et al: A large rubella outbreak with spread from the workplace to the community, JAMA 284:2733, 2000. 5. Centers for Disease Control and Prevention: Outbreak of poliomyelitis—Dominican Republic and Haiti, 2000, MMWR 49:1094, 2000.

6. Centers for Disease Control and Prevention: Two fatal cases of adenovirus-related illness in previously healthy young adults—Illinois, 2000, MMWR 50: 553, 2001. 7. Chen RT, Hibbs B: Vaccine safety: current and future challenges, Pediatr Ann 27:445, 1998. 8. Chen RT, Glasser JW, Rhodes PH, et al: Vaccine Safety Datalink Project: a new tool for improving vaccine safety monitoring in the United States, Pediatrics 99:765, 1997. 9. Jacobson RM, Adegbenro A, Pankratz VS, et al: Adverse events and vaccination: the lack of power and predictability of infrequent events in pre-licensure study, Vaccine 19:2428, 2001. Recent Changes in U.S. Vaccine Recommendations Related to Adverse Reactions 10. American Academy of Pediatrics, Committee on Infectious Diseases and Committee on Environmental Health: Thimerosal in vaccines: an interim report to clinicians, Pediatrics 104:570, 1999. 11. Centers for Disease Control and Prevention: Summary of the joint statement on thimerosal in vaccines, MMWR 49:622, 2000. 12. American Academy of Pediatrics, Committee on Infectious Diseases: Prevention of poliomyelitis: recommendations for use of only inactivated polio virus vaccine for routine immunization, Pediatrics 104:1404, 1999. 13. Centers for Disease Control and Prevention: Intussusception among recipients of rotavirus vaccine—United States, 1998–1999, MMWR 48:577, 1999. 14. Centers for Disease Control and Prevention: Withdrawal of rotavirus vaccine recommendation, MMWR 48:1007, 1999. IgE-Mediated Reactions to Vaccines 15. Kelso JM, Jones RT, Yunginger JW: Anaphylaxis to measles, mumps, and rubella vaccine, mediated by IgE to gelatin, J Allergy Clin Immunol 91:867, 1993. 16. Sakaguchi M, Nakayama T, Inouye S: Food allergy to gelatin in children with systemic immediate-type reactions, including anaphylaxis, to vaccines, J Allergy Clin Immunol 98:1058, 1996. 17. Patja A, Makinen-Kiljunen S, Davidkin I, et al: Allergic reactions to measles-mumps-rubella vaccination, Pediatrics 107:E27, 2001. 18. Sakaguchi M, Yamanaka T, Ikeda K, et al: IgE-mediated systemic reactions to gelatin included in the varicella vaccine, J Allergy Clin Immunol 99:263, 1997. 19. Singer S, Johnson CE, Mohr R, et al: Urticaria following varicella vaccine associated with gelatin allergy, Vaccine 17:327, 1999. 20. Sakaguchi M, Yoshida M, Kuroda W, et al: Systemic immediate-type reactions to gelatin included in Japanese encephalitis vaccines, Vaccine 15:121, 1997. 21. Nakayama T, Aizawa C: Change in gelatin content of vaccines associated with reduction in reports of allergic reactions, J Allergy Clin Immunol 106:591, 2000.

22. Kelso JM: The gelatin story, J Allergy Clin Immunol 103:200, 1999. 23. Pool V, Braun M, Kelso JM, et al: Prevalence of anti-gelatin IgE antibodies in people with anaphylaxis after measles-mumps-rubella vaccine in the U.S., Pediatrics 2000 (in press). 24. Kumagai T, Yamanaka T, Wataya Y, et al: A strong association between HLA-DR9 and gelatin allergy in the Japanese population, Vaccine 19:3273, 2001. 25. Herman JJ, Radin R, Schneiderman R: Allergic reactions to measles (rubeola) vaccine in patients hypersensitive to egg protein, J Pediatr 102:196, 1983. 26. O'Brien TC, Maloney CJ, Tauraso NM: Quantitation of residual host protein in chicken embryo derived vaccines by radial immunodiffusion, Appl Microbiol 21:780, 1971. 27. James JM, Burks AW, Roberson PK, et al: Safe administration of the measles vaccine to children allergic to eggs, N Engl J Med 332:1262, 1995. 28. Baxter DN: Measles immunization in children with a history of egg allergy, Vaccine 14:131–134, 1996. 29. Package insert: Fluogen (Parkedale). In Physicians' desk reference, Montvale, 2001, Medical Economics. 30. Package insert: FluShield (Wyeth-Ayerst). In Physicians' desk reference, Montvale, 2001, Medical Economics. 31. Package insert: Fluzone (Aventis Pasteur). In Physicians' desk reference, Montvale, 2001, Medical Economics. 32. James JM, Zeiger RS, Lester MR, et al: Safe administration of influenza vaccine to patients with egg allergy, J Pediatr 133:624, 1998. 33. Murphy KR, Strunk RC: Safe administration of influenza vaccine in asthmatic children hypersensitive to egg proteins, J Pediatr 106:931, 1985. 34. Bierman CW, Shapiro GG, Pierson WE, et al: Safety of influenza vaccination in allergic children, J Infect Dis 136:S652, 1977. 35. Miller JR, Orgel HA, Meltzer EO: The safety of egg-containing vaccines for egg-allergic patients, J Allergy Clin Immunol 71:568, 1983. 36. Yamane N, Uemura H: Serological examination of IgE- and IgG-specific antibodies to egg protein during influenza virus immunization, Epidemiol Infect 100:291, 1988. 37. Anolik R, Spiegel W, Posner M, et al: Influenza vaccine testing in egg sensitive patients, Ann Allergy 68:69, 1992. 38. Swartz H: Systemic allergic reaction induced by yellow fever vaccine, J Lab Clin Med 28:1663, 1943. 39. Sprague HB, Barnard JH: Egg allergy: significance in typhus and yellow fever immunization, U S Navy Med Bull 45:71, 1945. 40. Rubin SS: An allergic reaction following typhus-fever vaccine and yellow-fever vaccine due to egg yolk sensitivity, J Allergy 17:21, 1946. 41. Kelso JM, Mootrey GT, Tsai TS: Anaphylaxis from yellow fever vaccine, J Allergy Clin Immunol 103:698, 1999.

42. Kelso JM: Raw egg allergy: a potential issue in vaccine allergy, J Allergy Clin Immunol 106:990, 2000. 43. Lear JT, English JSC: Anaphylaxis after hepatitis B vaccination, Lancet 345:1249, 1995. 44. Primeau MN, Adkinson NF Jr, Hamilton RG: Natural rubber pharmaceutical vial closures release latex allergens that produce skin reactions, J Allergy Clin Immunol 107:958, 2001. 45. Nagel J, Svec D, Waters T, et al: IgE synthesis in man I: Development of specific IgE antibodies after immunization with tetanus-diphtheria (Td) toxoids, J Immunol 118:334, 1977. 46. Nagel JE, White C, Lin MS, et al: IgE synthesis in man II: comparison of tetanus and diphtheria IgE antibody in allergic and nonallergic children, J Allergy Clin Immunol 63:308, 1979. 47. Mark A, Bjorksten B, Granstrom M: Immunoglobulin E responses to diphtheria and tetanus toxoids after booster with aluminium-adsorbed and fluid DTvaccines, Vaccine 13:669, 1995. 48. Stratton KR, Howe CJ, Johnston RB: Adverse events associated with childhood vaccines: evidence bearing on causality, Washington, DC, 1994, National Academy Press. 49. Centers for Disease Control and Prevention: Update: vaccine side effects, adverse reactions, contraindications, and precautions—recommendations of the Advisory Committee on Immunization Practices (ACIP), MMWR 45(RR-12):1, 1996. 50. Skov PS, Pelck I, Ebbesen F, et al: Hypersensitivity to the diphtheria component in the Di-Te-Pol vaccine. A type I allergic reaction demonstrated by basophil histamine release, Pediatr Allergy Immunol 8:156, 1997. 51. Nelson MR, Oaks H, Smith LJ, et al: Anaphylaxis complicating routine childhood immunization: Haemophilus influenza b conjugated vaccine, Pediatr Asthma Allergy Immunol 14:315, 2000. 52. Grabenstein JD: Clinical management of hypersensitivities to vaccine components, Hosp Pharm 32:77, 1997. 53. Milstien JB, Gross TP, Kuritsky JN: Adverse reactions reported following receipt of Haemophilus influenzae type b vaccine: an analysis after 1 year of marketing, Pediatrics 80:270, 1987. 54. Sakaguchi M, Inouye S: Two patterns of systemic immediate-type reactions to Japanese encephalitis vaccines, Vaccine 16:68, 1998. 55. Sakaguchi M, Miyazawa H, Inouye S: Specific IgE and IgG to gelatin in children with systemic cutaneous reactions to Japanese encephalitis vaccines, Allergy 56:536, 2001. 56. Centers for Disease Control and Prevention: Inactivated Japanese encephalitis virus vaccine: recommendations of the Advisory Committee on Immunization Practices (ACIP), MMWR 42(RR-1):1, 1993. 57. Berg SW, Mitchell BS, Hanson RK, et al: Systemic reactions in U.S. Marine Corps personnel who received Japanese encephalitis vaccine, Clin Infect Dis 24:

265, 1997. 58. Plesner AM, Ronne T: Allergic mucocutaneous reactions to Japanese encephalitis vaccine, Vaccine 15:1239, 1997. 59. Ruff TA, Eisen D, Fuller A, et al: Adverse reactions to Japanese encephalitis vaccine, Lancet 338:881, 1991. 60. Plesner A, Ronne T, Wachmann H: Case-control study of allergic reactions to Japanese encephalitis vaccine, Vaccine 18:1830, 2000. 61. Aukrust L, Almeland TL, Refsum D, et al. Severe hypersensitivity or intolerance reactions to measles vaccine in six children: Clinical and immunological studies, Allergy 35:581, 1980. 62. Van Asperen PP, McEniery J, Kemp AS: Immediate reactions following live attenuated measles vaccine, Med J Aust 2:330, 1981. 63. McEwen J: Early-onset reaction after measles vaccination: further Australian reports, Med J Aust 2:503, 1983. 64. Pollock TM,. Morris J: A 7-year survey of disorders attributed to vaccination in North West Thames region, Lancet 1:753, 1983. 65. Puvvada L, Silverman B, Bassett C, et al: Systemic reactions to measles-mumps-rubella vaccine skin testing, Pediatrics 91:835, 1993. 66. Thurston A: Anaphylactic shock reaction to measles vaccine, J R Coll Gen Pract 37:41, 1987. 67. Kalet A, Berger DK, Bateman WB, et al: Allergic reactions to MMR vaccine, Pediatrics 89:168, 1992. 68. Fasano MB, Wood RA, Cooke SK, et al: Egg hypersensitivity and adverse reactions to measles, mumps, and rubella vaccine, J Pediatr 120:878, 1992. 69. Hedenskog S, Bjorksten B, Blennow M, et al: Immunoglobulin E response to pertussis toxin in whooping cough and after immunization with a whole-cell and an acellular pertussis vaccine, Int Arch Allergy Appl Immunol 89:156, 1989. 70. Torre D, Issi M, Chelazzi G, et al: Total serum IgE levels in children with pertussis, Am J Dis Child 144:290, 1990. 71. Duchen K, Granstrom M, Hedenskog S, et al: Immunoglobulin E and G responses to pertussis toxin in children immunised with adsorbed and non-adsorbed whole cell pertussis vaccines, Vaccine 15:1558, 1997. 72. Edelman K, Malmstrom K, He Q, et al: Local reactions and IgE antibodies to pertussis toxin after acellular diphtheria-tetanus-pertussis immunization, Eur J Pediatr 158:989, 1999. 73. Odelram H, Granstrom M, Hedenskog S, et al: Immunoglobulin E and G responses to pertussis toxin after booster immunization in relation to atopy, local reactions and aluminium content of the vaccines, Pediatr Allergy Immunol 5:118, 1994.

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74. Gifford CG, Gonsior EC, Villacorte GV, et al: Pertussis booster vaccination and immediate hypersensitivity, Ann Allergy 54:483, 1985. 75. Assa'ad A, Lierl M: Effect of acellular pertussis vaccine on the development of allergic sensitization to environmental allergens in adults, J Allergy Clin Immunol 105:170, 2000. 76. Centers for Disease Control and Prevention: Systemic allergic reactions following immunization with human diploid cell rabies vaccine, MMWR 33:185, 1984. 77. Dreesen DW, Bernard KW, Parker RA, et al: Immune complex-like disease in 23 persons following a booster dose of rabies human diploid cell vaccine, Vaccine 4:45, 1986. 78. Swanson MC, Rosanoff E, Gurwith M, et al: IgE and IgG antibodies to beta-propiolactone and human serum albumin associated with urticarial reactions to rabies vaccine, J Infect Dis 155:909, 1987. 79. Anderson MC, Baer H, Frazier DJ, et al: The role of specific IgE and beta-propiolactone in reactions resulting from booster doses of human diploid cell rabies vaccine, J Allergy Clin Immunol 80:861, 1987. 80. Fishbein DB, Yenne KM, Dreesen DW, et al: Risk factors for systemic hypersensitivity reactions after booster vaccinations with human diploid cell rabies vaccine: a nationwide prospective study, Vaccine 11:1390, 1993. 81. Package insert: Imovax Rabies Vaccine (Aventis Pasteur). In Physicians' desk reference, Montvale, 2001, Medical Economics. 82. Package insert: RabAvert (Chiron). In Physicians' desk reference, Montvale, 2001, Medical Economics. 83. Matuhasi T, Ikegami H: Elevation of levels of IgE antibody to tetanus toxin in individuals vaccinated with diphtheria-pertussis-tetanus vaccine, J Infect Dis 146: 290, 1982. 84. Cogne M, Ballet JJ, Schmitt C, et al: Total and IgE antibody levels following booster immunization with aluminum absorbed and nonabsorbed tetanus toxoid in humans, Ann Allergy 54:148, 1985. 85. Aalberse RC, van Ree R, Danneman A, et al: IgE antibodies to tetanus toxoid in relation to atopy, Int Arch Allergy Immunol 107:169, 1995. 86. Carey AB, Meltzer EO: Diagnosis and “desensitization” in tetanus vaccine hypersensitivity, Ann Allergy 69:336, 1992. 87. Wise RP, Salive ME, Braun MM, et al: Postlicensure safety surveillance for varicella vaccine, JAMA 284:1271, 2000. 88. Package insert: YF-VAX (Aventis Pasteur). In Physicians' desk reference, Montvale, 2001, Medical Economics. 89. Pickering LK, editor: 2000 Red book—report of the Committee on Infectious Diseases, ed 25, Elk Grove Village, IL, 2000, American Academy of Pediatrics. Non–IgE-Mediated Reactions to Vaccines

90. Centers for Disease Control and Prevention: General recommendations on immunization, MMWR 51(RR-1):1, 2002. 91. Prystowsky SD, Allen AM, Smith RW, et al: Allergic contact hypersensitivity to nickel, neomycin, ethylenediamine, and benzocaine: relationships between age, sex, history of exposure, and reactivity to standard patch tests and use tests in a general population, Arch Dermatol 115:959, 1979. 92. Goh CL: Anaphylaxis from topical neomycin and bacitracin, Aust J Dermatol 27:125, 1986. 93. Rietschel RL, Bernier R: Neomycin sensitivity and the MMR vaccine, JAMA 245:571, 1981. 94. Cox NH, Forsyth A: Thimerosal allergy and vaccination reactions, Contact Dermatitis 18:229, 1998. 95. Rietschel RL, Adams RM: Reactions to thimerosal in hepatitis B vaccines, Dermatol Clin 8:161, 1990. 96. Noel I, Galloway A, Ive FA: Hypersensitivity to thiomersal in hepatitis B vaccine, Lancet 338:705, 1991. 97. Kirkland LR: Ocular sensitivity to thimerosal: a problem with hepatitis B vaccine? South Med J 83:497, 1990. 98. Aberer W: Vaccination despite thimerosal sensitivity, Contact Dermatitis 24:6, 1991. 99. Redd SC, Markowitz LE, Katz SL: Measles vaccine. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders. 100. Barlow WE, Davis RL, Glasser JW, et al: The risk of seizures after receipt of whole-cell pertussis or measles, mumps, and rubella vaccine, N Engl J Med 345:656, 2001. 101. Beeler J, Varricchio F, Wise R: Thrombocytopenia after immunization with measles vaccines: review of the vaccine adverse events reporting system (1990 to 1994), Pediatr Infect Dis J 15:88, 1996. 102. Howson CP, Howe CJ, Fineberg HV: Adverse effects of pertussis and rubella vaccines, Washington, DC, 1991, National Academy Press. 103. Plotkin SA: Rubella vaccine. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders. 104. Edwards KM, Decker MD, Mortimer EA: Pertussis vaccine. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders Co. 105. Rosenthal S, Chen R, Hadler S: The safety of acellular pertussis vaccine vs whole-cell pertussis vaccine: a postmarketing assessment, Arch Pediatr Adolesc Med 150:457, 1996. 106. Cody CL, Baraff LJ, Cherry JD, et al: Nature and rates of adverse reactions associated with DTP and DT immunizations in infants and children, Pediatrics 68:650, 1981. 107. Shields WD, Nielsen C, Buch D, et al: Relationship of pertussis immunization to the onset of neurologic disorders: a retrospective epidemiologic study, J Pediatr 113:801, 1988. 108. DuVernoy TS, Braun MM: Hypotonic-hyporesponsive episodes reported to the Vaccine Adverse Event Reporting System (VAERS), 1996–1998, Pediatrics

106:E52, 2000. 109. Heijbel H, Ciofi degli Atti MC, Harzer E, et al: Hypotonic hyporesponsive episodes in eight pertussis vaccine studies, Dev Biol Stand 89:101, 1997. 110. Stratton KR, Howe CJ, Johnston RB: DPT vaccine and chronic nervous system dysfunction: a new analysis, Washington, DC, 1994, National Academy Press. 111. Wassilak SG, Orenstein WA, Sutter RW: Tetanus toxoid. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders Co. 112. Pounder DJ: Sudden, unexpected death following typhoid-cholera vaccination, Forensic Sci Int 24:95, 1984. 113. Kelleher PC, Kelley LR, Rickman LS: Anaphylactoid reaction after typhoid vaccination, Am J Med 89:822, 1990. 114. Rone JK, Friedstrom S: Severe systemic reactions to typhoid vaccination: two cases and a review of the literature, Mil Med 155:272, 1990. 115. Hoyt RE, Herip DS: Severe systemic reactions attributed to the acetone-inactivated parenteral typhoid vaccine, Mil Med 161:339, 1996. 116. Package insert: Varivax (Merck). In Physicians' desk reference, Montvale, 2001, Medical Economics. 117. Sharrar RG, LaRussa P, Galea SA, et al: The postmarketing safety profile of varicella vaccine, Vaccine 19:916, 2000. 118. Monath TP: Yellow fever. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders. 119. Vasconcelos PF, Luna EJ, Galler R, et al: Serious adverse events associated with yellow fever 17DD vaccine in Brazil: a report of two cases, Lancet 358:91, 2001. 120. Chan RC, Penney DJ, Little D, et al: Hepatitis and death following vaccination with 17D-204 yellow fever vaccine, Lancet 358:121, 2001. 121. Martin M, Tsai TF, Cropp B, et al: Fever and multisystem organ failure associated with 17D-204 yellow fever vaccination: a report of four cases, Lancet 358:98, 2001. Adverse Reactions to Vaccines for Biological Agents Used as Weapons 122. Franz DR, Jahrling PB, Friedlander AM, et al: Clinical recognition and management of patients exposed to biological warfare agents, JAMA 278:399, 1997. 123. Package insert: Anthrax vaccine absorbed (Bioport). 124. Centers for Disease Control and Prevention: Use of anthrax vaccine in the United States: recommendations of the Immunization Practices Advisory Committee (ACIP), MMWR 49(RR-15):1, 2000. 125. Fulco GE, Liverman CT, Sox HC: Gulf War and health, Washington, DC, 2000, National Academy Press. 126. Memorandum from the Secretary of Defense on implementation of the anthrax vaccination program for the total force, dated May 18, 1998.

127. Friedlander AM, Pittman PR, Parker GW: Anthrax vaccine: evidence for safety and efficacy against inhalational anthrax, JAMA 282:2104, 1999. 128. Fenner F, Henderson DA, Arita I, et al: Smallpox and its eradication, Geneva, 1988, World Health Organization. 129. Henderson DA, Inglesby TV, Bartlett JG, et al: Smallpox as a biological weapon: medical and public health management, JAMA 281:2127, 1999. 130. Centers for Disease Control and Prevention: Vaccinia (smallpox) vaccine: recommendations of the Immunization Practices Advisory Committee (ACIP), 2001, MMWR 50(RR-10):1, 2001. 131. Centers for Disease Control and Prevention: Interim smallpox response plan and guidelines, draft 2.0 11/21/01 http://www.bt.cdc.gov/DocumentsApp/Smallpox/ RPG/index.asp. 132. Inglesby TV, Dennis DT, Henderson DA, et al: Plague as a biological weapon: medical and public health management, JAMA 283:2281, 2000. 133. Arnon SS, Schechter R, Inglesby TV, et al: Botulinum toxin as a biological weapon: medical and public health management, JAMA 285:1059, 2001. 134. Dennis DT, Inglesby TV, Henderson DA, et al: Tularemia as a biological weapon: medical and public health management, JAMA 285:2763, 2001. Controversies Regarding Long-Term Consequences of Vaccination 135. Björkstén B: The intrauterine and postnatal environments, J Allergy Clin Immunol 104:1119, 1999. 136. Magnus P, Jaakkola JJ: Secular trend in the occurrence of asthma among children and young adults: critical appraisal of reported cross-sectional surveys, BMJ 314:1795, 1997. 137. Hertzen LC: The hygiene hypothesis in the development of atopy and asthma: still a matter of controversy? Q J Med 91:767, 1998. 138. Shirakawa T, Enomoto T, Shimazu S, et al: The inverse association in tuberculin responses and atopic disorder, Science 275:77, 1997. 139. Alm JS, Lilja G, Pershagen G, et al: Early BCG vaccination and development of atopy, Lancet 350:400, 1997. 140. Gruber C, Kulig M, Bergmann R, et al: Delayed hypersensitivity to tuberculin, total immunoglobulin E, specific sensitization, and atopic manifestation in longitudinally followed early Bacille Calmette-Guerin-vaccinated and nonvaccinated children, Pediatrics 107:E36, 2001. 141. Shaheen SO, Aaby P, Hall AJ, et al: Measles and atopy in Guinea-Bissau, Lancet 347:1792, 1996. 142. Paunio M, Heinonen OP, Virtanen M, et al: Measles history and atopic diseases: a population based cross-sectional study, JAMA 283:343, 2000. 143. Farooqi IS, Hopkin JM: Early childhood infection and atopic disorder, Thorax 53:927, 1998. 144. Nilsson L, Kjellman NI, Björkstén B: A randomized controlled trial of the effect of pertussis vaccines on atopic disease, Arch Pediatr Adolesc Med 152:734, 1998.

145. Anderson HR, Poloniecki JB, Strachan DP, et al: Immunization and symptoms of atopic disease in children: results from the International Study of Asthma and Allergies in Childhood, Am J Public Health 91:1126, 2001.

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146. Wakefield AJ, Murch SH, Anthony A, et al: Ileal-lymphophoid-nodular hyperplasia, nonspecific colitis, and pervasive developmental disorder in children, Lancet 351:637, 1998. 147. Taylor B, Miller E, Farrington CP, et al: Autism and measles, mumps, and rubella vaccines: no epidemiological evidence for a causal association, Lancet 353: 2026, 1999. 148. Dales L, Hammer SJ, Smith NJ: Time trends in autism and in MMR immunization coverage in California, JAMA 285:1183, 2001. 149. Stratton K, Gable A, Shetty P, et al: Immunization safety review: measles-mumps-rubella vaccine and autism, Washington, DC, 2001, National Academy Press. 150. Fourrier A, Touze E, Alperovitch A, et al: Association between hepatitis B vaccine and multiple sclerosis: a case-control study, Pharmacoepidemiol Drug Saf 8: S140, 1999. 151. Monteyne P, Andre FE: Is there a causal link between hepatitis B vaccination and multiple sclerosis? Vaccine 18:1994, 2000. 152. Ascherio A, Zhang SM, Hernan MA, et al: Hepatitis B vaccination and the risk of multiple sclerosis, N Engl J Med 344:327, 2001. 153. Confavreux C, Suissa S, Saddier P, et al: Vaccinations and the risk of relapse in multiple sclerosis, N Engl J Med 344:319, 2001. Vaccination Relative to Immunocompromise and Immunoglobulin Preparations 154. Moss W, Lederman H: Immunization of the immunocompromised host, Clinical Focus on Primary Immune Deficiencies 1:1, 1998. 155. Fedson DS, Musher DM, Eskola J: Pneumococcal vaccine. In Plotkin SA, Orenstein WA, editors: Vaccines, ed 3, Philadelphia, 1999, WB Saunders. 156. Ochs HD, Davis SD, Wedgwood RJ: Immunologic responses to bacteriophage phi X 174 in immunodeficiency diseases, J Clin Invest 50:2559, 1971. 157. Pyun KH, Ochs HD, Wedgwood RJ, et al: Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody, Clin Immunol Immunopathol 51:252, 1989.

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Chapter 92 - Drug Allergy

N. Franklin Adkinson Jr.

Adverse reactions to pharmaceutical and diagnostic products constitute a major hazard in the practice of medicine[

1] [2] [3] [4] [5] [6]

and are responsible for substantial

7 8 9 10 cost.[ ] [ ] [ ] [ ]

morbidity and One useful classification of adverse drug reactions distinguishes between reactions that can affect many if not most patients, given sufficient therapeutic intensity, and reactions usually conceptualized as hypersensitivity, the risk for which is restricted to a small subset of treated patients ( Table 921 ). One subset is patients who experience pharmacologically predictable side effects and toxicity of one or more drugs, but at low and sometimes even subtherapeutic doses. This putatively reflects altered drug metabolism or end-organ hyperacuity. Patients with this type of broad drug intolerance are easily identified by careful documentation of drug experiences. Idiosyncratic drug reactions are qualitatively distinct from the known pharmacologic toxicity profiles. Such reactions may result from a defined genetic defect, as in the well-studied example of primaquine-sensitive hemolytic anemia, which depends on deficiency of the enzyme 11

glucose-6-phosphodehydrogenase (G6PD).[ ] The mechanism of most idiosyncratic drug reactions remains obscure and often reflects a complex interaction of metabolic and constitutional factors (e.g., radiocontrast media reactions). Drug reactions resulting from consequences of a drug-specific immune response constitute immunologic drug reactions, usually referred to as drug allergy. The distinction between idiosyncratic drug reactions and drug allergy can be difficult to discern clinically, but it is of considerable importance because diagnosis and management will differ. This chapter reviews current understanding of the ways drugs can sensitized the immune system, the factors that influence drug immunogenicity, and the specificity of drug-induced immune responses. The effector mechanisms recognizable in drug allergy syndromes are distinguished from idiosyncratic reactions that mimic drug allergy (“pseudoallergy”). An approach to the diagnosis of drug allergy is offered, followed by a discussion of the management of drug allergic states, with a focus on the issues of prophylaxis and readministration to previously reactive patients.

CATEGORIES OF DRUG ALLERGENS 12

Karl Landsteiner first proposed an essential requirement for multivalency in the initiation of immune responses to foreign substances.[ ] The current paradigm insists that with few exceptions, an antigen must be presented to the immune system in multivalent form both to elicit a specific immune response (sensitization) and to activate immunopathologic mechanisms (effector functions) ( Figure 92-1 ). Some macromolecules used therapeutically meet this requirement intrinsically by virtue of large molecular weight with multiple repeating epitopes ( Table 92-2 ). A few interesting small molecules have multiple recurrences of a single epitope and thus qualify as “complete” allergens. The best-studied example is quaternary ammonium epitopes, which render drugs such as succinylcholine bivalent and related neuromuscular blockers as multivalent.[ qualify as drug allergens.

13] [14] [15] [16]

Most pharmaceutical agents are simple in structure and of small molecular weight; fortunately, most do not

There are two ways in which small chemicals (1•ng/ml) and plasma histamine (>10•nM/L) taken within 4 hours of 116 117

118]

a putative acute allergic event suggest mast cell and basophil activation.[ ] [ ] Mild anaphylaxis without hemodynamic changes may still be falsely negative.[ Total hemolytic complement (CH50 ) and major complement proteins (C3 and C4) are useful indicators of the presence and severity of complement activation in

immune complex disorders. The diagnostic utility of anaphylatoxin measurements (C3a, C4a, and C5a) and eosinophilic cationic protein (ECP) remains to be defined. [119] [120] [121]

However, these and other markers of inflammation only identify pathologic processes; they are less likely to be diagnostic of specific drug allergy

states. Immunohistochemical methods can assess activated inflammatory cells, lymphocyte subsets, and their products. [

122] [123]

1687

Figure 92-6 Approach to establishing current hypersensitivity status in patients with prior drug reactions to known agents.

Such techniques hold promise for

TABLE 92-8 -- Predictive Value of Penicillin Skin Testing

Prior Reaction

Penicillin Skin Tests

IgE-Dependent Reaction (%)

Any

Positive

50–80

All histories

Negative

2.6

• Anaphylaxis

3.6

†

*

Any Immunologic Reaction (%) — 5.4 7.1

•Urticaria

4.7

7.1

•Exanthem

2.0

6.8

•Other/unknown

1.1

3.0

None

Positive

50

Negative * Limited data from inadvertent treatment or provocational challenge.[

0.4

—

‡

†

1.0

174] 95

† Data from large, outpatient, sexually transmitted disease clinical study.[ ] ‡ Data unavailable for major and minor determinant testing. Early studies with penicilloyl polylysine in history-negative subjects indicated a low reaction rate 175]

(5.5%). [

1688

drugs, neuromuscular blockers, thiobarbiturates, some anticonvulsants, and local anesthetics. Insulin and other recombinant or native proteins (e.g., heteroantisera) are complete antigens and thus can engender an immune response under appropriate 135]

circumstances.[

Exceptions include protamine, a highly charged foreign protein that does not reliably provoke wheal-and-flare responses in all patients who have

circulating IgE antibody.[

136] [137]

Solid-phase immunoassays (RAST, ELISA) have been established for a wide variety of drug allergies, using sera from skin-test-positive patients for standardization. These serologic tests for IgE antibody have been useful in confirming positive skin tests, in evaluating allergenic specificities and the contributions of various macromolecular carriers, in evaluating cross-reactivity with similar compounds, and in longitudinal studies of the fluxes of such antibodies over time.[ penicillin allergy have in vitro test results been systematically compared with skin penicilloyl-IgE of 65% to 85% compared with penicilloyl-polylysine skin 140 challenge.[ ]

139 140 tests.[ ] [ ]

139 tests[ ]

138]

Only with

The consistent finding has been diagnostic sensitivity for

and 32% to 50% compared with a combination of skin testing and prevocational

Minor determinant penicillin IgE antibodies are not reliably detected by available radioallergosorbent (RAST)-type immunoassays. For these reasons, 141]

intradermal skin testing remains the diagnostic procedure of choice for IgE-dependent penicillin allergy.[

Immunoassays for IgG, IgM, or IgA responses to drug allergens have not proven to be clinically useful. IgG immune responses to the penicilloyl determinant occur

42

in half of patients receiving high-dose therapy,[ ] but the IgG response is not associated with drug allergy or risk for re-treatment. IgG antidrug can attenuate the biologic activity of complete drug allergens such as antisera, hormones, and cytokines. Earlier lymphocyte activation tests using drugs as stimulants were often strongly positive in drug-allergic subjects, but the response usually was not distinguishable from patients receiving equally intense and recent therapy but without 142 143

144

reactions.[ ] [ ] Studies have reported more favorable results with a variety of drugs.[ ] The lymphocyte transformation test was positive in 78% of patients classified as highly likely to be drug allergic on clinical grounds. Overall specificity was 85%; however false-positive results were observed, especially with NSAIDs. [

144]

This study was retrospective and did not include recently treated nonreactive patients as controls, but these favorable results indicate the need for

reevaluation of this and other in vitro T cell assays, especially those involving drug-stimulated interleukin-5 (IL-5) production. [

145] [146]

In vivo assays of cutaneous responses to putative drug allergens may also have diagnostic utility. Late-onset maculopapular rash may be the most common form of drug allergy, and increasing evidence indicates that these rashes are likely T cell mediated.[

122]

Aminopenicillins induce delayed exathema with a frequency threefold 46

to fivefold higher than with other penicillins, and evidence suggests that the risk of rash is related to class II MHC genes.[ ] Recent studies suggest that both patch tests and intradermal tests with delayed cutaneous readouts are useful in evaluating nonimmediate reactions to aminopenicillins, and that both can reliably predict the 147

results of rechallenge.[ ] Additional studies are required to confirm and extend these results to other drug allergies and to define more precisely the clinical correlation and predictive value for re-treatment.

MANAGEMENT OF DRUG ALLERGY Acute Treatment The management of acute allergic drug reactions involves identification and withdrawal of the offending agent; introduction of required supportive, suppressive, or remittive therapy, and consideration of whether and how the incriminating drug should be substituted. An approach to discovering the offending drug when multiple drugs are co-administered is discussed earlier. Withdrawal of the identified drug allergen is almost always prudent and may immediately attenuate the reaction. There are some exceptions to this rule. In patients with life-threatening enterococcal endocarditis who require long-term treatment with high-dose penicillin to effect a cure, “treating through” isolated episodes of urticaria and generalized pruritus may be necessary. Experience suggests that most such episodes are self-limited and will remit with continuous therapy. Antihistamines can be used to suppress symptoms while careful surveillance is established for multisystem immunopathology, such as drug fever, hepatitis, and renal damage. When treating through mild type I reactions, it is imperative to avoid lapses in treatment because restarting treatment after a lapse may invoke anaphylaxis. Late-occurring maculopapular exanthems with minimal or no pruritus is another setting for continuing treatment through relatively mild reactions, provided there is a compelling clinical need to do so. A high success rate with spontaneous resolution of ampicillin rashes despite continuation of 59

therapy has been reported in children,[ ] but anecdotal experience suggests that this is not always achievable in adults or with other drugs. Careful monitoring for fever, eosinophilia, proteinuria, arthralgia, lymphadenopathy, and hepatitis (if the drug is metabolized in the liver) is warranted, with prompt cessation of therapy if new signs or symptoms appear. Severe exfoliative syndromes, including Stevens-Johnson and toxic epidermal necrolysis, as well as any drug rash involving mucosal surfaces, warrant immediate drug withdrawal and often hospitalization. Drug-induced allergic syndromes are pharmacologically treated similar to non-drug-induced immunopathology. Treatment of urticaria and angioedema, anaphylaxis, serum sickness, immune cytopenias, and contact sensitivity is detailed in other chapters. Serum sickness syndromes due to immune complexes can often be

13 148

attenuated by plasmapheresis. Antibody-mediated haptenic drug allergy can be abrogated rapidly by infusion of a monovalent drug hapten,[ ] [ ] but no licensed pharmaceutical products of this type are currently available. Severe drug-induced immunopathology of any type usually prompts the use of corticosteroids. Whether 6] [149]

high-dose steroids is an effective remittive agent for exfoliative derma titis syndromes is controversial.[ with apparent success in a few patients.

Intravenous immune globulin (IVIG) has been used

[87] [150]

When ambiguity surrounds which drug induced a severe immunologic reaction, plans should be made to pursue a definitive diagnosis after the patient's convalescence. Any type I drug skin testing should be delayed at least 2 to 4 weeks after an acute anaphylactic episode to avoid testing during a refractory period. When the drug culprit is identified, the patient should be provided instruction on avoidance, including a written list of cross-reactive drugs. The use of wallet cards, identification jewelry, and registry services

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(e.g., Medic-Alert) should be recommended for patients with documented severe reactions. Alternatives for Drug-Allergic Patients There are three alternative approaches to providing acceptable pharmacotherapy for the underlying condition in drug-allergic patients. The most common approach is an unrelated alternative medication that is safe and effective for the disease requiring treatment. Careful attention should be given to the risks of second-line therapy, especially treatment failure with antibiotics, and the toxicity and cost of alternative regimens. For example, an increasing number of alternative antibiotics are becoming available for substitution in penicillin-allergic patients. However, many alternative choices produce higher treatment failure rates and inflict more toxicity than β-lactams, and some increase drug treatment costs many fold. For most common outpatient infections, alternative antibiotics provide a reasonable choice for the penicillin-allergic patient. For hospitalized patients with serious systemic infections, such as enterococcal endocarditis, bacterial meningitis, and brain abscesses, the risk of treatment failure with a second-line drug is an important consideration. The second alternative for drug-sensitive patients is to receive a medication not identical to but potentially cross-reactive with the offending drug allergen. Common clinical examples are the use of cephalosporins and penems in penicillin-allergic subjects,[

151]

drugs containing a sulfa moiety (e.g., sulfonylureas, celecoxib,

152 sulfamethoxazole,[ ]

153

sulfasalazine) in patients reactive to and alternative anticonvulsants within a broadly cross-reactive group.[ ] Cross-reactivity among βlactam antibiotics is discussed earlier (see Figure 92-3 ). Although the absolute risk of cross-allergic reactions is small (8-mm wheal is observed. Test is positive if both duplicate wheals increase significantly (>2–3•mm) 20 minutes after placement compared to diluent control. 2. Prepare sufficient quantities of drug solution/suspension for desensitization regimen in half-log10 dilutions (threefold and tenfold dilutions from concentrate, 1–3•mg/ml).

Procedure 1. Establish baseline monitoring of patient in medical setting appropriate for patient's clinical conditions and nature/severity of prior reaction. Start a secure intravenous infusion. 2. Starting dose: If skin test negative and test is unvalidated, begin with 0.1•ml of 1–3••g/ml solution/suspension; if skin test positive, begin 100-fold below dose that produces midpoint reaction (5- to 8-mm wheal). 3. Route: Oral by ingestion or nasogastric tube in 30•ml water; parenteral by intradermal (0.6•ml) injection. 4. Dosing interval: 15–20 minutes for parenteral doses; 20–30 minutes for oral dosing. Repeat dose for mild systemic reaction; drop back two doses (tenfold) for moderate reactions and further for any reaction producing hemodynamic changes. 5. Dose escalation: half-log10 (∼threefold) increments (1, 3, 10, 30, 100••g, etc.) 6. If IV therapy is indicated, begin infusion to deliver a dose equivalent to last oral/parenteral dose slowly over 1 hour. Double the infusion rate every hour until target therapeutic dosing is achieved.

Follow-up 1. If intravenous therapy follows desensitization, continuous 24-hour drug infusion is preferable if feasible. If not, avoid rapid infusion of intermittent drug doses. 2. If drug skin test was positive, repeat after desensitization to document shift in skin sensitivity. 3. Avoid lapses in therapeutic doses. 4. Treat through select mild to moderate reactions (e.g., urticaria) to avoid need to repeat desensitization. 5. Before subsequent courses of therapy, repeat desensitization if skin tests remain positive; desensitization therapy is dose and drug dependent.

Readministration of Previously Offending Agents Until relatively recently, the medical culture regarded readministration of a medicinal agent to a patient who previously reacted adversely to the drug as strongly contraindicated and fraught with concerns about both violation of the Hippocratic oath and medicolegal liability. Experience now teaches that dismissing the possibility of re-treatment often does the patient a disservice by withholding indicated therapy that may be well tolerated the second time. There are multiple reasons why a previously reactive patient may tolerate re-treatment. The most common is misdiagnosis of the original reaction based on a temporal association that was not causally linked. IgE antibody responses dissipate over time, especially for haptenic drugs, and as amply demonstrated for penicillins, a large majority of patients may lose sensitivity over time. Some pseudoallergic reactions result from psychologic overgeneralization of an initial frightening or life-threatening drug treatment episode. Also, biologic states required for reactivity may wane over time and render the patient less vulnerable. For example, aspirin-like drugs often aggravate chronic idiopathic urticaria but are well tolerated once the urticaria remits. These factors provide opportunities for safe re-treatment of a surprisingly large number of patients with adverse drug experiences, both allergic and idiosyncratic. When drugs being withheld are uniquely beneficial, or if alternative therapy has failed, risks need to be weighed against anticipated benefits. With a few exceptions, the possibility of readministration can be tested safely using gradual dose escalation. Factors to consider include the severity of the prior reaction and how recently it occurred, as well as the mechanism of immunopathology, if known. For IgE-dependent reactions, the principal concern is anaphylaxis, and lower initial doses are warranted. For some life-threatening reactions, such as exfoliative dermatitis syndromes and dermatoses with mucous membrane lesions (Stevens-Johnson like), readministration of any dose is strongly contraindicated. Otherwise, initial doses for re-treatment can generally begin at 1% of the desired therapeutic dose. The oral route is preferred, especially for haptenic drugs. At appropriate intervals, as determined by the pharmacology of the drug and the patient's prior experiences with the agent, half-log10 dose escalations (about threefold)

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are appropriate. The strategy is to use incremental doses and intervals such that a very mild clinical reaction will be elicited if the patient remains sensitive. Once the full therapeutic dose has been achieved without incident, continuous therapy should be begun immediately with appropriate monitoring. When there is clinical suspicion of anticipatory reactions (classic conditioning) or strong emotional elements are present, the inclusion of placebos in serial challenges is advisable. A decision tree helps to outline the prerequisites for readministration (see Figure 92-6 ). As discussed earlier, premedication with antihistamines and corticosteroids has been shown to reduce the incidence and severity of acute pseudoallergic reactions to radiocontrast media (see Table 92-7 ). Such regimens have not been systematically studied for the prevention of IgE-mediated anaphylaxis. Numerous anecdotes attest to the failure of steroids and antihistamines to prevent serious anaphylactic episodes. Premedication may mask early symptoms and allow dosing to proceed more rapidly than advisable. Drugs reinstituted under the cover of corticosteroids may still be problematic when steroids are withdrawn. For these reasons, we prefer not to use premedication before gradual dose escalation of drugs implicated in type I allergy, or when undertaking drug desensitization. Recently developed antileukotriene drugs are very effective in blocking asthma induced by NSAIDs[

171]

and are additive with antihistamines in attenuating antigen-induced pulmonary

172 reactions,[ ]

but their utility in preventing type I drug allergy has yet to be studied. As other new antagonists for inflammatory mediators become available, more effective premedication regimens may be developed for a variety of immunopathologic reactions. Prophylaxis in Drug Allergy–Prone Patients Some individuals are vulnerable to allergic drug reactions as a result of genetic or metabolic abnormalities (e.g., multiple drug allergy syndrome), frequent and recurrent drug exposure (e.g., antibiotics in cystic fibrosis), or certain disease states related to immune dysfunction (e.g., AIDS). Such patients are likely to benefit from a thorough and proactive evaluation that documents sensitivities ( Box 92-4 ). Ongoing reevaluation will help to keep the list of usable drugs from becoming unacceptably limited. In a drug reaction–prone population, pharmacotherapy requires special management to avoid adverse events and to prevent (to the degree possible) sensitization or resensitization. Preventing recurrent infections is a prime objective in patients with multiple antibiotic sensitivities. Aggressive management of rhinosinusitis can help minimize recurrent otitis media and sinusitis. Up-to-date vaccination is highly desirable. Avoidance of unnecessary exposures to contagious diseases may be helpful, especially in children. Low-dose prophylactic antibiotics may paradoxically reduce drug allergy if they prevent recurrent infections. Stringent indications for antibiotics, including a requirement for positive cultures, will help to limit cumulative drug exposures. These approaches can have substantial clinical impact on antimicrobial allergy, especially in patients constitutionally predisposed to haptenic drug allergy. For drug-intolerant patients, new drug therapy should begin at lower doses than required for therapy. Administration under medical observation is advisable when risk is considered substantial or the patient requires reassurance. Consideration

Box 92-4. Evaluation and Management of Multidrug-Sensitive Patient

Evaluation 1. Careful drug sensitivity data with focus on common features (groups of related drugs, similar time to reaction, commonality of syndrome elements). 2. Skin test evaluation for drugs with validated skin test (e.g., proteins, penicillin, quaternary ammonium compounds, cisplatin), or as preliminary evaluation to provocational testing. 3. Provocational testing for unconvincing reports, or mild symptoms, especially if the drug therapy remains clinically indicated. 4. Placebo-controlled challenges when the reaction patterns are similar for unrelated drugs, to demonstrate operant conditioning in suspected cases.

Management 1. Measures to prevent infections (if antimicrobials are implicated). 2. Stringent criteria for new drug use. 3. For intolerant patients, initiation of therapy at one-third to one-tenth the usual dose. 4. Medical observation after first dose of new or suspect drugs. 5. Careful monitoring and encouragement to allow treating through selected mild or atypical reactions. 6. Gradual dose escalation or desensitization as warranted. 7. Monitoring for, and discontinuing drug at earliest signs of severe cutaneous reactions (Stevens-Johnson syndrome, exfoliative dermatitis, toxic epidermal necrolysis).

should be given to treating through mild cutaneous reactions, including urticaria, especially early in therapy when an alternative antibiotic will usually be required. High-risk patients should also be advised to monitor for constitutional signs such as fever or early evidence of mucositis as warning signs. Aggressive management by a knowledgeable physician can greatly expedite the long-term care of drug allergy–prone patients by preserving and protecting therapeutic options.

SUMMARY

Drug hypersensitivity states present an often frustrating challenge for most practicing physicians. Knowledge of the natural history and pathogenesis of many immunologic and idiosyncratic drug reactions remains superficial and incomplete. Because immunodiagnostic tests for drug allergy are limited in number and require some sophistication to interpret, many practitioners have concluded that the only reasonable option for drug-reactive patients is permanent and total avoidance of putative offenders. In the extreme, patients with multiple drug hypersensitivity syndromes are sometimes abandoned by their primary physicians, or they are told to do without all drug therapy. Armed with an understanding of the distinction between drug allergy and idiosyncrasy, the risk factors for drug allergy,

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and the pharmacoepidemiology of sensitizing drugs, a medical practitioner can safely provide useful drug therapy for a surprisingly large number of drug-sensitive patients. For allergy/immunology specialists, the willingness to undertake this task is usually appreciatively obliged by other professionals, who readily refer drugsensitive patients and are grateful for the assistance received. Medical progress in understanding and managing drug hypersensitivity states often requires the collaborative efforts of multiple disciplines, including basic immunology, pharmacology and toxicology, genetics, biochemistry, pathology, and epidemiology. The high morbidity and cost associated with drug hypersensitivity 7]

makes this set of disorders a high priority for future research investment.[

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109. Enright T, Chua-Lim A, Duda E, Lim DT: The role of a documented allergic profile as a risk factor for radiographic contrast media reaction, Ann Allergy 62:302, 1989. 110. Lieberman PL, Seigle RL: Reactions to radiocontrast material: anaphylactoid events in radiology, Clin Rev Allergy Immunol 17:469, 1999. 111. Lang DM, Alpern MB, Visintainer PF, Smith ST: Elevated risk of anaphylactoid reaction from radiographic contrast media is associated with both beta-blocker exposure and cardiovascular disorders, Arch Intern Med 153:2033, 1993. 112. Kowalski ML: Aspirin sensitive rhinosinusitis and asthma, Allergy Proc 16:77, 1995. 113. Schatz M: Skin testing and incremental challenge in the evaluation of adverse reactions to local anesthetics, J Allergy Clin Immunol 74:606, 1984. 114. Gall H, Kaufmann R, Kalveram CM: Adverse reactions to local anesthetics: analysis of 197 cases, J Allergy Clin Immunol 97:933, 1996. 115. Aldrete JA, Johnson DA: Evaluation of intracutaneous testing for investigation of allergy to local anesthetic agents, Anesth Analg Curr Res 49:173, 1970. Diagnosis of Drug Allergy 116. Schwartz LB, Bradford TR, Rouse C, et al: Development of a new, more sensitive immunoassay for human tryptase: use in systemic anaphylaxis, J Clin Immunol 14:190, 1994. 117. Lin RY, Schwartz LB, Curry A, et al: Histamine and tryptase levels in patients with acute allergic reactions: an emergency department-based study, J Allergy Clin Immunol 106:65, 2000. 118. Schwartz LB, Metcalfe DD, Miller JS, et al: Tryptase levels as an indicator of mast-cell activation in systemic anaphylaxis and mastocytosis, N Engl J Med 316:1622, 1987. 119. Tomassini M, Magrini L, De Petrillo G, et al: Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure, J Allergy Clin Immunol 97:1350, 1996. 120. Dykewicz MS, Rosen ST, O'Connell MM, et al: Plasma histamine but not anaphylatoxin levels correlate with generalized urticaria from infusions of antilymphocyte monoclonal antibodies, J Lab Clin Med 120:290, 1992. 121. Szebeni J: Complement activation-related pseudoallergy caused by liposomes, micellar carriers of intravenous drugs, and radiocontrast agents, Crit Rev Ther Drug Carrier Syst 18:567, 2001. 122. Yawalkar N, Pichler WJ: Immunohistology of drug-induced exanthema: clues to pathogenesis, Curr Opin Allergy Clin Immunol 14:299, 2001. 123. Posadas SJ, Padial A, Torres MJ, et al: Delayed reactions to drugs show levels of perforin, granzyme B, and Fas-L to be related to disease severity, J Allergy Clin Immunol 109:155, 2002. 124. Sullivan TJ, Wedner HJ, Shatz GS, et al: Skin testing to detect penicillin allergy, Allergy Clin Immunol 68:171, 1981.

125. Salkind AR, Cuddy PG, Foxworth JW: The rational clinical examination: is this patient allergic to penicillin? An evidence-based analysis of the likelihood of penicillin allergy, JAMA 285:2498, 2001. 126. Pichichero ME, Pichichero DM: Diagnosis of penicillin, amoxicillin, and cephalosporin allergy: reliability of examination assessed by skin testing and oral challenge, J Pediatr 132:137, 1998. 127. Macy E, Richter PK, Falkoff R, Zeiger R: Skin testing with penicilloate and penilloate prepared by an improved method: amoxicillin oral challenge in patients with negative skin test responses to penicillin reagents, J Allergy Clin Immunol 100:586, 1997. 128. Arroliga ME, Wagner W, Bobek MB, et al: A pilot study of penicillin skin testing in patients with a history of penicillin allergy admitted to a medical ICU, Chest 118:1106, 2000. 129. Mendelson LM, Ressler C, Rosen JP, Selcow JE: Routine elective penicillin allergy skin testing in children and adolescents: study of sensitization, J Allergy Clin Immunol 73:76, 1984. 130. Solensky R, Earl HS, Gruchalla RS: Penicillin allergy: prevalence of vague history in skin test-positive patients, Ann Allergy Asthma Immunol 85:195, 2000.

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131. Parker PJ, Parrinello JT, Condemi J, Rose SI: Penicillin resensitization among hospitalized patients, J Allergy Clin Immunol 88:213, 1991. 132. Reed MA, Lopez M, Gutierrez E, et al: Quantitation of sulfamethoxazole-human serum albumin SMX-HSA IgE antibodies in HIV+ subjects, J Allergy Clin Immunol 87:230, 1991 (abstract). 133. Macy E: Current sulfamethoxazole skin test reagents fail to predict recurrent adverse reactions, J Allergy Clin Immunol 95:121, 1995 (abstract). 134. Gruchalla RS, Sullivan TJ: Detection of human IgE to sulfamethoxazole by skin testing with sulfamethoxazoly poly-l-tyrosine, J Allergy Clin Immunol 88:784, 1991. 135. Patterson R, Roberts M, Grammer LC: Insulin allergy: re-evaluation after two decades, Ann Allergy 64:459, 1990. 136. Weiss ME, Adkinson NF Jr: Allergy to protamine, Clin Rev Allergy 9:339, 1991. 137. Weiss ME, Chatham F, Kagey-Sobotka A, Adkinson NF Jr: Serial immunological investigations in a patient who had a life-threatening reaction to intravenous protamine, Clin Exp Allergy 20:713, 1990. 138. Adkinson NF Jr: Tests for immunological reactions to drugs and occupational allergens. In Rose NR, Conway de Macario E, Folds JD, et al, editors: Manual of clinical laboratory immunology, Washington, DC, 1997, American Society of Microbiology, p 893.

139. Garcia JJ, Blanca M, Moreno F, et al: Determination of IgE antibodies to the benzylpenicilloyl determinant: a comparison of the sensitivity and specificity of three radioallergosorbent test methods, J Clin Lab Anal 11:251, 1997. 140. Blanca M, Mayorga C, Torres MJ, et al: Clinical evaluation of Pharmacia CAP System RAST FEIA amoxicilloyl and benzylpenicilloyl in patients with penicillin allergy, Allergy 56:862, 2001. 141. Executive summary of disease management of drug hypersensitivity: a practice parameter. Joint Task Force on Practice Parameters, American College of Allergy, Asthma and Immunology, American Academy of Allergy, Asthma and Immunology, Joint Council of Allergy, Asthma and Immunology, Ann Allergy Asthma Immunol 83:665, 1999. 142. Stejskal VD, Olin RG, Forsbeck M: The lymphocyte transformation test for diagnosis of drug-induced occupational allergy, J Allergy Clin Immunol 77:411, 1986. 143. Gruchall RS, Sullivan TJ: In vivo and in vitro diagnosis of drug allergy, Immunol Allergy Clin North Am 11:595, 1991. 144. Nyfeler B, Pichler WJ: The lymphocyte transformation test for the diagnosis of drug allergy: sensitivity and specificity, Clin Exp Allergy 27:175, 1997. 145. Sachs B, Erdmann S, Malte BJ, et al: Determination of interleukin-5 secretion from drug-specific activated ex vivo peripheral blood mononuclear cells as a test system for the in vitro detection of drug sensitization, Clin Exp Allergy 32:736, 2002. 146. Brugnolo F, Annunziato F, Sampognaro S, et al: Highly Th2-skewed cytokine profile of beta-lactam-specific T cells from nonatopic subjects with adverse drug reactions, J Immunol 163:1053, 1999. 147. Romano A, Quaratino D, Di Fonso M, et al: A diagnostic protocol for evaluating nonimmediate reactions to aminopenicillins, J Allergy Clin Immunol 103:1186, 1999. Management of Drug Allergy 148. De Weck AL, Schneider CH: Specific inhibition of allergic reactions to penicillin in man by a monovalent hapten. II. Clinical studies, Int Arch Allergy 42:798, 1972. 149. Patterson R, Miller M, Kaplan M, et al: Effectiveness of early therapy with corticosteroids in Stevens-Johnson syndrome: experience with 41 cases and a hypothesis regarding pathogenesis, Ann Allergy 73:27, 1994. 150. Tristani-Firouzi P, Petersen MJ, Saffle JR, et al: Treatment of toxic epidermal necrolysis with intravenous immunoglobulin in children, J Am Acad Dermatol 47:548, 2002. 151. Kelkar PS, Li JT: Cephalosporin allergy, N Engl J Med 345:804, 2001. 152. Knowles S, Shapiro L, Shear NH: Should celecoxib be contraindicated in patients who are allergic to sulfonamides? Revisiting the meaning of “sulfa” allergy, Drug Saf 24:239, 2001.

153. Schlienger RG, Shear NH: Antiepileptic drug hypersensitivity syndrome, Epilepsia 39(suppl 7):3, 1998. 154. Vaillant L, Camenen I, Lorette G: Patch testing with carbamazepine: reinduction of an exfoliative dermatitis, Arch Dermatol 125:299, 1989. 155. Stark BJ, Earl HS, Gross GN, et al: Acute and chronic desensitization of penicillin-allergic patients using oral penicillin, J Allergy Clin Immunol 79:523, 1987. 156. Wendel GD, Stark BJ, Jamison RB, et al: Penicillin allergy and desensitization in serious infections during pregnancy, N Engl J Med 312:1229, 1985. 157. Graybill JR, Sande MA, Reinarz JA, Shapiro SR: Controlled penicillin anaphylaxis leading to desensitization, South Med J 67:62, 1974. 158. Sobotka AK, Dembo M, Goldstein B, Lichtenstein LM: Antigen-specific desensitization of human basophils, J Immunol 122:511, 1979. 159. Sullivan TJ: Antigen-specific desensitization of patients allergic to penicillin, J Allergy Clin Immunol 69:500, 1982. 160. MacGlashan D Jr, Lichtenstein LM: Basic characteristics of human lung mast cell desensitization, J Immunol 139:501, 1987. 161. Sullivan TJ: Drug allergy. In Middleton E Jr, Reed CE, Ellis EF, et al, editors: Allergy: principles and practice, ed 4, St Louis, 1993, Mosby, p 1726. 162. Patterson R, DeSwarte RD, Greenberger PA, et al: Drug allergy and protocols for management of drug allergies, Allergy Proc 15:239, 1994. 163. Brown LA, Goldberg ND, Shearer WT: Long-term ticarcillin desensitization by the continuous oral administration of penicillin, J Allergy Clin Immunol 69:51, 1982. 164. Smith RM, Iwamoto GK, Richerson HB, Flaherty JP: Trimethoprim-sulfamethoxazole desensitization in the acquired immunodeficiency syndrome, Ann Intern Med 106:335, 1987. 165. Jung AC, Paauw DS: Management of adverse reactions to trimethoprim-sulfamethoxazole in human immunodeficiency virus-infected patients, Arch Intern Med 154:2402, 1994. 166. Carr A, Cooper DA: Pathogenesis and management of HIV-associated drug hypersensitivity, AIDS Clin Rev, 1995, p 65. 167. Absar N, Daneshvar H, Beall G: Desensitization to trimethoprim/sulfamethoxazole in HIV-infected patients, J Allergy Clin Immunol 93:1001, 1994. 168. Taffet SL, Das KM: Desensitization of patients with inflammatory bowel disease to sulfamethoxazole, Am J Med 73:520, 1982. 169. Purdy BH, Philips DM, Summers RW: Desensitization to sulfasalazine skin rash, Ann Intern Med 100:512, 1984. 170. Wong IC, Mawer GE, Sander JW: Factors influencing the incidence of lamotrigine-related skin rash, Ann Pharmacother 33:1037, 1999. 171. Israel KS, Fischl MA, Rosenberg SA, et al: The pivotal role of 5-lipoxygenase products in the reaction of aspirin-sensitive asthmatics to aspirin, Am Rev Respir Dis 148:1447, 1993.

172. Roquet A, Dahlen B, Kumlin M, et al: Combined antagonism of leukotrienes and histamine produces predominant inhibition of allergen-induced early and late phase airway obstruction in asthmatics, Am J Respir Crit Care Med 155:1856, 1997. 173. Greenberger PA, Patterson GA, Radin RC: Two pretreatment regimens for high-risk patients receiving radiographic contrast media, J Allergy Clin Immunol 74:540, 1984. 174. Levine BB, Zolov DM: Prediction of penicillin allergy by immunological tests, J Allergy 43:231, 1969. 175. Rytel MW, Klion FM, Arlander TR, Miller LF: Detection of penicillin hypersensitivity with penicilloyl-polylysine, JAMA 186:894, 1963. 176. Adkinson NF Jr, Swabb EA, Sugerman AA: Immunology of the monobactam aztreonam, Antimicrob Agents Chemother 25:93, 1984.

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Chapter 93 - Sensitivity to Aspirin and Nonsteroidal Antiinflammatory Drugs

Donald D. Stevenson Ronald A. Simon Bruce L. Zuraw

Acetylsalicylic acid (ASA) has been used for treatment of human diseases for more than 100 years, and other nonsteroidal antiinflammatory drugs (NSAIDs) for almost 40 years. NSAIDs share pharmacologic effects with ASA by inhibiting cyclooxygenase enzymes (COX-1 and COX-2).[

1] [2] [3] [4]

These shared effects of 5 6

ASA and other NSAIDs are important in their efficacy as antiinflammatory and analgesic agents and also in some of their adverse reactions.[ ] [ ] ASA and other NSAIDs induce gastritis through at least two mechanisms: inhibition of COX-1, with loss of prostaglandin E2 (PGE2) protection of the gut; and the direct toxic 4 7 8 9 10

effects of ASA/ NSAIDs on mucosal cells.[ ] [ ] [ ] [ ] [ ] ASA/NSAIDs induce a number of other adverse reactions, including but not limited to hepatotoxicity, interstitial nephritis, bleeding disorders, anemia, thrombocytopenia, erythema multiforme, fixed drug eruptions, toxic epidermal necrolysis, erythema nodosum, 5] [11]

maculopapular eruptions, bullous leukocytoclastic vasculitis, and Stevens-Johnson syndrome.[

TYPES OF PSEUDOALLERGIC AND ALLERGIC REACTIONS TO ASA/NSAIDS

Eight types of pseudoallergic and allergic reactions to ASA and other NSAIDs have been identified. The first four reactions are closely linked to the shared effects of ASA/NSAIDs (inhibition of COX-1). The remaining reactions are drug specific, independent of COX inhibition, and probably immune mediated. In an attempt to bring further clarity to the understanding and descriptions of NSAID-induced reactions, a new classification system was proposed in an editorial in September 2001. [11]

The classification used in this chapter is based on the new system.

Type 1: NSAID-Induced Asthma and Rhinitis in Asthmatic Patients In a subset of asthmatic patients with a high frequency of nasal polyps and sinusitis, ingestion of ASA or cross-reacting NSAIDs can induce combinations of intense 12]

rhinorrhea, nasal congestion, injection of the conjunctivae, periorbital edema, laryngospasm, and bronchospasm.[ tract

13 reactions,[ ]

and others react only in the nose, sinuses, and

14 eyes,[ ]

Some patients experience only lower respiratory

but the majority experience both upper and lower respiratory tract reactions.[

15]

A few

[16]

patients simultaneously experience extrapulmonary reactions (e.g., gastrointestinal, cutaneous, vascular).

The provoking dose of ASA/NSAIDs is the single most important factor influencing the severity of ASA-induced respiratory reactions. Small doses of ASA/NSAIDs may produce no reaction or minimal reactions, sometimes limited to the upper respiratory tract. However, therapeutic doses of ASA/NSAIDs may induce such severe bronchospasm that hospitalization and even intubation with mechanical ventilation may be required to maintain respiratory function. Deaths have resulted from ingestion of full doses of ASA or NSAIDs in patients with ASA-sensitive asthma. Type 2: NSAID-Induced Urticaria/Angioedema in Patients with Chronic Urticaria Susceptible patients, usually patients with active chronic urticaria, develop hives and sometimes angioedema 2 to 4 hours after ingestion of either ASA or NSAIDs 17] [18] [19]

that inhibit COX-1.[

Reactions are dose dependent, and the degree of activity of the underlying chronic urticaria is also important. The more active the 20]

chronic urticaria, the more likely is the ASA/NSAID to induce a further flare or extension of the hives.[

Antihistamines that block histamine1 (H1 ) receptors, H2

blockers, and leukotriene receptor antagonists (LTRAs) prevent or modify these reactions. Usually one blocking drug is insufficient, and all three drugs may be required to prevent these reactions. Type 3: ASA/NSAID-Induced Cross-reacting Urticaria in Otherwise Normal Individuals A few seemingly normal patients without underlying chronic urticaria develop urticaria/angioedema after treatment with more than one of the NSAIDs that inhibit 21 22 23

COX-1.[ ] [ ] [ ] Some of these patients develop chronic idiopathic urticaria at a later time; some have unexplained urticarial episodes at times when they are not exposed to ASA/NSAIDs; and others never have urticaria except after ingestion of ASA/NSAIDs. This pattern of cross-reacting urticaria in individuals without chronic idiopathic urticaria is unusual. Type 4: Blended Reactions in Otherwise Normal Individuals

Sometimes reactions to NSAIDs cannot be classified in any recognizable pattern. Reactions to one or more NSAIDs may have features of urticaria/angioedema, rhinitis, and bronchospasm.

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Rather than inaccurately forcing such reactions into the wrong classification, it seems better to place them in their own category.[ several reaction mechanisms are operating independently or that several shock organs are responding simultaneously.

11]

These reactions suggest that

Type 5: Single-NSAID–Induced Urticaria/Angioedema in Otherwise Normal Subjects After prior sensitization to ASA or a specific NSAID, otherwise healthy individuals may develop urticaria/angioedema on re-exposure to the same drug.[ type of reaction, cross-reactivity between ASA and NSAIDs does not

24 25 occur.[ ] [ ]

12]

In this

In a study of 1974 normal adults, only 2 patients reported acute urticaria after 26]

ASA ingestion (incidence, 0.1%). Among 618 normal children, parents reported that only 2 children (0.32%) developed acute urticaria after ingestion of ASA.[ some studies, atopy appeared to be a risk factor for single-NSAID-induced urticarial to nimesulide but not for acetaminophen-induced

28 reactions.[ ]

22 27 reactions.[ ] [ ]

In

In another study atopy was a risk factor for urticarial reactions

From an on-line computerized Medicaid pharmaceutical analysis and surveillance system, Strom,

[29]

Carson, and Schinnar conducted a two-phase retrospective cohort study in three states to assess the relative risk of hypersensitivity reactions to six common NSAIDs. Of the 128,344 study subjects who were users of one NSAID, the average relative risk of urticaria/angioedema to one NSAID was 1.1% for those who used their NSAIDs chronically and 3.6% for those who took their NSAID intermittently for acute pain. Type 6: Single-NSAID–Induced Anaphylaxis and Anaphylactoid Syndromes Anaphylaxis is an immunoglobulin E (IgE)-mediated, drug-specific, severe systemic reaction characterized by multiorgan responses to release of mediators from mast cells and basophils. In a retrospective study of 266 subjects brought to an emergency room with anaphylaxis, 52 patients (20%) were suspected to be having a 30]

reaction to a single drug.[

Of these 52 patients, 27 reacted to ASA and 7 to another NSAID (ibuprofen, indomethacin, or naproxen). Therefore, more than half of 29] [31]

the drug-induced anaphylaxis episodes were attributed to ASA or another NSAID. Anaphylactic reactions to other specific NSAIDs have also been reported,[ [32]

included fenoprofen,[

[35] [36]

37 tolmetin,[ ]

29]

piroxicam, [

and acetaminophen

ASA, or acetaminophen without adverse

29]

sulindac,[

29]

29]

meclofenamate,[

38 39 (paracetamol),[ ] [ ] 25 38 40 affects.[ ] [ ] [ ]

indo-methacin, [

31]

31]

zomepirac,[

naproxen,[

33]

34]

diclofenac,[

ketorolac tromethamine,

among others. Patients with such reactions can be challenged with structurally different NSAIDs,

The report of celecoxib-induced anaphylaxis was particularly interesting, because this selective COX-2 41

inhibitor does not cross-react through inhibition of COX-1. [ ] However, like many drugs, it can function as a drug hapten to induce immune sensitization, followed by a probable IgE-mediated reaction, including near-fatal anaphylaxis. Anaphylactoid reactions are clinically indistinguishable from anaphylactic reactions, but the mechanisms by which mast cells and basophils discharge mediators are

nonimmunologic.[

42]

It is possible, although extremely rare, for ASA and NSAIDs to participate in anaphylactoid reactions, probably through inhibition of COX-1.

[43]

There are several clinical settings in which cross-reacting anaphylactoid reactions occur. First, during oral ASA challenge, a small number of patients with asthma and nasal polyps experience typical respiratory reactions but also have systemic reactions beyond the respiratory tract, including flush, urticaria, hypotension, abdominal pain, and/or diarrhea.[

16]

Second, although we have never encountered such patients, there are reports in the literature of normal individuals who

experienced anaphylactoid reactions after ingestion of structurally dissimilar NSAIDs on different occasions.[

21] [44] [45]

Third, sulindac, tolmetin, and zomepirac

37

have a common acetic acid structure that provides a common antigen and a basis for immunologic cross-reactivity.[ ] Fortunately, multiple-NSAID-induced anaphylactoid reactions and cross-reacting anaphylactic reactions are very rare, with most severe systemic reactions induced by only one NSAID. Type 7: Aseptic Meningitis Caused by a Specific NSAID A small number of patients develop aseptic meningitis after ingestion of a specific NSAID. This is a diagnosis of exclusion, because infectious and autoimmune meningitis must be ruled out. However, recurrent bouts of meningitis within hours (usually 12 to 24 hours) after ingestion of the same NSAID; association with neck stiffness, headache, fever, nausea, abdominal pain, arthralgias, and rash; and disappearance of all signs and symptoms after discontinuation of the NSAID, leads to 46

46] [47]

the diagnosis. [ ] The four NSAIDs reported to have caused aseptic meningitis are ibuprofen, sulindac, tolmetin, and naproxen. [ NSAIDs did not occur, and ASA has not been reported to cause aseptic meningitis.

Cross-reactions among

Type 8: Hypersensitivity Pneumonitis Caused by a Specific NSAID Since the mid-1970s, individual cases of pulmonary infiltrates coinciding with the ingestion of a specific NSAID, usually on multiple occasions, have been reported. [48] [49] [50]

51

In 1983, the first controlled oral challenge with naproxen was conducted.[ ] Within 24 hours after challenge, the patient developed fever, cough, pulmonary infiltrates, and peripheral eosinophilia. Subsequently, three other controlled oral challenges with naproxen in sensitive patients were reported with similar 48 50 52

53

responses.[ ] [ ] [ ] A number of NSAIDs have been reported to cause hypersensitivity pneumonitis.[ ] However, no cases of ASA-induced hyper-sensitivity pneumonitis or cross-reactivity among NSAIDs have been reported. Disappearance of the pulmonary infiltrates over a number of weeks, and more rapidly if systemic 49]

glucocorticoid therapy is used, characterizes the clinical course. Residual pulmonary damage has not been documented during long-term follow-up.[

UNDERLYING DISEASES: RISK FACTORS FOR ASA/NSAID CROSS-REACTIONS Aspirin-Exacerbated Respiratory Disease Historically, aspirin-exacerbated respiratory disease (AERD) has been called Samter's triad, aspirin intolerance or idiosyncrasy, aspirin sensitivity, and aspirininduced asthma. However,

1697

aspirin does not induce the disease, and “sensitivity” refers to all types of reactions. Instead, ingestion of any NSAID that inhibits COX-1 temporarily increases formation or release of mediators, with exacerbation of disease in the respiratory tract. Of 300 patients with the AERD syndrome referred to Scripps Clinic between 54

1995 and 2001, 171 (57%) were female, and nasal polyps or asthma first appeared at an average age of 34 years.[ ] Respiratory disease severity was the same in both genders. Positive wheal and flare skin tests were recorded in 66% of our 300 patients with AERD. The European Network of Aspirin-Induced Asthma combined 55

their data from 16 centers and analyzed 500 patients with AERD.[ ] In this European population, persistent rhinitis appeared at a mean age of 30 years, followed by asthma, ASA/NSAID-induced respiratory reactions, and then nasal polyps. Females outnumbered males by 2.3:1 and tended to have earlier onset and more severe disease. Atopy was present in one third of the patients, and a family history of AERD was present in 6%. 14

Avoiding ASA/NSAIDs does not change the relentless course of the respiratory disease. Inflammation may be isolated to the upper respiratory tract,[ ] producing ASA-exacerbated rhinosinusitis. More commonly, upper airway disease is accompanied by lower respiratory tract inflammation, which produces asthma. This “intrinsic” type of asthma progresses and persists, regardless of environmental exposures. The clinical setting in which the physician should be most suspicious of AERD is an asthmatic patient with relentless formation of nasal polyps and pansinusitis by imaging procedures. In our 300 patients with AERD, 99% had abnormal sinus radiographs or computed tomography scans. The diagnosis of AERD cannot be made from clinical presentation alone. Approximately two thirds of patients 56

with an identical clinical picture never react to ASA or other NSAIDs.[ ] Most ASA-tolerant asthmatic patients with nasal polyps and pansinusitis have IgEmediated rhinitis, usually to dust mites, and a few are sulfite sensitive. The remaining patients have intrinsic rhino-sinusitis and asthma of unknown cause. A small number of patients eventually develop AERD.[

15]

57] [58] [59] [60]

The prevalence of AERD in adult asthma populations, when oral ASA challenges are used to detect sensitivity, is between 9% and 20%. [ adult asthmatics who were identified as also having nasal polyps and pansinusitis, the prevalence of AERD increased to

61 34%.[ ]

Adult asthmatics who gave a

history of a prior ASA/NSAID-induced respiratory reaction were found to have positive oral challenges to ASA between 66% and 97% of the time.[ children, the prevalence of sensitivity is generally lower (0% to 21%), but after puberty the prevalence

Among

15] [58] [59]

In

62 63 64 65 increases.[ ] [ ] [ ] [ ]

Chronic Idiopathic Urticaria A subpopulation of patients with chronic urticaria experiences flares of hives after ingestion of ASA or NSAIDs. Other than chronic idiopathic urticaria, additional distinguishing clinical features have not been identified. Avoidance of ASA/NSAIDs does not change the course of the chronic urticaria in these patients, other than to eliminate acute flares caused by ASA/NSAIDs.[

13]

The prevalence of ASA-induced urticaria in patients with chronic idiopathic urticaria that is quiescent at the 17] [18] [19] [20]

time of challenge has been reported to be between 21% and 30%.[

20 Stevenson[ ]

The more active the underlying urticaria, the more likely it is that ASA/NSAIDs

will cause urticarial exacerbations. Mathison, Lumry, and reported that patients with active chronic urticaria reacted with additional hives during double-blind oral ASA challenges 80% of the time. By contrast, those with relapsing urticaria that was quiescent at the time of oral ASA challenge reacted to ASA only 28% of the time.

DETECTION OF ASA/NSAID SENSITIVITY

Currently, acceptable, reproducible, and practical in vitro tests for the identification of patients at risk for AERD or NSAID-induced urticaria or anaphylaxis do not exist. Therefore the gold standard for identifying such patients is through ASA challenges. Oral ASA Challenges In the United States, only oral ASA challenges are available. It should be emphasized that oral challenges, with increasing doses of ASA or NSAIDs, can induce severe bronchospastic and nasal reactions, as well as extrapulmonary effects. Therefore, physicians conducting these challenges should be experienced in their proper performance and prepared to aggressively treat reactions. 15

At the time of challenge, unstable asthma is associated with more severe ASA-induced reactions.[ ] Oral and inhaled cortico-steroids, intranasal corticosteroids, theophylline, and leukotriene modifier drugs (LTMDs) should be continued. Despite treatment with theophylline and oral and inhaled corticosteroids, ASA- and 15

66

NSAIDs-induced reactions routinely occur.[ ] Nizankowska and Szczeklik[ ] reported that pretreatment with systemic corticosteroids reduced the degree of bronchospasm during oral ASA challenges and may be responsible for a few false-negative oral ASA challenge results. However, for most AERD asthmatic patients, discontinuation of systemic corticosteroids increases bronchial irritability to the point where ASA challenges cannot be accurately and safely performed. During oral 67 68

ASA challenges, LTMDs attenuate and sometimes block bronchospastic responses but do not inhibit ASA-induced upper respiratory tract reactions. [ ] [ ] Among 271 patients undergoing oral ASA challenges at the Scripps Clinic, 163 were taking inhaled and systemic corticosteroids and 108 were also taking an LTMD. The LTMD-treated group had a statistically significant decrease in asthmatic reactions (21% versus 39%) and an increase in pure upper respiratory reactions (49% versus 32%), compared with the control group. However, the numbers of patients originally suspected of having AERD who experienced negative challenges to ASA were 69

the same in both groups (12% versus 15%).[ ] Therefore, taking an LTMD during oral ASA challenges appears to protect the lower respiratory tract without diminishing the capacity of the challenge to induce nasoocular reactions. During oral ASA challenges, pretreatment with cromolyn has minor inhibitory effects on the severity of the ASA-induced lower respiratory reactions.[

70]

13]

However, pretreatment with cromolyn delays the onset of ASA-induced asthmatic reactions. [

Pretreatment with an antihistamine, clemastine, prevented nasal and ocular reactions to ASA but had no effect on ASA-induced bronchospasm.[ sodium salicylate prevented most ASA-induced respiratory reactions, presumably by interfering with the interaction of ASA and

71]

Pretreatment with

72 COX-1.[ ]

1698

TABLE 93-1 -- Oral Challenge Protocol to Detect Respiratory Reactions Induced by Acetylsalicylic Acid (ASA) ASA doses (mg) Time

Day 1

Day 2

*

†

Day 3

7 am

Placebo

30

100–150

10 am

Placebo

45–60

150–325

1 pm

Placebo

60–100

650

* Start challenges, if baseline forced expiratory volume in 1 second (FEV1 ) is 70% or greater than predicted, without bronchodilator. Alternatively, if the absolute FEV1 value is greater than 1.5 L and represents the best prior FEV1 value, proceed with oral challenge. On the placebo day, FEV1 values should vary by 15% decline in FEV1 values

Bronchospasm alone (lower respiratory)

No nasoocular reaction but >20% decline in FEV1 values

Rhinitis and conjunctivitis (upper respiratory)

Nasoocular reactions and