Middleton's Allergy 2-Volume Set: Principles and Practice [9 ed.] 032354424X, 9780323544245

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MIDDLETON’S

ALLERGY Principles and Practice

MIDDLETON’S

ALLERGY Principles and Practice Ninth Edition A. Wesley Burks, MD

Stuart Bondurant Distinguished Professor Dean and CEO, UNC School of Medicine and UNC Health Care The University of North Carolina at Chapel Hill Chapel Hill, NC, USA

Stephen T. Holgate, CBE, FMedSci

MRC Professor of Immunopharmacology, Clinical and Experimental Sciences Faculty of Medicine Southampton University and General Hospital Southampton, UK

Robyn E. O’Hehir, FRACP, PhD, FRCPath

Professor and Director, Department of Respiratory Medicine, Allergy and Clinical Immunology (Research) Central Clinical School, Monash University, and Alfred Hospital Melbourne, VIC, Australia

David H. Broide, MB ChB Professor of Medicine Department of Medicine University of California, San Diego La Jolla, CA, USA

Leonard B. Bacharier, MD

Robert C. Strunk Endowed Chair for Lung and Respiratory Research Professor of Pediatrics and Medicine Clinical Director, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine Washington University in St. Louis St. Louis, MO, USA

Gurjit K. Khurana Hershey, MD, PhD

Kindervelt Endowed Chair in Asthma Research Professor of Pediatrics, University of Cincinnati College of Medicine Director, Division of Asthma Research Co-Director, Office of Pediatric Clinical Fellowships Attending Physician, Allergy and Immunology Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

R. Stokes Peebles, Jr., MD

Elizabeth and John Murray Professor of Medicine Professor Pathology, Microbiology, and Immunology Division of Allergy, Pulmonary and Critical Care Medicine Vanderbilt University School of Medicine Nashville, TN, USA For additional online content visit ExpertConsult.com

Edinburgh London New York Oxford Philadelphia St Louis Sydney 2020

© 2020, Elsevier Inc. All rights reserved. First edition 1978 Second edition 1983 Third edition 1988 Fourth edition 1993 Fifth edition 1998 Sixth edition 2003 Seventh edition 2009 Eighth edition 2014

ISBN: 978-0-323-54424-5 Volume 1 ISBN: 978-0-323-75935-9 Volume 2 ISBN: 978-0-323-75937-3

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. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). The following chapters are in the public domain: Chapter 73: Eosinophilia and Eosinophil-Related Disorders, Paneez Khoury, Praveen Akuthota, Peter F. Weller, Amy D. Klion Chapter 74: Mastocytosis, Dean D. Metcalfe

Notices Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein.

Content Strategist: Robin Carter Content Development Specialist: Joanne Scott Project Manager: Joanna Souch Design: Brian Salisbury Illustration Manager: Paula Catalano Illustrators: Oxford Illustrators, Chartwell, Dartmouth Publishing Inc., MPS North America LLC Marketing Manager: Michele Milano Printed in China Last digit is the print number: 9 8 7 6 5 4 3 2 1

F O R E WO R D I am honored to provide this foreword to the Elsevier text: Middleton’s Allergy: Principles and Practice, edited by Professors Burks, Holgate, O’Hehir, Broide, Bacharier, Khurana Hershey, and Peebles. Not because I am an allergist or immunologist—I am a clinical informatician, and academic general internist—nor because it will pad my curriculum vitae with another piece of writing. Rather, it gives me an opportunity to honor my father Elliott Middleton, Jr., MD, and to share with readers of this text a glimpse into the man who inspired me and many others to pursue a career in medicine, and for his many Fellows and trainees a career in allergy and immunology. My dad had a passion both for life and his work in clinical medicine, his research, and teaching and a humility based upon a desire to help patients, those with asthma especially, and the myriad problems that may be associated with allergic disorders and the diseases of clinical immunology. As I was growing up my father always worked hard, but always seemed to be having fun. There were frequent rounds in hospital early and late in the day, and extra time often spent in the lab on Saturday mornings to “catch up” after a busy week. Most nights were spent with the family for dinner, and then afterwards several hours on the living room couch editing a manuscript or chapter or sorting slides on one of those now old-fashioned slide view boxes, preparing for a lecture. For many years, it was a chapter for the text now known as Middleton’s Allergy: Principles and Practice. He absolutely loved putting this text together: crafting the outline of chapters, meeting with the coeditors, and reviewing chapters and occasionally cajoling authors to get things done. It may be his proudest accomplishment. On a couple of occasions we met at one of his national meetings and I saw him in his element: gregarious, well known, interested in others, and respected. The passion my dad had about his work was infectious: He would always take the time to explain in simple terms

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what was going on with his laboratory and clinical investigations— whether it was the early work on mast cell functions and histamine release, leukotrienes, or bioflavonoids (we all came to recognize the terms quercetin, rutin, and others) and their impact on inflammation, reverse transcriptase, or free radicals. Equally importantly, we came to know the good dietary sources for these compounds. He instilled in me the sense of wonder not only about the incredible processes of chemistry and biology, which affect the human system and may manifest when aberrant as disease states, or worse, the human interaction with the natural world that seemed filled with potential allergens—pollen, grasses, sage, cat dander, and more (his Fellows have told me his “weed walks” were fun and informative). He viewed his research—whether it was basic laboratory investigation or clinical trials—from the patient’s perspective, and honored the patient as the center of his clinical attention. One cannot really know the perception people have of one’s parents as we as children are always too close, too intertwined with them. Even as adults as we try to both distance ourselves from them as well as to take from them all that they have to offer that is good as we come to define ourselves. In my training, I have been surprised to see people’s faces light up and smile if the occasion ever arose for me to acknowledge that Elliott Middleton, Jr., was my dad. It is a delight—he left an indelible mark on the world that I aspire to leave as well. Blackford Middleton, MD, MPH, MSc, FACP, FACMI, FHIMSS Harvard T.H. Chan School of Public Health Chief Informatics & Innovation Officer, Apervita, Inc. Past Chairman, American Medical Informatics Association Past Chairman, Healthcare Information Management & Systems Society

P R E FA C E Since 1978, when the late Elliott Middleton, Jr., together with the founding editors Elliot Ellis and Charles Reed, published the comprehensive book Allergy: Principles and Practice, these two volumes have been the definitive text on allergy practice and disease mechanisms worldwide. As the recognized definitive text of allergy, the tome is now in its ninth edition. Over the last decade, understanding of allergic diseases and their diagnosis, prevention, and management has advanced considerably. In parallel, the prevalence, spectrum, and severity of allergy have also increased, such that allergic disorders are a major public health issue affecting a high proportion of the global population with a substantial socioeconomic burden. In some quarters there is a perception that allergy has little impact on the lives of sufferers, but nothing could be further from the truth. Diseases such as asthma, food, drug, and insect allergy can be life threatening if not diagnosed and treated properly. Allergy often affects multiple organs in the same individual, magnifying the overall health burden that patients experience. Of note, allergy manifests in all age groups being influenced by strong genetic, environmental, and epigenetic drivers. In recent years there has been a major change in the practice of medicine with the advent of precision medicine and the development and clinical application of a wide array of immunologic therapies. The biologics, including humanized monoclonal antibody therapies, are now applied in most disciplines of internal medicine with impressive results. In this ninth edition of Middleton’s Allergy, new topics of Innate Lymphoid Cells, Systems Biology, Epidemic Thunderstorm Asthma, and Precision Medicine in Allergic Disorders have been included. The many advances around Primary Immunodeficiencies have allowed the expansion of coverage. In creating this ninth edition, the authors were asked to update relevant sections in Middleton’s Allergy, Eighth Edition, and include references and a number of assessment questions, with brief explanations of the correct answers. Naturally, such a text must be selective, but the editors hope that the topics covered address the needs of trainee and established practitioners across the healthcare sector to enable them to access novel and useful information to inform their practice.

The editors wish to express their sincere thanks to all of the authors for the very considerable amount of work they have undertaken to produce this new edition of Middleton’s Allergy. With great sadness the editors acknowledge the passing during the production of this book of three of the highly respected authors, Dr Michael M. Frank (US), Professor Anthony J. Frew (UK), and Professor William T. Shearer (US). The late Michael Frank was senior author on the chapter on Immune Complexes and Allergic Disease, the late Tony Frew was senior author on the chapter on Sublingual Immunotherapy for Inhalant Allergens, and the late Bill Shearer was senior author for the chapter on HIV Infection and Allergic Disease and a previous contributor to the chapter on Adaptive Immunity. Their legacies contribute to the success of this new ninth edition. The final product would not be possible without the diligence of Joanne Scott, Joanna Souch, and Robin Carter with their publishing team at Elsevier who have worked seamlessly with the editors throughout the commissioning and editorial process. As editors of Middleton’s Allergy, Ninth Edition, we express our very sincere thanks to all the editors and authors of the previous editions but especially to the three founders who had the vision to produce such a consistent beacon of success in the field of allergy. We are also grateful to Professor Blackford Middleton—son of Elliott Middleton, Jr.—for writing the Foreword to this edition. The best testament to Elliott and his co-authors would be the widespread use of the wealth of information in these pages, and its impact on how allergy therapy is delivered to our patients worldwide; after all, it is our patients who continue to motivate us to achieve ever better care. A. Wesley Burks Stephen T. Holgate Robyn E. O’Hehir David H. Broide Leonard B. Bacharier Gurjit K. Khurana Hershey R. Stokes Peebles, Jr. November 2019

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LIST OF CONTRIBUTORS The editor(s) would like to acknowledge and offer grateful thanks for the input of all previous editions’ contributors, without whom this new edition would not have been possible.

Seema S. Aceves, MD, PhD

Leonard B. Bacharier, MD

Neal P. Barney, MD

Professor, Pediatrics and Medicine Director, Eosinophilic Gastrointestinal Disorders Clinic Division of Rheumatology, Allergy and Immunology University of California, San Diego Rady Children’s Hospital, San Diego La Jolla, CA, USA

Robert C. Strunk Endowed Chair for Lung and Respiratory Research Professor of Pediatrics and Medicine Clinical Director, Division of Pediatric Allergy, Immunology, and Pulmonary Medicine Washington University in St. Louis St. Louis, MO, USA

Professor, Emeritus, Department of Ophthalmology and Visual Sciences University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Ian M. Adcock, PhD

Claus Bachert, MD, PhD

Professor, National Heart and Lung Institute Imperial College London London, UK

Professor of Medicine Chief of Clinics Head, Upper Airway Research Laboratory Ear, Nose, and Throat Department Ghent University Hospital Ghent, Belgium

Cezmi A. Akdis, MD Swiss Institute of Allergy and Asthma Research (SIAF), Davos, Switzerland Medical Faculty, University of Zurich, Zurich Christine Kühne – Center for Allergy Research and Education Davos, Switzerland

Mübeccel Akdis, PD, MD, PhD Head of Immunodermatology, Swiss Institute of Allergy and Asthma Research Department of Immunodermatology University of Zürich Zürich, Switzerland

Praveen Akuthota, MD Associate Professor of Medicine Pulmonary and Critical Care University of California, San Diego San Diego, CA, USA

Katherine J. Allnutt, MBBS (Hons), BMedSc (Hons), DRANZCOG Education and Research Fellow Skin and Cancer Foundation Inc. Melbourne, VIC, Australia

Katherine J. Baines, PhD Post-Doctoral Research Fellow Department of Respiratory and Sleep Medicine University of Newcastle Hunter Medical Research Institute Newcastle, NSW, Australia

Mark Ballow, MD Professor of Pediatrics Division of Allergy & Immunology Department of Pediatrics University of South Florida Morsani College of Medicine Tampa, FL, USA

Lora Bankova, MD Assistant Professor of Medicine Division of Allergy and Clinical Immunology Department of Medicine Brigham and Women’s Hospital Boston, MA, USA

Peter J. Barnes, FMedSci, FRS Andrea J. Apter, MD, MSc, MA Professor of Medicine Division of Pulmonary, Allergy, and Critical Care Medicine Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA, USA

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Head of Respiratory Medicine National Heart and Lung Institute Imperial College London London, UK

Fuad M. Baroody, MD, FACS, FAAAAI Professor of Otolaryngology-Head and Neck Surgery and Pediatrics The University of Chicago Medicine Chicago, IL, USA

Nora Barrett, MD Assistant Professor of Medicine Division of Allergy and Clinical Immunology Department of Medicine Brigham and Women’s Hospital Boston, MA, USA

Joseph L. Baumert, PhD Associate Professor Department of Food Science and Technology Co-Director, Food Allergy Research and Resource Program Department of Food Science and Technology The University of Nebraska- Lincoln Lincoln, NE, USA

Bruce G. Bender, PhD Professor of Pediatrics and Psychiatry Head, Division of Pediatric Behavioral Health National Jewish Health Denver, CO, USA

M. Cecilia Berin, PhD Professor of Pediatrics Division of Allergy and Immunology Department of Pediatrics Icahn School of Medicine at Mount Sinai New York, NY, USA

Jocelyn M. Biagini Myers, PhD Associate Professor of Pediatrics Division of Asthma Research Department of Pediatrics University of Cincinnati College of Medicine Cincinnati, OH, USA

List of Contributors

Kathryn V. Blake, Pharm.D, BCPS, FCCP, CIP

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Peter Bradding, BM, DM, FRCP

A. Wesley Burks, MD

Director, Center for Pharmacogenomics and Translational Research Principal Research Scientist Nemours Children’s Specialty Care Jacksonville, FL, USA

Professor of Respiratory Medicine Department of Infection, Immunity, and Inflammation Institute for Lung Health University of Leicester Leicester, UK

Stuart Bondurant Distinguished Professor Dean and CEO, UNC School of Medicine and UNC Health Care The University of North Carolina at Chapel Hill Chapel Hill, NC, USA

Bruce S. Bochner, MD

John D. Brannan, PhD

Robert K. Bush, MD

Samuel M. Feinberg Professor of Medicine Division of Allergy and Immunology Department of Medicine Northwestern University Feinberg School of Medicine Chicago, IL, USA

Scientific Director Department of Respiratory and Sleep Medicine John Hunter Hospital Newcastle, NSW, Australia

Professor Emeritus Department of Medicine Division of Allergy, Immunology, Pulmonary, and Critical Care Medicine University of Wisconsin Madison, WI, USA

Jennifer L. Bridgewater, MPH Mark Boguniewicz, MD Professor Division of Allergy–Immunology Department of Pediatrics, National Jewish Health University of Colorado School of Medicine Denver, CO, USA

Associate Director for Regulatory Policy Division of Bacterial, Parasitic and Allergenic Products Office of Vaccines Research and Review Center for Biologics Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD, USA

William W. Busse, MD

Christopher E. Brightling, MD, PhD, FCCP

Carlos A. Camargo, Jr., MD, DrPH

Professor of Medicine Department of Medicine Allergy, Pulmonary, and Critical Care Medicine University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Larry Borish, MD Professor of Medicine and Microbiology Asthma and Allergic Disease Center, Carter Immunology Center University of Virginia Health System Charlottesville, VA, USA

Louis-Philippe Boulet, MD, FRCPC Professor of Medicine Department of Medicine Laval University Québec Heart and Lung Institute Québec City, QC, Canada

Jean Bousquet, MD Honorary Professor of Pulmonology University Hospital of Montpellier Montpellier, France

Joshua A. Boyce, MD Albert L. Sheffer Professor of Medicine in the Field of Allergic Diseases Jeff and Penny Vinik Center for Allergic Disease Research Harvard Medical School Director, Inflammation and Allergic Disease Research Section Division of Rheumatology, Immunology, and Allergy Brigham and Women’s Hospital Boston, MA, USA

Welcome Senior Research Fellow Clinical Professor in Respiratory Medicine Institute for Lung Health Department of Infection, Inflammation, and Immunity University of Leicester Glenfield Hospital Leicester, UK

Professor of Emergency Medicine & Medicine Harvard Medical School Professor of Epidemiology Harvard T.H. Chan School of Public Health Conn Chair of Emergency Medicine Massachusetts General Hospital Boston, MA, USA

Matthew Camiolo, MD, PhD David H. Broide, MB ChB Professor of Medicine Department of Medicine University of California, San Diego La Jolla, CA, USA

Fellow Division of Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh Pittsburgh, PA, USA

Rebecca H. Buckley, MD

Brendan J. Canning, PhD

J. Buren Sidbury Distinguished Professor of Pediatrics Department of Pediatrics Professor of Immunology Department of Immunology Duke University Medical Center Durham, NC, USA

Professor of Medicine Department of Medicine Division of Allergy and Clinical Immunology Johns Hopkins University School of Medicine Baltimore, MD, USA

Supinda Bunyavanich, MD, MPH, MPhil

Thomas B. Casale, MD

Associate Director Jaffe Food Allergy Institute Associate Professor Department of Pediatrics Department of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai New York, NY, USA

Professor of Medicine and Pediatrics Division of Allergy and Immunology University of South Florida Tampa, FL, USA

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List of Contributors

Mario Castro, MD, MPH

Ariella T. Cohain, PhD

Thomas L. Diepgen, MD

Professor of Medicine, Pediatrics and Radiology Department of Medicine Washington University School of Medicine St. Louis, MO, USA

Post-Doctoral Fellow Department of Genetics and Genomic Sciences Icahn Institute for Genomics and Multiscale Biology Icahn School of Medicine at Mount Sinai New York, NY, USA

Professor and Chairman Department of Clinical Social Medicine Center of Occupational and Environmental Dermatology Ruprecht Karl University of Heidelberg Heidelberg, Germany

Christina Chambers, PhD, MPH Professor Departments of Pediatrics and Family Medicine and Public Health University of California, San Diego La Jolla, CA, USA

Javier Chinen, MD, PhD Department of Pediatrics, Immunology, Allergy and Rheumatology Division Baylor College of Medicine Texas Children’s Hospital The Woodlands The Woodlands, TX, USA

Ivan Chinn, MD Assistant Professor Department of Pediatrics Section of Immunology, Allergy, and Retrovirology Director, Immunogenetics Program Texas Children’s Hospital Baylor College of Medicine Houston, TX, USA

Taylor A. Doherty, MD Lauren Cohn, MD Associate Professor Section of Pulmonary and Critical Care Medicine Department of Internal Medicine Yale University School of Medicine New Haven, CT, USA

Associate Professor Division of Rheumatology, Allergy and Immunology Department of Medicine University of California, San Diego La Jolla, CA, USA

Myrna B. Dolovich, B Eng (Elec), P Eng Scott P. Commins, MD, PhD Associate Professor of Medicine and Pediatrics Thurston Research Center University of North Carolina School of Medicine Chapel Hill, NC, USA

Professor Department of Medicine, Faculty of Health Sciences Michael DeGroote School of Medicine McMaster University Hamilton, ON, Canada

Jo A. Douglass, FRACP, MD Jonathan Corren, MD, FAAAAI Associate Clinical Professor of Medicine and Pediatrics Divisions of Allergy and Immunology David Geffen School of Medicine at UCLA Los Angeles, CA, USA

Head, Department of Immunology and Allergy Royal Melbourne Hospital Clinical Professor Department of Medicine The University of Melbourne Parkville, VIC, Australia

David J. Cousins, BSc, PhD

Stephen C. Dreskin, MD, PhD

Department of Respiratory Sciences NIHR Leicester Biomedical Research Centre – Respiratory Leicester Institute for Lung Health Leicester Institute for Precision Medicine University of Leicester Glenfield Hospital Leicester, UK

Professor of Medicine and Immunology Division of Allergy and Clinical Immunology Department of Medicine University of Colorado Denver School of Medicine Aurora, CO, USA

Anca Mirela Chiriac, MD Allergologist, Department of Respiratory Medicine and Addictology Arnaud de Villeneuve Hospital University Hospital of Montpellier Montpellier, France

Sandra C. Christiansen, MD Professor of Medicine Division of Rheumatology, Allergy and Immunology University of California, San Diego La Jolla, CA, USA

K. Fan Chung, MD, DSc, FRCP Professor of Respiratory Medicine Head of Experimental Studies National Heart and Lung Institute Imperial College London Consultant Respiratory Physician Royal Brompton and Harefield Foundation NHS Trust London, UK

Sandy R. Durrani, MD Adnan Custovic, MD, PhD Clinical Professor of Paediatric Allergy Imperial College London London, UK

Janet M. Davies, BSc, PhD, GAICD Professor School of Biomedical Science Institute of Health and Biomedical Innovation Queensland University of Technology Office of Research Metro North Hospital and Health Services Brisbane, QLD, Australia

Donald W. Cockcroft, BSc, MD, FRCP Professor Division of Respiratory, Critical Care and Sleep Medicine Department of Medicine University of Saskatchewan Saskatoon, SK, Canada

Pascal Demoly, MD, PhD Professor of Respiratory Medicine Department of Respiratory Medicine and Addictology Arnaud de Villeneuve Hospital University Hospital of Montpellier Montpellier, France

Assistant Professor Department of Pediatrics Division of Allergy and Immunology Cincinnati Children’s Hospital Medical Center University of Cincinnati School of Medicine Cincinnati, OH, USA

Mark S. Dykewicz, MD Raymond and Alberta Slavin Endowed Professor in Allergy & Immunology Chief, Section of Allergy & Immunology Division of Infectious Diseases, Allergy and Immunology Department of Internal Medicine Director, Allergy & Immunology Fellowship Program Saint Louis University School of Medicine St. Louis, MO, USA

List of Contributors

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Ronald Eccles, DSc

Sean B. Fain, PhD

Maureen George, PhD, RN, AE-C FAAN

Director, Common Cold Centre Cardiff School of Biosciences Cardiff University Cardiff, UK

Professor and Director of Lung Imaging Research Departments of Radiology and Medical Physics University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Associate Professor of Nursing Columbia University School of Nursing New York, NY, USA

Alan M. Edwards, MA, MB, BChir Clinical Assistant (retired) The David Hide Asthma and Allergy Research Centre St Mary’s Hospital Newport, Isle of Wight, UK

Thomas Eiwegger, MD Staff, Clinical Scientist, Division of Immunology and Allergy, Food Allergy and Anaphylaxis Program Department of Pediatrics The Hospital for Sick Children Scientist, Research Institute The Hospital for Sick Children, Translational Medicine Program Associate Professor Department of Immunology and Department of Pediatrics The University of Toronto Toronto, ON, Canada

Pablo Engel, MD, PhD Professor of Immunology Head of the Immunology Unit Department of Biomedical Sciences Faculty of Medicine University of Barcelona Barcelona, Spain

Renata J.M. Engler, MD, FAAAAI, FACAAI, FACP Colonel (retired), Medical Corps, US Army Professor of Medicine and Pediatrics F. Edward Hebert School of Medicine – Uniformed Services University Consultant, Allergy-ImmunologyImmunizations, Cardiovascular Immunology, Integrative Medicine and Research Immunization Healthcare Branch, Defense Health Agency Walter Reed National Military Medical Center Bethesda, MD, USA

Joseph R. Fontana, MD Lieutenant Commander, U.S. Public Health Service Senior Research Clinician Pulmonary Branch, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, MD, USA

Assistant Professor of Medicine Division of Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh School of Medicine Pittsburgh, PA, USA

Chairman, Medical Education Professor in Rhinology/Allergy Department of Otorhinolaryngology Ghent University Ghent, Belguim

Peter G. Gibson, MBBS Professor Department of Respiratory and Sleep Medicine University of Newcastle Newcastle, NSW, Australia

Michael M. Frank†, MD Samuel L. Katz Professor of Pediatrics, Medicine and Immunology Department of Pediatrics Duke University Medical Center Durham, NC, USA

Krystelle Godbout, MD, FRCPC

Anthony J. Frew†, MD, FRCP

David B.K. Golden, MD

Professor of Allergy and Respiratory Medicine Department of Respiratory Medicine Royal Sussex County Hospital Brighton, UK

Associate Professor of Medicine Division of Allergy and Clinical Immunology Department of Medicine Johns Hopkins University School of Medicine Baltimore, MD, USA

Michael Fricker, PhD Post-Doctoral Research Fellow NHMRC Centre of Excellence in Severe Asthma Priority Research Centre for Healthy Lungs University of Newcastle Hunter Medical Research Institute Newcastle, NSW, Australia

Holger Garn, PhD Head of Research Institute of Laboratory Medicine and Pathobiochemistry – Molecular Diagnostics Center for Tumor- and Immunobiology Medical Faculty Philipps University of Marburg Marburg, Germany

Marc Gauthier, MD Merritt L. Fajt, MD, FAAAAI

Philippe Gevaert, MD, PhD

Assistant Professor of Medicine Division of Pulmonary, Allergy and Critical Care Medicine University of Pittsburgh School of Medicine Pittsburgh, PA, USA

†

Deceased

Institut Universitaire de Cardiologie et de Pneumologie de Québec (IUCPQ) Université Laval Québec City, QC, Canada

Anete S. Grumach, MD, PhD Clinical Immunology ABC School of Medicine Santo Andre, SP, Brazil

Theresa W. Guilbert, MD, MS Professor of Pediatrics Department of Pediatrics University of Cincinnati Pulmonary Division Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Natasha Gunawardana, MBBS, MA, MRCP Allergy Registrar Royal Brompton Hospital London Clinical Research Fellow Imperial College London London, UK

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List of Contributors

Sudhir Gupta, MD, PhD, MACP

C. Garren Hester, BS

Daniel J. Jackson, MD

Professor of Medicine, Pathology & Laboratory Medicine, and Microbiology & Molecular Genetics Chief of Basic and Clinical Immunology Director, Jeffrey Modell Diagnostic Center for Primary Immunodeficiencies Director, Programs in Primary Immunodeficiency and Aging Medical Sciences, University of California, Irvine Irvine, CA, USA

Research Analyst Department of Pediatrics Duke University Medical Center Durham, NC, USA

Associate Professor of Pediatrics Department of Allergy & Immunology University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Teal S. Hallstrand, MD, MPH Associate Professor Division of Pulmonary, Critical Care and Sleep Medicine University of Washington Seattle, WA, USA

Mark Hew, MBBS, PhD, MScEBHC, FRACP Head of Allergy, Asthma and Clinical Immunology Alfred Health Associate Professor Public Health and Preventative Medicine Monash University Melbourne, VIC, Australia

Professor of Medicine and Pathology Division of Allergy and Clinical Immunology Department of Medicine Johns Hopkins University School of Medicine Baltimore, MD, USA

Hamida Hammad, PhD

Stephen T. Holgate, MD, DSc, FMedSci

Associate Professor of Medicine Department of Molecular Biomedical Research VIB–Ghent University Ghent, Belgium

MRC Professor of Immunopharmacology, Clinical and Experimental Sciences Faculty of Medicine Southampton University and General Hospital Southampton, UK

Michelle L. Hernandez, MD Assistant Professor Department of Pediatrics Division of Allergy, Immunology and Rheumatology University of North Carolina School of Medicine Chapel Hill, NC, USA

Consultant Department of Immunology Gene and Mary Lou Kurtz Professor of Multiple Myeloma Research Professor of Immunology Department of Immunology Dean for Research Mayo Clinic Scottsdale, AZ, USA

Stacie M. Jones, MD Professor of Pediatrics and Physiology/ Biophysics Department of Pediatrics Division of Allergy and Immunology University of Arkansas for Medical Sciences Chief, Allergy and Immunology Dr. and Mrs. Leeman King Chair in Pediatric Allergy Arkansas Children’s Hospital Little Rock, AK, USA

John W. Holloway, PhD Professor of Allergy and Respiratory Genetics Department of Human Development and Health Faculty of Medicine University of Southampton Southampton, UK

Gurjit K. Khurana Hershey, MD, PhD Kindervelt Endowed Chair in Asthma Research Professor of Pediatrics, University of Cincinnati College of Medicine Director, Division of Asthma Research Co-Director, Office of Pediatric Clinical Fellowships Attending Physician, Allergy and Immunology Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Associate Professor of Medicine Division of Pulmonary, Critical Care and Sleep Medicine Department of Medicine National Jewish Health Denver, CO, USA

Diane F. Jelinek, PhD Jeremy Hirota, PhD Canada Research Chair in Respiratory Mucosal Immunology Firestone Institute for Respiratory Health – St. Joseph’s Healthcare Assistant Professor of Medicine McMaster University Affiliate Professor of Medicine The University of British Columbia Adjunct Professor of Biology University of Waterloo Hamilton, ON, Canada

Robert G. Hamilton, PhD, D(ABMLI)

William J. Janssen, MD

David A. Kaminsky, MD Professor of Medicine Pulmonary and Critical Care Medicine University of Vermont Larner College of Medicine Burlington, VT, USA

Margaret M. Kelly, MBChB, PhD, FRCPC Charles G. Irvin, PhD, DE, ATSF, FERS Director, Vermont Lung Center Professor Departments of Medicine and Molecular Physiology, and Biophysics Larner College of Medicine University of Vermont Burlington, VT, USA

Richard S. Irwin, MD Chair, Critical Care Operations UMass Memorial Medical Center Professor of Medicine and Nursing Department of Medicine University of Massachusetts Medical School Worcester, MA, USA

Associate Professor Department of Pathology and Laboratory Medicine Cumming School of Medicine University of Calgary Calgary, AB, Canada

John M. Kelso, MD Division of Allergy, Asthma, and Immunology Scripps Clinic Clinical Professor of Pediatrics and Internal Medicine University of California, San Diego School of Medicine San Diego, CA, USA

List of Contributors

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Paneez Khoury, MD

Tanya M. Laidlaw, MD

Robert F. Lemanske, Jr., MD

Human Eosinophil Section National Institute of Allergic and Infectious Diseases National Institutes of Health Bethesda, MD, USA

Assistant Professor of Medicine Department of Medicine Harvard Medical School Director of Translational Research in Allergy Division of Rheumatology, Immunology and Allergy Brigham and Women’s Hospital and Jeff and Penny Vinik Center Boston, MA, USA

Professor of Pediatrics and Medicine Department of Pediatrics Associate Dean Clinical and Translational Research University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Hirohito Kita, MD Professor of Medicine and Immunology Departments of Medicine, Immunology, and Otorhinolaryngology Mayo Clinic Rochester, MN, USA

Amy D. Klion, MD Senior Clinical Investigator Head, Human Eosinophil Section Laboratory of Parasitic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA

Darryl Knight, PhD Professor and Head School of Biomedical Sciences and Pharmacy University of Newcastle Callaghan, NSW, Australia

Marek L. Kowalski, MD, PhD Chairman, Department of Immunology and Allergy Chair of Clinical Immunology and Rheumatology Director, Healthy Ageing Research Center (HARC) Medical University of Łódź Łódź, Poland

Cynthia J. Koziol-White, PhD Instructor Department of Pharmacology Rutgers Institute of Translational Medicine and Science Robert Wood Johnson Medical School, Rutgers University New Brunswick, NJ, USA

Gideon Lack, MD Professor of Pediatric Allergy Pediatric Allergy Group, Department of Women and Children’s Health, Peter Gorer Department of Immunobiology, School of Life Course Sciences, Faculty of Life Sciences & Medicine King’s College London Children’s Allergy Service, Guy’s and St. Thomas’ NHS Foundation Trust, London London, UK

Bart N. Lambrecht, MD, PhD Director, VIB Inflammation Research Center Ghent University Professor of Medicine Department of Respiratory Medicine University Hospital Ghent, Belgium

Carol A. Langford, MD, MHS Director, Center for Vasculitis Care and Research Department of Rheumatic and Immunologic Diseases Cleveland Clinic Cleveland, OH, USA

Beth L. Laube, PhD Professor of Pediatrics Eudowood Division of Pediatric Respiratory Sciences Johns Hopkins University School of Medicine Baltimore, MD, USA

Stephen B. LeBlanc, MD Assistant Professor of Allergy and Clinical Immunology Department of Medicine Division of Allergy and Clinical Immunology University of Mississippi Medical Center Jackson, MS, USA

Jennifer W. Leiding, MD Associate Professor Director, Multidisciplinary Immunology Service Division of Allergy and Immunology Department of Pediatrics, Morsani College of Medicine University of South Florida Tampa, FL, USA

Heather K. Lehman, MD, FAAAAI Assistant Professor of Pediatrics Division of Allergy, Immunology and Rheumatology Department of Pediatrics Jacobs School of Medicine and Biomedical Sciences University of Buffalo Buffalo, NY, USA

Catherine Lemière, MD, MSc Professor Department of Medicine Université de Montréal Montréal, QC, Canada

Donald Y.M. Leung, MD, PhD Edelstein Family Chair of Pediatric Allergy and Immunology National Jewish Health Professor, Department of Pediatrics University of Colorado School of Medicine Denver, CO, USA

James T. Li, MD, PhD Professor of Medicine Division of Allergy and Immunology Mayo Clinic Rochester, MN, USA

Xiu-Min Li, MD, MS Professor Department of Microbiology and Immunology Department Otolaryngology New York Medical College Valhalla, NY, USA

Clare Lloyd, PhD Wellcome Senior Research Fellow in Basic Biomedical Science Professor of Respiratory Immunology Head of Leukocyte Biology Section National Heart and Lung Institute Faculty of Medicine, Imperial College London London, UK

Andrew D. Luster, MD, PhD Persis, Cyrus, and Marlow B. Harrison Professor of Medicine Harvard Medical School Chief, Division of Rheumatology, Allergy, and Immunology Director, Center for Immunology and Inflammatory Diseases Massachusetts General Hospital Boston, MA, USA

xvi

List of Contributors

Eric Macy, MS, MD, FAAAAI

Tesfaye B. Mersha, PhD

Paul M. O’Byrne, MB, FRCP(C), FRSC

Southern California Permanente Medical Group Department of Allergy Kaiser Permanente Medical Center San Diego, CA, USA

Associate Professor Cincinnati Children’s Hospital Medical Center Department of Pediatrics University of Cincinnati Cincinnati, OH, USA

Dean and Vice-President Faculty of Health Sciences Michael G. DeGroote School of Medicine Distinguished University Professor Department of Medicine McMaster University Hamilton, ON, Canada

J. Mark Madison, MD Professor of Medicine and Microbiology and Physiological Systems Department of Medicine University of Massachusetts Medical School Worcester, MA, USA

Aaron R. Mangold, MD Assistant Professor of Dermatology Mayo Clinic Alix School of Medicine Consultant, Department of Dermatology Mayo Clinic Arizona Scottsdale, AZ, USA

Rebecca A. Marsh, MD Associate Professor University of Cincinnati Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Gailen D. Marshall, Jr., MD, PhD, FACP, DFACAAI, FAAAAI The R. Faser Triplett, Sr. MD Chair of Allergy and Immunology Medical Director, UMMC Clinical Research Support Program/Clinical Research and Trials Units Professor of Medicine, Pediatrics, Pathology and Population Science Vice Chair for Research, Department of Medicine Director, Division of Clinical Immunology and Allergy Chief, Laboratory of Behavioral Immunology Research The University of Mississippi Medical Center Editor-in-Chief Annals of Allergy, Asthma and Immunology Jackson, MS, USA

Elizabeth Matsui, MD, MHS Professor of Population Health and Pediatrics Director of Clinical and Translational Research Dell Medical School The University of Texas at Austin Austin, TX, USA

Dean D. Metcalfe, MD Chief Mast Cell Biology Section Laboratory of Allergic Diseases National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA

Hans C. Oettgen, MD, PhD

Zamaneh Mikhak, MD

Robyn E. O’Hehir, FRACP, PhD, FRCPath

Senior Director Clinical Research & Development Kiniksa Pharmaceuticals Lexington, MA, USA

Professor and Director, Department of Respiratory Medicine, Allergy and Clinical Immunology (Research) Central Clinical School, Monash University, and Alfred Hospital Melbourne, VIC, Australia

E.N. Clare Mills, PhD Professor of Molecular Allergology Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Manchester Academic Health Sciences Centre Manchester Institute of Biotechnology The University of Manchester Manchester, UK

Harold S. Nelson, MD Professor of Medicine Division of Allergy and Immunology Department of Medicine, National Jewish Health University of Colorado Denver School of Medicine Denver, CO, USA

Rosemary L. Nixon, MPH, FACD, FAFOEM Adjunct Clinical Associate Professor Monash University Clinical Associate Professor University of Melbourne Director, Occupational Dermatology Research and Education Centre Skin and Cancer Foundation Melbourne, VIC, Australia

Associate Chief Division of Immunology Boston Children’s Hospital Professor of Pediatrics Harvard Medical School Boston, MA, USA

Jordan S. Orange, MD, PhD Reuben S. Carpentier Professor and Chairman Department of Pediatrics, Vagelos College of Physicians and Surgeons Columbia University Pediatrician-In-Chief, Morgan Stanley Children’s Hospital of New York Presbyterian New York, NY, USA

Dennis R. Ownby, MD Betty B. Wray Professor of Pediatrics Professor of Internal Medicine Chief, Division of Allergy, Immunology, and Rheumatology Department of Pediatrics Georgia Regents University Augusta, GA, USA

Clive P. Page, OBE, PhD Professor of Pharmacology Sackler Institute of Pulmonary Pharmacology Institute of Pharmaceutical Science School of Biomedical Science Kings College London London, UK

Anna Nowak-Węgrzyn, MD, PhD Professor of Pediatrics Icahn School of Medicine at Mount Sinai Jaffe Food Allergy Institute Kravis Children’s Hospital New York, NY, USA

Reynold A. Panettieri, Jr., MD Professor of Medicine, Robert Wood Johnson Medical School Vice Chancellor, Clinical & Translational Science Director, Rutgers Institute for Translational Medicine & Science Rutgers, The State University of New Jersey New Brunswick, NJ, USA

List of Contributors

Hae-Sim Park, MD, PhD Professor Department of Allergy and Clinical Immunology Ajou University School of Medicine Suwon, South Korea

Mary E. Paul, MD Associate Professor of Pediatrics Baylor College of Medicine Chief of Service, Retrovirology and Global Health Texas Children’s Hospital Houston, TX, USA

David B. Peden, MD, MS Andrews Distinguished Professor of Pediatrics Senior Associate Dean for Translational Research Director, The Center for Environmental Medicine, Asthma and Lung Biology Chief, Division of Allergy, Immunology & Rheumatology Department of Pediatrics The School of Medicine The University of North Carolina at Chapel Hill Chapel Hill, NC, USA

R. Stokes Peebles, Jr., MD Elizabeth and John Murray Professor of Medicine Professor of Pathology, Microbiology, and Immunology Division of Allergy, Pulmonary, and Critical Care Medicine Vanderbilt University Medical Center Nashville, TN, USA

Stephen P. Peters, MD, PhD, ATSF, FAAAAI, FACP, FCCP, FCPP Thomas H. Davis Chair in Pulmonary Medicine Department of Internal Medicine Chief, Section on Pulmonary, Critical Care, Allergy & Immunologic Diseases Professor of Internal Medicine, Pediatrics and Translational Science Executive Director, Respiratory Service Line Wake Forest Baptist Health Winston Salem, NC, USA

Elizabeth J. Phillips, MD, FRCPC, FRACP, FIDSA, FAAAI

Professor of Internal Medicine Division of Pulmonary and Critical Care Medicine and Graduate Program in Immunology University of Michigan Medical School Ann Arbor, MI, USA

Samriddha Ray, PhD

Professor of Medicine and Pharmacology Professor of Pathology, Microbiology and Immunology Department of Medicine Vanderbilt University Medical Center Nashville, TN, USA

Research Associate The Perinatal Institute and Section of Neonatology, Perinatal and Pulmonary Biology Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Mark R. Pittelkow, MD

Harald Renz, MD

Robert S. Totz, MD Professor of Dermatology Departments of Dermatology and Biochemistry and Molecular Biology Mayo Clinic Alix School of Medicine Consultant and Chair Department of Dermatology Mayo Clinic Arizona Scottsdale, AZ, USA

Professor and Director Institute of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics Philipps University Marburg University Hospital Giessen and Marburg GmbH Marburg, Germany

Clive Robinson, PhD, FHEA, FSB, FRSA Thomas A.E. Platts-Mills, MD, PhD, FRS Professor and Division Chief Department of Internal Medicine University of Virginia Health Science Center Charlottesville, VA, USA

Professor of Respiratory Cell Science Institute for Infection and Immunity St. George’s University of London London, UK

Chen E. Rosenberg, MD Susan L. Prescott, PhD, MD Professor Division of Pediatrics School of Medicine University of Western Australia Pediatric Allergist and Immunologist Perth Children’s Hospital Perth, WA, Australia

Clinical Fellow Division of Allergy and Immunology Department of Pediatrics Cincinnati Children’s Hospital Medical Center University of Cincinnati College of Medicine Cincinnati, OH, USA

Ronald L. Rabin, MD

Marc E. Rothenberg, MD, PhD

Chief, Laboratory of Immunobiochemistry Division of Bacterial, Parasitic and Allergenic Products Center for Biologics Evaluation and Research Office of Vaccines Research and Review U.S. Food and Drug Administration Silver Spring, MD, USA

Professor of Pediatrics Department of Pediatrics University of Cincinnati College of Medicine Director, Division of Allergy and Immunology Director, Cincinnati Center for Eosinophilic Disorders Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Hengameh H. Raissy, PharmD Research Professor of Pediatrics Department of Pediatric University of New Mexico School of Medicine Albuquerque, NM, USA

Anuradha Ray, PhD Marc Peters-Golden, MD

xvii

Professor of Medicine and Immunology Departments of Medicine and Immunology University of Pittsburgh School of Medicine Pittsburgh, PA, USA

Brian H. Rowe, MD, MSc Scientific Director Institute of Circulatory and Respiratory Health, Canadian Institutes of Health Research Professor Department of Emergency Medicine University of Alberta Edmonton, AB, Canada

xviii

List of Contributors

Sejal Saglani, MD, MRCPCH

Guy W. Scadding, MRCP, PhD

George K. Siberry, MD, MPH

Professor of Pediatric Respiratory Medicine Department of National Heart & Lung Institute Consultant in Respiratory Pediatrics, Royal Brompton Hospital Imperial College London Honorary Consultant in Respiratory Pediatrics Royal Brompton Hospital London London, UK

Honorary Clinical Senior Lecturer Allergy and Clinical Immunology Imperial College London London, UK

Medical Officer, Adult Clinical Branch, Office of HIV/AIDS Division of Prevention, Care & Treatment United States Agency for International Development (USAID) Arlington, VA, USA

Ozlem Sahin, MD Doctoral Student Department of Microbiology and Immunology New York Medical College Valhalla, NY, USA

Eric E. Schadt, PhD Dean for Precision Medicine Jean C. and James W. Crystal Professor of Genomics Department of Genetics and Genomic Sciences Icahn School of Medicine at Mount Sinai New York, NY, USA

Professor of Pediatrics Chief, Division of Allergy Director, Jaffe Food Allergy Institute Department of Pediatrics Icahn School of Medicine at Mount Sinai New York, NY, USA

Michael Schatz, MD, MS Department of Allergy Kaiser Permanente Medical Center San Diego, CA, USA

Sarbjit S. Saini, MD Professor of Medicine Division of Allergy and Clinical Immunology Johns Hopkins University School of Medicine Baltimore, MD, USA

Scott H. Sicherer, MD

Eric Schauberger, DO, PhD Assistant Professor of Pediatrics Division of Pediatric Allergy, Immunology, and Rheumatology University of Wisconsin School of Medicine and Public Health Madison, WI, USA

F. Estelle R. Simons, MD, FRCPC Professor, Department of Pediatrics Department of Immunology University of Manitoba Winnipeg, MB, Canada

Jodie L. Simpson, PhD, FThorSoc Priority Research Centre for Healthy Lungs School of Medicine and Public Health The University of Newcastle Newcastle, NSW, Australia

Hirohisa Saito, MD, PhD Advisor to the Executive Director National Research Institute for Child Health and Development Tokyo, Japan

Hugh A. Sampson, MD Kurt Hirschhorn Professor of Pediatrics Department of Pediatrics Icahn School of Medicine at Mount Sinai New York, NY, USA

Imran Satia, MA, MB, BChir, PhD, MRCP Post-Doctoral Clinical Fellow Department of Medicine Division of Respirology McMaster University Honorary Senior Lecturer Division of Infection, Immunity and Respiratory Medicine University of Manchester and Manchester Academic Health Science Center Manchester, UK

John T. Schroeder, PhD

Jay E. Slater, MD

Associate Professor of Medicine Division of Allergy and Clinical Immunology Johns Hopkins University School of Medicine Baltimore, MD, USA

Director Division of Bacterial, Parasitic, and Allergenic Products Office of Vaccines Research and Review Center for Biologics Evaluation and Research U.S. Food and Drug Administration Silver Spring, MD, USA

Christine M. Seroogy, MD, FAAAAI Professor Department of Pediatrics University of Wisconsin School of Medicine and Public Health Madison, WI, USA

William T. Shearer†, MD, PhD Professor of Pediatrics and Pathology and Immunology Distinguished Service Professor Baylor College of Medicine Allergy and Immunology Service Texas Children’s Hospital Houston, TX, USA

Ian Sayers, BSc, PhD

James H. Shelhamer, MD

Professor of Respiratory Molecular Genetics Deputy Director of Doctoral Programmes for the School of Medicine Division of Respiratory Medicine NIHR Nottingham Biomedical Research Centre University of Nottingham Queens Medical Centre Nottingham, UK

Special Volunteer Critical Care Medicine Department Clinical Center National Institutes of Health Bethesda, MD, USA

†

Deceased

Peter D. Sly, AO, MBBS, MD, DSc Director Children’s Health and Environment Program The University of Queensland Brisbane, QLD, Australia

Helen E. Smith, DM, FFPHM Professor Department of Family Medicine & Primary Care Lee Kong Chian School of Medicine Nanyang Technological University Singapore

Philip H. Smith, MD Associate Professor, Pediatrics and Medicine, Division of Allergy and Immunology Departments of Pediatrics and Medicine Medical College of Georgia Children’s Hospital of Georgia Georgia Regents University Charlie Norwood Veterans Administration Medical Center Augusta, GA, USA

List of Contributors

Caroline L. Sokol, MD

Jeffrey R. Stokes, MD

Karen S. Tuano, MD

Assistant Professor of Medicine Harvard Medical School Division of Rheumatology, Allergy and Immunology Center for Immunology and Inflammatory Diseases Massachusetts General Hospital Boston, MA, USA

Professor of Pediatrics Department of Pediatrics Division of Pediatric Allergy, Immunology & Pulmonary Medicine Washington University School of Medicine St. Louis, MO, USA

Assistant Professor Department of Pediatrics Section of Immunology, Allergy, Rheumatology and Retrovirology Baylor College of Medicine Houston, TX, USA

Kathleen E. Sullivan, MD, PhD

Bradley J. Undem, PhD

Professor of Pediatrics Department of Pediatrics The Children’s Hospital of Philadelphia Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA, USA

Professor of Medicine Department of Medicine Division of Allergy and Clinical Immunology Johns Hopkins University School of Medicine Baltimore, MD, USA

Roland Solensky, MD Allergist/ Immunologist Division of Allergy & Immunology The Corvallis Clinic Lecturer Oregon State University/ Oregon Health and Science University College of Pharmacy Corvallis, OR, USA

P. Sriramarao, PhD Professor Department of Microbiology and Immunology School of Medicine Vice President for Research and Innovation Virginia Commonwealth University Richmond, VA, USA

Kamal Srivastava, PhD Assistant Professor Department of Microbiology & Immunology New York Medical College Valhalla, NY, USA

John W. Steinke, PhD Associate Professor, Department of Medicine Asthma and Allergic Diseases Center Carter Center for Immunology Research University of Virginia Charlottesville, VA, USA

Tunn Ren Tay, MBBS, MRCP Respiratory and Intensive Care Consultant Physician Department of Respiratory & Critical Care Medicine Changi General Hospital Singapore

Director Institute for Respiratory Health Nedlands Perth, WA, Australia

Jenny M. Stitt, MD Assistant Professor Division of Allergy and Immunology Department of Medicine University of Colorado Aurora, CO, USA

Olivier Vandenplas, MD, PhD Professor of Medicine Department of Chest Medicine Centre Hospitalier Universitaire de Mont-Godinne Université Catholique de Louvain Yvoir, Belgium

Steve L. Taylor, PhD Professor Department of Food Science and Technology, Food Allergy Research, and Resource Program University of Nebraska Lincoln, NE, USA

Bruce Thompson, CRFS, FANZSRS, FThorSoc, FAPSR, PhD Head, Physiology Service Allergy, Immunology and Respiratory Medicine The Alfred Hospital Adjunct Professor Central Clinical School Monash University Melbourne, VIC, Australia

Alkis Togias, MD Geoffrey A. Stewart, BSC, PhD

xix

Branch Chief, Allergy, Asthma and Airway Biology Division of Allergy, Immunology and Transplantation National Institute of Allergy and Infectious Diseases National Institutes of Health Bethesda, MD, USA

Euan Tovey, MD Associate Professor Woolcock Institute of Medical Research The University of Sydney School of Medicine Sydney, NSW, Australia

Menno C. van Zelm, PhD Associate Professor and Deputy Head of Department (Research) NHMRC Senior Research Fellow Department of Immunology and Pathology, Monash University Department of Allergy, Immunology and Respiratory Medicine The Alfred Hospital Melbourne, VIC, Australia

Ravi K. Viswanathan, MD Assistant Professor (CHS) Section of Allergy, Pulmonary and Critical Care Department of Medicine University of Wisconsin School of Medicine and Public Health Madison, WI, USA

Richard W. Weber, MD Professor of Medicine National Jewish Health University of Colorado School of Medicine Denver, CO, USA

xx

List of Contributors

Peter F. Weller, MD, FACP, FAAAAI

Robert A. Wood, MD

Nan Zhang, MD

William B. Castle Professor of Medicine Department of Medicine Harvard Medical School Professor of Immunology and Infectious Diseases Department of Immunology and Infectious Diseases Harvard T. H. Chan School of Public Health Chief, Allergy and Inflammation Division Chief, Infectious Diseases Division, Department of Medicine Beth Israel Deaconess Medical Center Boston, MA, USA

Professor of Pediatrics Director, Pediatric Allergy and Immunology Johns Hopkins University School of Medicine Johns Hopkins Hospital Baltimore, MD, USA

Professor in Rhinology Upper Airway Research Laboratory Ghent University Ghent, Belgium

Sally E. Wenzel, MD Professor of Medicine and Immunology Chair, Department of Environmental and Occupational Health Director, University of Pittsburgh Asthma Institute at UPMC UPMC Chair in Translational Airway Biology Pittsburgh, PA, USA

Jeffrey A. Whitsett, MD Kindervelt Professor of Pediatrics Executive Director, Perinatal Institute Chief, Section of Neonatology, Perinatal and Pulmonary Biology Cincinnati Children’s Hospital Medical Center Cincinnati, OH, USA

Leman Yel, MD, FAAP, FAAAAI Vice President Head of Clinical Medicine, Global R&D, Plasma Derived Therapies The Takeda Group of Companies Cambridge, MA, USA Emeritus Associate Professor of Clinical Medicine Division of Basic and Clinical Immunology Department of Medicine University of California, Irvine Irvine, CA, USA

Robert S. Zeiger, MD, PhD Senior Adjunct Physician Investigator Kaiser Permanente Southern California Region University of California, San Diego San Diego, CA, USA

Bruce L. Zuraw, MD Professor of Medicine Department of Medicine University of California, San Diego Veterans Affairs San Diego Healthcare La Jolla, CA, USA

SECTION A  Basic Sciences Underlying Allergy and Immunology

1  Innate Immunity Lora Bankova, Nora Barrett

CONTENTS Introduction, 1 Microbial Pattern Recognition by the Innate Immune System, 1 Resident Cellular Responses of Innate Immunity, 6 Infiltrative Cellular Responses of Innate Immunity, 8

SUMMARY OF IMPORTANT CONCEPTS • The innate immune system is composed of receptors, cells, and antimicrobial molecules that provide rapid protection from potential pathogens before adaptive immunity is established. • Pathogens are identified by a limited number of key conserved molecular components that are not made by the host. These pathogen-associated molecular patterns (PAMPs) are recognized by germline-encoded pattern recognition receptors (PRRs). • In addition to its sentinel function, the innate immune system activates and instructs the adaptive immune system for antigen-specific T and B lymphocyte responses and the development of immunologic memory. • Innate immune defenses are highly efficient and include homeostatic mechanisms that downregulate inflammation to optimize the health of the host. • Like antimicrobial immunity, allergen recognition and uptake and allergic sensitization, inflammation, and disease originate in the innate immune system.

INTRODUCTION The innate immune system has a long evolutionary heritage, with elements shared by most vertebrates, plants, and insects. This group of conserved receptors, barrier and immune cells, and antimicrobial molecules are required to provide immediate protection from potential pathogens. An effective innate immune response must include immune recognition of pathogens by discriminating “self ” from “nonself,” rapid induction of effector mechanisms for pathogen containment and clearance, stimulation of long-term adaptive immunity so that subsequent exposures are handled more efficiently, and regulation of the response to prevent damage to the host. Innate immune responses are initiated by recognition of molecular components of microorganisms that are foreign to the host, so-called

Innate Instruction of Adaptive Immune Responses, 10 Homeostasis in the Innate Immune System, 10 Innate Immunity and Allergy, 12 Summary, 15

pathogen-associated molecular patterns (PAMPs). They can also be activated by host molecules that are released by damaged cells or produced by host cells during an inflammatory response, so-called damageassociated molecular patterns (DAMPs). The sensing receptors activated by PAMPs and DAMPs are termed pattern recognition receptors (PRRs). These receptors are germline encoded and therefore present at birth; they are not generated, tailored, or expanded by clonal selection in response to antigen, as are the recognition receptors of T and B lymphocytes in acquired immunity. Because innate immunity is “inborn,” it plays a critical role early in the life of an organism when the adaptive repertoire has not yet been shaped and continues to provide immediate protection throughout the life of an organism, bridging the gap to adaptive immune responses, which require days to amplify and become effective. In addition to its sentinel detection and first-responder roles, the innate immune system activates and instructs adaptive immunity, regulates inflammation, and maintains homeostasis to allow the organism to develop, grow, and thrive in its environment. Unfortunately, allergic sensitization, inflammation, and disease may originate in aberrant innate immune development. The innate roots of allergy are considered in this chapter.

MICROBIAL PATTERN RECOGNITION BY THE INNATE IMMUNE SYSTEM The germline-encoded PRRs (Table 1.1) of the innate immune system have genetically predetermined specificities for microbial constituents. Natural selection has formed and refined the repertoire of these receptors to recognize microbe-specific PAMPs. Although different PAMP structures are biochemically distinct from each other, they share common features: • PAMPs are produced only by microbes, not by their hosts. • PAMPs are common molecular structures, typically shared by entire classes of pathogens. • PAMPs are usually fundamental to the integrity, survival, and pathogenicity of the microbe.

1

2

SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 1.1  Innate Pattern Recognition Receptors in Humans Pattern Recognition Receptors Secreted Antimicrobial peptides   α- and β-defensins   Cathelicidin (LL-37)  Dermcidin  RegIIIγ Collectins   Mannose-binding lectin   Surfactant proteins A and D Pentraxins   C-reactive protein

Pathogen-Associated Molecular Pattern Structures Recognized

Functions

Microbial membranes (negatively charged)

Opsonization, microbial cell lysis, immune cell chemoattractant

Microbial mannan

Opsonization, complement activation, microbial cell lysis, chemoattraction, phagocytosis

Bacterial cell wall lipids; viral coat proteins

Opsonization, killing, phagocytosis, proinflammatory and antiinflammatory mediator release

Bacterial phospholipids (phosphorylcholine)

Opsonization, complement activation, microbial cell lysis, chemoattraction, phagocytosis

Secreted and Membrane Bound CD14 Endotoxin

TLR4 signaling

LPS binding protein

Endotoxin

TLR4 signaling

MD-2

Endotoxin

TLR4 coreceptor

Membrane Bound Toll-like receptorsa

Microbial PAMPs

Immune cell activation

  Mannose receptor (CD206)

Microbial mannan

Cell activation, phagocytosis, proinflammatory mediator release

 DECTIN-1

β-1,3-Glucan

Cell activation, phagocytosis, proinflammatory mediator release

 DECTIN-2

Fungal mannose

Cell activation, phagocytosis, proinflammatory mediator release

 DC-SIGN

Microbial mannose, fucose

Immunoregulation, IL-10 production

 Siglecs

Sialic acid containing glycans

Cell inhibition, endocytosis

Peptidoglycans from gram-negative bacteria

Cell activation

 NOD-2

Bacterial muramyl dipeptides

Cell activation

 NLRP1

Anthrax lethal toxin

PAMP recognition in inflammasome

  NLRP3 (cryopyrin)

Microbial RNA

PAMP recognition in inflammasome

 NLRC4

Bacterial flagellin

PAMP recognition in inflammasome

RIG-I and MDA5

Viral double-stranded RNA

Type 1 IFN responses

Cyclic GMP-AMP synthase (cGAS)

Cytosolic double-stranded DNA

Type 1 IFN responses

C-type lectin receptors

Cytosolic NOD-like Receptors  NOD-1

DC-SIGN, Dendritic cell–specific intracellular adhesion molecule 3 (ICAM-3)–grabbing nonintegrin; DECTIN, dendritic cell–specific receptor; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MD-2, myeloid differentiation factor 2 (also called lymphocyte antigen 96 [LY98]); MDA5, melanoma differentiation–associated 5 (also called interferon induced with helicase domain 1 [IFIH1]); NLR, NOD-like receptor; NOD, nucleotidebinding oligomerization domain protein; PAMP, pathogen-associated molecular pattern; RegIIIγ, regenerating islet-derived 3 γ (REG3G); RIG-I, retinoic acid–inducible 1 (also called DDX58); Siglecs, sialic acid–binding immunoglobulin-like lectins; TLR, toll-like receptor. a See Table 1.2.

For example, bacterial endotoxin is a lipopolysaccharide (LPS) PAMP that makes up most of the outer membrane layer of all gram-negative bacteria. Lipid A, a highly conserved component of LPS that confers much of endotoxin’s biologic activities, is the recognized target of tolllike receptor 4 (TLR4) (Fig. 1.1).1 Other PAMPs include common microbial cell membrane components and nucleic acids with molecular features distinct from those of animals or humans.

This approach to microbial recognition by PRRs in innate immunity is fundamentally different from the development of microbial recognition in the adaptive immune system by T and B lymphocytes. Each T and B lymphocyte acquires a structurally unique receptor during somatic recombination, generating a very diverse and almost limitless repertoire of antigen specificities (approximately 1014 different immunoglobulin receptors and 1018 different T cell receptors), from which the useful

3

CHAPTER 1  Innate Immunity Macrophages

LPS

Neutrophils

LBP MD-2 CD14

TLR4 Toll-like receptors

TIRAP MYD88

MYD88-dependent pathway

Antimicrobial peptides Collectins

TRAM Epithelial cells

TRIF

TRIF-dependent pathway

C-type lectin receptors

NOD-like receptors

C-reactive protein

Mast cells

Dendritic cells Proinflammatory cytokines

Type 1 interferons

Fig. 1.1  Endotoxin recognition and cell activation through toll-like receptor 4 (TLR4). Lipopolysaccharide (LPS) (i.e., endotoxin) from the outer cell wall of gram-negative bacteria is a prototypical microbial pathogenassociated molecular pattern (PAMP) that is bound by soluble LPS-binding protein (LBP) and CD14 and transferred to myeloid differentiation factor 2 (MD-2). MD-2 specifically binds LPS and forms signal-transducing multimers with TLR4. Four signal-transducing adaptor proteins are recruited to the LPS–MD-2–TLR multimers: MYD88 and TIRAP of the MYD88-dependent pathway and TRIF and TRAM of the TRIF-dependent pathway. The MYD88-dependent pathway induces the expression of inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) and costimulatory molecules (e.g., CD80). The TRIF-dependent pathway mediates the induction of type 1 interferons and interferon-inducible genes. IL, Interleukin; MD-2, myeloid differentiation factor 2 (also called lymphocyte antigen 96 [LY98]); MYD88, myeloid differentiation primary response gene 88; TIRAP, toll–interleukin-1 receptor (TIR) domain-containing adaptor protein; TNF, tumor necrosis factor; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain–containing adaptor protein inducing interferon β (also called toll-like receptor adaptor protein 1 [TICAM1]). (Adapted from Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine 2008;42:145-51.)

receptors (e.g., those specific for microbial pathogens rather than self) are selected for clonal expansion. This process allows for greater diversification, specificity, and affinity of antibodies over the life of an organism but cannot be passed on to progeny. By contrast, the innate immune system relies on evolutionarily conserved PRRs, which are not individualized for each host but which are passed on to progeny.2

Pattern Recognition Receptors PRRs of the innate immune system can be divided into two groups: secreted receptors and transmembrane signal-transducing receptors (Table 1.1). Secreted PRRs typically have multiple effects in innate immunity and host defense, including direct microbial killing, serving as helper proteins for transmembrane receptors, opsonization for phagocytosis, and chemoattraction of innate and adaptive immune effector cells. Transmembrane PRRs such as TLRs are expressed on many innate immune cell types, including macrophages, dendritic cells (DCs),

Innate lymphoid type 2 cell Fig. 1.2  Main categories of pattern recognition receptors and the innate immune cell types that express them. NOD, Nucleotide-binding oligomerization domain protein. (Adapted from Liu AH. Innate microbial sensors and their relevance to allergy. J Allergy Clin Immunol 2008;122:846-58.)

monocytes, and B lymphocytes—the professional antigen-presenting cells (Fig. 1.2). Innate immune efficiency is achieved in part by the constitutive expression of some of these sentinel receptors and the rapid upregulation of others with innate immune activation. Notably, although most of the PRRs reviewed here are well-characterized, recent studies have demonstrated important antimicrobial functions of yet additional receptor classes, such as taste receptors,3 highlighting the emerging nature of this field and the additional work on PRRs to be done.

Antimicrobial Peptides Antimicrobial peptides (AMPs) are highly diverse small cationic peptides with broad antimicrobial activity that are secreted by activated innate immune cells. A fundamental shared feature of all AMPs is their amphipathic structure: clustering hydrophobic and cationic amino acids in discrete regions of the molecule (Fig. 1.3). This structure allows AMPs to interact with negatively charged phospholipids in microbial cell membranes, integrate into the membranes, and disrupt them.4 Importantly, while AMPs have antimicrobial activity against a broad range of bacteria, fungi, and enveloped viruses, they do not interact with the cell membranes of plants and animals, which lack polar phospholipids.4 AMPs are produced by hematopoietic cells and epithelial cells in the airway, skin, intestine, and urinary tract.5,6 In humans, the two main categories of AMPs are defensins (α and β classes) and the cathelicidin LL-37 (Table 1.1). There are six human α-defensins (HD1-6) and four well-characterized human β-defensins (HBD1-4), with computational genetic approaches predicting many more.4 Their production can be constitutive (e.g., HBD1) or inducible (e.g., HBD2, HBD3, HBD4) through multiple innate signaling pathways. For example, HBD2 expression can be induced by bacterial PAMPs signaling through TLR2 or TLR4 or by innate inflammatory cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α).4–6

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Hydrophobic interactions

Antimicrobial peptide + +

Inner leaflet –

Chemoattraction

Neutrophil

Dendritic cell

Strong

Weak Outer leaflet

Electrostatic and hydrophobic interactions

+

–

+

Mast cell –

– –

–

+ + – – –

T lymphocyte

Monocyte

–

Prototypic plasma membrane of a Bacterial cytoplasmic membrane multicellular animal Outside Inside

Opsonization

Cell lysis

Fig. 1.3  Mechanism of antimicrobial peptide-mediated host defense. Antimicrobial peptides (AMPs) are amphipathic, containing a discrete cationic region of the molecule. They target the exposed outer membrane of bacteria that is dense with negatively charged phospholipid head groups. This is different from the cell membranes of plants and animals that are spared AMP binding, because their outer cell membrane lipids have no net charge. AMPs integrate into bacterial membranes and form holes that physically disrupt membrane integrity and lyse target cells. AMPs are chemoattractive for a variety of immune cells, while also carpeting and opsonizing bacterial targets for recognition and uptake by phagocytes bearing AMP receptors. (Adapted from Zasloff M. Antimicrobial peptides of multicellular organisms. Nature 2002;415:389-95.)

Human cathelicidin LL-37 is released from neutrophils, mast cells, epithelial cells, and keratinocytes, and it exhibits a broad range of antimicrobial activities. LL-37 is induced by vitamin D; the gene encoding LL-37 has a vitamin D receptor binding site. In keratinocytes and macrophages, stimulation of TLR2 results in the induction of CYP27B1, the cytochrome P-450 enzyme that converts 25-hydroxyvitamin D3 (25-OH-D) to the active form of 1,25-dihydroxyvitamin D3 (1,25-OHD), which induces LL-37 expression. By this route, vitamin D can influence microbicidal defenses of the skin and circulating phagocytic cells. It has been proposed that certain human infections, such as by Mycobacterium tuberculosis, might be more prevalent among populations with inadequate plasma levels of vitamin D.5,7 Other types of AMPs include dermcidin in sweat and the lectin protein RegIIIγ (REG3G) in the intestine, which serves to both bind pathogens and alter the distribution of luminal mucus, segregating bacteria away from the epithelial cell surface.5 In addition to their direct bactericidal activities, AMPs also contribute to host defense through the control of cytokine/chemokine production, cell migration, and maintenance of skin barrier function. Both human α- and β-defensins act as chemoattractants for immature DCs and peripheral blood T cells, thereby enhancing antigen-specific adaptive immune responses. LL-37 attracts neutrophils, monocytes, mast cells, and T lymphocytes through formyl peptide-like receptor 1 (FPRL1), a PRR that also binds bacterial formyl peptides.5 Importantly, failure to upregulate AMP production has been linked to the increased Staphylococcus aureus colonization and susceptibility to viral skin infections in patients with atopic dermatitis.5

To summarize, when AMPs are induced at a site of injury, they act directly to destroy microbial invaders and to attract an array of defensive cells that provide backup support to defend the breached barrier. AMPs probably control commensal relationships to maintain health in the gut and maybe elsewhere. Inflammation results when the AMP-based defenses have proved inadequate and robust secondary defensive responses are mobilized.

Collectins Collectins are secreted C-type lectin receptors (CLRs) that are structurally similar to the transmembrane CLRs (discussed later) and contain a collagenous domain. Mannose-binding lectin (MBL) is an acute-phase reactant that recognizes terminal mannose residues of carbohydrates on gram-positive and gram-negative bacteria, fungi, yeast, and some viruses and parasites.8 MBL is structurally similar to the complement component C1q, and, like C1q, it activates the classic complement cascade through MBL-associated serine proteases that are related to C1r and C1s and cleave C4, C2, and C3, leading to amplified opsonization, membrane pore formation, cell lysis, and neutrophil chemoattraction.9 Two of the four pulmonary surfactant proteins, SP-A and SP-D, are collectins with similar structures and multiple innate immune functions. They share carbohydrate-binding domains that bind oligosaccharides specific for a variety of microbes (e.g., gram-positive and gram-negative bacteria, viruses, fungi). They recognize a wide variety of PAMPs, such as bacterial LPS, mycobacterial lipoarabinomannan, other bacterial glycolipids, and common viral glycoproteins, such as influenza hemagglutinin and neuraminidase envelope glycoproteins and respiratory

CHAPTER 1  Innate Immunity syncytial virus (RSV) G (attachment) and F (fusion) proteins.9 SP-A and SP-D mediate multiple antimicrobial functions. They aggregate and opsonize microbes for phagocytosis by alveolar macrophages, monocytes, neutrophils, and DCs. They also trigger nuclear factor-κB (NF-κB) activation and cytokine production through TLR4 and TLR2. SP-A induces the expression of scavenger and mannose receptors on phagocytes, thereby improving phagocytosis. SP-A and SP-D have direct bactericidal and fungicidal properties, and they help to dampen inflammatory responses by enhancing the clearance of proinflammatory apoptotic cells by macrophages.10

Pentraxins Pentraxins are acute-phase reactant PRRs that are secreted in response to TLR activation or proinflammatory cytokines.11 C-reactive protein (CRP) was the first PRR and the first pentraxin to be described. CRP specifically binds bacterial phospholipids (e.g., phosphorylcholine) and the complement factor C1q, thereby opsonizing bacteria and activating the classic complement cascade. CRP also directly binds Fcγ receptors on phagocytes, further promoting phagocytosis.

Toll-Like Receptors (Table 1.2) The immediate cellular responders of the innate immune system (e.g., epithelial cells, monocytes, macrophages, DCs, mast cells, neutrophils) and other cell types express a family of transmembrane PRRs with functional roots found in the toll receptor of Drosophila. These toll-like receptors (TLRs) are structurally similar, with large, leucine-rich extracellular domains and cytoplasmic domains that are similar to those of the mammalian IL-1 receptor (Table 1.2).12 The IL-1 receptor and TLRs share a signaling pathway that leads to NF-κB activation through the adaptor protein myeloid differentiation primary response protein 88 (MYD88), described further below. TLR4 was the first human TLR identified, and it is specific for bacterial endotoxin. Endotoxin, a prototypical PAMP, is a gram-negative bacterial cell wall LPS with a highly conserved lipid A moiety. Very small amounts of endotoxin (i.e., picogram amounts, estimated to equal about 10 LPS molecules/cell) are immunostimulatory. This very high sensitivity for endotoxin-mediated cell activation can be attributed to the endotoxin receptor complex (Fig. 1.1). Lipopolysaccharide binding

protein (LBP) and CD14 are soluble proteins that capture and transfer LPS to the MD-2/TLR4 complex. MD-2 specifically binds LPS and forms signal-transducing multimers with TLR4.1,13 Although LBP and CD14 are not classic PRRs in that their binding specificity is not limited to PAMPs, they improve cellular detection of and sensitivity to endotoxin.14 Conversely, repeated, prolonged, or high-level endotoxin exposure induces cellular unresponsiveness or tolerance.15 Ligand-induced oligomerization of TLR4 induces the recruitment of four intracellular signal-transducing adaptor proteins through their toll/IL-1 receptor (TIR) domains: MYD88 and TIR domain-containing adaptor protein (TIRAP) of the MYD88-dependent pathway and TIR domain–containing adaptor protein–inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM) of the MYD88-independent or TRIF-dependent pathway.16 Different TLRs use different combinations of adaptor proteins for downstream signaling; TLR4 is the only known TLR that uses all four of these adaptor proteins. The MyD88dependent pathway induces the expression of costimulatory molecules (e.g., CD80) and inflammatory cytokines (e.g., TNF-α, IL-1, IL-6, IL-8) through a series of signal-transducing intermediates that lead to the nuclear translocation of transcription factors NF-κB and activator protein 1 (AP-1). The TRIF-dependent pathway mediates the induction of type 1 interferons and interferon-inducible genes through activation of the transcription factor interferon regulatory factor 3 (IRF3). Ten human TLRs have been identified (Table 1.2). They collectively recognize a diverse range of microbial cell wall components, proteins, and nucleic acids, the classic PAMPs.17 Unlike gram-negative bacteria, the cell walls or membranes of other bacteria (e.g., gram-positive bacteria, mycoplasma) do not contain endotoxin, but they contain peptidoglycan and lipoproteins that are recognized by TLR2, TLR1, TLR6, and possibly TLR10. TLR5 recognizes bacterial flagellin. The cytosinephosphate-guanine (CpG) sequences of bacterial and viral DNA are unmethylated, distinguishing microbial DNA from mammalian DNA; microbial unmethylated CpG is recognized by TLR9. TLR7 and TLR8 are closely related to TLR9 and recognize virus-derived, single-stranded RNA. Double-stranded RNA, unique to certain viruses, is recognized by TLR3. TLR2 and TLR4 also bind members of the family of alarmins, proteins that are passively released from necrotic cells during infection or tissue injury, thereby eliciting inflammation.18

TABLE 1.2  Toll-Like Receptors in Humans Toll-like Receptors

Cell Location

Ligands

Microbial Sources

TLR1

Surface

Lipoproteins, lipoteichoic acid

Gram-positive bacteria, mycoplasma

TLR2

Surface

Lipoproteins, alarmins Peptidoglycan, lipoteichoic acid Zymosan Lipoarabinomannan

Bacterial cell walls and membranes Gram-positive bacteria cell walls Fungi and mycobacteria cell walls

TLR3

Cytosol

Double-stranded RNA

Viral RNA

TLR4

Surface

Endotoxin, alarmins, viral coat proteins

Gram-negative bacteria cell walls Respiratory syncytial virus

TLR5

Surface

Flagellin

Bacteria

TLR6

Surface

Lipoproteins, lipoteichoic acid

Gram-positive bacteria cell walls and membranes

TLR7

Cytosol

Single-stranded RNA

Viral RNA

TLR8

Cytosol

Single-stranded RNA

Viral RNA

TLR9

Cytosol

Unmethylated CpG DNA

Bacterial and viral DNA

TLR10

Surface

Lipoproteins

Bacterial cell walls and membranes

CpG, Cytosine-phosphate-guanine oligonucleotide.

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C-Type Lectin Receptors Transmembrane C-type lectin receptors (CLRs) are defined by their lectin structure, although they bind additional moieties beyond glycans. They are expressed widely on innate immune cells and epithelial cells. They function as sensors for diverse microbes, but several play a particularly important role in fungal recognition and immunity, including the mannose receptor, dendritic cell–specific ICAM-3–grabbing nonintegrin (DC-SIGN), Mincle, Dectin-1, and Dectin-2.19 CLR activation elicits several antimicrobial functions. Some receptors such as the mannose receptor (CD206), DEC-205, and DC-SIGN function as endocytic receptors, facilitating DC and macrophage antigen uptake, recycling, and presentation. Many elicit the activation of NF-κB with attendant proinflammatory cytokine production. Importantly, several receptors including Dectin-1, Dectin-2, and Mincle elicit immunoreceptor tyrosinebased activating motif (ITAM)–based signaling to activate spleen tyrosine kinase, elicit a calcium flux, and trigger NFAT activation. This signaling pathway is not shared by TLRs and is critical for generation of IL-6, IL-10, and IL-23; shaping subsequent adaptive immunity with skewing of naïve T cells to Th17; and generation of proinflammatory lipid mediators, the cysteinyl leukotrienes.20,21 CLR activation also plays a role in activating the inflammasome for the production of mature IL-1β.19 The structure of the inflammasome is reviewed below (see NucleotideBinding Oligomerization Domain–Like Receptors).

Sialic Acid–Binding Immunoglobulin-Like Lectins Sialic acid–binding immunoglobulin-like lectins (Siglecs) are a family of receptors that bind sialic acid–containing glycans expressed on cell surfaces. Siglecs promote cell-cell interactions, regulate cell functions, and mediate microbial endocytosis.22 Different Siglecs are expressed by different immune cell types. For example, Siglec-1 (CD169 or sialoadhesin) is macrophage specific, Siglec-2 (CD22) is B lymphocyte specific, and the CD33-related Siglecs are specific for innate immune cells and resident macrophages, including microglia. Eosinophils, basophils, and mast cells express Siglec-8, whereas monocytes, dendritic cells, natural killer cells, and neutrophils express Siglec-9.22 Siglecs typically are inhibitory receptors. Some (e.g., sialoadhesin, CD33-related Siglecs) recognize sialic acid–expressing microbes, mediate their endocytosis, and dampen inflammatory and immune responses to these pathogens. Specific Siglecs have been implicated for their role in limiting tissue damage from activated granulocytes. Activation of Siglec-8 and Siglec-9 has differential effects on granulocytes leading to apoptosis of cytokine-primed eosinophils and neutrophils and inhibition of FcεR1-mediated activation of mast cells.23

Nucleotide-Binding Oligomerization Domain–Like Receptors Nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs) are cytosolic PRRs that are structurally similar and recognize microbial PAMPs that find their way into the cytoplasm. The human NLR family has 23 members that can be conceptually organized in two groups. The best characterized, NOD1 and NOD2, recognize different core motifs of bacterial peptidoglycans. NOD1 is specific for a core motif of peptidoglycans from primarily gram-negative bacteria. NOD2 detects the peptidoglycan muramyl dipeptide, present in all gram-positive and gram-negative bacteria.24 NOD2 has been of particular interest, because mutations in the human NOD2 gene are associated with an increased risk of Crohn disease.25 A different set of NLRs (including NLRP1, NLRP3, and NLRC4) are the sensing portion for the cytosolic protein complex termed the inflammasome. These NLRs recognize a diverse range of microbial PAMPs that find their way into cellular cytoplasm, including anthrax lethal

toxin (NLRP1), bacterial flagellin (NLRC4), bacterial and viral RNA (NLRP3), and bacterial pore-forming toxins such as nigericin and maitotoxin (NLRP3). NLRP3 is likely the most important among them, because it is activated in response to a broad array of diverse stimuli, perhaps through a common mediator of cellular stress.26 On activation, NLRP3 associates with several other proteins to form the inflammasome and activate the caspase-1-dependent cleavage of proIL-1β and proIL-18 to their mature active forms. Notably, caspase-1 also cleaves gasdermin D, leading to cell death after activation of the inflammasome.26

Additional Cytosolic Nucleic Acid Receptors The RNA helicases retinoic acid–inducible protein 1 (RIG-I), melanoma differentiation–associated 5 (MDA5, now called interferon induced with helicase domain 1 [IFIH1]), and RIG-I-like receptor LGP227 are another class of cytosolic PRRs that recognize double-stranded RNA viruses and mediate type 1 interferon antiviral responses.28 Cytosolic doublestranded DNA also triggers the generation of type 1 interferons, and many DNA sensors have been proposed.29 GMP-AMP synthase (cGAS) is a double-stranded DNA sensor that is activated to generate a cyclic dinucleotide second messenger and, through a variety of signaling intermediates, cGAS activates the TBK1-IRF3-dependent production of interferons.30 The roles of other sensors and additional downstream signaling pathways are still emerging.

RESIDENT CELLULAR RESPONSES OF INNATE IMMUNITY Tissue-resident innate immune cells serve as critical first responders in host defense. This includes epithelial cells, DCs, macrophages, mast cells, and innate lymphoid cells (ILCs) (Fig. 1.4). Epithelial cells provide a first line of host defense by maintaining a barrier function, trapping and killing potential pathogens, and activating additional innate immune cells. Their physical barrier to the external environment is achieved through a network of junctional complexes including tight junctions and underlying junction adherens.31 An additional layer of defense is provided by the mucociliary apparatus. Cellassociated and secreted mucins trap pathogens in the conducting airways and act in concert with antimicrobial peptides and the ciliary apparatus to clear pathogens. In addition to its barrier role, the homeostatic mucin MUC5B regulates alveolar macrophage function, indicating an unexpected role for mucins in regulating innate immune responses.32 Epithelial cells secrete many of the AMPs reviewed previously and orchestrate innate immunity through inflammatory cytokine generation elicited by PRR signaling. Perhaps the best characterized are TLR4 and TLR2, which are expressed in epithelial cells from the lung, skin, and gut and which mediate NF-κB-dependent production of proinflammatory cytokines in response to both pathogens and commensal organisms.33,34 DCs are key sentinels of the innate immune system that link innate and adaptive immunity through their unique capacity to potently activate naïve T cells. DCs can be subdivided into classic myeloid (mDC) and plasmacytoid (pDC) types, which are thought to originate from a common DC precursor in the bone marrow.35 The mDCs are recruited from the blood to histologic sites with high levels of antigen exposure (e.g., skin, mucosal surfaces, lymph nodes, spleen). With their long dendrites and their PRR-rich cell surfaces, mDCs form a subepithelial web that is sensitive to microbes, inflammation, and cellular stress. In the airways and intestine, antigens are immediately captured by mucosal mDCs that extend dendrites into the lumen for antigen sampling. After activation, mDCs quickly alert and instruct the immune system by secreting proinflammatory cytokines such as interferons and interleukin-12 and migrating to draining lymph nodes for T lymphocyte instruction.

CHAPTER 1  Innate Immunity

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Innate immunity Infiltrative • Neutrophils • Monocytes • Dendritic cells • NK cells • Eosinophils Chemoattraction • Basophils IFNs Cytokines • NKT , MAIT ILs Immediate (resident) Acquired • AMPs Microbes (adaptive) • Phagocytes: Antigen presentation • T lymphocytes – Macrophages instruction • B lymphocytes – Dendritic cells – antibody • Epithelial cells • ILC2s • Mast cells Homeostatic • Macrophages • Monocytes • Dendritic cells • Epithelial cells • Regulatory lymphocytes

Fig. 1.4  Innate immune responses to microbes can be broadly characterized as antimicrobial or homeostatic. Antimicrobial responses begin with protective layers of antimicrobial peptides and detection by immune cells residing at the epithelial interface. Often, these immediate responses sufficiently protect the host. If this first layer of host defense is inadequate, the frontline responders attract infiltrative innate immune cells that are activated as they approach the source of inflammation. Immediate and infiltrating immune cells stimulate adaptive immune responses and educate lymphocytes through antigen presentation and costimulation. Homeostatic responses by innate immune cells downregulate inflammatory and antimicrobial immune responses when they are no longer needed to optimize the use of resources and well-being of the host. AMP, Antimicrobial peptide; IFN, interferon; IL, interleukin; NK, natural killer.

The functions of mDCs are developmentally related.36 They migrate from the bone marrow to peripheral tissues in an immature form, at which stage their role is primarily sentinel detection. They readily sense, sample, and process incoming antigen through dense PRR expression (i.e., TLR1 through TLR6), but they have a poor ability to stimulate T lymphocytes. After sensing environmental microbial PAMPs or inflammatory stress, mDCs become activated scavengers of antigen, and they subsequently return to draining lymph nodes. They mature during this migration. As mature mDCs, their antigen uptake and processing functions are shut down, and large amounts of processed antigen are displayed in cell surface major histocompatibility complex (MHC) molecules with a battery of costimulatory factors for T lymphocyte education. The central role of DCs in directing T lymphocyte development in health (see Innate Instruction of Adaptive Immune Responses) and in allergic and asthmatic disease (see Innate Immunity and Allergy) is addressed later in this chapter. The mDCs also can be superior stimulators of natural killer (NK) and natural killer T (NKT) cells by virtue of their robust IL-12 production.36 Compared with mDCs, pDCs are sentinel antiviral responders, expressing TLR7 and TLR9 for recognizing viral infections37 and releasing large amounts of interferon α (IFN-α) to limit viral replication.38 They can also act as antigen-presenting cells and control T lymphocyte responses.39 Langerhans cells, although similar to DCs in their function, seem to originate from an embryonic precursor that populates the epidermis before birth, differentiates and self-renews in situ, and proliferates during inflammation.40

Macrophages share many features with DCs, including their enrichment in tissue areas with high environmental antigen exposure, cell surface expression of PRRs, capacity to phagocytose and digest organisms, and presentation of antigens to lymphocytes. However, macrophages have a limited capacity to migrate to regional lymph nodes and cannot stimulate naïve T cell proliferation as strongly as DCs.41,42 By contrast, macrophage phagolysosomes reach a lower pH than that of DCs, endowing them with increased killing capacity.43 Recent work in mouse and human cells has underscored the considerable heterogeneity of tissueresident macrophages, likely reflecting their development from local tissue progenitors seeded in embryonic life from the fetal yolk sac.44 In addition to their phagocytic and killing capacity, macrophages secrete more than 100 proteins that mediate host defense and inflammation, and macrophages play important roles in removal of dead tissue and apoptotic cells, metabolism, tissue development, wound healing, and homeostasis (reviewed in reference 45) (see Homeostasis in the Innate Immune System). Mast cells are evolutionarily ancient immune cells and the only granulocyte that resides primarily in peripheral tissues. With granules containing preformed mediators including proteases (tryptase, chymase), heparin, histamine, platelet activating factor, AMPs (cathelicidin), defensins, and some cytokines (TNF-α), they are poised to rapidly neutralize microbial invaders. While the best-characterized pathway for mast cell activation is the IgE-dependent crosslinking of FcεRI, highlighting its effector function in amplifying adaptive immunity, mast cells are also activated through a wide variety of classical PRRs, including TLR1,

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TLR2, TLR4, and TLR6; and complement receptors for C3a and C5a.46 Furthermore, mast cells can sense small molecules like substance 48/80 through the Mas-related G protein–coupled receptor MRGPRX247 and can sense some allergens in an FcεR1-independent fashion.48 On activation, mast cells can rapidly degranulate, generate lipid mediators such as cysteinyl leukotrienes and prostaglandin D2, and synthesize numerous proinflammatory cytokines. They are key sources of immediate release of TNF-α and IL-8, which are uniquely preformed in mast cells, and their immediate release of TNF-α may have a central role in effective antimicrobial responses to infections.46 Additionally, mast cells play a role in intestinal parasite clearance, and their proteases play a role in reducing the systemic toxicity of some venoms.49,50 Innate lymphoid cells (ILCs) are a recently described family of lymphoid cells derived from the common lymphoid progenitor. They lack the canonical T cell receptor and are instead activated by tissue-derived mediators in an antigen-independent manner.51 Largely following the nomenclature for T cells, ILCs have been subdivided into three subsets: ILC1, 2, and 3. Group 1 ILCs include NK cells and ILC1s; the transcription factors that regulate their terminal development are Eomes and T-bet, respectively. They are activated by IL-12, IL-15, and IL-18 secreted in response to intracellular pathogens and generate high amounts of interferon γ (IFN-γ). ILC2s, on the other hand, are regulated by Gata-3, ROR-α, Gfi1, and T cell factor 1 and respond to epithelial cytokines and products of the arachidonic acid pathway generated in the setting of cellular injury from helminths or allergens.52,53 Activation of ILC2s leads to the production of high amounts of IL-4, IL-5, and IL-13. They also play homeostatic roles in barrier repair responses through the production of amphiregulin and in regulating thermogenesis in adipose tissue.54 Lastly, ILC3s respond mainly to IL-1β and IL-23 produced by myeloid cells in response to bacterial and fungal infection and are transcriptionally controlled by RORγt.53 Two additional subsets of lymphoid cells sit at the interface of innate and adaptive immunity. Invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells express invariant T cell receptors with highly restricted diversity of the T cell receptor alpha chain.55,56 They are unique because of their ability to recognize nonpeptide antigens: iNKT cells respond to glycolipids, and mucosalassociated invariant T cells (MAIT cells) recognize microbial metabolites. These antigens are presented to them by nonclassical antigen-presenting molecules: CD1d for iNKT cells and MR1 for MAIT cells.55 In addition to activation through the T cell receptor, MAIT and iNKT cells can respond rapidly to cytokine stimulation, similarly to ILCs.56 Activated MAIT cells produce IFN-γ, whereas iNKT cells produce high levels of many cytokines including IFN-γ, TNF-α, IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-13, IL-17, IL-21, and GM-CSF. Although much less is known about MAIT cells, studies in mice suggest that both MAIT and iNKT cells can be powerful modulators of the immune system, supplying critical cytokines before the generation of adaptive immunity.

INFILTRATIVE CELLULAR RESPONSES OF INNATE IMMUNITY Infiltrative cellular responses are potent antimicrobial effectors that usually are recruited by an innate immune intermediary to induce the full weight of their response, but they can respond directly to microbial stimuli through their own surface-expressed PRRs (Fig. 1.4). Neutrophils, the most abundant circulating phagocytes in the human host, are recruited to sites of infection and inflammation where they are activated to degranulate, phagocytose, and release neutrophil extracellular traps (NETs), to kill microorganisms. Circulating neutrophils are short-lived (approximately 24 hours), and about 1011 cells die each day.57 This constant stream of neutrophil death would be potently

inflammatory without the extraordinarily efficient uptake and processing of apoptotic neutrophils by macrophages and DCs to prevent release of toxic constituents, a process called efferocytosis.58 In response to tissue infection, circulating neutrophils adhere to adjacent vascular endothelium, extravasate across it, and migrate to the site. Neutrophils have a number of receptors for diverse chemoattractants, including bacterially derived N-formyl oligopeptides and hostderived C5a, IL-8, and leukotriene B4, secreted by activated innate immune cells. These neutrophil chemoattractants diffuse from the site of infection to provide a chemotactic gradient for neutrophil migration and further neutrophil activation.59,60 Neutrophils have several modes of killing. On reaching the infected site, they can phagocytose invading microorganisms that are opsonized by complement C3 fragments (e.g., C3b, iC3b) and immunoglobulin G (IgG).61 After phagocytosis, ingested microbes are killed almost immediately through several mechanisms. Microbicidal products such as α-defensins (HD 1-4) are released into the phagosome from intracellular granules. Additionally, highly reactive oxidizing agents (e.g., O2−, H2O2, hypochlorous acid) are generated by myeloperoxidase and membrane NADPH oxidase,62 which has an essential role in killing and preventing infection with certain common organisms (e.g., Staphylococcus aureus, Serratia, enteric bacteria, Aspergillus).63 An increased role for neutrophil granule proteases (i.e., neutrophil elastase and cathepsin G) has been recognized. These cationic proteases are released and activated with alkalinization and K+ ion fluxes into phagocytic vacuoles. These pH and potassium requirements for protease solubilization and activity restrict their toxicity to phagocytic vacuoles and limit damage to host tissues.62 Finally, neutrophils can form extracellular traps (NETs) in response to gram-positive and gram-negative bacteria and upon stimulation with LPS or interleukin-8. NETs consist of extruded DNA of either nuclear or mitochondrial origin, histones, neutrophil granule proteins, and antimicrobial peptides.64,65 They have direct bactericidal activity and trap bacteria in the extracellular space, preventing their spread.66 NK cells are an innate immune cell type with unique features. Similar to ILCs, they are lymphoid cells that are not activated through antigenspecific receptors such as the T cell receptor or surface immunoglobulin. Although NK cells express PRRs such as TLR2, TLR3, TLR4, TLR5, TLR7, and TLR8 and recognize and respond to the respective TLR ligands directly,67,68 they are best known for responding in an antigenindependent manner to help contain viral infections (especially herpes­ virus infections) and malignant tumors by recognizing aberrant host cells for elimination. NK cells distinguish healthy host cells through inhibitory receptors such as the killer cell immunoglobulin-like receptor (KIR) and CD94/NKG2A receptors that recognize MHC class I molecules expressed on healthy cells (Fig. 1.5).69,70 Binding of these receptors inhibits NK cell–mediated lysis and cytokine secretion. Virus-infected and malignant cells often downregulate MHC class I molecules, rendering them susceptible to attack by NK cells.71 These inhibitory receptors on NK cells are counterbalanced by activating receptors, such as the NKG2D receptor that recognizes stress ligands expressed on cell surfaces in response to intracellular DNA damage.72 Recruited and activated NK cells mediate antimicrobial activities by induction of apoptosis in target cells and cytokine secretion, which promotes innate immune functions and contributes to adaptive immune responses. Target cell apoptosis results from granule exocytosis and death-receptor engagement. NK cell granules contain perforins and granzymes that are released on activation into the synapse between target and effector cell, disrupting target cell membranes and inducing apoptosis (Fig. 1.5).73 NK cells also mediate apoptosis by expressing FAS ligand (FASLG) and TNF-related apoptosis-inducing ligand (TRAIL, now called tumor necrosis factor superfamily member 10 [TNFSF10]), which bind the FAS and TNFRSF10 receptors, respectively, on target cells.74

CHAPTER 1  Innate Immunity Normal state

NK

NKG2A

MHC I

KIR

MHC I

Healthy cell

Activation

Infected or cancerous cell

NKG2D

Killing Granzymes Perforin

Apoptosis FASLG TRAIL

FAS TRAILR

IFN-γ

Th1

IFN-γ Cytotoxic T lymphocyte

Fig. 1.5  Natural killer (NK) cells recognize and target infected or malignant cells in an antigen-independent manner. They distinguish healthy host cells by receptors on NK cells (e.g., KIR, NKG2A/CD94) that interact with MHC class I molecules on host cells to inhibit NK cell activation. Pathogen-infected or malignant cells typically downregulate MHC class I molecule expression while concurrently expressing stress ligands that are recognized by activating receptors on NK cells (e.g., NKG2D). These changes in the balance of activationto-inhibition receptor engagement lead to NK cell activation and targeting for killing. NK cells induce target cell apoptosis by the release of toxic granules containing granzymes and perforins that disrupt cell membranes. The cells also express apoptosis-inducing FAS ligand (FASLG) and TRAIL that interact with their counterparts on target cells (FAS and TRAILR, respectively). Activated NK cells produce stimulatory cytokines and chemokines and are a rich source of IFN γ that augment innate and adaptive cytotoxic T lymphocyte and type 1 helper T lymphocyte (Th1) immune responses. IFN, Interferon; KIR, killer cell immunoglobulin-like receptor; MHC, major histocompatibility complex; NKG2A, natural killer cell receptor (also called 159A); NKG2D, natural killer receptor (now called killer cell lectin-like receptor subfamily member 1 [KLRK1]); TRAIL, tumor necrosis factor–related apoptosis-inducing ligand (now called TNFSF10); TRAILR, TRAIL receptor. (Adapted from Orange JS, Ballas ZK. Natural killer cells in human health and disease. Clin Immunol 2006;118:1-10.)

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Activated NK cells are known for their secretion of IFN-γ in particular, but they also secrete TNF-α, growth factors, IL-5, IL-10, IL-13, and chemokines.75 DCs recruit, interact with, and activate NK cells through cytokines (e.g., type I interferons, IL-12, IL-18) and cell-to-cell surface interactions.75 NK cells can activate bystander immature DCs by producing TNF-α and IFN-γ along with cell-cell contact.76 Reciprocal NK-DC interactions occur in secondary lymphoid organs, where NK cells respond to IL-12 produced by mature DCs by producing IFN-γ and promoting the development of helper T cell type 1 (Th1) and cytotoxic T lymphocytes.77–79 Eosinophils are terminally differentiated circulating granulocytes derived from the common granulocyte-monocyte precursor (GMP). The eosinophil precursor was recently identified as Gata1+GMP distinct from the Gata1+GMP that gives rise to neutrophils, monocytes, and macrophages.80 Human eosinophil granules are rich in four cationic proteins: major basic protein 1 (MBP1; also known as MBP and PRG2), eosinophil cationic protein (ECP; also known as RNase3), eosinophilderived neurotoxin (EDN; also known as RNase2) and eosinophil peroxidase (EPX; also known as EPO).81 In addition, mature eosinophil granules contain preformed stores of cytokines and chemokines including IFN-γ, IL-4, IL-6, TNF, IL-10, IL-12, and IL-13.82 Notably, rather than receptor-mediated activation, the most common pathways for eosinophil granule extrusion are cytolysis and/or piecemeal degranulation. Eosinophils and their extruded granules increase in number in patients with atopic disease or helminth infection, likely because of increased levels of IL-5 and GM-CSF/IL-3 that expand and activate the population, respectively.83 This observation, and data demonstrating that eosinophils can kill helminths in vitro, led to speculation that eosinophils are important in antihelminth immunity. Although murine models have not born this out, they have demonstrated roles for them in regulating mucosal barrier integrity and the intestinal microbiome through regulation of secretory IgA production. Additional studies have demonstrated largely homeostatic roles for IL-4-secreting eosinophils in beige fat biogenesis and thermoregulation83 (see Homeostasis in the Innate Immune System). Basophils are circulating granulocytes derived from a common GMP shared with mast cells. Unlike mast cells, they lose surface expression of the kit receptor as they differentiate in the bone marrow under the direction of IL-3 and are released in the circulation as mature granulocytes.84 Basophil granules contain histamine, serine proteases including cathepsin G, granzyme B, and a basophil-specific mediator, basogranulin. Upon activation, basophils generate cysteinyl leukotrienes and high levels of IL-4 and IL-13. In murine models, basophils are an important source of serine protease-elicited IL-4,85 leading to a polarization of the immune response to type 2 immunity. Because serine proteases are shared by multiple allergens and some helminths, basophils may play a role in initiating and amplifying type 2 immunity, but a definitive role in humans has not been established.85

INNATE INSTRUCTION OF ADAPTIVE IMMUNE RESPONSES The immediate and infiltrative responses of innate immunity set the stage for their instruction of adaptive immunity and the maintenance of immunologic memory—long-lived memory T lymphocytes and a persistent antibody response. Because the adaptive immune system has a near limitless antigen receptor repertoire, instruction is necessary to guide adaptive antimicrobial immune responses toward pathogens and not self-antigens or harmless environmental antigens. Microbial pattern recognition by innate immune cells controls the activation of adaptive immune responses by directing microbial antigens linked to TLRs through

the cellular processes leading to antigen presentation and the expression of costimulatory molecules (e.g., CD80 with CD86). A legacy of research on the prototypical PAMP endotoxin is helpful in understanding PAMP control of adaptive immunity. Endotoxin can be used as an essential adjuvant in the induction of antigen-specific T cell memory. Although T cells mount a short-lived proliferative response to protein antigens alone, classic immunologic memory depends on immunization with an adjuvant such as endotoxin. Endotoxin potently induces IL-12 and IFN-γ secretion, which are key regulators of memory Th1-type immune development.86 LPS strongly influences innate antigenpresenting cells (especially DCs) to produce IL-12 and to costimulate naïve T lymphocytes to become effector T lymphocytes that primarily secrete IFN-γ.87–89 In a reciprocal manner, IFN-γ primes innate immune cells to produce greater amounts of IL-12 in response to stimulation, fostering a positive feedback relationship between the innate and adaptive immune compartments for Th1-type immune development. Among antigen-presenting cells, DCs are the most efficient educators of T lymphocytes. Immature DCs are activated and recruited to epithelial surfaces, where they scavenge antigen and migrate to draining lymph nodes. During their migration, they mature and redirect their processes to MHC class II antigen presentation, and they express cytokines and cell surface molecules to attract antigen-specific T lymphocytes and direct their maturation and differentiation to helper T cell subsets (e.g., Th1, Th2, Th17) or regulatory T (Treg) cells (Fig. 1.6). The nature of DC instruction is affected by the dose and types of the PAMPs and PRRs involved, the duration of exposure, and the microenvironment in which the DCs are activated and located. For example, different cytokines influence TLR4-activated DCs to skew their subsequent instruction of T lymphocyte differentiation as follows: TGF-β and IL-10 induce DCs to instruct Treg development; IL-12 promotes Th1 immune development; thymic stromal lymphopoietin (TSLP) and IL-33 promote Th2 immune responses; and IL-23, TGF-β, IL-6, and IL-1β induce Th17 development.90,91 These activated and differentiated T lymphocytes subsequently migrate to other lymph nodes and back to the mucosa, where they again interact with and are sustained by mature mDCs in the subepithelial periphery. This peripheral tissue-specific interaction between mature mDCs and progeny effector T lymphocytes may underlie the persistence of organ-specific immune memory. Although immunologic memory is considered a function of longlived T and B lymphocytes, innate immune cells also show altered responsiveness, termed trained immunity, after exposure to certain pathogens or cytokines.92 Notably, immunologic training in this setting does not involve genetic rearrangement but rather stable alterations in gene expression mediated by epigenetic changes. Moreover, this process is not antigen specific, such that exposure to one pathogen may alter subsequent responsiveness to an unrelated one. Although some innate immune cells have a short half-life, environmentally driven gene changes would seem likely to influence long-lived hematopoietic stem cells or stromal or hematopoietic populations that self-renew in peripheral tissues.

HOMEOSTASIS IN THE INNATE IMMUNE SYSTEM The breadth and depth of innate immune activities that defend the host in its microbe-laden environment are silent from a clinical perspective. In health, inflammation is the exception rather than the norm. This immune tranquility of the well-defended host is testament to the seamless efficiency of frontline defenses combined with active homeostatic processes that closely regulate inflammatory responses within the innate immune system. Macrophages have an essential role in maintaining immune homeostasis (Fig. 1.7). Airway macrophages exemplify this antiinflammatory

11

CHAPTER 1  Innate Immunity Dendritic cell uptake of Ag in mucosa Local Ag presentation by DC to Teffector

Effector site DC migration maturation Teffector

PAMP-Ag Toll-like receptor Pathogen

Cytokines Quiescent T cell (interleukin-1, 6, 12) CD28 B7

Tmemory

Naïve T Draining mode

Activation

Other nodes/spleen

T cell receptor Peptide Mature dendritic cell

MHC class II molecule

Clonal selection Teffector Proliferation T Differentiation (to Teffector and Tmemory) memory

Ovalbumin

T cell

Dendritic cell

Eosinophil

Epithelial cell

Goblet cell

Fig. 1.6  Innate immune instruction of adaptive immunity is exemplified by dendritic cells (DCs) in the lung. Microbial stimuli activate immature DCs in the periphery to take up and process antigen, migrate to draining lymph nodes, and mature as they migrate. On reaching lymph nodes, DCs secrete chemokines that attract T lymphocytes. The activities of mature DCs—presenting processed antigen, secreting cytokines, and expressing costimulatory molecules—induce antigen-specific T lymphocyte activation, proliferation, and differentiation. The cytokine microenvironment of peripheral tissues from which DCs migrate biases them to direct naïve T cells in different directions (e.g., Th1, Th2, Th17, Treg). Memory T lymphocytes migrate to other lymph nodes, and effector T lymphocytes migrate to peripheral tissues. Mature DCs also migrate to peripheral tissues, where their interactions with effector T lymphocytes are thought to underlie tissue-specific immune memory. Ag, Antigen; DC, dendritic cell; MHC, major histocompatibility complex; PAMP, pathogen-associated molecular pattern; Th, helper T cell (types 1, 2, and 17); Treg, regulatory T cell. (Adapted from Lambrecht BN. Dendritic cells and the regulation of the allergic immune response. Allergy 2005;60:271-82; inset from Medzhitov R, Janeway C Jr. Innate immunity. N Engl J Med 2000;343:338-44. Copyright 2000 by Massachusetts Medical Society.)

role, actively suppressing DC maturation and antigen presentation in mouse airways, which is revealed when they are depleted.93,94 PAMPPRR activation temporarily diverts alveolar macrophages from their antiinflammatory mode and primes them for antimicrobial functions.95,96 Classic activation of macrophages induces both proinflammatory (e.g. TNF-α) and antiinflammatory mediators (e.g., IL-10, TGF-β, and prostaglandin E2 [PGE2]) that downregulate macrophage and DC functions (Fig. 1.7). Thus microbe-induced activation of the innate immune system is tightly linked to concurrent induction of downregulatory mechanisms to regain immune homeostasis. An alternative pathway of macrophage activation is mediated by IL-4 and IL-13. This modified response modestly downregulates macrophage and DC function and induces antiinflammatory IL-10, IL-1 receptor antagonist (IL-1RA), and the decoy, nonsignaling, type II IL-1 receptor (IL1R2) while promoting MHC class II antigen presentation and antibody production.97 Macrophages also control inflammation through their constitutive ability to rapidly ingest and clear apoptotic cells (i.e., efferocytosis) (Fig. 1.7).58,98 The efficiency of this process is illustrated by the observation that more than 1011 circulating neutrophils are eliminated each day without a trace of inflammation.58 Macrophages recognize apoptotic cells by molecular pattern recognition reminiscent of microbial recognition by innate immune cells. The plasma membrane of viable cells

actively maintains an asymmetric phospholipid distribution such that phosphatidylserine is kept on the inner side of the membrane bilayer. Apoptosis perturbs this asymmetry and exposes phosphatidylserine on the cell’s outer surface, leading to their recognition by macrophages bearing phosphatidylserine receptors.99,100 On recognition of apoptotic cells, macrophages release antiinflammatory IL-10, PGE2, and TGF-β to complete the task of maintaining immune homeostasis. The complement component C1q and the collectins MBL and surfactant proteins A and D bind to apoptotic cells and mediate their clearance.58 Components of the innate immune system accomplish this through PRRs that recognize both microbes and distinct apoptotic cell–associated molecular patterns (ACAMPs).101 Although macrophages have received most of the attention in mediating efferocytosis in the immune system, epithelial cells, endothelial cells, fibroblasts, and stromal cells can also contribute. During resolution of acute inflammation, neutrophils switch their production of proinflammatory lipid mediators (e.g., leukotrienes, prostaglandins) to other molecular families of fatty acid–derived mediators (e.g., lipoxins, resolvins, protectins) that are potently antiinflammatory and injury resolving.102 They restore tissue homeostasis by stimulating efferocytosis of apoptotic or necrotic neutrophils, blocking neutrophil infiltration, and reducing vascular permeability.

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SECTION A  Basic Sciences Underlying Allergy and Immunology Macrophage Neutrophil PS-R PS

IL-1RA Antiinflammatory IL-10 TGF-β PGE2

Apoptotic neutrophil Efferocytosis Antiinflammatory Lipoxins Resolvins Protectins

Dendritic cell Fig. 1.7  Homeostasis in innate immunity. Macrophages have specialized regulatory functions that prevent inflammatory responses. Activation of macrophages induces antiinflammatory mediators such as interleukin-1 receptor antagonist (IL-1RA), interleukin-10 (IL-10), prostaglandin E2 (PGE2), and transforming growth factor β (TGF-β), which are thought to downregulate dendritic cell maturation and function. Macrophages also control inflammation by rapidly ingesting apoptotic cells to prevent their inflammatory rupture in the microenvironment, a process known as efferocytosis. The cell membranes of apoptotic cells have externalized phosphatidylserine (PS) that is actively maintained by healthy cells on the inner side of cell membranes. Macrophages have PS receptors (PS-R) that recognize apoptotic cells, triggering their ingestion and the release of antiinflammatory cytokines. During the resolution of inflammation, neutrophils switch their production of proinflammatory lipid mediators to antiinflammatory lipoxins, resolvins, and protectins. They help to restore tissue homeostasis by stimulating efferocytosis and blocking neutrophil infiltration.

Epithelial cells also maintain homeostasis through several lines of defense. Poor expression of MD-2, a component of TLR4, on airway epithelial cells is likely an important adaptation to prevent excessive NF-κB-dependent airway inflammation. Furthermore, a recent study examining the protective effect of farm dust on type 2 inflammation (reviewed further in Environmental Determinants of Atopy: the Hygiene Hypothesis and the Microbiome) found a critical role for epithelial cell expression of A20, an inhibitor of NF-κB in preventing lung disease elicited by environmental antigen.103 Epithelial cell–derived cytokines also have a demonstrated role in restoration of tissue homeostasis. For example, IL-33–mediated activation of ILC2s leads to production of amphiregulin, an EGFR agonist that promotes restoration of epithelial integrity.104,105 Finally, an emerging theme from preclinical studies is the innate immune control of metabolic functions. For example, a well-studied murine model of homeostasis involving type 2 immunity is in visceral adipose tissue. In response to cold exposure, IL-33–mediated activation of ILC2s, eosinophils, and alternatively activated macrophages elicits transformation of white adipose tissue into metabolically active beige adipose tissue, essential for cold adaptation and thermogenesis.54,106 In mouse models of obesity-induced insulin resistance, eosinophils, alternatively activated macrophages, ILC2s, and Foxp3+ T regulatory cells are protective in maintaining insulin sensitivity.107–110 Thus the function of innate type 2 immunity in maintaining homeostasis may be considerably more complex than we have understood.

INNATE IMMUNITY AND ALLERGY The innate immune system of the airway, gastrointestinal tract, and skin is continuously exposed to potential allergens. Like microbial antigens, allergens can engage innate PRRs and be processed by innate immune cells. In the case of allergic disease, this recognition leads to generation of antigen-specific IgE; overexpression of type 2 cytokines (IL-4, IL-5, IL-13); and tissue injury, dysfunction, and aberrant remodeling. Although the circumstances leading to allergic immunity in humans are not clear, evidence suggests that allergic susceptibilities can originate in the innate immune system,111–113 with key roles for barrier epithelial cell programs that condition DCs for adaptive memory Th2 responses (Fig. 1.8). Indeed, GWAS studies in asthma114 and expression analyses in atopic patients115–118 point to key roles for epithelial-derived innate type 2 cytokines and DC activation programs that underlie the atopic march. Notably, recent research has demonstrated that barrier epithelial cells may also activate innate cells such as ILC2s and mast cells to make substantial quantities of type 2 cytokines, indicating the possibility that type 2 inflammatory diseases could be generated or perpetuated in the absence of classical memory Th2 cells. The importance of these pathways in humans has yet to be clarified.

Allergen Recognition By the Innate Immune System Because of the structural diversity of allergens that elicit IgE in humans, it is likely that type 2 immunity arises from several complex recognition programs, the subjects of ongoing study. Although some allergens contain structural motifs that are recognized by PRRs, others may have shared function such as the protease-mediated breakdown of tissue barriers and elicitation of aberrant tissue repair mechanisms. The allergenicity of major allergens results in part from their recognition by PRRs. Allergen-elicited TLR4 signaling on airway epithelial cells plays a key role. In the case of house dust mite, activation of TLR4 elicits the generation of epithelial-derived innate type 2 cytokines such as GM-CSF and IL-33, promoting downstream allergic lung inflammation in mouse models.119 The major dust mite allergen, Der p 2, is a structural and functional homolog of MD-2, the TLR4 coreceptor responsible for binding LPS, and thereby augments TLR4 signaling.120 This facilitation is critical, because TLR4 signaling in human airway epithelial cells is limited by normally low MD-2 expression.121 Whether other major allergens can substitute for MD-2 is unknown, but like Der p 2, many are lipid-binding proteins (e.g., dust mite Der p 7, cat Fel d 1, lipocalins mouse Mus m 1, and horse Equ c 1). Finally, other allergens appear to facilitate epithelial cell TLR4 activation by binding to MD-2, including pollen allergens from diverse families of trees (Cottonwood, Walnut), grasses (Bermuda, Timothy, Rye), and weeds (Ragweed, Pigweed, Thistle).122 This again can drive TLR4-dependent allergic airway inflammation, highlighting the importance of TLR4 activation of airway epithelial cells in the allergic process. Some CLRs recognize carbohydrate PAMPs common to fungi, pollens, and helminths but not mammals and mediate Th2 responses. DC-SIGN binds fucosylated glycan moieties on some major allergens (e.g., peanut Ara h 1, mite Der p 2, Bermuda grass BG60).123,124 The mannose receptor binds carbohydrate moieties on diverse major allergens (e.g., mite Der p 1 and Der p 2, dog Can f 1, cockroach Bla g 2, peanut Ara h 1).125 Of particular interest in allergen-induced disease mechanisms, Dectin-2 on DCs binds glycans in house dust mite and Aspergillus extracts, inducing cysteinyl leukotriene production and allergen-specific Th2 inflammatory responses in the lung.20,126 The β glucan receptor Dectin-1, expressed on DCs, can also facilitate murine allergic airway inflammation elicited by Aspergillus127 and by house-dust mite,128 but the ligand in dust mite extracts has not been characterized.

CHAPTER 1  Innate Immunity Adaptive Th2

13

Innate Type 2

Peripheral tissue Epithelial cells

Dendritic cell IL-25 TSLP IL-33 Mast cell

Dendritic cell conditioning

Lymph node DC Th0 Mast cell

ILC2

Basophil

Type 2 cytokine generation Th2

B cell

Th2 priming

Class switching to IgE

Fig. 1.8  Epithelial control of type 2 immunity. After encounter with allergens, barrier epithelial cells generate the canonical innate type 2 cytokines, IL-33, IL-25, and thymic stromal lymphopoietin (TSLP). On the left, these cytokines can activate dendritic cells (DCs), conditioning them to upregulate cell surface expression of OX40L. On arrival in the tissue-draining lymph node, these DCs promote the skewing of T helper type 2 (Th2) cells from naïve (Th0) cells and the generation of type 2 cytokines. Additionally, B cells (B) are activated to undergo antibody class switching and the generation of IgE+ B cells. On the right, these same innate cytokines can directly activate tissue-resident and recruited innate effector cells such as mast cells (MCs), basophils (Ba), and group 2 innate lymphoid cells (ILC2s) to make type 2 cytokines.

Many allergens, including mite, cockroach, fungi, and grass and weed pollen, have protease activity that is associated with their allergenicity,129 and some can cleave and activate protease-activated receptors (PARs) expressed by innate immune cells.130 House dust mite proteases Der p 1, Der p 3, and Der p 9 stimulate proinflammatory cytokine release by airway epithelial cells through PAR2.131 In the case of house dust mite,132 cockroach,133 and the mold Alternaria,134 PAR2 mediates allergic airway inflammation, likely through the protease-mediated release of IL-33 from airway epithelial cells.134

Allergen-Elicited Innate Inflammation The barrier epithelial cell program activated in response to allergens is still emerging but includes the generation of canonical “innate type 2 cytokines” IL-33, IL-25, and thymic stromal lymphopoietin (TSLP). These cytokines activate mDCs to upregulate membrane-bound OX40L and generate CCL17 and CCL22 to induce allergen-specific Th2 differentiation.135–137 They also elicit expansion of cytokine-producing ILC2s138–140 and prime and activate innate effectors, such as mast cells.141,142 Although these epithelial cell cytokines are detected in response to protease-containing allergens, viruses, and impaired epithelial cell integrity, the mechanisms by which they are generated, released, and regulated remains poorly understood. Recent work suggests that

DAMPs or “alarmins” released in the setting of cellular stress or nonprogrammed cell death such as uric acid, IL-1α, ATP, and High Mobility Group Box 1 (HMGB1) may play a key role in eliciting innate type 2 cytokines,143–147 but how this occurs in humans is not well substantiated, and what prevents this from happening in all people remains unknown.

Environmental Determinants of Atopy: The Hygiene Hypothesis and the Microbiome The hygiene hypothesis was originally proposed to explain the inverse relationship between the development of hay fever and family size or birth order148 and suggested that the epidemic of atopic diseases noted in developed countries might be related to a decreased incidence of childhood infections. As further epidemiologic studies noted a reduced incidence of atopic diseases in children with early life exposures to farms,149,150 dogs,151,152 endotoxin,153 and pests,154 among other exposures, the hygiene hypothesis has been revised to propose that exposure to a diverse microbiome in neonatal life, and not specific infections per se, may mediate a protective effect. Support for a rich or diverse microbiome mediating a protective effect has come from multiple studies. First, epidemiologic factors that protect against the development of atopy and atopic diseases profoundly alter the microbial composition

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SECTION A  Basic Sciences Underlying Allergy and Immunology

of house dust.155,156 More importantly, in European farming studies, increased microbial diversity in environmental samples of house dust was inversely related to atopy and asthma development,157 and in an American birth cohort of inner city children at high risk for asthma and allergy development, reduced bacterial diversity at 1 year of age was associated with atopy and recurrent wheeze by age 3 and asthma diagnosis by age 7.154,158 These studies indicate that an altered microbial community is associated with the expression of atopic disease in genetically diverse populations. An increasing literature also supports an association between atopic disease and alterations in human microbiota. Human microbial communities in adult skin, gastrointestinal tract, and airway demonstrate relative stability over time with greater variations between individuals than within an individual.159,160 However, within the first year of life, these same communities exhibit greater variability161 and appear to be more susceptible to outside insults, suggesting there is a unique time window in early life during which microbial communities are established and can influence the developing immune system. Prior to the development of the current sequencing technology, sensitization and atopic dermatitis were reported to be associated with altered infant intestinal flora in the first year of life.162,163 This was later confirmed in multiple sequencing-based studies that showed reduced diversity of the infant intestinal microbiome or loss of specific genera associated with sensitization and allergic rhinitis,164 eczema,165,166 and asthma.167 Although less is known about the relationship between microbial communities in the skin, lung, and nasopharynx and the development of atopy, dysbiosis accompanies clinical flares of atopic disease at each site. Some studies have hinted at specific microbes that confer risk for or protection from the development atopic disease. The gastrointestinal microbiota of infants who subsequently develop allergy can have more Clostridia species and S. aureus, but nonallergic infants can have more enterococci, bifidobacteria, lactobacilli, and bacteroides.162,163,168,169 Similarly, the risk of atopic disease is reduced in those with parasitic infestations, Ascaris and hookworm, and Trichuris trichiura (whipworm).170 In the airways, an intriguing birth cohort study observed that newborn infants with nasopharyngeal colonization with common bacterial pathogens (e.g., Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae) had a greater risk of developing recurrent wheezing in later life (i.e., 5 years of age).171 Ultimately, although this field is still in its infancy, the microbial differences uncovered by the use of advanced molecular techniques in atopic disease have compelled investigators to understand how microbiomes are regulated and how they interact with and contribute to healthful immune development and responses.

Environmental Determinants of Atopy: Endotoxin, PAMPs, and Additional Products Disease-associated changes in the microbial communities noted previously lend credence to the hypothesis that conserved structural components in some bacterial communities likely act as PAMPs and productively educate the developing immune system. Because mice with globally impaired TLR responses (i.e., MYD88-deficient mice) are skewed to IgE/Th2 immune responses, it is reasonable to suspect that TLR ligand exposure protects against allergies.172 However, research has revealed that the relationship between TLR ligand exposure and allergy is more complex than a unimodal protective effect. Endotoxin has been a prototypical TLR ligand for understanding the importance of dosage and timing of microbial exposures on allergy and asthma outcomes in laboratory and clinical settings. In short-term rodent models of allergic sensitization and asthma, experimental allergen without concurrent TLR stimulation (i.e., ovalbumin stripped of endotoxin) does not induce a persistent immune response173; however, ovalbumin with low-level endotoxin induces IgE-mediated allergic sensitization and a

Th2-type allergic inflammatory response.174 In contrast, higher levels of endotoxin with allergen trigger DC production of IL-12 and lead to nonpathogenic IgG and Th1-type immune responses specific to the allergen. The timing of endotoxin exposure relative to allergen exposure also alters allergy and asthma outcomes. Chronic endotoxin exposure in naïve hosts, similar to what is encountered in farming communities, induces upregulation of the A20 enzyme in airway epithelial cells, reducing epithelial cell cytokine generation and subsequent development of type 2 lung inflammation in response to allergen.103 By contrast, endotoxin exposure in sensitized hosts amplifies the pathogenic allergic immune response on reexposure to allergen. Human epidemiologic studies of ambient home endotoxin exposure and allergy typically reveal a picture consistent with the basic science and animal model evidence for an atopy-protective influence, with higher home endotoxin levels associated with less atopic dermatitis,175–177 less inhalant allergen sensitization, less allergic rhinitis, and less atopy-associated asthma153,175 in infancy and childhood. Other naturally occurring microbial PAMPs may protect against the development of allergy, such as unmethylated CpG motifs. These signatures of microbial DNA are PAMPs that are distinguished from nonmicrobial DNA and recognized by TLR9.178 DNA from dust samples from farm homes, farm barns, and rural homes in India (where allergy tends to be less common) was found to have a higher proportion of microbial DNA.179 When combined with a small amount of endotoxin, DNA extracted from dust from farm barns, but not from metropolitan homes, augmented IL-10 and IL-12 production by peripheral blood mononuclear cells in vitro. This is consistent with the basic scientific understanding of the effect of combinations of sterile PAMP exposures on immune regulatory and Th1 responses and development, and it suggests that environments that are rich and diverse in PAMPs may strengthen innate antimicrobial responses and steer adaptive immunity away from pathogenic atopy. Finally, microbial communities associated with health may confer this benefit through their metabolic products. Short chain fatty acids (SCFAs) such as butyrate, propionate, and acetate are generated by microbial breakdown of dietary fiber and have antiinflammatory properties, inducing IL-10 generation from colonic T regulatory cells through activation of the free fatty acid receptor 2, G protein-coupled receptor 43.180,181 A provocative study recently demonstrated that mice fed a high-fiber diet, but not a low-fiber diet, had suppression of type 2 inflammation in a house dust mite asthma model, which was associated with an increase in the relative abundance of intestinal Bacteroidaceae and Bifidobacteriaceae species, an increase in SCFAs, and the induction of both circulating DC precursors and colonic Tregs, here mediated through the free fatty acid receptor 3.182 Although there are limited reports of alterations in SCFAs associated with human atopy,167,183 this emerging area of research demonstrates how microbiome-mediated gastrointestinal metabolism is likely to have systemic immune regulatory effects. Emerging research suggests that the health outcomes of microbial exposures may be mediated by epigenetic mechanisms in both innate and adaptive arms of the immune system. Epigenetic mechanisms, such as DNA methylation that downregulates gene expression, can be modified by environmental stimuli and alter atopic outcomes. For example, the differential immune responses of macrophages to endotoxin (i.e., proinflammatory to low doses; tolerogenic to high doses) is epigenetically controlled,184 as is the differentiation of naïve T lymphocytes into Th1, Th2, Th17, or Treg cells.185 To summarize, the evidence suggests that natural microbial environments that are rich, diverse, and nonpathogenic may strongly influence allergic and asthmatic outcomes. This is consistent with an innate immune developmental paradigm positing that inadequate environmental microbial conditions may result in inadequate development of

CHAPTER 1  Innate Immunity

15

innate homeostatic responses, allowing development of effector Th2 responses to nonpathogenic allergens with allergic consequences. Powerful microbial genomic techniques can fully speciate and quantify human and environmental microbiomes without the limitations and selection biases of culture-based methods. These methods can provide an understanding at the species level of microbial protectors and pathogens in allergy and asthma.

ligand mannoside and administering this modified extract before sensitization reduced the anaphylactic response to food allergen challenge.196 Mannoside coating targeted the food allergen to SIGNR1-expressing lamina propria DCs, leading to IL-10 expression and the generation of type 1 Tregs, which produce IL-10 and IFN-γ. These observations reveal specific innate sensors and tolerogenic versus allergic pathways in the gut and highlight their potential for clinical translation.

Impairment of Innate Antimicrobial Responses by Allergy

SUMMARY

Allergic immune responses can impair innate antimicrobial immunity. For example, atopic dermatitis is a Th2-type disease characterized by overexpression of IL-4 and IL-13. Atopic dermatitis lesions are distinguished by a decrease in innate immune cell signaling, decreased AMP production (i.e., HBD2 and cathelicidin), and barrier protein deficiencies (e.g., filaggrin).186,187 The susceptibility of atopic dermatitis skin to colonization with S. aureus and complications of herpesvirus infections (e.g., eczema herpeticum, eczema vaccinatum) in part result from impairment in the innate immune responses to these microbes. Interference with Th2 cytokines restores AMP production and S. aureus killing in affected skin.188 The susceptibility to common respiratory viruses that underlies asthma exacerbations is thought to originate in the innate immune system. Allergic asthmatics have diminished pDC antiviral IFN-α responses associated with impaired cross-linking of IgE bound to pDC FcεR1.189 Humanized anti-IgE therapy (i.e., omalizumab) in humans, which reduces the frequency of respiratory virus– associated severe asthma exacerbations,190 increases airway antiviral pDCs while reducing airway mDCs, and reduces FcεR1 on mDCs.191,192 These observations are consistent with an evolving understanding that adaptive immunity can strongly influence innate immune development and antimicrobial responses for better or worse.

Innate Homeostasis in Resistance to Allergic Disease Studies have begun to reveal the homeostatic mechanisms that protect against allergic sensitization. Families of host proteins, including lipoxins, resolvins, protectins, pentraxins, ficolins, and collectins, recognize and eliminate large groups of microbial molecules while downregulating inflammatory responses to maintain homeostasis. They can do the same for allergens. DCs and macrophages are thought to have central roles in the development of tolerogenic versus inflammatory responses to allergen. In murine models of lung immunity, partial activation of myeloid DCs or elimination of the population abrogates allergenmediated inflammation in the lungs.193 In these models, depletion of pDCs leads to an allergic asthma phenotype when tolerance would be the usual outcome; conversely, adoptive transfer of allergen-pulsed pDCs prevents the induction of asthma. Thus pDCs and mDCs may have competing roles in the development of allergic responses in the lungs. Airway macrophages have an important role in mediating allergic responses in the airways. In murine models, depletion of alveolar macrophages during exposure to harmless antigens greatly enhances primary and secondary immune responses.93 Allergen-pulsed alveolar macrophages do not promote a pathogenic Th2-type immune response in the airways, as do allergen-pulsed, mature mDCs; instead, a protective Th1-type immune response is induced.194 A paradigm in macrophage biology derived primarily from mouse studies distinguishes macrophages that are alternatively activated and promote Th2 immune responses from classically activated, Th1-promoting macrophages.195 The characterization of macrophages as classically activated, alternatively activated, or regulatory is paradigmatically appealing, although an oversimplification. Tolerogenic DCs have a similar role in the gut, where they are marked by expression of the CLR SIGNR1, the murine homolog of human DC-SIGN. In a murine model of food allergy, coating food allergen with the SIGNR1

Studies over the past two decades have greatly advanced our understanding of innate immune function in host defense and have expanded the repertoire of innate immune cells. Additionally, they have highlighted the numerous innate immune pathways implicated in the pathogenesis of allergic diseases. As it is for microbes, the innate immune system is the gateway to allergen recognition and immune responses, and barrier epithelial cells appear to play a central role in initiating, amplifying, and coordinating type 2 immunity. Investigations of the environmental conditions and microbiomes that cause or protect against the development of allergic disease are poised to provide novel approaches to prevent and reverse allergic disease through the innate immune system.

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SECTION A  Basic Sciences Underlying Allergy and Immunology

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143. Kool M, Willart MA, van Nimwegen M, et al. An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma. Immunity 2011;34:527–40. 144. Willart MA, Deswarte K, Pouliot P, et al. Interleukin-1alpha controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33. J Exp Med 2012;209:1505–17. 145. Hara K, Iijima K, Elias MK, et al. Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa. J Immunol 2014;192:4032–42. 146. O’Grady SM, Patil N, Melkamu T, et al. ATP release and Ca2+ signalling by human bronchial epithelial cells following Alternaria aeroallergen exposure. J Physiol 2013;591:4595–609. 147. Ullah MA, Loh Z, Gan WJ, et al. Receptor for advanced glycation end products and its ligand high-mobility group box-1 mediate allergic airway sensitization and airway inflammation. J Allergy Clin Immunol 2014;134:440–50. 148. Strachan DP. Hay fever, hygiene, and household size. BMJ 1989;299:1259–60. 149. Riedler J, Braun-Fahrländer C, Eder W, et al. Exposure to farming in early life and development of asthma and allergy: a cross-sectional survey. Lancet 2001;358:1129–33. 150. Genuneit J. Exposure to farming environments in childhood and asthma and wheeze in rural populations: a systematic review with meta-analysis. Pediatr Allergy Immunol 2012;23:509–18. 151. Ownby DR, Johnson CC, Peterson EL. Exposure to dogs and cats in the first year of life and risk of allergic sensitization at 6 to 7 years of age. JAMA 2002;288:963–72. 152. Fall T, Lundholm C, Örtqvist AK, et al. Early exposure to dogs and farm animals and the risk of childhood asthma. JAMA Pediatr 2015; 169:e153219. 153. Braun-Fahrländer C, Riedler J, Herz U, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869–77. 154. Lynch SV, Wood RA, Boushey H, et al. Effects of early-life exposure to allergens and bacteria on recurrent wheeze and atopy in urban children. J Allergy Clin Immunol 2014;134:593–601.e12. 155. Fujimura KE, Johnson CC, Ownby DR, et al. Man’s best friend? The effect of pet ownership on house dust microbial communities. J Allergy Clin Immunol 2010;126:410–12.e1–3. 156. Maier RM, Palmer MW, Andersen GL, et al. Environmental determinants of and impact on childhood asthma by the bacterial community in household dust. Appl Environ Microbiol 2010;76:2663–7. 157. Ege MJ, Mayer M, Normand AC, et al. Exposure to environmental microorganisms and childhood asthma. N Engl J Med 2011;364:701–9. 158. Lynch MD, McFadden JP, White JM, et al. Age-specific profiling of cutaneous allergy at high temporal resolution suggests age-related alterations in regulatory immune function. J Allergy Clin Immunol 2017;140:1451–3.e5. 159. Oh J, Byrd AL, Park M, et al. Temporal stability of the human skin microbiome. Cell 2016;165:854–66. 160. Costello EK, Lauber CL, Hamady M, et al. Bacterial community variation in human body habitats across space and time. Science 2009;326:1694–7. 161. Chu DM, Ma J, Prince AL, et al. Maturation of the infant microbiome community structure and function across multiple body sites and in relation to mode of delivery. Nat Med 2017;23:314–26. 162. Bjorksten B, Sepp E, Julge K, et al. Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 2001;108:516–20. 163. Kalliomäki M, Kirjavainen P, Eerola E, et al. Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol 2001;107:129–34. 164. Bisgaard H, Li N, Bonnelykke K, et al. Reduced diversity of the intestinal microbiota during infancy is associated with increased risk of allergic disease at school age. J Allergy Clin Immunol 2011;128:646–52.e1–5. 165. Abrahamsson TR, Jakobsson HE, Andersson AF, et al. Low diversity of the gut microbiota in infants with atopic eczema. J Allergy Clin Immunol 2012;129:434–40.e1–2.

CHAPTER 1  Innate Immunity 166. Wang M, Karlsson C, Olsson C, et al. Reduced diversity in the early fecal microbiota of infants with atopic eczema. J Allergy Clin Immunol 2008;121:129–34. 167. Arrieta MC, Stiemsma LT, Dimitriu PA, et al. Early infancy microbial and metabolic alterations affect risk of childhood asthma. Sci Transl Med 2015;7:307ra152. 168. Sjogren YM, Jenmalm MC, Bottcher MF, et al. Altered early infant gut microbiota in children developing allergy up to 5 years of age. Clin Exp Allergy 2009;39:518–26. 169. Vael C, Nelen V, Verhulst SL, et al. Early intestinal Bacteroides fragilis colonisation and development of asthma. BMC Pulm Med 2008;8:19. 170. Amoah AS, Boakye DA, Yazdanbakhsh M, et al. Influence of parasitic worm infections on allergy diagnosis in Sub-Saharan Africa. Curr Allergy Asthma Rep 2017;17:65. 171. Bisgaard H, Hermansen MN, Buchvald F, et al. Childhood asthma after bacterial colonization of the airway in neonates. N Engl J Med 2007;357:1487–95. 172. Schnare M, Barton GM, Holt AC, et al. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001;2:947–50. 173. Akbari O, DeKruyff RH, Umetsu DT. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2001;2:725–31. 174. Eisenbarth SC, Piggott DA, Huleatt JW, et al. Lipopolysaccharide-en hanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 2002;196:1645–51. 175. Gehring U, Bischof W, Fahlbusch B, et al. House dust endotoxin and allergic sensitization in children. Am J Respir Crit Care Med 2002;166:939–44. 176. Perzanowski MS, Miller RL, Thorne PS, et al. Endotoxin in inner-city homes: associations with wheeze and eczema in early childhood. J Allergy Clin Immunol 2006;117:1082–9. 177. Phipatanakul W, Celedón JC, Raby BA, et al. Endotoxin exposure and eczema in the first year of life. Pediatrics 2004;114:13–18. 178. Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002;20:709–60. 179. Roy SR, Schiltz AM, Marotta A, et al. Bacterial DNA in house and farm barn dust. J Allergy Clin Immunol 2003;112:571–8. 180. Maslowski KM, Vieira AT, Ng A, et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009;461:1282–6. 181. Furusawa Y, Obata Y, Fukuda S, et al. Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 2013;504:446–50.

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182. Trompette A, Gollwitzer ES, Yadava K, et al. Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat Med 2014;20:159–66. 183. Song H, Yoo Y, Hwang J, et al. Faecalibacterium prausnitzii subspecies-level dysbiosis in the human gut microbiome underlying atopic dermatitis. J Allergy Clin Immunol 2016;137:852–60. 184. Foster SL, Hargreaves DC, Medzhitov R. Gene-specific control of inflammation by TLR-induced chromatin modifications. Nature 2007;447:972–8. 185. Kanno Y, Vahedi G, Hirahara K, et al. Transcriptional and epigenetic control of T helper cell specification: molecular mechanisms underlying commitment and plasticity. Annu Rev Immunol 2012;30:707–31. 186. Ong PY, Ohtake T, Brandt C, et al. Endogenous antimicrobial peptides and skin infections in atopic dermatitis. N Engl J Med 2002;347:1151–60. 187. De Benedetto A, Agnihothri R, McGirt LY, et al. Atopic dermatitis: a disease caused by innate immune defects? J Invest Dermatol 2009;129:14–30. 188. Kisich KO, Carspecken CW, Fieve S, et al. Defective killing of Staphylococcus aureus in atopic dermatitis is associated with reduced mobilization of human beta-defensin-3. J Allergy Clin Immunol 2008;122:62–8. 189. Gill MA, Bajwa G, George TA, et al. Counterregulation between the FcepsilonRI pathway and antiviral responses in human plasmacytoid dendritic cells. J Immunol 2010;184:5999–6006. 190. Busse WW, Morgan WJ, Gergen PJ, et al. Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children. N Engl J Med 2011;364:1005–15. 191. Foster B, Metcalfe DD, Prussin C. Human dendritic cell 1 and dendritic cell 2 subsets express FcepsilonRI: correlation with serum IgE and allergic asthma. J Allergy Clin Immunol 2003;112:1132–8. 192. Prussin C, Griffith DT, Boesel KM, et al. Omalizumab treatment downregulates dendritic cell FcepsilonRI expression. J Allergy Clin Immunol 2003;112:1147–54. 193. Lambrecht BN, Salomon B, Klatzmann D, et al. Dendritic cells are required for the development of chronic eosinophilic airway inflammation in response to inhaled antigen in sensitized mice. J Immunol 1998;160:4090–7. 194. Tang C, Inman MD, van Rooijen N, et al. Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism. J Immunol 2001;166:1471–81. 195. Mantovani A, Sica A, Sozzani S, et al. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677–86. 196. Zhou Y, Kawasaki H, Hsu SC, et al. Oral tolerance to food-induced systemic anaphylaxis mediated by the C-type lectin SIGNR1. Nat Med 2010;16:1128–33.

CHAPTER 1  Innate Immunity

19.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following statements is accurate? a. MyD88/TRAM pathway is required for all TLR signaling. b. The TRIF/TRAM pathway leads to activation of NF-κB. c. Inflammasome activation and the generation of mature IL-1B is elicited by TLR signaling. d. Viral nucleic acid is recognized by both NLRs and RIG-I. e. Endocytosis is the primary function of C-type lectin receptors. 2. Which neutrophil granule product exploits a structural difference in the cellular membrane organization between bacterial and mammalian cells for its microbicidal activity? a. Myeloperoxidase b. Human α-defensins c. Neutrophil elastase d. Cathepsin G

3. Which cytokine is released by necrotic epithelial cells but has also been shown to play a role in restoration of epithelial integrity and thermogenesis? a. IL-33 b. IL-25 c. Thymic stromal lymphopoietin (TSLP) d. IL-8 4. Which is a bacterial metabolic by-product with antiinflammatory properties linked to dietary fiber breakdown in the intestine? a. Long chain fatty acids b. Butyrate c. Alpha ketoglutarate d. Citric acid

2  Adaptive Immunity Karen S. Tuano, Javier Chinen

CONTENTS Introduction, 20 The Adaptive Immune Response in Allergic Disease, 20 Components of the Adaptive Immune System, 21 Features of the Adaptive Immune Response, 24

SUMMARY OF IMPORTANT CONCEPTS • The elements of the immune system that are responsible for the specific recognition of antigens to deliver an effective immune response are collectively known as the adaptive immunity. • Immunologic memory is the ability of the adaptive immunity to identify a previously encountered antigen and develop an efficient immune response. • Adaptive immunity is based on the capacity of T and B cell receptors to recognize a wide variety of unique antigens. • Helper T (Th) cells determine the type of immune response: the Th1 response mediates cell-mediated immunity; the Th2 response mediates elimination of helminth infections and promotes allergic diseases; Th17 responses are characterized by inflammation and often involved in autoimmune diseases. In combination, these immune responses are primarily directed to eradicate pathogens. • B cells differentiate into plasma cells and produce antibodies. • There are five classes of antibodies, immunoglobulin (Ig) M, IgG, IgD, IgE, and IgA. IgG is the most abundant and is distributed throughout the body. IgA is mostly located in the mucosa. • Regulatory T cells ensure that immune responses are not overactivated and limit damage to host tissues. • IgE mediates immediate hypersensitivity responses by triggering release of mast cell mediators after allergen binding. • Mechanisms of immune-mediated hypersensitivity responses are immediate hypersensitivity, antibody-mediated cytotoxicity, immune complex reactions, delayed hypersensitivity reactions, antibody-mediated biologic function, cell-mediated cytotoxicity, and granulomatous reaction.

INTRODUCTION The immune system is a sophisticated network of cells and tissues responsible for discriminating the diverse variety of proteins and other substances that our body encounters and to decide whether to develop an immune response against these substances, known as antigens, or develop tolerance. The immune response can be conceptually separated into two complementary networks of immunologic processes. One network provides an immediate, rapid, and hard-wired response called innate

20

Mechanisms of Diseases Involving Adaptive Immunity: Hypersensitivity Reactions, 27 Conclusion, 28

immunity, and the second network provides a highly specific response to each antigen, resulting in a long-lasting defense, known as adaptive immunity. Innate immune responses are discussed in Chapter 1. Adaptive immunity has evolved to increase the efficiency of immune responses to pathogens, including the provision of specific memory (recall) of antigens previously encountered. This property allows prompt elimination of these pathogens before they can produce disease. However, these responses need to be regulated, or they might lead to chronic illnesses. Allergic and autoimmune diseases are significant, potentially debilitating, and often severe conditions resulting from immune responses to innocuous elements, such as pollen or host tissues, respectively. The classic immediate allergic response refers to hypersensitivity reactions directed at allergens, mediated by biologic processes that are designed to provide host protection from parasitic infections.

THE ADAPTIVE IMMUNE RESPONSE IN ALLERGIC DISEASE Adaptive immunity is a feature of the immune system in vertebrates and is central to the capacity to distinguish nonself-molecules, produced by pathogens, from self-molecules. This immunologic property exists in a delicate balance between developing immune tolerance or an immune response. In autoimmunity and allergy, this balance is disrupted. Autoimmunity defines a state in which immunologic tolerance to one or several self-antigens is lost, and the immune response is activated against host tissues, such as the pancreatic beta cells in type 1 insulin-dependent diabetes mellitus. Allergic or immediate hypersensitivity reactions are the result of immune responses to innocuous nonself-molecules referred to as allergens. This immediate response is activated by immunoglobulin E (IgE) antibody specific to an allergen. Allergen binding to allergenspecific IgE on the surface of mast cells and basophils triggers a series of cellular and molecular events that produce the clinical manifestations of allergic disease.11 IgE-mediated immunity is critical for defense against parasites; however, the low prevalence of parasitic infections in modern societies has turned the attention to the role of IgE in allergic disorders. Moreover, it has been suggested that parasitic infections occurring early in life result in regulatory mechanisms that reduce the development of allergic hypersensitivity.2 Adaptive immunity is designed to distinguish each of a large number of molecules and to amplify the response with recurrent exposures to

CHAPTER 2  Adaptive Immunity the same pathogen or molecule. Recognition of a particular pathogen by adaptive immunity may result in enhanced killing by phagocytes. A remarkable property of the adaptive immune system is its memory, which provides efficient protection against repeated exposures to harmful microbial agents by accelerating and magnifying the response even if the events are separated by decades. Immunologic memory is made possible by the clonal expansion of lymphocytes in response to specific antigen (including allergen) stimulation. From the time the human immune system begins to differentiate in fetal life, lymphocytes possessing unique reactivity are created by the recombination of genes encoding antigen receptors, which are then expressed on the lymphocyte cell membrane. These unique receptors provide each lymphocyte with the ability to bind to and become activated by a specific antigen. Interaction with antigen results in the development of effector T and B cells and generates long-lived, antigen-specific memory cell clones. When the same antigen enters the body again, there is prompt recognition by these memory cells. Cellular and humoral responses to the antigen are produced more rapidly, and more memory cells are generated than in the first encounter. This process of expansion of clonal populations of antigen-specific lymphocytes explains the B cell origin of antibody diversity and applies to cellular (T cell) immune responses.3

Environmental and Genetic Factors Affecting the Allergic Immune Response The prevalence of allergic disorders in urban communities is increasing.4,5 Possible causes include environmental factors, such as ambient pollution,6 increased concentration of indoor allergens, diet, and decreased exposure to microbes because of improved sanitation. The human microbiome is thought to play a role early in life and influence genetically susceptible individuals to develop allergic disease. Diversity of an infant’s gut microbiome has been shown to be inversely associated to allergic conditions, by promoting a balance of helper T cell and regulatory T cell responses.7 The hygiene hypothesis, which attempts to explain the increasing prevalence of allergy, is based on the immunomodulation induced by bacterial and viral infections early in infancy. Reduction or absence of infections are thought to decrease Th1 cytokines, resulting in a Th2 cytokine, and therefore favor the development of allergic responses. However, environmental factors do not fully explain the increase of allergic disease.8 Allergists have known for decades that children of allergic parents are more likely to develop allergic disease.9 Genetic studies, including linkage analyses of large families, have identified several loci containing candidate genes that may confer increased susceptibility to allergic disease, modify its severity,10 or affect the response to medications.11 For example, the R130Q polymorphism in the gene encoding IL-13 results in increased ligand-receptor interaction and confers risk of allergic rhinitis and asthma.12 Epigenetics refers to modifications of DNA that regulate gene expression, such as DNA methylation and histone acetylation, occurring secondary to environmental stimuli. The relevance of epigenetics is illustrated by the association of exposure to air pollutant particles and methylation changes in the IL-4 gene observed in pediatric asthma patients, who show increased IL-4 expression and Th2 bias.13 Allergic disorders, with their variety of presentations, result from a complex interplay between the genetic predisposition of particular populations and specific environmental factors.

COMPONENTS OF THE ADAPTIVE IMMUNE SYSTEM All cells of the immune system are derived from the pluripotent hematopoietic stem cells found in the bone marrow. This pluripotent stem

21

cell gives rise to lymphoid stem cells and myeloid stem cells. The common lymphoid stem cell differentiates into three types of cells—T cell, B cell, and natural killer (NK) cell—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 these committed stem cells depends on an array of cytokine and cell-cell interactions. An ever-increasing number of biologically important surface membrane proteins have been 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, and the designations are useful for identifying cells and their developmental stage (see Appendix A).

T Cells T cell progenitors derived from the common lymphoid progenitor cells leave the bone marrow through the bloodstream and home to the thymus gland, guided by the expression of cell adhesion proteins.14,15 T cell progenitors entering the thymus lack surface antigens, including the T cell antigen receptor (TCR) complex and mature T cell markers (e.g., CD4, CD8) that are associated with specific effector functions. These double-negative thymocytes (CD4−/CD8−) are induced to express CD1, CD2, CD5, CD6, CD7, and interleukin-2 receptor (IL-2R) molecules, which serve as critical receptor-ligand functions during early ontogeny. Subsequently, these progenitor T cells undergo rearrangement of α and β (or γ and δ) genes of the TCR, generating immature T cells. These precursor cells develop into mature T cells, which emerge from the thymus gland with distinct surface antigens and functional characteristics. T cell development within the thymus involves positive and negative selection based on TCR affinity for antigens that ultimately results in the release of mature T cells with the capacity to respond to nonselfantigens presented in the context of self–major histocompatibility complex (MHC) molecules. This educational process is under the control of specialized cortical cells of the thymus and depends on cell-cell contact and the secretion of cytokines, with subsequent elimination of most precursors that enter the thymus. Two main types of T cells leave the thymus to circulate in the peripheral blood, lymphatic system, and tissues: TCR-αβ+/CD4+ T cells and TCR-αβ+/CD8+ T cells. Less than 10% of mature T cells emerge from the thymus as TCR-γδ+ T cells, which are predominantly CD4−/CD8−. TCR-αβ+ T cell activation occurs when the TCR binds to an immunogenic peptide displayed on the surface of a cell. CD8+ T cells recognize antigenic peptides displayed in the context of class I MHC molecules, which are expressed on virtually all nucleated cells, whereas CD4+ T cells recognize antigen peptides presented in the context of class II MHC molecules, which are found on a limited range of cells that are referred to as antigen-presenting cells (APCs) and that include monocytes, macrophages, B cells, and especially dendritic cells. TCR-γδ+ T cells are most important in the recognition of lipid-containing antigens, are more abundant in mucosa, and have less diversity range than the TCR-αβ+ T cells. On antigen recognition, the CD3 complex of proteins transduces a signal through the cell membrane lipid bilayer to the cell interior and nucleus. The CD3 complex is composed of four distinct polypeptide chains; epsilon (CD3ε), gamma (CD3γ), delta (CD3δ), and zeta (CD3ζ), which are expressed together as three pairs of dimers (εγ, εδ, ζζ). Essential to the proliferation of antigen-activated T cells is their expression of CD25 (IL-2R α chain, p55), which combines with the β chain (CD122, p75) 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 activation and proliferation. Several accessory glycoprotein adhesion molecules stabilize the binding of T cells to the APC during the recognition phase or to the target cell in the

22

SECTION A  Basic Sciences Underlying Allergy and Immunology

effector phase, and they provide a costimulatory signal required for T cell activation (Table 2.1). The process of TCR-αβ binding to a specific peptide presented in the context of an appropriate MHC molecule requires a second costimulatory signal to initiate cell activation. Cell activation proceeds through several steps: (1) hydrolysis of the phospholipid component of the lipid bilayer, phosphatidyl inositol bisphosphate, into inositol triphosphate (IP3) and diacylglycerol (DAG); (2) elevation of intracellular calcium levels, produced partly by IP3; (3) activation of protein kinase C (PKC) by interaction with DAG; and (4) phosphorylation and activation of tyrosine kinases (Fig. 2.1). These activation events convey messages to the cell nucleus and appropriate target genes for the nuclear factor of activated T cells (NFAT) and the proto-oncogene FOS or transcriptional regulator proteins, such as activator protein 1 (AP-1), which together regulate cell activity and cytokine expression. Cytokine expression and secretion (i.e. IL-2) regulates T cell activation, survival, and proliferation. The intricate T cell activation events are important to the clinical practice of allergy and immunology, because they 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 cyclosporine and tacrolimus inhibit calcineurin, a serine-threonine–specific protein phosphatase, blocking

TABLE 2.1  Major Receptor-Ligand Pairs

That Participate in T Cell Binding to AntigenPresenting Cells T Cell

Antigen-Presenting Cell

T cell receptor

HLA and peptide

CD4

HLA-DR, HLA-DQ, HLA-DP (class II MHC)

CD8

HLA-A, HLA-B, HLA-C (class I MHC)

CD11a (LFA-1α), CD18

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

CD2 (LFA-2)

CD58 (LFA-3)

CD40L

CD40

CD28

CD80 (B7-1), CD86 (B7-2)

CD152 (CTLA-4)

CD80 (B7-1), CD86 (B7-2)

CD278 (ICOS)

CD275 (ICOSL)

CD279 (PD-1)

CD274 (PDL-1), CD273 (PDL-2)

CD, Cluster of differentiation cell surface marker; CTLA-4, cytotoxic T lymphocyte–associated protein 4; HLA, human leukocyte antigen; ICAM, intercellular adhesion molecule; ICOS, inducible T cell costimulator; ICOSL, ICOS ligand; LFA, leukocyte function–associated antigen; MHC, major histocompatibility complex; PD-1, programmed cell death protein 1; PDL, PD-1 ligand.

APC MHC class II

CD4 Lipid bilayer

TCR

lck

CD3 complex

Activation of phospholipase C

Phosphorylation of tyrosine kinase: ZAP-70

T cell

RAS activation MAP kinase cascade

AP-1

Inositol triphosphate (IP3) Increased calcium Calcineurin activation

NFAT +

Diacylglycerol Protein kinase C

NF-κB

Fig. 2.1  Activation of CD4 T cells, with binding of the T cell antigen receptor (TCR) to the antigen-presenting cell (APC) as the first signal, and to accessory molecules as the second signal. Cross-linking of the TCR causes aggregation with the CD3 complex containing ζ, δ, and ε chains. AP-1, NFAT, and NF-κB are transcription factors that activate genes necessary for cell proliferation and differentiation. AP-1, Activator protein 1; lck, tyrosine kinase; MAP, mitogenic-associated proliferation; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T cells; NF-κB, nuclear factor κ-light-chain enhancer of activated B cells, ZAP-70, zeta-associated protein 70.

CHAPTER 2  Adaptive Immunity

23

TABLE 2.2  Characteristics of B Cell Differentiation Stages Parameter

Stem Cell

Pre-B Cell

Immature B Cell

Mature Naïve B Cell

Activated Memory B Cell

Antibody-Secreting B (Plasma) Cell

Antigen dependency

−

−

−

−

+

+

Compartment

BM

BM

BM + PB

PB

PB

PB

Intracellular proteins

RAG-1, RAG-2 µ chain, TdT

Heavy-chain genes

Germline

VDJ

VDJ

VDJ

Isotype switch

Isotype switch

Light-chain genes

Germline

Germline

VJ

VJ

VJ

VJ

Surface markers

CD45 CD34

CD45R MHC II CD10 CD19 CD20 CD38 CD40

CD45R MHC II IgM CD19 CD20 CD22 CD40

CD45R MHC II IgM, IgD CD19 CD20 CD21, CD22 CD40, CD27

CD45R MHC II IgM CD19 CD20 CD21, CD22 CD40

PC-1 CD20 CD38 CD138

Immunoglobulin production

None

Cytoplasm µ chain

Membrane IgM

Membrane IgD, IgM

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

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

BM, Bone marrow; D, diversity gene segment; Ig, immunoglobulin; J, joining gene segment; MHC II, major histocompatibility complex class II; PB, peripheral blood; PC-1, plasma cell antigen 1; RAG, recombinase activating gene–encoded protein; TdT, terminal deoxynucleotidyl transferase; V, variable gene segment; −, no; +, yes. (Only representative stages are described.)

the dephosphorylation and translocation to the nucleus of NFAT and, consequently, inhibits the expression of cytokines such as IL-2 and IL4 and cell membrane proteins such as CD40L.

B Cells The B cell matures in the bone marrow, but during fetal life, maturation occurs in the liver. During differentiation of the B cell, a series of DNA rearrangements of immunoglobulin heavy-chain genes and light-chain genes precedes the production of membrane-bound and secreted immunoglobulin molecules (Table 2.2). As the pre-B cell matures, it undergoes µ-chain gene rearrangement that combines with a surrogate invariant light chain (λ5 and Vpre-B) necessary for effective transport of the µ chain to the cell surface, expressed as the pre-B cell receptor. Association of the pre-B cell receptor with the Igα and Igβ proteins provides the means for signal transduction that facilitates further maturation of the pre-B cell, associated with kappa (κ) or lambda (λ) light-chain gene rearrangements. These events ultimately result in the expression of a transmembrane version of an IgM molecule on the cell surface.16 Mature B cells co-express surface IgM and IgD after heavy-chain mRNA splicing. Up to this stage, the B cell maturation process is antigen independent, and subsequent differentiation of the IgM+/IgD+ mature B cells circulating in the periphery is antigen driven. Activation of mature B cells into immunoglobulin-secreting B cells or long-lived memory B cells and final differentiation into plasma cells are also antigen dependent. Isotype switching involves further rearrangement of 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 nine switch regions of the heavy-chain DNA sequence that enable transcription of all immunoglobulin subclass genes and leads to production of IgG, IgA, or IgE. These immunoglobulins express the same heavy-chain variable regional sequences and light-chain sequences, maintaining antigen specificity.17

Immunoglobulin gene rearrangements and expression of surface immunoglobulin by developing B cells are concomitant with the appearance of certain B cell markers. B cell markers include the variable expression of terminal deoxynucleotidyl transferase (TdT); MHC 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 CD27, a costimulatory molecule that is characteristic of memory B cells. The plasma cell 1 (PC1), CD20, CD38, and CD138 surface markers are expressed on the antibody-producing cell. The phenotypic expression of markers can distinguish the stage of B cell differentiation (see Table 2.2). The maturation and differentiation of B cells is under the control of cytokines. For example, IL-2 promotes B cell activation and growth, and IL-4 induces IgE isotype switch. Antibody diversity results from the recombination of immunoglobulin genes, generating millions of different immunoglobulin molecules. 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 using intracellular processes that are similar to those for TCR. The heavy-chain variable region is encoded by variable (V), diversity (D), and joining (J) gene segments, which are then juxtaposed to specific constant (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. The resulting antibody specificity is further modified in the germinal centers of lymphoid tissues by somatic hypermutation to generate antibodies of higher affinity. Antigen activation of B cells is initiated by ligation of membrane-bound immunoglobulin, which has a cytoplasmic tail consisting of only three amino acids, which is inadequate for signal transduction. A complex of molecules noncovalently associated with membrane immunoglobulin has cytoplasmic tails sufficient to be phosphorylated and initiate signal transduction. These molecules include

24

SECTION A  Basic Sciences Underlying Allergy and Immunology

an IgM-specific, 32-kD µ chain and an IgD-specific, 33-kD δ chain, each of which is disulfide-linked with an α chain (CD79A, Igα) and a β chain (CD79B, Igβ). This membrane immunoglobulin-associated complex is analogous to the CD3 complex protein of the TCR. The process of B cell activation events occurs by many of the same signal transduction pathways described for T cells, including key tyrosine kinases.

Antigen-Presenting Cells: Monocytes, Macrophages, and Dendritic Cells Although not programmed with immune memory, the circulating peripheral blood monocyte, tissue macrophage, and dendritic cell are essential components of adaptive immune responses because of their role of presenting antigens to T cells. These antigen-presenting cells (APCs) contain several important receptors specific for the Fc region of IgG molecules and the third complement component that facilitate antigen presentation.18 APCs express class I and class II MHC molecules and present antigenic epitopes to TCR-αβ+ receptors on CD8+ or CD4+ T cells during antigen recognition. Dendritic cells are present in skin and mucosal areas, and they are recognized by their characteristic projections and surface phenotype. Monocytes are produced in the bone marrow, circulate in the blood for a few days, and mature into macrophages or dendritic cells in different tissues, such as the liver and the spleen. The contributions of the monocyte-macrophage system to the general inflammatory response and the specific immune response are diverse, including functioning as phagocytic cells for intracellular pathogens and as cytotoxic effector cells, particularly as effectors of antibodydependent cellular cytotoxicity (ADCC). These cells also produce multiple cytokines, including IL-1, IL-12, and tumor necrosis factor (TNF), that are central to inflammatory immune responses and induce an extra­ ordinary diversity of effects on hematopoietic and nonhematopoietic cells and tissues. Follicular dendritic cells are stromal cells found interacting with B cells in secondary lymphoid tissues. They are similar to DCs in their cell morphology and the ability to present antigens with antigen–antibody complexes and are essential for the maturation of B cells that produce high-affinity antibodies.

Cytokines and Chemokines Cytokines stimulate T cells needed for the development, activation, and differentiation of lymphocytes. On binding of APCs to resting (G0) T cells, several interleukins (e.g., IL-1, IL-4, IL-6) and TNF-α facilitate the transformation to activated T cells (G1), which secrete IL-2 and upregulate the expression of their IL-2 receptors. IL-2 produced by G1-phase T cells stimulates other T cells to express additional IL-2 receptors on cell surfaces, enhancing further activation. G1-phase T cells under the continuing stimulation by IL-2 become actively replicating cells that begin to secrete additional cytokines that modulate immune and inflammatory responses. IL-7 is another significant cytokine with a role in T cell differentiation and survival, preferentially naïve T cells. It is produced by stromal cells in the bone marrow and the thymus. Several T cell–derived cytokines (i.e., IL-3, IL-4, IL-5, and IL-13) have profound effects on immediate hypersensitivity reactions and IgEderived reactions (Table 2.3).19 IL-4 is important during B cell activation and induces switching of immunoglobulin production to IgE. IL-3 stimulates the proliferation of basophils. IL-3, granulocyte macrophage colony–stimulating factor (GM-CSF), and IL-5 stimulate the growth and survival of eosinophils. Moreover, IL-6 acts as a stimulant to the polyclonal production of immunoglobulin by B cells and may enhance IL-4–induced IgE production. Cytokines are attractive targets for pharmacologic intervention in allergic diseases. Because the immune system has such a powerful capacity to stimulate itself, it is essential that a counter-regulatory side exists to reduce or

TABLE 2.3  Effects of Cytokines on Human

Cells Involved in Immediate Hypersensitivity (IgE-Induced) Reactions Activity

IL-3 IL-4 IL-5 IL-9 IL-13 IL-33 SCF

B cell proliferation

−

++

−

+

++

−

−

IgE production

−

+

−

+

++

−

−

MHC class II

−

+

−

+

+

−

−

CD23 (FcεRII)

−

+

−

+

+

−

−

B cell differentiation

+

++

−

+

+

−

−

Mast cell maturation

+

+

−

+

+

++

+++

Eosinophil maturation + Basophil maturation

++

+

+++

+

+

++

−

+

+

+

+

++

+

IgE, Immunoglobulin E; IL, interleukin; MHC, major histocompatibility complex; SCF, stem cell factor; −, no effect; +, small effect; ++, moderate effect; +++, large effect.

turn off immune responses. The immune networks that inhibit excessive stimulation use cytokines (e.g., TGF-β, IL-10) and regulatory T cells (Tregs). These networks allow for managing the magnitude and duration of an immune response and appear to depend in part on a process referred to as programmed cell death (i.e., apoptosis). The two general pathways of apoptosis at play in the immune system are a passive process of cell death initiated by the removal of life-sustaining growth factors and an active (extrinsic) pathway that involves the interaction between specialized receptors and their ligands that actively induce cell death. Apoptosis is a mechanism for controlling a specific immune response, and it is a central process in the elimination of autoreactive lymphocytes. Apoptosis also appears to be a mechanism for controlling other inflammatory cells involved in the immune response and an effector pathway for cytolytic lymphocytes.20 In barrier sites such as the skin and mucosal surfaces, epithelial cells secrete IL-33, IL-25, and thymic stromal lipoprotein (TSLP), which are involved in atopic dermatitis, food allergy, and allergic asthma. IL-33 interacts with dendritic cells, Th2 cells, follicular T cells, and regulatory T cells (Tregs), resulting in chronic airway inflammation and remodeling.21,22 Similar to IL-33, IL-25 acts on Th2 cells and promotes eosinophilic infiltration.23 TSLP interacts with DCs to promote cytokine secretion and induce Th2 differentiation in CD4+ T cells and also directly act on CD8+ T cells and Tregs, supporting cell proliferation and survival.24

FEATURES OF THE ADAPTIVE IMMUNE RESPONSE Antigen Presentation APCs (e.g., dendritic cells, monocytes, macrophages) process and present antigen within an antigen-binding cleft of MHC molecules. These events start at the APC cell surface with the capture and endocytosis of antigens, followed by a complex sequence of enzymatic activities leading to the association of antigenic peptides with MHC molecules and expression back to the cell surface. CD4+ T cells recognize antigenic peptides when presented in the context of a class II MHC molecule (Fig. 2.2) together with the appropriate costimulatory signal or signals and become activated in response to monocyte-derived IL-1 and other cytokines, including autocrine stimulation by IL-2. The activated CD4+ T cell affects additional CD4+ or CD8+ T cells through the secretion of IL-2 and activates B cells by secreting B cell growth and differentiation factors (i.e., IL-2, IL-4, IL-6, and interferon-γ [IFN-γ]). CD4+ T cells augment immune responses by stimulating B cells activated by antigen and by

CHAPTER 2  Adaptive Immunity

APC

α3

HLA class I

CD8 α2

β1 α1

TCR

T cell

Fig. 2.2  Interaction of a human leukocyte antigen (HLA) class I molecule on an antigen-presenting cell (APC) with a CD8+ T cell. The antigen receptor (i.e., T cell receptor [TCR]) complex (purple) recognizes a combination of an antigen peptide (red) and an HLA molecule (brown and orange). The CD8 molecule (aqua blue) in the T cells interacts with the α3 domain of the HLA molecule. HLA class II molecules present antigen peptides to CD4+ T cells in a similar manner, interacting with the TCR and the CD4 molecules.

stimulating CD8+ T cells sensitized by binding of specific antigenic peptide in the context of class I MHC molecules. B cells also can present antigens. The different environments in which antigen presentation occurs are modified by cytokines and cell-to-cell contact signals, influencing T cell activation and the quality of the immune response that follows.

Antigen Recognition The biologic basis for antigen recognition in the context of MHC molecules is the distinction between self and nonself. In humans, the MHC gene complex is located on chromosome 6 and comprises genes that code for human leukocyte antigens (HLAs). Class I MHC molecules (e.g., 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 (e.g., HLA-DR, DP, DQ) are composed of two, noncovalently linked variant chains, a 34-kD protein and a 29-kD protein. Class I MHC molecules usually present endogenously derived antigenic peptides after antigen processing, such as viral epitopes, to CD8+ T cells, whereas class II MHC molecules present exogenously derived antigenic peptide, such as soluble bacterial protein–derived antigenic peptide, to CD4+ T cells. The class I and class II MHC gene products exhibit simple mendelian inheritance with codominant expression. Single cells from any individual typically express pairs of the MHC gene products corresponding to the maternal and paternal alleles. The biologic role of the MHC molecules is to display antigenic peptides to interact with appropriate TCRs. The strength of this interaction appears to affect the direction of the immune response; low affinity is followed by weak cell activation, and very high affinity results in relative tolerance requiring high antigen expression to be activated. 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

25

CD8+ T cells recognizes antigenic peptide in association with these epitopes. The α3 domain has been implicated in the interaction with CD8 (Fig. 2.2). Each of the class II MHC α and β chains has two immunoglobulin-like external domains. The crystalline structure of class II molecules has a putative antigen-binding cleft on the distal face of the molecule. The appropriate TCR on CD4+ T cells recognizes the antigenic peptide in this binding cleft, whereas the CD4 molecule binds to a nonpolymorphic epitope or epitopes on the class II MHC molecule. Unlike antigenic peptides, certain microbial antigens referred to as superantigens can activate large numbers of T cells by direct interaction with class II MHC molecules and the ζ chain of the TCR. This interaction may influence the risk of severe inflammatory responses, which can be more significant than the microbial infection. Antigen-induced activation of T cells requires a combination of two signals. The first is provided by a TCR-based interaction between the receptor and the appropriate MHC–antigenic peptide complex. A second or costimulatory signal is required for antigen-induced T cell activation; for example, the interaction between CD28 on the T cell and CD80 on the APC provides a costimulatory signal for T cell activation. Because generation of an effector immune response depends on helper T (Th) cell function mediated by CD4+ T cell TCR recognition of an antigenic peptide presented in the groove of a class II MHC molecule, the potential to mount an immune response depends on the class II MHC gene repertoire and an appropriate T cell receptor gene repertoire. Changes as subtle as a single amino acid at a critical site within the class II MHC molecule can alter its capacity to present an immunogenic peptide sequence derived from the intact antigen. These changes within class II MHC molecules occur within the amino terminal domain and can be associated with a responder or nonresponder state, and they may translate into disease resistance or susceptibility. For example, disease occurrence and progression for juvenile-onset diabetes is associated with HLA-DR3-DQ2 or HLA-DR4-DQ8 haplotypes.25 Similarly, susceptibility for development of rheumatoid arthritis and other autoimmune diseases is strongly associated with specific amino acid residues in the HLA-DR4B1 haplotype.26 Efforts to identify HLA susceptibility genes for allergic disorders have had mixed results, with associations found for hypersensitivity to drugs such as the human immunodeficiency virus (HIV) inhibitor abacavir27 and not conclusive for food allergies.28

Th1, Th2, and Th17 Responses The immune regulation of allergic processes includes recruitment of cells involved in their pathogenesis (e.g., eosinophils, mast cells) and regulation of IgE production. These processes depend on T cell control, as observed in the initial descriptions of human T cell deficiency diseases, in which patients’ IgE levels were extremely elevated and returned to normal after T cell reconstitution after bone marrow transplantation. Because a significant portion of the clinical practice of allergy deals with IgE responses, it is appropriate for allergists to understand the fundamental pathways resulting in IgE biosynthesis. Th cell subsets dictate cytokine production involved in the regulation of immune responses.29 At a conceptual level, the Th1 cells are associated with cell-mediated or DTH immune responses mediated by IFN-γ and IL-12, and Th2 inflammatory immune responses involve IgE production and eosinophilic infiltration as a result of the actions of IL-4, IL-5, and IL-13. The Th17 immune response is also associated with inflammation, but it is mediated by IL-17A, IL-17F, IL-21, and IL-22. Th1 cells generate an immunologic response that provides an effective defense against viral infections and other intracellular pathogens and that depends on T cell and monocyte or dendritic cell interactions. Th2 cells exert their effect on immunologic responses to parasites and allergens with augmentation of inflammation from IgE production and

26

SECTION A  Basic Sciences Underlying Allergy and Immunology

favoring eosinophilic infiltration. 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 when promoted by the presence of Th2 cytokines in the cell milieu. IL-33 is a potent inducer of Th2 responses in the lung and plays a central role in asthma. An additional subset, Th9 cells, express IL-9 and participates in lung inflammation, although in humans, their action requires concomitant Th2 cell responses.30 Th17 cells are involved in protection against extracellular pathogens associated with neutrophilic infiltration, and mucocutaneous host defense against fungal and staphylococcal infections. They are also involved in the pathogenesis of autoimmune disorders. In the intimate interactions of these three Th cell subsets, Th1 cells primarily inhibit Th2 cells with the same antigen specificity. The functional compartmentalization of T cells regulates the balance of IL-4 and IFN-γ, which has a profound effect on determining IgE production by B cells. Intracellular molecular pathways are unique to these cytokine expression patterns, each triggered by the activation of transcription factors: T-bet, GATA-3, and RORγ for Th1, Th2, and Th17, respectively. These transcription factors are targets of specific signal transducer and activator of transcription (STAT) proteins, which transmit cytokine signals and have essential roles in T helper cell differentiation. For example, STAT4 is activated by IL-12 and T-bet, resulting in Th1 polarization. IL-4 activates STAT6, which targets GATA-3 and induces Th2 polarization. Th17 differentiation is induced by STAT3, which is activated by IL-6 and in turn activates RORγ. Follicular T helper cells (TFH) constitute a subset of T cells with a central role in the differentiation of B cells into plasma and memory cells.31 They are differentiated after exposure to Th1 and Th17 cytokines and are found in the germinal center of secondary lymphoid organs. TFH cells express CXCR5 and other chemokines, inducible costimulatory (ICOS) and the T cell inhibitory receptor PD-1. The importance of ICOS is demonstrated by patients with ICOS deficiency, who present with hypogammaglobulinemia, decreased memory B cells, and impaired germinal centers in lymphoid tissues.

Immune Tolerance and Regulatory T Cells The defensive capacity of the immune system needs a mechanism to counterbalance its proinflammatory response and to minimize unnecessary

tissue damage. Several processes ensure that the different immune effector cells are not activated against host tissues and innocuous substances and that they can downregulate a response after the threat is resolved. All of these processes underlie immune tolerance, which is defined as central when occurring in primary lymphoid organs or as peripheral when occurring in other tissues. An example of central tolerance is the deletion of self-reactive T cells in the thymus or B cells in the bone marrow on the basis of expression of high-affinity antigen receptors for self-antigens. The autoimmune regulator (AIRE) protein is a transcription factor in thymic epithelia that promotes the expression of genes that are characteristically expressed in other organs, presumably to present self-antigens to newly formed T cells, and it facilitates the elimination of self-reactive T cells as part of central immune tolerance.31 AIRE-deficient patients develop autoimmune polyendocrinopathy, candidiasis, and ectodermal dystrophy (APECED) syndrome, a severe form of autoimmunity involving multiple endocrine organs. Because not all self-antigens are expressed during the central induction of tolerance, other self-reactive T cells may need to be inactivated in the periphery. In lymphoid organs, lymphocytes with high affinity for antigens are deleted on encountering self-antigens. In other peripheral tissues, immune tolerance might occur by the induction of a state of unresponsiveness called anergy. T cells might become anergic by insufficient costimulation in the presence of antigen stimulation. T cells expressing CD25 (IL-2Rα chain) and the transcription factor forkhead box P3 protein (FOXP3) have been identified as regulatory T cells or Tregs, because they suppress the function of other T cells when present in the same site (Fig. 2.3).32,33 T cell proliferation and cytokine responses are blunted in the presence of Tregs. In experimental models, Tregs can function through cell-cell contact, and in other models, they function through the secretion of IL-10 or TGF-β, or both, inducing activation-induced cell death or anergy. A rare human disorder caused by mutations in the FOXP3 gene, the immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, is characterized by a deficiency of CD4+CD25+ Tregs. The clinical phenotype consists of severe autoimmunity, including insulin-dependent diabetes mellitus, inflammatory colitis, atopic dermatitis, and asthma.34 Other types of Tregs that are not FOXP3+ have been described. Type 1 Tregs (Tr1) are peripherally differentiated cells that suppress T cell responses by secreting TGF-β and IL-10, and also by cell-mediated cytotoxicity.35 T regulatory

Dendritic cell

cell

IL-10 TGF-β IFN-α

Low-dose antigen

CD4+ CD25+ T cell

CD4+ CD25+ FOXP3+

Chronic antigen exposure Allergen immunotherapy

IL-10

Th1 or Th2 cell Fig. 2.3  Regulatory T cells are generated by the interaction of antigen-presenting cells and T cells, mediated by the cytokines interleukin-10 (IL-10), transforming growth factor β (TGF-β), and interferon α (IFN-α). These cytokines are secreted when the antigen is presented under certain conditions, such as when administering allergen immunotherapy at very low concentration. Regulatory T cells secrete IL-10 and inhibit effector T cells that share similar antigen specificity.

CHAPTER 2  Adaptive Immunity

BOX 2.1  Pathogenic Mechanisms of

Immune Reactions

IgE-mediated allergic reactions Cytotoxic or cytolytic antibody reactions Immune complex reactions Delayed hypersensitivity reactions Inactivation or activation antibody reactions T cell cytotoxic reactions Granulomatous reactions

Tregs suppress both autoimmunity and graft rejection. Tregs expanded in vitro are being studied for the treatment and prevention of organ transplantation.36 Allergen immunotherapy is another therapy aimed to induce immune tolerance. T cells are induced to turn off allergenspecific IgE responses, a process that likely involves a switch from Th2 to Th1 allergen-specific T cells and the development of regulatory T cells.37,38

MECHANISMS OF DISEASES INVOLVING ADAPTIVE IMMUNITY: HYPERSENSITIVITY REACTIONS The immune system is designed for protection against disease; however, immune responses might also produce undesirable manifestations, commonly referred as hypersensitivity reactions. Clinicians caring for a patient presenting with these reactions might diagnose an allergic or an autoimmune condition. The Gell and Coombs classification has enjoyed wide acceptance as a guide to understanding complex immunologic reactions and lists four types of hypersensitivity reactions: type I, immediate (IgE mediated); type II, cytotoxic (IgG and IgM mediated); type III, immune complexes (IgG and IgM immune complex mediated); and type IV, delayed-type hypersensitivity (T-cell mediated). Type IV hypersensitivity might be further sub-classified according to the predominant T cell involved. We endorse an expanded classification system proposed by Sell that categorizes hypersensitivity reactions on the basis of seven immunopathologic mechanisms (Box 2.1). Several of these mechanisms may apply simultaneously; for example, patients who present with an allergic reaction because of taking a medication (e.g., penicillin) may display symptoms of anaphylaxis and those associated with immune complexes.

Gell and Coombs Type I: Immunoglobulin E– Mediated Reactions The type I hypersensitivity reaction involving the release of mast cell or basophil mediators is characterized by immediate responses to allergens, such as anaphylaxis to penicillin or allergic rhinitis to ragweed pollen. Allergen-induced cross-linking of IgE bound to a high-affinity IgE receptor (FcεRI) on mast cells triggers a cascade of intracellular signals leading to gene transcription of IL-4, TNF, and IL-6; synthesis of prostaglandins and cysteinyl leukotrienes; and release of preformed mediators that include histamine, proteases, and proteoglycans.1 These mediators cause immediate vasodilation, tissue edema, mucus production, and smooth muscle constriction. In some cases, the immediate reaction is followed by a delayed late-phase response (8 to 24 hours) that presents as recurrence of airflow obstruction and cardiovascular, gastrointestinal, and cutaneous symptoms. At least four cell types (i.e., mast cell, basophil, eosinophil, and neutrophil) may participate in the full expression of type I or anaphylactic reactions.

27

Gell and Coombs Type II: Antibody-Mediated Cytolytic Reactions The type II immune reactions involve IgG, IgM, and to a lesser extent IgA, which are 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. Three distinct immune reactions might be induced: The first occurs by opsonization, which is facilitated by complement activation; the second induces complement-mediated lysis; and the third is antibody-dependent cell-mediated cytotoxicity or ADCC. These mechanisms afford protection against infections and eradication of malignant cells but can also result in damage to various tissues associated with responses to self-antigens. An example of opsonization is phagocytic cell destruction of antibody-coated platelets, causing immune thrombocytopenia. The second category is demonstrated by penicillin binding to the surfaces of erythrocytes, creating a nonself-antigen composed of penicillinmodified erythrocyte cell surfaces. 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 membrane attack complex (MAC, C5 to C9). Clinically, this condition is known as penicillin-induced autoimmune hemolytic anemia. Other clinical examples of this reaction include quinidine-induced autoimmune thrombocytopenia and ceftriaxone-induced autoimmune hemolytic anemia.39 ADCC is the process by which NK cells and other cells recognize IgG bound to target cells, such as neoplastic cells, and triggers the release of cytotoxic granules.

Gell and Coombs Type III: Immune Complex–Mediated Reactions The massive formation of immune complexes, by IgM or IgG and a soluble antigen in plasma or tissues, triggers the classical complement pathway and induces immune responses, causing a condition called serum sickness. Recognition of this immunologic disease was first reported 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 became seriously ill or died. 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 and seem to be involved in vasculitis, certain glomerulonephritis, autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus, and even acute respiratory distress syndrome.40 Highly sensitized patients sustain the immune complex–mediated reaction 10 to 14 days after exposure to antigen, and this delayed reaction is often heralded by a classic immediate allergic reaction.

Gell and Coombs Type IV: Delayed Hypersensitivity Reaction The cellular hypersensitivity response is best represented by the delayed hypersensitivity reaction, which is elicited by sensitized T cells, particularly the CD4+ T cell population. Delayed reactions develop slowly, taking 1 to 3 days to be clinically evident after initial contact with antigen. The most common reactions involve the skin, although other organ systems can be involved. A clinical example of these responses is contact dermatitis from exposure to Rhus toxicodendron (poison ivy) antigen. This form of hypersensitivity reaction, which is typically considered an allergic reaction, is a CD4+ T cell–mediated, Th1 type of response (rather than a Th2 response).

28

SECTION A  Basic Sciences Underlying Allergy and Immunology

Antibody-Induced Activation or Inactivation of a Biologic Function Antibodies to a hormone, hormone receptor, blood clotting factor, growth factor, enzyme, or drug may cause disease or treatment failure by inactivating the vital biologic functions of these molecules. 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. Two examples of nondestructive and paradoxical antibody-mediated reactions are characterized by target cell stimulation or by negative signaling or ligand blockade. Antibody to the thyrotropin receptor on thyroid epithelial cells is an autoantibody that can act equivalent to the ligand for the hormone receptor, yielding a positive signal to the cell and resulting in Graves’ disease.41

Cell-Mediated Cytotoxicity CD4+ T cells, CD8+ T cells, or NK cells can cause immunopathogenic reactions and play an important role in immune surveillance against certain viruses and tumor cells. Th17 cytokines promote cell-mediated cytotoxicity and granuloma formation. An example of cell-mediated cytotoxicity is the T cell–induced damage to bone marrow in hemophagocytic lymphohistiocytosis.42 CD8+ T cell cytolytic responses to viruses and alloantigens are clinical examples of this type of immune reaction.

Granulomatous Reactions Granulomas are focal collections in tissues of inflammatory cells, including macrophages, histiocytes, epithelioid cells, and giant cells, as well as lymphocytes and plasma cells surrounded by various amounts of fibrous tissue. Granulomatous reactions are cellular responses to irritating, persistent, and poorly soluble substances such as foreign bodies or infections.43 These reactions are characteristically initiated by sensitized lymphocytes reacting with antigen but may also occur in response to antigen-antibody complexes that persist locally. Clinical conditions involving granulomatous reactions include tuberculosis, leprosy, parasitic diseases, berylliosis, and asbestosis.

CONCLUSION Understanding the elements and mechanisms of the adaptive immunity is fundamental for the approach to the management of allergic and immunologic diseases. Central to the adaptive immune response are T and B cells, which characteristically can recognize each of a universe of antigens to develop one of several different immune responses, including immune tolerance. Allergic conditions are explained by an exaggerated Th2-type immune response. Cytokines that induce this response are IL-4 and IL-13, after binding to their receptors on cell membrane and activating signal transducers such as STAT6. Signal transducers activated from antigen receptor binding together with STAT6 promote the expression of several proteins that produce the allergic response, including GATA3, an essential transcription factor for Th2 cell differentiation. All these molecular processes are potential therapeutic targets for the management of allergic diseases.

REFERENCES 1. Oettgen HC. Fifty years later: emerging functions of IgE antibodies in host defense, immune regulation, and allergic diseases. J Allergy Clin Immunol 2016;137:1631–45. 2. Cruz AA, Cooper PJ, Figueiredo CA, et al. Global issues in allergy and immunology: parasitic infections and allergy. J Allergy Clin Immunol 2017;140:1217–28.

3. Carbone FR. Tissue-resident memory. T cells and fixed immune surveillance in nonlymphoid organs. J Immunol 2015;195:17–22. 4. Platts-Mills TA. The allergy epidemics: 1870-2010. J Allergy Clin Immunol 2015;136:3–13. 5. Anvari S, Chokshi NY, Kamili QU, et al. Evolution of guidelines on peanut allergy and peanut introduction in infants: a review. JAMA Pediatr 2016;171:77–82. 6. Burbank AJ, Sood AK, Kesic MJ, et al. Environmental determinants of allergy and asthma in early life. J Allergy Clin Immunol 2017;140:1–12. 7. Johnson CC, Ownby DR. The infant gut bacterial microbiota and risk of pediatric asthma and allergic diseases. Transl Res 2017;179:60–70. 8. Liu AH. Revisiting the hygiene hypothesis for allergy and asthma. J Allergy Clin Immunol 2015;136:860–5. 9. Ferreira MA, Vonk JM, Baurecht H, et al. Shared genetic origin of asthma, hay fever and eczema elucidates allergic disease biology. Nat Genet 2017;49(12):1752–7. 10. Nyenhuis SM, Krishnan JA, Berry A, et al. Race is associated with differences in airway inflammation in patients with asthma. J Allergy Clin Immunol 2017;140:257–65. 11. Ortega VE, Meyers DA. Pharmacogenetics: implications of race and ethnicity on defining genetic profiles for personalized medicine. J Allergy Clin Immunol 2014;136:16–26. 12. Vladich FD, Brazille SM, Stern D, et al. IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation. J Clin Invest 2005;115:747–54. 13. Yang IV, Pedersen BS, Liu A, et al. DNA methylation and childhood asthma in the inner city. J Allergy Clin Immunol 2015;136(1):69–80. 14. Krueger A, Ziętara N, Łyszkiewicz M. T Cell Development by the Numbers. Trends Immunol 2017;38:128–39. 15. Verma NK, Kelleher D. Not just an adhesion molecule: LFA-1 contact tunes the T lymphocyte program. J Immunol 2017;199:1213–21. 16. Melchers F. Checkpoints that control B cell development. J Clin Invest 2015;125:2203–10. 17. Tong P, Wesemann DR. Molecular mechanisms of IgE class switch recombination. Curr Top Microbiol Immunol 2015;388:21–37. 18. Jakubzick CV, Randolph GJ, Henson PM. Monocyte differentiation and antigen-presenting functions. Nat Rev Immunol 2017;17:349–62. 19. Paller AS, Kabashima K, Bieber T. Therapeutic pipeline for atopic dermatitis: end of the drought? J Allergy Clin Immunol 2017;140:633–43. 20. Voss K, Larsen SE, Snow AL. Metabolic reprogramming and apoptosis sensitivity: defining the contours of a T cell response. Cancer Lett 2017;408:190–6. 21. Drake LY, Kita H. IL-33: biological properties, functions, and roles in airway disease. Immunol Rev 2017;278:173–84. 22. Muehling LM, Lawrence MG, Woodfolk JA. Pathogenic CD4+ T cells in patients with asthma. J Allergy Clin Immunol 2017;140:1523–40. 23. Xu M, Dong C. IL-25 in allergic inflammation. Immunol Rev 2017;278:185–91. 24. Mitchell PD, O’Byrne PM. Epithelial-derived cytokines in asthma. Chest 2017;151:1338–44. 25. Pociot F, Lernmark Å. Genetic risk factors for type 1 diabetes. Lancet 2016;387:2331–9. 26. Viatte S, Barton A. Genetics of rheumatoid arthritis susceptibility, severity, and treatment response. Semin Immunopathol 2017;39: 395–408. 27. Khan D. Pharmacogenomics and adverse drug reactions: primetime and not ready for primetime tests. J Allergy Clin Immunol 2016;138:943–55. 28. Li J, Maggadottir SM, Hakonarson H. Are genetic tests informative in predicting food allergy? Curr Opin Allergy Clin Immunol 2016;16:257–64. 29. Hirahara K, Nakayama T. CD4+ T-cell subsets in inflammatory diseases: beyond the Th1/Th2 paradigm. Int Immunol 2016;28:163–71. 30. Moldaver DM, Larché M, Rudulier CD. An update on lymphocyte subtypes in asthma and airway disease. Chest 2017;151:1122–30. 31. Ueno H, Banchereau J, Vinuesa CG. Pathophysiology of T follicular helper cells in humans and mice. Nat Immunol 2015;16:142–52. 32. Proekt I, Miller CN, Lionakis MS, et al. Insights into immune tolerance from AIRE deficiency. Curr Opin Immunol 2017;49:71–8.

CHAPTER 2  Adaptive Immunity 33. Huehn J, Beyer M. Epigenetic and transcriptional control of Foxp3+ regulatory T cells. Semin Immunol 2015;27:10–18. 34. Verbsky JW, Chatila TA. Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) and IPEX-related disorders: an evolving web of heritable autoimmune diseases. Curr Opin Pediatr 2013;25:708–14. 35. Roncarolo MG, Gregori S, Bacchetta R, et al. Tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications. Curr Top Microbiol Immunol 2014;380:39–68. 36. Tang Q, Vincenti F. Transplant trials with Tregs: perils and promises. J Clin Invest 2017;127:2505–12. 37. Lawrence MG, Steinke JW, Borish L. Basic science for the clinician: mechanisms of sublingual and subcutaneous immunotherapy. Ann Allergy Asthma Immunol 2016;117:138–42.

29

38. Palomares O, Akdis M, Martín-Fontecha M, et al. Mechanisms of immune regulation in allergic diseases: the role of regulatory T and B cells. Immunol Rev 2017;278:219–36. 39. Neuman G, Boodhan S, Wurman I, et al. Ceftriaxone-induced immune hemolytic anemia. Ann Pharmacother 2014;48:1594–604. 40. Ward PA, Fattahi F, Bosmann M. New insights into molecular mechanisms of immune complex-induced injury in lung. Front Immunol 2016;9:86. 41. Smith TJ, Hegedüs L. Graves’ disease. N Engl J Med 2016;375:1552–65. 42. Morimoto A, Nakazawa Y, Ishii E. Hemophagocytic lymphohistiocytosis: pathogenesis, diagnosis, and management. Pediatr Int 2016;58:817–25. 43. Ye Z, Lin Y, Cao Q, et al. Granulomas as the most useful histopathological feature in distinguishing between Crohn’s disease and intestinal tuberculosis in endoscopic biopsy specimens. Medicine (Baltimore) 2015;94:e2157.

CHAPTER 2  Adaptive Immunity

29.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following has a major role in the development of protective immunity induced by childhood immunizations? a. Eosinophils b. B cells c. NK cells d. Neutrophils e. Basophils 2. Which class of antibodies trigger a peanut allergic reaction? a. IgA b. IgM c. IgD d. IgE e. IgG

3. Which of the following immune responses is most responsible for dermatitis associated with poison ivy exposure? a. Th1 b. Th2 c. Th3 d. Tregs e. Th17 4. Which cytokines are involved in the development of immune tolerance to a specific antigen? a. IL-10 and IL-17 b. IL-4 and IL-13 c. TNF-α and TGF-β d. IL-10 and TGF-β e. IFN-α and TNF-α

3  Immunoglobulin Structure and Function James T. Li, Diane F. Jelinek

CONTENTS Introduction, 30 B Lymphocytes and the Humoral Immune Response, 30 Immunoglobulin Structure and Gene Rearrangement, 34

SUMMARY OF IMPORTANT CONCEPTS • Antibodies specific for antigen mediate a variety of biologic effects, including neutralization, activation of complement, and interaction with specific cellsurface Fc receptors. • To construct functional light and heavy Ig chain genes, the discontinuous DNA coding sequences must first be rearranged. • Isotype class switching is highly T cell dependent and is regulated through CD40 and the actions of cytokines. • Monoclonal and polyclonal Igs are used therapeutically.

INTRODUCTION The hallmark features of the adaptive immune response are the B and T lymphocytes and their ability to recognize specific antigens. T cells express the T cell antigen receptor (TCR) only as a transmembrane mole­ cule; B cells initially express immunoglobulin (Ig) as a transmembrane molecule and later as a secreted molecule after differentiation into plasma cells. A monomeric antibody molecule consists of two identical heavy chains (HCs) and two identical light chains (LCs). The B cell antigen receptor (BCR) complex includes a transmembrane antibody, which confers antigenic specificity, and requisite accessory signaling molecules. The humoral immune response exhibits remarkable diversity. Humans typically generate 10 million different antibodies and have the potential to generate billions, each specific for a particular target. The antibody is elegantly constructed in a manner that allows it to serve two complementary functions. One region of the molecule confers the capability to recognize and bind an enormous variety of antigenic determinants, and the other region is involved in mediating a variety of diverse, isotypedependent biologic effects of the Ig, such as complement fixation or antibody-dependent cellular cytotoxicity (ADCC). The means by which Igs recognize antigens are significantly different from TCR antigen recognition. Secreted or transmembrane Ig recognizes antigens in their native, properly folded form, whereas the TCR recognizes only antigens that have been processed and presented in the context of the major histocompatibility complex (MHC) by an antigen-presenting cell. B and T cells specific for the same antigen are most likely recognizing different epitopes on that antigen. Knowledge of the structural

30

Immunoglobulin Function, 38 Immunoglobulins and Human Disease, 41 Summary, 42

features of antibody molecules and the genetics underlying the expansive Ig repertoire is essential to understanding antigen-antibody interactions and immunoregulation. The study of hypersensitivity and immunodeficiency rightfully includes a review of antibody structural diversity and function.

B LYMPHOCYTES AND THE HUMORAL IMMUNE RESPONSE B and T cells develop from bone marrow hematopoietic stem cells. Throughout life, the human body produces millions of new B cells every day. In specialized bone marrow microenvironments, precursor B cells proceed through an orderly process of antigen-independent development and Ig gene rearrangement. Expression of a functional BCR is essential for B cell development, maturation, and release from the bone marrow. The BCR also plays a critical role during antigeninduced activation of mature B cells in secondary lymphoid tissues.1 After differentiation into antibody-secreting plasma cells, Ig molecules are largely expressed as secreted molecules, and the rate of antibody production significantly increases. Plasma cells are capable of secreting thousands of specific antibody molecules per second. Transmembrane and secreted forms of Ig from the same B or plasma cell exhibit the same antigenic specificity, but they are different at the carboxyl (C) terminus due to alternative messenger RNA (mRNA) splicing that results in the presence or absence of a hydrophobic transmembrane region (tail). This tail anchors Ig to the membrane, and its absence enables Ig secretion (Fig. 3.1). B cell differentiation into plasma cells is accompanied by the preferential processing of mRNA transcripts that encode the secreted form.

B Cell Receptor Structure and Signaling Although the transmembrane forms of all Ig HC isotypes possess cytoplasmic tails, they lack signaling motifs due to their short length (3 to 28 amino acids). This limitation is overcome by the association of one transmembrane Ig molecule with one disulfide-linked heterodimer consisting of two polypeptides, Igα (CD79a) and Igβ (CD79b) (Fig. 3.2). Igα and Igβ are transmembrane glycoproteins characterized by an extracellular and intracellular C-terminal cytoplasmic domain that is obligatory for BCR signaling.2 The cytoplasmic domain of each Igα and Igβ molecule contains an immunoreceptor tyrosine-based activation

31

CHAPTER 3  Immunoglobulin Structure and Function

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9'-

&µ

&µ

&µ

&µ 73 70±±±&
>> C4a

Increases vascular permeability, mast cell degranulation, chemotaxis

48-kD protein

C5aR

C5a, C5a desArg

Chemotaxis, mast cell degranulation, increases vascular permeability

43-kD protein

ICAM, Intercellular adhesion molecule; MBL, mannose-binding lectin.

Antibody and C3b act as opsonins to enhance phagocytosis

C5a recruits neutrophils to the site of bacteria C3

C3a, C4a, C5a: Increase blood flow Increase permeability

Fig. 8.7  Complement plays an important role in inflammation. The anaphylatoxic activities of the classical pathway are very important in the initiation of the inflammatory process. The purpose of inflammation is to deliver cells and proteins that might participate in host defense to the site: C3a, C4a, and C5a enhance blood flow and vascular leak; C3b and antibody on the surface of bacteria enhance phagocytosis; and C5a plays an important role in the recruitment of neutrophils through its chemotactic effects.

expressed on hepatocytes, in lung endothelium, vascular smooth muscle as well as umbilical cord endothelium, and on astrocytes, microglial cells, and T cells.17,18 In addition, C5a is the most powerful endogenous chemotactic factor for neutrophils and is a chemotactic agent for monocyte-macrophages.19 Thus, the early inflammatory response mediated by complement anaphylatoxins, bradykinin, and other small molecule mediators serves to deliver increased blood flow to the area, thereby increasing extravasation of antibody and complement into the tissues, and then C5a provides directionality for the recruited neutrophils (see Fig. 8.7). This is a very efficient system for dealing with localized infections; however, in overwhelming sepsis, high levels of serum C5a

can cause neutrophil aggregation, blockage of pulmonary vessels, and acute respiratory distress syndrome (ARDS). Overproduction of C5a in sepsis also can lead to inhibition of neutrophil function, which may partly explain the paradoxical immunocompromise seen in sepsis in the face of high levels of inflammatory mediators.20 The remaining complement receptors recognize either intact complement components or the large cleavage products. Several poorly defined C1q receptors have been described. Their function appears to relate to phagocytosis and clearance of apoptotic and cell debris. C1qRp (CD93) is expressed on myeloid cells, endothelium, and platelets and appears to be capable of also binding MBL,21 although its function has been called into question.22 Two proteins that bind C1q, CRT (cC1qR/ calreticulin receptor) and the mitochondrial protein gC1qbp, are primarily intracellular but can appear on the surface after myeloid cell stimulation.23,24 Finally, the complement receptor CR1 (discussed subsequently) can bind C1q, although the exact role of the interaction remains to be defined. In general, C1q interactions with its receptors lead to enhanced phagocytosis, enhanced clearance of apoptotic cells, increased respiratory burst, and improved microbicidal activity. CR1 (CD35) has been difficult to study because it is present only in primates. In primates, it is widely expressed and is found on mast cells, basophils, eosinophils, monocyte-macrophages, glomerular cells, B cells, follicular dendritic cells, some T cells, and erythrocytes. CR1 binds primarily C3b, C4b, and iC3b, although in some systems it also may bind C1q. Engagement of the receptor enhances phagocytosis, but its most important role appears to be in the binding of immune complexes. Immune complexes are extremely potent stimuli for inflammation mediated by Fc receptors and complement receptors. In a normal host, immune complexes are maintained in a soluble state by complement through the binding of C3b, and it is this C3b that is recognized by the CR1 receptor. Maintaining the immune complexes in a soluble state prevents their access to the proinflammatory Fc receptors and complement receptors. Erythrocytes are extremely numerous and constitute a nonresponsive “sink” for immune complexes because they are incapable of generating an inflammatory response. The erythrocytes bind the immune complex and transport it to reticuloendothelial cells of the liver and spleen, where it is degraded.25-27 Two other functions of the CR1 receptor deserve mention. CR1 has a regulatory role in the inactivation of C3 and the destabilization of the C3 and C5 convertase complexes. It is not clear how important this function is in the regulation of the complement cascade. The other function relates to antigen processing. B cells and other professional

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SECTION A  Basic Sciences Underlying Allergy and Immunology

antigen-presenting cells may process antigen differently when it is bound by complement, and this could theoretically affect antigen presentation. In a more specialized antigen uptake scenario, CR1-bearing follicular dendritic cells trap antigen, providing a stimulus for B cells.28,29 A more speculative role for CR1-bearing cells in the bone marrow is that they may become tolerized on exposure to apoptotic cells bound by complement. Defects in early complement components could lead to lupus as a consequence of defective tolerization to apoptotic antigens. The CR2 receptor (CD21) is the receptor for the Epstein-Barr virus and is expressed on B cells, follicular dendritic cells, epithelial cells, and some T cells.30 CR2 favors downstream degradation products of C3 such as C3d. Antigen bound by C3d, as would normally occur on an antigen after complement activation, is far more stimulatory for B cells than antigen that is not bound by C3d. The activation threshold for B cells is dramatically lowered when both the B cell receptor and CR2 are simultaneously engaged.31,32 Thus, patients with deficiencies in C3 are compromised in terms of ability to produce antibody in response to ongoing infections. This impaired response also is seen in patients with deficiencies of the classical pathway activation arm; however, because C3 may still be cleaved through the alternative and lectin activation pathways, the compromise in antibody production is less severe. CR3 (CD11b, CD18) and CR4 (CD11c, CD18) are both members of the β2 integrin family.33 These receptors bind iC3b and C3b. The CR3 receptor also binds C3d. Although clearly complement receptors, these receptors act primarily as adhesion molecules. CR3 is expressed on monocytes, neutrophils, natural killer cells, and microglial cells and can mediate phagocytosis of particles opsonized with C3b and adhesion through binding to its ligand, ICAM (intracellular adhesion molecule). The β2 integrin family has only three members. Each member utilizes the same β chain, giving the family its name. The second β2 integrin is the complement receptor CR4. The role of CR4 as a complement receptor is not well understood. The main roles of complement are opsonization, initiation of inflammation, clearance of apoptotic debris, lysis, and modulation of the adaptive responses. All three activation arms lead to opsonization, and the attachment of C3b or iC3b to a particle enhances phagocytosis through an interaction with the CR1, CR3, or CR4 receptor. Residual C1q may be bound by the CR1 receptor and also contribute to enhanced phagocytosis. The importance of C3 in host defense is clear from the extremely high frequency of infections seen in patients with C3 deficiency. The infections arise from an inability to opsonize, an inability to generate the C5a chemotactic factor, and the impaired antibody response resulting from poor antigen delivery and failure of B cell costimulation through the CR2 receptor. This opsonization occurs in tissue space, not in the intravascular space, and in fact, neutrophils have a safety mechanism such that they are relatively resistant to activation unless bound to a surface. Neutrophils must pass out of the vascular space by adhering to the vascular endothelium. Complement plays a role in the early inflammatory response, which aids in the recruitment of neutrophils. The release of the anaphylatoxins leads to blood vessel dilation to deliver more cells to the location and increased leakage of plasma proteins such as antibody and additional complement into the tissue space, and activation of mast cells and platelets initiates the complex adhesion process that directs neutrophils to the site. C5a acts to guide the neutrophils to the pathogen by acting as a chemotactic gradient, which the neutrophil senses with its C5a receptor. The neutrophil rapidly digests the pathogen, and for many pathogens, the arrival of the neutrophil is the end of the threat. Although the human body is constantly inundated with bacteria, this early defense system is so effective that the bacteria rarely have an opportunity to proliferate and establish a clinical infection. In certain infections, the

neutrophil may not be capable of killing the pathogen, or the number of pathogens may overwhelm the system. Persistent antigen bound by C3d can stimulate B cells to produce antibody, which would aid clearance in subsequent infections. Although the alternative pathway and lectin activation arms are not dependent on antibody, it is important for defense against bacteria in two ways. The classical pathway is the most efficient of the activation arms; thus, the presence of antibody to a pathogen maximizes the complement cascade. Second, antibody is itself a powerful opsonin. Most myeloid cells have Fc receptors, which also mediate phagocytosis. Dual engagement of Fc receptors and complement receptors by a phagocytic cell results in a synergistic enhancement of phagocytosis.

REGULATION OF COMPLEMENT ACTIVATION Unregulated complement activation would lead to host cell lysis, the inappropriate production of inflammatory mediators, and aberrant B cell activation. Nearly as much metabolic energy is spent in the regulation of the complement system as in production of the main constituents of the complement cascade. Traditionally, the regulators of complement are divided into fluid-phase regulators and membrane-bound regulators; although this division is somewhat arbitrary, it is the format used in this chapter (Table 8.2). Another categorization strategy groups the regulators by targeted component. For example, C3 is the most important complement component and the most tightly regulated component. C3 is regulated by factor H, factor I, properdin, C4 binding protein, membrane cofactor protein (MCP), CR1, CR2, and decay accelerating factor (DAF) (i.e., CD55). Several of these molecules have a conserved motif of 60 amino acids termed the short consensus motif and belong to the RCA gene cluster on chromosome 1 (Fig. 8.8). C1 inhibitor is perhaps the most clinically important of the regulatory proteins. Heterozygous deficiencies of C1 inhibitor lead to hereditary angioedema (HAE). C1 inhibitor is a serine protease inhibitor that inhibits both the low-level autoactivation of C1 and the fluid phase activation of C1 (Fig. 8.9). Immune complex activation of C1 is preserved and is not affected by C1 inhibitor. The mechanism by which C1 inhibitor regulates C1 is by binding to C1s and C1r, leading to dissociation from C1q. C1 inhibitor also regulates MASP-1 and -2 through a similar mechanism. C1 inhibitor has important roles inhibiting factor XII (Hageman factor) and prekallikrein of the contact system of coagulation. Activation of these two pathways is thought to be of paramount importance for the clinical manifestations of C1 inhibitor deficiency. C1 inhibitor has also action in fibrinolytic system. Other regulators of C1 have been described but are less well characterized. Certain defensins appear to inhibit activation of C1, and a molecule termed factor J has inhibitory activity. Their roles are not understood. C4 binding protein is another fluid phase regulator of complement and also shares the short consensus motif configuration of many of the complement regulators. It acts to displace C2a and dissociates the classical pathway convertase. In addition, C4 binding protein is a cofactor for factor I cleavage of C4b (Fig. 8.9). Factor I, alongside factor H, regulates the alternative pathway (Fig. 8.10). Factor H identifies nonactivator surfaces though the recognition of nonpathogen oligosaccharides and displaces Bb from C3b on those surfaces. Factor H also can prevent factor B from ever binding to C3b in the first place. Factor I inactivates C3b by cleaving it to iC3b, and its activity is enhanced in the presence of factor H. Together, these two regulators ensure that the spontaneous activation of the alternative pathway remains at a low level unless an activator surface is available to support a more intensive and sustained activation. Factor I also acts to inhibit C4b from the classical pathway. In this setting, it interacts with C4 binding protein and cleaves C4b into C4c

CHAPTER 8  The Complement System CR1

117

Factor H C3b

C4b

C4 binding protein

C3b CR2

C4b C3b

C3d X7

MCP

DAF

Lipid bilayer Fig. 8.8  Complement regulatory proteins. The regulators of complement proteins all are composed of short consensus repeats (circles) and encoded on chromosomal locus 1q32. The binding sites for various ligands are indicated in the figure. CR1, CR2, Complement receptors 1 and 2; DAF, decay accelerating factor; MCP, membrane cofactor protein; X7, unnamed beta chain.

TABLE 8.2  Complement Regulatory Proteins Protein

Localization

Function

Comments

C1 inhibitor

Serum

Binds to C1r and C1s and dissociates the C1 complex

105-kD protein

C4 binding protein

Serum

Cofactor for factor I; cleavage of C4b

550-kD protein

Factor I

Serum

Cleaves C3b and C4b

Factor H

Serum

Defines activator surface

S-protein

Serum

Inhibits the insertion of the membrane attack complex into the cell membrane

Also known as vitronectin

Decay accelerating factor (DAF)

Ubiquitous/cell membrane

Dissociates both C3 and C5 convertases

GPI-anchored CD55

Membrane cofactor protein (MCP)

Hematopoietic cells except erythrocytes

Cofactor for C3b cleavage by factor I

CD46

C8 binding protein

Most hematopoietic cells

Binds to C8 and prevents interaction with C9

GPI-anchored

CD59

Hematopoietic cells, endothelial cells, epithelial cells, glomerular cells

Inhibits the membrane attack complex

GPI-anchored Also known as HRF20

GPI, Glycosylphosphatidylinositol.

and C4d. C4 binding protein has an interesting structure and regulates the complement cascade as well as the coagulation cascade, as is true for C1 inhibitor. C4 binding protein binds circulating protein S (anticoagulation pathway) and inhibits it. One last fluid phase complement regulatory protein should be mentioned. S-protein (distinct from protein S) is an inhibitor of the membrane attack complex. A majority of terminal complement inhibitors are membrane-bound, and this soluble inhibitor is thought to act as

the nucleus of a back-up strategy to prevent inappropriate lysis of host tissue. The membrane-bound regulators of complement consist of the ubiquitous 70-kD molecule DAF (CD55), MCP (CD46), C8 binding protein, and CD59. DAF, C8 binding protein, and CD59 are glycosylphosphatidylinositol (GPI)-linked proteins, and MCP is an integral membrane glycoprotein. DAF is expressed on most cells, C8 binding protein is a 65-kD protein expressed on all hematopoietic cells, and

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Activators Immune complexes Apoptotic cells Chromatin CRP Oxidized LDL Serum amyloid P SIGN-R1

C1 complex

C4

C2

C4b

C2a C4b2a

C1 inhibitor

C3

C4b2a3b

C4 BP

C4 BP Factor I DAF MCP CR1 Fig. 8.9  Regulation of the classical pathway. The classical activation pathway is shown, with the relevant regulatory proteins shown in green hexagons. C4 BP, C4 binding protein; CR1, complement receptor 1; CRP, C-reactive protein; DAF, decay accelerating factor; LDL, low-density lipoprotein; MCP, membrane cofactor protein.

CD59 is a 20-kD protein expressed on all cells in contact with blood and certain epithelial cells and brain cells. DAF serves to dissociate the C3 convertase, and MCP serves as a cofactor for factor I cleavage of C3b and C4b (Fig. 8.11; see also Figs. 8.9 and 8.10). CD59 is an important protein that, along with S-protein, inhibits the membrane attack complex. CD59 accomplishes this by becoming incorporated into the accumulating membrane attack complex and inhibiting C9 binding. C8 binding protein functions similarly but acts by binding to C8. The S-protein acts in the fluid phase as an inhibitor of C5b and prevents insertion into the cell membrane. Several receptors also act as complement regulatory proteins, and in these cases, the regulatory activity may be viewed as termination of the specific complement function after the signal has been delivered. CR1 binds C3b and C4b and serves as a cofactor for factor I–mediated cleavage. CR1 can further support additional cleavage steps leading to C3dg, which can be cleaved by serine proteases to C3d, the fragment capable of costimulating B cells.34 Finally, CR1 accelerates the decay of both C3 convertases. CR2 has a similar role supporting cleavage of C3b.

DISORDERS ASSOCIATED WITH COMPLEMENT ACTIVATION Complement is easily triggered and despite a broad range of control strategies can “feed back” on itself, engendering significant harm. The primary examples of wholesale systemic complement activation are post–cardiac bypass syndrome and immune complex diseases. In post– cardiac bypass syndrome, the bypass circuitry is an activator surface, and the alternative pathway is fully activated.35 This leads to activation of the kinin system, fibrinolysis, platelet activation, and the release of large amounts of C5a. C5a can directly stimulate release of tumor necrosis factor (TNF) and interleukins IL-1, IL-6, and IL-8, further fueling the inflammatory process. Neutrophils bind the C5a and become activated, forming aggregates. These aggregates interfere with circulation (often in the lung) and can release reactive oxygen species, as well as damaging lysosomal enzymes. The systemic consequences of this process are neutropenia and thrombocytopenia secondary to trapping, end organ dysfunction resulting from impaired circulation, and hypotension mediated by the action of the anaphylatoxins. In several preclinical trials,

CHAPTER 8  The Complement System

119

Activators Spontaneous activation Polysaccharides Endotoxin Nephritic factors Medical instrumentation

C3

C3(H2O)B Factor D

C3bBb

C3b

C3b2Bb

Properdin

Factor H Factor I DAF MCP CR1

Fig. 8.10  Regulation of the alternative pathway. The alternative activation pathway is shown, with the relevant regulatory proteins shown in the green hexagon. Hydrolyzed C3 is required to initiate the cascade. C3bBb is stabilized by properdin. CR1, Complement receptor 1; DAF, decay accelerating factor; MCP, membrane cofactor protein.

administration of a C5 inhibitor reduced mortality and morbidity in patients after cardiac bypass. Serum sickness is uncommon; however, it can occur when soluble antigen is produced at high levels (hepatitis C), when an antigen is administered (OKT3 administration), or when antibodies develop to a medication. Immune complexes contribute to the clinical features of a variety of autoimmune disorders in which autoantibodies are produced. The classic manifestations of serum sickness relate to the deposition of immune complexes onto vascular beds with either high oncotic pressure or tortuosity. Classic serum sickness consists of a vasculitic rash, glomerulonephritis, and arthritis. Immune complexes normally are maintained in a soluble state by C3. Recognition of C3b by CR1 allows the immune complexes to travel to the Kupffer cells of the liver via erythrocytes, where the immune complexes are taken up and degraded. Fc receptors in the liver and spleen also contribute to clearance. When the clearance pathway is overwhelmed, immune complexes deposit on endothelial cells wherever pressure or tortuosity allows (usually skin, glomeruli, and synovium). The deposited immune complexes initiate an inflammatory response by interacting with Fc receptors on the endothelial cell. Preexisting antibody can mediate an immediate response to the antigen, with abrupt onset of serum sickness. When a neoantigen is administered, initially no response is seen. After 10 to 14 days, however, an antibody response develops and the initial clinical signs of serum sickness appear. An urticarial or vasculitic rash often is the first sign, followed by adenopathy, fever, arthritis, and proteinuria.36,37 Laboratory studies demonstrate hypocomplementemia secondary to consumption and a leukocytoclastic vasculitis with deposition of C3, IgM, and IgG.

There are four disorders associated with limited complement activation from stabilization of complement components by autoantibodies. Nephritic factor is a set of autoantibodies that stabilize C3 convertases. The initial nephritic factor described was an autoantibody directed against C3bBb, the alternative pathway convertase. This nephritic factor stabilizes the active form and resists decay mediated by factors H and I. Thus the activity of the alternative pathway is prolonged in the presence of nephritic factor. The vast majority of patients with the classic C3bBb nephritic factor have membranoproliferative glomerulonephritis.38 Other types of nephritic factors have been described, including antibodies that stabilize the classical pathway convertase. Nephritic factors in general are associated with lipodystrophy and membranoproliferative glomerulonephritis. Nearly 80% of patients with dense deposit disease or membranoproliferative glomerulonephritis type II have a nephritic factor. Type III membranoproliferative glomerulonephritis is associated with a nephritic factor that stabilizes the alternative pathway convertase and the C5 convertase in 70% of the patients. Type I disease is associated with various nephritic factors in only 15% of cases. The age at onset typically is in late childhood or adolescence. The lipodystrophy is characterized by the progressive loss of subcutaneous tissues, leading to a sunken-eyed appearance. Often the fat below the waist is spared. The renal involvement may either precede or postdate the lipodystrophy by many years. A low C3 is seen, with preservation of C4 because the defect is distal to C4. The pathogenetic mechanism of the lipodystrophy involves lysis of factor D producing adipocytes.39 Why some regions of the body are spared is not understood. The pathomechanism underlying the membranoproliferative glomerulonephritis is not completely

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Activators Carbohydrates Agalactosyl IgG

MBL complex

C4

C2

C4b

C2a C4b2a

C4 BP Factor I DAF MCP CR1

C3

C4b2a3b

C4 BP CR1

Fig. 8.11  Regulation of the lectin activation pathway. The lectin activation pathway is initiated by mannosebinding lectin (MBL) and utilizes regulatory proteins that are nearly identical to those of the classical pathway. The regulatory proteins are shown in green hexagons. C4 BP, C4 binding protein; CR1, complement receptor 1; DAF, decay accelerating factor; IgG, immunoglobulin G; LDL, low-density lipoprotein; MBL, mannosebinding lectin; MCP, membrane cofactor protein.

understood. The nephritic factor may act as an immune complex and simply incite inflammation in the glomerulus as a consequence of its deposition. The fact that factor H deficiency can be associated with a similar phenotype suggests that there may be something specific about partial activation of the complement cascade in glomeruli. Renal podocytes have several complement receptors. Antibodies to C1q were first described in patients with hypocomplementemic urticarial vasculitis.40,41 They have since been described in patients with SLE. These IgG antibodies are directed against the collagenlike region of C1q. In lupus, the anti-C1q antibodies are preferentially found in renal tissue, and the presence of anti-C1q in the serum of patients is a very strong predictor of renal disease.42,43 The antibody appears to amplify any other injury to the glomerulus. In experimental animal models, antibodies to C1q caused pathologic changes only after preexisting injury. In this disorder, glomerulonephritis is seen; however, cutaneous manifestations such as urticaria and angioedema are the most prominent findings. Ocular inflammation also is common, and in patients who smoke, an aggressive obstructive lung disease is seen.44 Antibodies to factor H and C1 inhibitor, two complement regulatory proteins, have been described and cause significant disease. The features of the diseases are comparable to the inherited deficiencies of those proteins and are covered in the following section. In addition to systemic activation of the complement cascade, local activation can be associated with tissue damage and serious pathologic consequences. Pregnant women with antiphospholipid antibodies or antibodies associated with SLE are at risk for preeclampsia. In this condition, autoantibodies initiate complement-mediated damage to the placenta, leading to placental insufficiency, fetal growth retardation, and in some cases, fetal loss.45 Similarly, complement activation contributes to the pathophysiologic manifestations of burn injuries, pancreatitis, or crush injuries. In these cases, the tissues themselves are

altered so as to become activator surfaces. In the case of burns, early colonization of the wound with bacteria also contributes to complement activation.

DISORDERS ASSOCIATED WITH COMPLEMENT DEFICIENCY Acquired and inherited deficiencies of complement can have significant biologic effects or have very minor consequences. This section presents descriptions of various specific deficiencies (Table 8.3). In most cases, it is possible to predict the effects of a particular deficiency based on understanding the normal physiologic function of that protein. For example, the early classical pathway components function to aid apoptotic cell clearance, to activate C3, and to produce anaphylatoxic activity. The main phenotype associated with defects in C1, C2, and C4 is SLE. There is also an increase in the risk of infection, and atherosclerosis may be accelerated. The terminal component deficiencies lead to impaired lysis of gram-negative bacteria, and the predominant manifestation is an increased risk of neisserial disease. The vast majority of genetically determined complement deficiencies are inherited in an autosomal recessive fashion. Exceptions are properdin deficiency, which is X-linked, and C1 inhibitor deficiency, which is autosomal dominant. Most of the inherited complement disorders typically are associated with a very low CH50 or AH50. One CH50 unit is the volume of serum that lyses 50% of sensitized sheep erythrocytes in the reaction mixture for the classical activation pathway; likewise one unit in the AH50, an analogous test using rabbit or chicken cells to measure alternative pathway function, defines the volume of serum leading to 50% lysis. Specific component deficiencies can be specifically defined in reference laboratories using a complementation system whereby patient sera are added to mixes generated with a single missing

CHAPTER 8  The Complement System

TABLE 8.3  Inherited Complement

Deficiencies Deficiency

Clinical Features; Diagnostic Strategy

C1q

SLE, infections; CH50 near zero

C1r/s

SLE, infections; CH50 near zero

C4

SLE, infections; CH50 near zero

C2

SLE, infections, some asymptomatic; CH50 near zero

C3

Infections frequent and severe, glomerulonephritis; CH50 near zero

Factor D

Neisseria; AH50 near zero

Factor B

Neisseria; AH50 near zero

Properdin

Neisseria; AH50 diminished

MBL

Most asymptomatic infections, SLE; CH50 normal, MBL assay required

C5

Neisseria; CH50 near zero

C6

Neisseria; CH50 near zero

C7

Neisseria; CH50 near zero

C8

Neisseria; CH50 near zero

C9

Neisseria; CH50 diminished

Factor I

Neisseria, HUS; C3 may be diminished, many require mutation analysis

Factor H

Neisseria, HUS; C3 may be diminished, many require mutation analysis

MCP

HUS; mutation analysis required

C1 inhibitor

Angioedema; C1 antigen and functional levels

CR3/CR4

Leukocyte adhesion deficiency, very severe systemic infections, lack of pus; flow cytometry

CD59

Paroxysmal nocturnal hemoglobinuria; flow cytometry

AH50, Serum dilution that lyses 50% of a rabbit red cell suspension; CH50, serum dilution that lyses 50% of a sensitized sheep red cell suspension; HUS, hemolytic uremic syndrome; MBL, mannosebinding lectin; MCP, membrane cofactor protein; SLE, systemic lupus erythematosus.

component. Lysis is achieved except when the component missing in the patient is identical to the component missing in the lytic mix. Further studies may be done if required to define the mutation; however, this is seldom of any clinical utility. Identifying deficiencies of regulatory proteins is much more difficult, because mutations must be specifically sought in most cases. C1 inhibitor assays are widely available and are quite reliable, but for other complement regulatory proteins, the assays infrequently are available and diagnosis often relies on direct mutation detection.

C1 Deficiency Patients with C1q deficiency present almost uniformly with earlyonset SLE. C1q deficiency is the strongest known genetic risk factor for lupus. The manifestations of lupus in these individuals are similar to those in non–complement-deficient individuals. The autoantibody profile is similar, although anti-dsDNA antibodies may be somewhat less common.46,47 Clinically, the features have more dramatic cutaneous and CNS manifestations than in the typical patient with lupus, and the disease may be more severe. As supported by data for a small number of patients, the lupus seen in C1q-deficient individuals also is thought to be less steroid-responsive, with an earlier age at onset. Patients with C1q deficiency also report an increased rate of infection;

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this susceptibility no doubt relates to compromised opsonization and a mild decrease in B cell costimulation. Only a few patients with inherited deficiencies of C1r and C1s have been described.48-50 It is thought that neither component is stable without the other, so that a mutation in one often leads to diminished levels of both. Glomerulonephritis and lupus have been reported in C1r/C1sdeficient patients. The limited number of affected patients precludes definition of a clear phenotype.

C4 Deficiency The two genes for C4, C4A and C4B, are highly homologous, although C4A binds more avidly to protein, whereas C4B binds more avidly to carbohydrate. Within each C4 gene, there can be deletions or duplications or simple inactivating mutations.51,52 Thus interpretation of a serum level is difficult.53 A low C4 level may be related to consumption or to the inheritance of an inactive allele. Partial C4 deficiencies are extremely common. Approximately 1% to 2% of the general population and up to 15% of patients with SLE have complete C4A deficiency. Although C4A deficiency is a risk factor for SLE, the disease is often milder in patients with C4A deficiency than in complement-sufficient hosts. Approximately 1% to 2% of the population has complete C4B deficiency, but up to 15% of patients with invasive bacterial disease are deficient.54 In contrast to the common partial deficiencies, complete C4 deficiency caused by all inactive alleles is quite rare. Well over 50% of the C4-deficient individuals have SLE.55 The cutaneous manifestations are common and severe, and the age at onset is usually early. The usual array of autoantibodies are found, although anti-dsDNA antibodies may be somewhat less frequent in C4-deficient individuals compared with normal hosts. Infection appears to be a significant problem for patients with C4 deficiency, and infection is the major cause of death.56,57 The mechanisms underlying the predisposition to infection probably are related to impaired opsonization and a modestly compromised B cell response to antigen. Each of these defects is partial, because C3 may still be cleaved via the alternative pathway. The association with SLE is due to compromised clearance of apoptotic debris and impaired B cell tolerance. Persistence of apoptotic cells, with nuclear antigens displayed on the surface, can act as an antigen depot.

C2 Deficiency C2 deficiency is one of the more common complement deficiencies. Although rare in some ethnic groups, in whites it is found with a frequency of 1/10,000. Most C2-deficient individuals are asymptomatic, in contrast with those patients with C4 and C1 deficiency. Approximately half of C2-deficient individuals will develop lupus.56-58 The age at onset is early adulthood, as is true for SLE in the general population. Cerebritis, nephritis, and arthritis are less common in C2-deficient SLE patients than in the typical SLE patient; however, anti-Ro antibodies are extremely common in C2-deficient patients with SLE. Anti-dsDNA antibodies are infrequent. Other autoimmune disorders have been described in patients with C2 deficiency, although an element of ascertainment bias may be present in such cases, because CH50 assays are commonly run on patients with autoimmune disorders. Infections are increased in C2-deficient individuals, as would be expected, and the most common cause of death among C2-deficient patients is sepsis.58 Two-thirds of C2-deficient patients have a history of invasive bacterial disease.59 Other systemic infections such as meningitis, pneumonia, epiglottitis, and peritonitis have been described, and the most common organisms have been S. pneumoniae and H. influenzae. With improved vaccination against these organisms, the infection pattern could be improved. A third phenotype, which was identified only epidemiologically, is the presence of accelerated atherosclerosis in C2-deficient individuals. Although this phenotype was defined

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in this population, it is likely that all patients with early complement component deficiencies have a similar phenotype, because subsequent to the epidemiologic identification of accelerated atherosclerosis, studies have demonstrated an important role for complement cascade proteins and complement regulatory proteins in the regulation of low-density lipoprotein (LDL) and vascular injury.60

C3 Deficiency With C1, C4, and C2 deficiency, the phenotypic susceptibility to infection is not fully penetrant, and certain patients exhibit no obvious increase in infections. C3 deficiency is the rarest of the four early component deficiencies, with the most severe disease phenotype by far. Membranoproliferative glomerulonephritis is seen instead of lupus in approximately a third of the cases of C3 deficiency.56,57 All patients have a profound predisposition to infection, and the infections are sometimes characteristic of neutrophil dysfunction (abscesses), humoral deficiencies (sinopulmonary disease), and complement deficiencies (sepsis, meningitis). These types of infections reflect the various roles of C3. Staphylococcal abscesses probably reflect an inability to opsonize and to generate the C5a chemotactic factor. Recurrent sinopulmonary infections reflect a significant compromise in B cell costimulation. Systemic infections are due to a complete failure of C3b opsonization. These multiple defects contribute to the severe infectious manifestations of C3 deficiency.61 One other feature of C3 deficiency is unique. During infections, a vasculitic rash may appear and symptoms of serum sickness may occasionally be seen. These unusual findings are due to the lack of immune complex solubilization by C3. They typically are transient in nature but can cause confusion with lupus, particularly in the presence of glomerulonephritis. C3 deficiency is rare, with fewer than 30 cases reported in the literature. There is a founder effect in South Africa among the Afrikaansspeaking population.62 Slightly more common is a partial deficiency of C3, termed hypomorphic C3.63,64 This partial deficiency has been seen in a number of autoimmune disorders, but it is difficult to diagnose and probably is underascertained. Rare activating mutations of C3 are associated with atypical HUS.65

Mannose-Binding Lectin Deficiency MBL deficiency originally was identified in a cohort of hospitalized patients with a variety of infectious diseases ranging from tuberculosis to sepsis. It is now known that MBL deficiency is quite common, with a frequency of 2% to 7% in the general population.66 In this disorder, common structural mutations destabilize the higher-order complexes, leading to accelerated clearance and poor function.67 In addition, several promoter mutations lead to impaired production. Each type of mutation has a characteristic effect on function and serum levels, and because each one is relatively common, various combinations of mild mutations may be associated with complete loss of function. A large number of studies have attempted to characterize the phenotype of MBL-deficient individuals.66 MBL deficiency is not typically associated with absent levels, and it has been difficult to define the normal range in healthy people. The deficiency appears to represent a modest risk factor for infection, typically revealed in a high-risk setting. Similarly, it may subtly alter the course or contribute to the overall risk profile in common variable immunodeficiency, cystic fibrosis, hepatitis, and others.

Ficolin-3 Deficiency Ficolin-3 binds enteric bacteria and activates the lectin pathway. Individuals with ficolin-3 deficiency related to a homozygous frameshift mutation have been described.68 The clinical features were inconsistent, and one patient was asymptomatic. The allele frequency of the variant

is 0.01-0.02. Thus the clinical relevance of this deficiency is incompletely understood.

Mannose-Binding Lectin–Associated Serine Protease-2 Deficiency MASP-2 deficiency initially was described in a patient with serious infections and autoimmune disease.69 Subsequently, an association with pneumococcal infection came to be recognized. Asymptomatic individuals have been described, and it is now known to occur with a frequency of 6/10,000, suggesting that the phenotype is mild.70,71

Mannose-Binding Lectin–Associated Serine Protease-1 and -3 and Collectin Kidney-1 Deficiencies MASP-1 and MASP-3 are produced from splice variants of the MASP1 gene. The COLEC11 gene encodes the structurally and functionally related collectin kidney-1 protein. These three proteins appear to function in the lectin activation pathway, but deficiencies of these three proteins have now been associated with the 3MC syndrome, comprising the Malpuech, Michels, and Mingarelli-Carnevale (obstructive sleep apnea) syndromes.72,73 The clinical manifestations of the 3MC syndrome consist of developmental delay, facial dysmorphia, and various skeletal anomalies. The proposed mechanism was revealed by an unexpected role for these proteins in cuing neural crest cell migration.73

Factor B Deficiency Few cases of factor B deficiency have been reported.74 Two patients were identified after developing neisserial disease, and laboratory studies revealed an AH50 with nearly absent hemolysis. One other patient had aseptic meningitis. Gain of function mutations in factor B is associated with atypical hemolytic uremic syndrome (HUS).

Factor D Deficiency Neisserial infections are the most common manifestation of factor D deficiency.56,57,75 Systemic streptococcal infections have also been seen. Other complement levels typically are normal in factor D deficiency; however, the ability to activate the alternative pathway is minimal to absent.

Properdin Deficiency Properdin deficiency is the only X-linked complement deficiency. It is one of the more common complement deficiencies among Caucasian individuals. Approximately half of the properdin-deficient individuals have had one or more episodes of meningococcal disease. Other bacterial infections are also seen but are much less common. There is a particularly high fatality rate for meningococcal disease in properdindeficient patients, in contrast with the protection from early death seen in patients with terminal complement component deficiencies. This protection afforded by other complement deficiencies is thought to be due to diminished inflammation in the setting of deficient complementderived mediators, however, it is not clear why this would not be true for properdin deficiency as well. A founder effect in Tunisian Jewish people has been suggested; however, properdin deficiency is seen in persons of all ethnic backgrounds. The role of properdin is to stabilize C3bBb, and in the absence of this stabilizing function, activation of the alternative pathway is impaired.

C5 Deficiency C5 is the major endogenous chemotactic factor for neutrophils. Of note, however, C5-deficient patients have the same phenotypic susceptibility to neisserial infections as described for the other terminal component deficiencies.56,57,76 C5 deficiency is found in persons with a variety of ethnic and racial backgrounds. Although it has been detected in

CHAPTER 8  The Complement System patients with SLE and other autoimmune disorders, little rationale can be mustered for a mechanistic relationship, and these findings may represent ascertainment bias. C5 does play an important role in the defense against Neisseria, and the relationship of C5 deficiency to meningococcal and gonococcal disease is more assured.

C6 Deficiency C6 deficiency is one of the more common complement disorders and occurs more frequently in African Americans and in people from South Africa. As is true for the other terminal component deficiencies, C6 deficiency is known to be associated with meningococcemia, meningococcal meningitis, and disseminated gonococcal disease. The occasional reports of C6 deficiency associated with autoimmune disease are likely to represent ascertainment bias. Two variations on C6 deficiency have been described. In one case, a splice defect leads to a smaller than usual protein, C6SD.77 This protein functions less efficiently than wild-type C6; however, it is not clear whether bearing C6SD leads to compromised host defense. The other variation is combined C6 and C7 deficiency.78

C7 Deficiency C7 deficiency is not particularly common, and in the few reported cases, the clinical presentations have varied. The most common presentation was neisserial disease.

C8 Deficiency C8 is composed of three chains: α, β, and γ. The α and γ chains become covalently attached during synthesis and bind to the β chain. Of interest, it is the α and β chain genes that are in linkage disequilibrium and map to chromosomal locus 1p3.2. C8β deficiency is more common in Caucasians, whereas C8α-γ deficiency is more common among African Americans.79-82 A majority of the C8β mutations are due to a singlebase-pair transition leading to a premature stop codon. The most common C8α-γ mutation is caused by a 10-base-pair insertion leading to a stop codon. Regardless of the genetic basis, all types of C8 deficiency are associated with diminished bactericidal activity in vitro and clinical susceptibility to neisserial disease.56,57 Meningococcal meningitis, meningococcemia, and disseminated gonococcus have been described in affected patients. Rarely, C8 deficiency has been identified in a patient with SLE or other autoimmune disorder. The relationship of C8 deficiency to autoimmunity is not certain.

C9 Deficiency C9 deficiency is seen with high frequency in Japan and Korea.83,84 Approximately 1 patient in 1000 carries a homozygous common nonsense mutation.85 It is less often seen outside of Japan; however, it is more difficult to diagnose than most of the other complement deficiencies because the CH50 is diminished but not absent. Because lytic activity can be generated in the absence of C9, the CH50 typically is one-third to half of normal in patients with C9 deficiency. This CH50 level typically would not lead to further evaluation, because mildly diminished levels are commonly seen when the sample is poorly handled or in the presence of active disease leading to complement consumption. As is true for the other terminal complement component deficiencies, C9 deficiency is associated with neisserial disease, although the penetrance appears to be less than that with other terminal component deficiencies.86

C1 Inhibitor Deficiency HAE is the clinical entity that has been recognized in patients with heterozygous deficiencies of C1 inhibitor. Two kindreds with homozygous C1 inhibitor deficiency have been described, and these patients

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exhibited more extreme features of the disease.87,88 Patients with C1 inhibitor deficiency may have a mildly increased susceptibility to infection and have been clearly demonstrated to be at increased risk for development of SLE, presumably owing to chronic consumption of C2 and C4. The most common clinical presentation is angioedema. The angioedema has no distinguishing features. It is not associated with urticaria, although it often is preceded by a lacey reticular rash sometimes called serpiginous erythema. The historical features most helpful in identifying potential C1 inhibitor–deficient individuals are recurrent episodes of angioedema, involvement of the airway in the absence of anaphylaxis, a positive family history, and relationship to antecedent trauma. The focus of the following discussion is on inherited C1 inhibitor deficiency; a brief description of the acquired form also is included. C1 inhibitor deficiency often is categorized as type I or II (or, less commonly, III or with normal C1-INH). Type I deficiency, characterized by a concomitant decrease in protein levels and function, is the most common, accounting for approximately 85% of the inherited cases. Type II deficiency is associated with production of a quantitatively normal but dysfunctional protein. In patients with type II deficiency, the serum levels of C1 inhibitor detected antigenically are normal or elevated, although the function is diminished. For this reason, it is recommended that both antigenic and functional levels be obtained in testing for C1 inhibitor deficiency. A typical functional level is approximately 25% to 40% of normal in both types. Because this typically is a disorder due to heterozygous mutations, a level of 50% would be expected. If the functional allele produced normal amounts of C1 inhibitor, the serum level should be half that expected if two alleles were functional. The explanation for the lower-than-expected level of C1 inhibitor appears to be altered catabolism. The main manifestations of C1 inhibitor deficiency are recurrent episodes of submucosal or subcutaneous edema. Half of the patients have experienced episodes before the age of 10 years. Approximately 5% of people who carry a C1 inhibitor mutation are asymptomatic. The episodes may be as infrequent as one per year or as frequent as one per month. The frequency and the severity of episodes do not correlate with laboratory features and often are inconsistent within a family. The extremities, face, and genitalia are most often involved. Involvement of the gastrointestinal tract can lead to disabling abdominal pain. Abdominal episodes begin with pain, often accompanied by vomiting and more rarely by diarrhea. In one study, one-third of patients with C1 inhibitor deficiency had undergone an appendectomy or exploratory laparotomy for abdominal pain. The most feared type of angioedema is that involving the airway. Although the lungs are not involved, the upper airway can swell, leading to respiratory arrest. This complication can occur in as many as two-thirds of the patients with C1 inhibitor deficiency, although improved management has made it somewhat less common. Before the advent of modern management, slightly more than 10% of patients underwent a tracheostomy to manage airway episodes. The angioedema typically progresses for 1 to 2 days and resolves in another 2 to 3 days. Common precipitants are infections, hormonal fluctuations, trauma, and stress. Although many patients can identify triggers, many episodes have no identifiable trigger, which increases anxiety and contributes to feelings of loss of control. The C1 inhibitor promoter is androgen-responsive, which is why men have fewer problems in general than female patients.89-91 Androgen sensitivity also may explain the common complaint that symptoms vary with menstruation and provides the rationale for the use of attenuated androgens. The mechanism underlying the angioedema relates to the role of C1 inhibitor as an inhibitor of both the classical complement pathway activation and mainly the kinin pathway92 (Fig. 8.12). Treatment is therefore directed at either correcting the level of C1 inhibitor or interfering with kinin effects.

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SECTION A  Basic Sciences Underlying Allergy and Immunology

C1 inhibitor

Baseline complement activation

Factor XII activation

C2b Plasmin

Kallikrein

C2-kinin

Bradykinin

Angioedema

Fig. 8.12  The role of C1 inhibitor. C1 inhibitor deficiency is thought to lead to angioedema through loss of inhibitory activity for the intrinsic coagulation pathway. Factor XII (Hageman factor) is activated by negatively charged molecules, and this is most often the case when blood vessels are damaged and collagen is exposed. Factor XII activation leads to the activation of bradykinin, which is one of the most potent vasodilators known. In addition, bradykinin leads to vascular leak and hence angioedema. This pathway is thought to be the most important for the development of angioedema; however, a cleavage product of C2b, C2-kinin, is produced by plasmin. Plasmin is itself activated by factor XII. The C2-kinin has some effect on vasodilation.

Management typically is divided into four areas of intervention (Table 8.4). Ideally, the episodes of angioedema are prevented. The most common strategy for prevention had been the use of attenuated androgens, recently limited to the highest dosage of 200 mg per day. Tranexamic acid also has a long history of use, but it has limited efficacy. C1 inhibitor concentrate is licensed for use in Europe and the United States as a prophylactic agent. The use of C1 inhibitor has been associated with a markedly improved pattern of angioedema; however, it must be given intravenously twice a week when used as prophylaxis, which has limited its use to severely affected patients who are motivated and have the financial means. The newest agent is lanadelumab, a kallikrein inhibitor. This is give every two weeks as a subcutaneous injection. Short-term prophylaxis is used for dental procedures, surgical procedures, endoscopies, or other situations in which significant trauma may be expected. Attenuated androgens can be used for this indication; however, C1 inhibitor concentrates have largely supplanted androgens in this setting. Fresh frozen plasma (FFP) is another alternative in this setting. In addition to both long-term and short-term prophylaxis, treatment of an acute episode is often indicated. Despite prophylaxis, breakthrough episodes do occur. Episodes also occur in people who may not be on any active prophylaxis. Finally, acute episodes arise in the undiagnosed patient or in noncompliant patients. By general consensus, corticosteroids, epinephrine, and antihistamines are recognized to have no effect. Supportive care and close observation are essential,

because pharyngeal swelling can progress to airway compromise in a few hours. Narcotics are appropriate for management of abdominal pain episodes. For addressing the angioedema, numerous options exist. C1 inhibitor concentrate, a kallikrein inhibitor, ecallantide, and a bradykinin B2 receptor antagonist, icatibant are available for acute treatment.93-95 Each has benefits and limitations, and management should be tailored to the patient’s preference and circumstances. New drugs are under development and tested in protocols for prophylaxis. The last category of management is fertility and obstetric management. Polycystic ovary syndrome is seen in approximately a third of female patients with C1 inhibitor deficiency regardless of prior therapy. The typical endocrine findings of increased luteinizing hormone and testosterone are lacking. Ultrasound images demonstrate polycystic ovaries, and the only laboratory abnormality often is reduced levels of follicle-stimulating hormone. Menstrual irregularities are common, and the underlying pathogenesis involves the aberrant regulation of complement activation in follicular fluid.96 Unexpectedly, attenuated androgen therapy leads to lessened cyst production in ovaries. It is not yet known whether C1 inhibitor administration decreases the ovarian cystic changes, but estrogen-containing birth control pills are contraindicated in hereditary angioedema (HAE). Despite common menstrual irregularities, fertility is largely preserved, and pregnancy poses a particular risk of harm for both the mother and her fetus. The hormonal shifts of pregnancy lead to an increased risk of angioedema, although late pregnancy seems to offer some protection. An affected mother has a 50% chance of transmitting the disorder to her offspring. Potentially both mother and child could be at risk during delivery. C1 inhibitor can be administered to minimize associated risks, although vaginal delivery is not usually associated with flare. A third type of HAE referred to as HAE with normal C1 inhibitor, previously called type III, is not a complement deficiency and is mentioned here for completeness. It is characterized by normal serum levels and functional activity of C1 inhibitor. It has been described primarily in women and is associated with mutations in factor XII of the coagulation pathway in approximately 20% of the cases.97,98 Angiopoietin-1 mutations have also been described. Acquired C1 inhibitor deficiency is clinically indistinguishable from inherited C1 inhibitor deficiency except that onset is after 30 years of age.99 A distinction between acquired disease due to lymphoreticular malignancy and acquired disease seen in association with autoimmune disease has not proved practical, because anti-C1 inhibitor antibodies have been found in both. The laboratory features are similar to those of hereditary C1 inhibitor deficiency, except that C1q levels are diminished in these patients. All patients with acquired C1 inhibitor deficiency require careful surveillance for malignancy. B cell malignancies and monoclonal gammopathies are the most common. Only 14% of patients with acquired C1 inhibitor deficiency had no associated medical condition in one study. Malignancy, infection, and autoimmune disease were most commonly reported. Treatment for acquired C1 inhibitor deficiency is slightly different in that attenuated androgens are seldom helpful. C1 inhibitor has been used successfully, although the increased catabolism often mandates higher doses. Rituximab has been used successfully to treat the autoantibody and improve the angioedema. Antifibrinolytics have a long record of use in acquired C1 inhibitor deficiency but are difficult to obtain in the United States. Ecallantide and icatibant may have roles in the treatment of acquired C1 inhibitor deficiency.

C4 Binding Protein Deficiency A single kindred with C4 binding protein deficiency has been described.100 The proband presented with angioedema, vasculitis, and arthritis. The manifestations were thought to relate to uncontrolled activation of the classical pathway and release of anaphylatoxins.

CHAPTER 8  The Complement System

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TABLE 8.4  Therapeutic Options for C1 Inhibitor Deficiency* Treatment

Adult

Pediatric

Comments

Tranexamic acid (Cyclokapron)

1-3 g/day PO in divided doses for prophylaxis

25-50 mg/kg bid-tid as prophylaxis; 1.5 g/day for acute episodes (available as IV form)

Not available in the United States

Epsilon-aminocaproic acid (Amicar)

1 g PO tid as prophylaxis, 1 g/h as IV therapy for acute attacks

100 mg/kg q4-6h not to exceed 30 g/day as therapy. Oral syrup available for prophylaxis but doses not established; 6 g/day for children 11 y have been used successfully

The only antifibrinolytic available in the United States Has modest efficacy Cannot be used in neonates Oral dosing associated with significant GI side effects

Danazol (Danocrine)

200 mg PO qd as a starting point for prophylaxis (titrate to effect); 200 mg/day for short-term prophylaxis. It is not recommended for acute attacks.

50-200 mg PO qd as a starting point for prophylaxis (titrate to effect); consider qod or every 3 days in preadolescent children Can use up to 400 mg PO qd for short-term prophylaxis

Use of attenuated androgens in children limited by concerns about androgenization and premature closure of the epiphyses

Oxandrolone (Oxandrin)

2.5-20 mg PO tid as prophylaxis (titrate to effect); not proven effective for short-term prophylaxis or treatment

0.1 mg/kg/day as prophylaxis Not proven effective for short-term prophylaxis or treatment

Has less androgenizing effects than Danazol

Fresh frozen plasma (FFP)

2 U IV as short-term prophylaxis May be required for up to 36 hours after surgery

10-15 mL/kg as short term prophylaxis May be required for up to 36 hours after surgery

Not typically used for acute episodes owing to danger of accelerating angioedema; useful for short-term prophylaxis for surgery or dental extractions

C1 inhibitor concentrate

1000 U twice a week as prophylaxis; 20 U/kg as attack treatment

10-30 U/kg as treatment (up to 500-1000 U total)

Very rapid effect, especially useful in pregnancy. Subcutaneous form available

Ecallantide (Kalbitor)

30 mg SC (as three 10-mg injections)

Not yet established

Possible occurrence of anaphylaxis; requires medical oversight

Icatibant (Fyrazyr)

30 mg SC

Not yet established

Bradykinin receptor antagonist Approved in the United States

Lanadelumab-flyo (Takhzyro)

300 mg SC every two weeks

As adult dose for children >12 y

Self administered

*Airway protection, fluid replacement, and pain relief are of paramount importance. Some episodes require no intervention. For long-term androgen prophylaxis, monitoring of liver function by ultrasound and blood studies may be considered. Although epinephrine is ineffective when given systemically, it may provide some benefit when used topically in airway obstruction. bid, Twice daily; GI, gastrointestinal; IV, intravenous; PO, orally; qd, daily; qod, every other day; SC, subcutaneously; tid, three times a day.

Factor I Deficiency Three disease phenotypes have been recognized for factor I deficiency. The first phenotype described, marked susceptibility to infections, relates to the role of factor I as a cofactor for C3bBb dissociation. When factor I is lacking, C3bBb continues to cleave C3 unabated, and a secondary deficit in C3 occurs. Both the CH50 and the AH50 are depressed but not absent, and C3 antigen levels are low. The infectious consequences of low C3 are similar to those seen in true C3 deficiency. Neisserial disease has been reported, as well as infections with encapsulated organisms such as S. pneumoniae and H. influenzae. Partial factor I deficiency has been described.101 As is true for inherited C3 deficiency, some patients have developed a serum sickness–like picture. The second disease phenotype is atypical hemolytic uremic syndrome (HUS), or membranoproliferative glomerulonephritis II.102-104 This unusual phenotype has now been described in patients with several types of regulatory defects. It has been hypothesized that these complement regulatory proteins protect vascular endothelium from activating complement after microtrauma. HUS is characterized by microangiopathic hemolytic anemia, renal disease, and hypertension. These cases are “atypical” in that they lack the common trigger of infectious diarrhea. Toxins elaborated by certain E. coli are typical triggers for HUS. These cases of factor I deficiency are difficult to identify, because complement studies often give normal results. C3 may be depressed but is not

necessarily affected. The factor I level typically is normal because the mutations are not null—they simply inactivate certain binding sites. It is thought that the mutations in the regulatory proteins adversely affect binding to surface-bound C3b and polyanion surfaces such as endothelium. The fenestrated endothelium of the glomerulus represents a landscape of polyanions in which the basement membrane is exposed by the fenestration. Thus the effect of complement regulatory proteins would be greatest on those surfaces. Lack of protection due to lack of complement regulatory proteins or to enhanced activation of C3 by C3 nephritic factor would lead to endothelial damage in the glomerulus. A third phenotype resembles an autoinflammatory process and has been described in a small number of patients. Central nervous system inflammation has been the hallmark.

Factor H Deficiency Infections, atypical HUS, glomerulonephritis, and macular degeneration are the main disease phenotypes seen in patients with factor H deficiency. As is true for factor I deficiency, the first cases described were those involving infections, which are secondary to consumption of C3 with consequent partial deficiency.61 This type of factor H deficiency can be readily suspected from diminished C3 levels and low but not absent CH50 and AH50, and the antigenic levels of factor H typically are low. Factor H–deficient pigs were known to develop membranoproliferative glomerulonephritis, and several people with membranoproliferative

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glomerulonephritis were identified with factor H deficiency. Finally, factor H deficiency was found to be the underlying basis for the pathophysiologic changes in 15% to 30% of patients with atypical HUS. Both autosomal recessive and heterozygous mutations have been seen. The age at onset is quite young in most cases, and the disease is recurrent. Death is not uncommon. These patients have a diminished C3 level, although the antigenic level of factor H typically is normal or elevated. Normal C3 levels are sometimes seen, and the only way in which this disorder can be confidently identified is with direct mutation analysis. The basis for the HUS in factor H deficiency is thought to be an inability to protect fenestrated endothelium in the glomerulus from complement-mediated damage.105 Microtrauma arises frequently as a consequence of the high oncotic pressure, and the basement membrane is able to support complement activation if not protected. Of interest, recurrent atypical HUS also has been seen in patients with antibodies to factor H, defining an acquired form as well. This form may be slightly more amenable to therapy. A common tyrosine-histidine polymorphism of factor H was identified as a significant risk factor for macular degeneration in a genomewide linkage study.106 Homozygous bearers of this polymorphic variant have a relative risk of 7.4 for the development of macular degeneration. Macular degeneration is the leading cause of blindness in the United States and many other developed countries. The central region of the retina is gradually destroyed by a process that leaves deposits of protein termed drusen. These deposits contain factor H and terminal complement components. It has been hypothesized that the abnormal factor H provides less protection to the choroidal vessels, allowing smoldering complement activation with gradual damage to the endothelium.

Membrane Cofactor Protein (CD46) Deficiency Deficiencies of MCP are associated with a later onset of atypical HUS than that for factor H and factor I deficiencies.107-109 MCP mutations are thought to account for approximately 10% of all cases of atypical HUS. There is no other known phenotype for MCP deficiency. Because MCP is a widely expressed membrane protein, this defect is intrinsic to the kidney not the serum. In contrast with factor H and factor I deficiencies, renal transplantation can be successful although it is not yet clear if other vascular lesions arise in other organs later in life. Findings on traditional complement analysis are normal, although the mechanism is thought to be the same as for factor H and factor I deficiencies.

CD59 Deficiency and Paroxysmal Nocturnal Hemoglobinuria CD59 deficiency is associated with chronic hemolytic anemia and recurrent stroke.110 The most severe manifestations are early ischemic stroke and neuropathy. CD59 is expressed on most hematopoietic cells and endothelial cells, where it confers protection from intravascular complement-mediated lysis. This defect in CD59 was suspected in cases of chronic hemolysis because of the phenotypic resemblance to paroxysmal nocturnal hemoglobinuria (PNH). PNH is characterized by recurrent episodes of hemoglobinuria secondary to intravascular hemolysis. Thrombosis occurs for unknown reasons, and aplastic anemia can both predate and postdate the PNH. PNH is caused by acquired somatic mutations of PIG-A or PIG-M in a clone of bone marrow progenitor cells.111 The protein product of PIG-A is required for GPI-anchored proteins, and C8 binding protein, DAF, and CD59 are GPI-anchored proteins that protect hematopoietic cells from complement-mediated lysis. The red cells are the most vulnerable because they have no ability to repair membrane damage. When the cells develop from the mutation-bearing progenitor, they lack all GPI-anchored membrane proteins, although the major features relate to loss of CD59. Because DAF deficiency does not have a hemolytic

phenotype, it would appear that CD59 is the more important of the two. The diagnosis of PNH is made by flow cytometry for CD59 or CD55 (DAF). Monitoring of the red cell expression is warranted, because spontaneous remissions have been described. This same test can screen for congenital CD59 deficiency.

Decay Accelerating Factor (CD55) Deficiency DAF deficiency is also termed the Inab blood group phenotype. The Cromer blood group antigens reside on DAF, and the null phenotype is referred to as the Inab phenotype. In certain kindreds, DAF deficiency has been associated with protein-losing enteropathy, whereas in others, all of the members have been completely healthy, with the deficiency being identified at the time of blood donation or cross-matching for a transfusion. None of the patients have had hemolysis, suggesting that CD59 is substantially more important in regulating red cell lysis by complement.

CR1 Deficiency No cases of complete inherited CR1 deficiency have been reported; however, acquired mild C1R deficiency is quite common in patients with immune complex diseases and serum sickness. The mechanism appears to involve internalization of immune complexes subsequent to binding, leading to a temporary lack of surface expression. This mechanism appears to be physiologic, although long-term internalization presumably could lead to a secondary inability to clear immune complexes, further contributing to the inflammatory consequences. Similarly, a polymorphic variant of CR1 with diminished levels and function has been described, although it does not appear to be a risk factor for autoimmune disease.112,113

CR3/CR4 Deficiency CR3/CR4 deficiency is a defect in the three β2 integrin adhesion molecules. The more common designation for this β2 integrin deficiency is leukocyte adhesion deficiency type I (LAD-I). Mutations in the common β chain (CD18) lead to failure to express adequate α chains: CD11a, CD11b, and CD11c. These three proteins are perhaps better known as LFA-1, CR3, and CR4. Most patients with LAD-I do not have complete null mutations, and the severity of the disease relates to the residual level of protein expression on the surface. LAD-I is a very serious disorder associated with high mortality. Among patients with no residual expression of β2 integrins, the mortality rate is quite high, and bone marrow transplantation often is recommended. These patients have very high resting white blood cell counts, frequent necrotic skin infections without pus formation, a delayed separation of the umbilical cord, and assorted other serious bacterial and fungal infections. The infections are characteristic in that necrosis predominates, with little in the way of neutrophilic infiltrate. An important predilection is for spontaneous peritonitis, which is seldom seen in other immune deficiencies. With some residual β2 integrin expression, the patient may be able to survive without a bone marrow transplant; infections are common, however, and the associated morbidity can compromise the quality of life. The manifestations of the disorder are due to the combined effects of ineffective opsonization and an inability to traverse the vascular endothelium to phagocytose bacteria. β2 integrins are essential for the firm adhesion step and diapedesis. Lacking β2 integrins, the neutrophils remain in the vascular space, where they are unable to participate in the defense against bacteria. This also explains the lack of pus at sites of active infection. Two other forms of leukocyte adhesion deficiency are recognized: LAD-II is related to a defect in fucosylation of selectin ligands and is not discussed further in this chapter. LAD-III is caused by an activation

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defect of integrins. The major manifestations are the infection pattern just described and a moderate to severe bleeding tendency secondary to impaired activation of platelet adhesion molecules.

possibility of hepatic transplantation could be contemplated as a curative strategy.

MANAGEMENT OF COMPLEMENT DEFICIENCIES

No prospective study of MBL deficiency has been performed. Of the many millions of people with MBL deficiency, it is not known what proportion suffer from significant infections, although the number is expected to be low. In those in whom infection develops, do certain cofactors synergize with MBL deficiency, thereby leading to recurrent infections? If this is the case, management is best directed at the cofactor. In current clinical practice, cofactors for infection should be addressed when possible, and the use of prophylactic antibiotics should be considered.

The management of complement deficiencies is completely dependent on the type of defect. In some cases, the management is critically dependent on knowing the precise defect present. For example, in inherited forms of atypical HUS, renal transplantation is indicated for MCP deficiency but not for factor H or factor I deficiency. For this reason, each class of defect is discussed separately in this section. Of note, with few exceptions, there are no trials supporting the management strategies presented here. The management tools offered in this chapter represent possible interventions based on current literature. Because this is a rapidly moving field, the wise clinician will seek out expert advice on encountering a complement-deficient patient.

Early Classical Component Deficiencies The major features of early classical component deficiencies are SLE and infection. Therapy for infection is not standardized, nor have clinical trials demonstrated the benefits of intervention; however, patients often are given vaccines to raise titers of antibodies to encapsulated organisms to high levels. In the case of terminal component deficiencies, high levels of antibody have been shown to partially compensate for the complement deficiency. For early complement component deficiencies, the major risks seem to be infections with S. pneumoniae and H. influenzae.56-58 Vaccines effective against these entities are available, and some evidence suggests that high titers of antibody may offer protection. The other strategy to mitigate infection risk is prophylactic antibiotics. Just as with postsplenectomy sepsis, antibiotics may offer additional protection from serious infection. In one study, half of the C2-deficient patients had serious infections such as sepsis, and infection was the leading cause of death.58 The range of ages at death was quite broad, suggesting that lifelong antibiotic prophylaxis might be required. An additional one-quarter of the patients had meningitis. Thus prevention of infection is desirable, and vaccination and prophylactic antibiotics should be given consideration. Patients on immunosuppressive medication for rheumatologic disorders may require yet more vigilance. Management of cardiac risk factors is of heightened importance in early complement component–deficient individuals owing to their accelerated atherosclerosis.59 C1q deficiency represents a unique consideration. Prognosis is poor, and bone marrow transplantation has been curative. C1q, unlike the other early classical pathway components, is produced to large extent by myeloid cells. Therefore bone marrow transplantation can be considered for this specific deficiency.

C3 Deficiency With a very limited number of reported cases of C3 deficiency, it is extremely difficult to define optimal therapy for affected patients. Their infections are the most severe of any of the complement deficiencies, and management must address loss of opsonization, loss of B cell costimulation, and loss of immune complex solubilization.61 The use of intravenous immune globulin (IVIG) to compensate for the compromised B cell function could be considered, and prophylactic antibiotics could ameliorate some of the infections. The membranoproliferative glomerulonephritis seen in C3-deficient patients responds to no specific intervention. Renal transplantation has been attempted. The recurrence risk has not been characterized; however, some degree of risk can be anticipated. Nevertheless, because membranoproliferative glomerulonephritis does not develop in all C3-deficient patients, renal transplantation should be considered in those with end-stage renal disease. The

Mannose-Binding Lectin Deficiency

Factor D and Properdin Deficiencies Patients with factor D and properdin deficiencies have manifestations related to secondary consumption of C3. Accordingly, neisserial disease is common, and infections with S. pneumoniae and H. influenzae also are seen. Vaccination to achieve high titers of antibody to those entities could theoretically be of benefit. Traditionally, prophylactic antibiotics have been used for some patients in an effort to prevent infections.

Terminal Complement Component Deficiencies Deficiencies of C5, C6, C7, C8, and C9 all are associated with an increased risk of neisserial disease. Meningococcal disease is by far the most common, but disseminated gonococcal infections have been seen with significant frequency, and patients should be warned about the possibility. The prevention of meningococcal disease has been studied thoroughly in Russia and Europe, and two things have emerged from these studies. Patients with terminal complement component deficiencies have a rather abrupt onset of meningococcal disease but have a shorter and milder course ultimately. Thus, patients in rural areas may be at increased risk owing to potential delay in the initiation of treatment. The other important lesson is that vaccination every 3 years with the polysaccharide meningococcal vaccine decreases the frequency of meningococcal episodes but does not eliminate them.114-116 The frequency is decreased to 20% of that for nonvaccinated individuals. No study has examined prophylactic antibiotics or the use of the new conjugated vaccines.

C1 Inhibitor Deficiency Management of this regulatory protein defect has been discussed earlier. Medical interventions for short-term prophylaxis, long-term prophylaxis, and acute therapy are given in Table 8.4. A number of strategies are available and should be reviewed with each patient. Genetic counseling should be offered to the patient’s family. The use of bracelets with medical information is a very individual choice. For patients with anticipated travel or events that might lead to isolation from friends and family members familiar with their disorder, bracelets may have some role to inform paramedics and physicians in the event of an emergency. Patients should be counseled to avoid certain medications. Angiotensinconverting enzyme (ACE) inhibitors can induce angioedema, as can estrogen-containing birth control pills or postmenopausal hormone replacement. In men, hormonal modulation also may affect their angioedema. Vaccination against hepatitis B is recommended due to the use of FFP, although there is control for the transmission of infections.

Factor H, Factor I, and Membrane Cofactor Protein Deficiencies Certain mutations in factor H and factor I predispose to meningococcal disease, whereas others predispose to atypical HUS. In kindreds with meningococcal disease, the same strategies utilized for patients with terminal complement component deficiencies would be expected to be

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of benefit. In kindreds with HUS, optimal management is less clear. As is done for thrombocytopenic purpura (TTP), some patients receive pheresis and FFP replacement for acute episodes.107,117 One study evaluating factor H replacement demonstrated benefit, suggesting that FFP alone might be of benefit for prophylaxis. In the case of MCP deficiency, in which the affected protein is membrane-bound, it is less clear that pheresis and FFP approach would provide benefit, but this strategy could potentially act to clear inciting agents or complement activation products. With end-stage renal disease, the rate of recurrence of the renal defect in patients with factor H or factor I deficiency is unacceptably high, and renal transplantation is not recommended. By contrast, renal disease in MCP deficiency typically does not recur in the transplanted kidney. Eculizumab, an antibody to C5, has been used to treat the renal disease of atypical HUS. Its exact role in long-term management has not been fully defined.118,119 Similarly, the role of liver transplantation has not yet been formally examined in a trial, but this appears to be a promising option.

LABORATORY ASSESSMENT OF COMPLEMENT Several themes arise from discussion of laboratory assessment of complement. An important issue is to define the population of patients who would benefit from complement screening analyses. A second major issue is to match the appropriate study to the suspected complement deficiency. For neither of these questions has a perfect answer emerged; however, some specific considerations with relevant recommendations have been identified for commonly encountered patient populations, as discussed next.

Indications Patients with recurrent sinopulmonary infections often are referred for an immunologic evaluation. Complement deficiencies will be found infrequently in this population. For patients with recurrent sepsis or recurrent systemic infections, particularly on a background of autoimmune disease (or a family history of autoimmune disease), the frequency of identifying a complement defect is probably higher, although data to support this approach are lacking. A reasonable evaluation would include CH50 and AH50 assays for these patients.120 Patients with a single meningococcal infection, either meningitis or meningococcemia, probably deserve an evaluation in nonendemic areas.121-123 The evaluation would include CH50 and AH50 assays. The frequency with which complement-deficient individuals are identified in this population may be as high as 18%.122 The rationale for evaluating such patients is that identification of a complement deficiency would lead to vaccination and prevention of future episodes. By contrast, the general consensus is that patients with meningococcal disease with an unusual serotype (serotype X, Y, Z, W135, or 29E in the United States), or those with meningococcal disease on a background of a positive family history or with recurrent meningococcal disease, should have an evaluation with CH50 and AH50 assays. In these patient groups, the frequency of complement deficiency ranges from 10% to 50%.122,124,125 Chronic meningococcemia appears to be another condition associated with a high frequency of complement deficiency.126 Patients with lupus often are tested for complement deficiency inadvertently. Until recently, it was common to follow patients with serial CH50 assays as a measure of complement activation. For this reason, in most white lupus cohorts, approximately 1% to 2% of the patients have been found to have complement deficiency,127 most often C2 deficiency. In view of the high rate of infection and accelerated atherosclerosis, identification of these patients is of clinical importance. CH50 assays are not performed as widely as they once were, and a reasonable strategy is to consider which populations of patients with SLE might

benefit from CH50 screening for complement deficiency. Because patients with C1 and C4 deficiency tend to have severe disease with early presentations, it is possible that testing pediatric-onset severe SLE might be revealing of complement deficiencies. An additional category in which a CH50 assay might be considered is in the evaluation of patients with clinical symptoms suggestive of SLE but with negative results on antinuclear antibody (ANA) and anti-dsDNA assays. Although such antibodies often are thought of as important indicators of SLE, they are less frequently detected in complement-deficient patients, and confirmation of presence of a complement deficiency might support the diagnosis of SLE. Membranoproliferative glomerulonephritis and HUS are of more clear-cut clinical significance. All patients with atypical HUS should have a complement evaluation, and a recent set of recommendations has included pregnancy-associated HUS.107-109,128 Another study has suggested that patients with severe preeclampsia might be another group with a high rate of regulatory gene defects.129 An initial screen would include a CH50 assay, an AH50 assay, and factor H, factor I, factor B, and C3 levels. Flow cytometry for MCP on neutrophils should be performed if available. In most clinical settings, ADAMTS13 activity level and antifactor H antibody measures would be appropriate. In many cases, results will be normal, and factor H, factor I, and MCP mutation analyses will most often be required. Patients with membranoproliferative glomerulonephritis type II also should be evaluated for a nephritic factor. Angioedema presentations were discussed earlier. Several considerations arise in the evaluation of a patient with angioedema; when it occurs in the setting of a known allergic response, it is much less likely to be related to C1 inhibitor deficiency. Patients with recurrent angioedema in the absence of allergic reactions, patients with a family history of angioedema, patients in whom angioedema is preceded by a reticular rash, and patients who experience angioedema after trauma all should have an evaluation. A simple but rather insensitive screen is to measure C4 levels. C4 typically is decreased at baseline but is diminished even more during an acute attack owing to consumption. A superior strategy is to measure C1 inhibitor antigen and functional levels.

Complement Laboratory Analyses A CH50 assay consists of adding dilutions of patient serum to sensitized sheep red cells. The antibody on the sensitized sheep cells initiates complement activation and, when all components are present, leads to lysis. The assay result reports the dilution of serum capable of lysing 50% of the sheep cells. Similarly, rabbit red cells are used to measure the intactness of the alternative pathway. Of note, all components for the activation arm through the terminal components must be intact for a normal CH50 or AH50. With the exception of C9 deficiency, deficiencies of all of the cascade components lead to a CH50 of zero or near zero. With low levels of CH50 or AH50, the assays should be repeated, because mishandling of the serum is an extremely common problem, leading to diminished complement levels. Other causes of low but not absent CH50 results are complement consumption resulting from active immune complex disease, diminished hepatic production related to liver disease, and immaturity of hepatic production seen in young infants. Less common but more medically important are the regulatory protein defects leading to consumption of C3, such as factor D, factor H, and factor I deficiencies. C9 deficiency also leads to a reduction in both CH50 and AH50. An alternative assay popular in Europe is a plate-based activation assay. Once an abnormal CH50 or AH50 has been confirmed, nephelometry is used to define the serum levels of certain components (C1q, C3, and C4 primarily). Enzyme-linked immunosorbent assays (ELISAs) and radial immunodiffusion are available for certain other components, and for others, the only strategy is a laborious add-back hemolytic

CHAPTER 8  The Complement System assay. These assays are not widely available except through reference laboratories. These assays will lead to the identification of a component that is absent or markedly diminished. Once the specific diagnosis is established, the management path can be defined. Screening with hemolytic assays is not adequate for C9, properdin, MBL, MASP-2, or ficolin deficiencies. In patients with these defects, the hemolytic assay value may be minimally decreased or normal. Increasingly, genetics is supplanting specific component assays, but in many cases, the mutations are not straightforward to identify: deletion, gene conversion, copy number effects.

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38. West CD, Witte DP, McAdams AJ. Composition of nephritic factor-generated glomerular deposits in membranoproliferative glomerulonephritis type 2. Am J Kidney Dis 2001;37:1120–30. 39. Mathieson PW, Wurzner R, Oliveria DB, et al. Complement-mediated adipocyte lysis by nephritic factor sera. J Exp Med 1993;177:1827–31. 40. Wisnieski JJ, Jones SM. IgG autoantibody to the collagen-like region of Clq in hypocomplementemic urticarial vasculitis syndrome, systemic lupus erythematosus, and 6 other musculoskeletal or rheumatic diseases. J Rheumatol 1992;19:884–8. 41. Wisnieski JJ, Naff GB. Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome. Arthritis Rheum 1989;32:1119–27. 42. Trendelenburg M, Lopez-Trascasa M, Potlukova E, et al. High prevalence of anti-C1q antibodies in biopsy-proven active lupus nephritis. Nephrol Dial Transplant 2006;21:3115–21. 43. Marto N, Bertolaccini ML, Calabuig E, et al. Anti-C1q antibodies in nephritis: correlation between titres and renal disease activity and positive predictive value in systemic lupus erythematosus. Ann Rheum Dis 2005;64:444–8. 44. Wisnieski JJ, Baer AN, Christensen J, et al. Hypocomplementemic urticarial vasculitis syndrome. Clinical and serologic findings in 18 patients. Medicine (Baltimore) 1995;74:24–41. 45. Holers VM, Girardi G, Mo L, et al. Complement C3 activation is required for antiphospholipid antibody-induced fetal loss. J Exp Med 2002;195:211–20.

Disorders Associated With Complement Deficiency 46. Bowness P, Davies KA, Norsworthy PJ, et al. Hereditary C1q deficiency and systemic lupus erythematosus. Q J Med 1994;87:455–64. 47. Walport MJ, Davies KA, Botto M. C1q and systemic lupus erythematosus. Immunobiology 1998;199:265–85. 48. Lee SL, Wallace SL, Barone R, et al. Familial deficiency of two subunits of the first component of complement: C1r and C1s associated with a lupus erythematosus-like disease. Arthritis Rheum 1978;21:958–67. 49. Dragon-Durey MA, Quartier P, Fremeaux-Bacchi V, et al. Molecular basis of a selective C1s deficiency associated with early onset multiple autoimmune diseases. J Immunol 2001;166:7612–16. 50. Rich KC Jr, Hurley J, Gewurz H. Inborn C1r deficiency with a mild lupus-like syndrome. Clin Immunol Immunopathol 1979;13:77–84. 51. Ballow M, McLean R, Einarson M. Hereditary C4 deficiency—genetic studies and linkage to the HLA. Trans Proc 1979;11:1710–12. 52. Nordin Fredrikson G, Truedsson L, Sjöholm AG, et al. DNA analysis in a MHC heterozygous patients with complete C4 deficiency— homozygosity for C4 gene deletion and C4 pseudogene. Exp Clin Immunogen 1991;8:29–37. 53. Wilson WA, Armatis PE, Perez MC. C4 concentrations and C4 deficiency alleles in systemic lupus erythematosus. Ann Rheum Dis 1989;48:600–4. 54. Bishof NA, Welch TR, Beischel LS. C4B deficiency: a risk factor for bacteremia with encapsulated organisms. J Infect Dis 1990;162:248–50. 55. Colten HR, Rosen FS. Complement deficiencies. Annu Rev Immunol 1992;10:809–34. 56. Ross SC, Densen P. Complement deficiency states and infection: epidemiology, pathogeneisis and consequences of neisserial and other infections in an immune deficiency. Medicine (Baltimore) 1984;63:243–73. 57. Figueroa JE, Densen P. Infectious diseases associated with complement deficiencies. Clin Microbiol Rev 1991;4:359–95. 58. Jonsson G, Truedsson L, Sturfelt G, et al. Hereditary C2 deficiency in Sweden: frequent occurrence of invasive infection, atherosclerosis, and rheumatic disease. Medicine (Baltimore) 2005;84:23–34. 59. Sjoholm AG, Jonsson G, Braconier JH, et al. Complement deficiency and disease: an update. Mol Immunol 2006;43:78–85. 60. Haskard DO, Boyle JJ, Mason JC. The role of complement in atherosclerosis. Curr Opin Lipidol 2008;19:478–82. 61. Reis ES, Falcao DA, Isaac L. Clinical aspects and molecular basis of primary deficiencies of complement component C3 and its regulatory proteins factor I and factor H. Scand J Immunol 2006;63:155–68. 62. Botto M, Fong KY, So AK, et al. Homozygous hereditary C3 deficiency due to a partial gene deletion. Proc Natl Acad Sci USA 1992;89:4957–61.

63. McLean RH, Bryan RK, Winkelstein J. Hypomorphic variant of the slow allele of C3 associated with hypocomplementemia and hematuria. Am J Med 1985;78:865–8. 64. McLean RH, Weinstein A, Damjanov I, et al. Hypomorphic variant of C3, arthritis, and chronic glomerulonephritis. J Pediatr 1978;93:937–43. 65. Fremeaux-Bacchi V, Miller EC, Liszewski MK, et al. Mutations in complement C3 predispose to development of atypical hemolytic uremic syndrome. Blood 2008;112:4948–52. 66. Thiel S, Frederiksen PD, Jensenius JC. Clinical manifestations of mannan-binding lectin deficiency. Mol Immunol 2006;43:86–96. 67. Garred P, Larsen F, Seyfarth J, et al. Mannose-binding lectin and its genetic variants. Genes Immun 2006;7:85–94. 68. Munthe-Fog L, Hummelshoj T, Honore C, et al. Immunodeficiency associated with FCN3 mutation and ficolin-3 deficiency. N Engl J Med 2009;360:2637–44. 69. Stengaard-Pedersen K, Thiel S, Gadjeva M, et al. Inherited deficiency of mannan-binding lectin-associated serine protease 2. N Engl J Med 2003;349:554–60. 70. Garcia-Laorden MI, Sole-Violan J, Rodriguez de Castro F, et al. Mannose-binding lectin and mannose-binding lectin-associated serine protease 2 in susceptibility, severity, and outcome of pneumonia in adults. J Allergy Clin Immunol 2008;122:368–74. 71. Olesen HV, Jensenius JC, Steffensen R, et al. The mannan-binding lectin pathway and lung disease in cystic fibrosis—dysfunction of mannan-binding lectin-associated serine protease 2 (MASP-2) may be a major modifier. Clin Immunol 2006;121:324–31. 72. Sirmaci A, Walsh T, Akay H, et al. MASP1 mutations in patients with facial, umbilical, coccygeal, and auditory findings of Carnevale, Malpuech, OSA, and Michels syndromes. Am J Hum Genet 2010;87:679–86. 73. Rooryck C, Diaz-Font A, Osborn DP, et al. Mutations in lectin complement pathway genes COLEC11 and MASP1 cause 3MC syndrome. Nat Genet 2011;43:197–203. 74. Densen P, Weiler J, Ackermann L, et al. Functional and antigenic analysis of human factor B deficiency. Mol Immunol 1996;33(Suppl. 1):68. 75. Kluin-Nelemans H, van Velzen-Blad H, van Helden HP, et al. Functional deficiency of complement factor D in a monozygous twin. Clin Exp Immunol 1984;58:724–30. 76. Wang X, Fleischer DT, Whitehead WT, et al. Inherited human complement C5 deficiency. Nonsense mutations in exons 1 (Gln1 to Stop) and 36 (Arg1458 to Stop) and compound heterozygosity in three African-American families. J Immunol 1995;154:5464–71. 77. Wurzner R, Hobart MJ, Fernie BA, et al. Molecular basis of subtotal complement C6 deficiency. J Clin Invest 1995;95:1877–83. 78. Fernie BA, Wurzner R, Morgan BP, et al. Molecular basis of combined C6 and C7 deficiency. Mol Immunol 1996;33(Suppl. 1):59. 79. Kojima T, Horiuchi T, Nishizaka H, et al. Genetic basis of human complement C8 alpha-gamma deficiency. J Immunol 1998;161:3762–6. 80. Kotnik V, Luznik-Bufon T, Schneider PM, et al. Molecular, genetic, and functional analysis of homozygous C8 beta-chain deficiency in two siblings. Immunopharmacology 1997;38:215–21. 81. Kaufmann T, Hansch G, Rittner C, et al. Genetic basis of human complement C8 beta deficiency. J Immunol 1993;150:4943–7. 82. Komatsu M, Yamamoto K, Mikami H, et al. Genetic deficiency of complement component C8 in the rabbit: evidence of a translational defect in expression of the alpha-gamma subunit. Biochem Genet 1991;29:271–4. 83. Kang HJ, Kim HS, Lee YK, et al. High incidence of complement C9 deficiency in Koreans. Ann Clin Lab Sci 2005;35:144–8. 84. Hayama K, Sugai N, Tanaka S, et al. High-incidence of C9 deficiency throughout Japan: there are no significant differences in incidence among eight areas of Japan. Int Arch Allergy Appl Immunol 1989;90:400–4. 85. Kira R, Ihara K, Takada H, et al. Nonsense mutation in exon 4 of human complement C9 gene is the major cause of Japanese complement C9 deficiency. Hum Genet 1998;102:605–10. 86. Fukumor Y, Yoshimura K, Ohnoki S, et al. A high incidence of C9 deficiency among healthy blood donors in Osaka, Japan. Int Immunol 1989;1:85–9.

CHAPTER 8  The Complement System 87. Blanch A, Roche O, Urrutia I, et al. First case of homozygous C1 inhibitor deficiency. J Allergy Clin Immunol 2006;118:1330–5. 88. Lopez-Lera A, Favier B, de la Cruz RM, et al. A new case of homozygous C1-inhibitor deficiency suggests a role for Arg378 in the control of kinin pathway activation. J Allergy Clin Immunol 2010;126:1307–10. 89. Prada AE, Zahedi K, Davis AE 3rd. Regulation of C1 inhibitor synthesis. Immunobiology 1998;199:377–88. 90. Lener M, Vinci G, Duponchel C, et al. Molecular cloning, gene structure and expression profile of mouse C1 inhibitor. Eur J Biochem 1998;254:117–22. 91. Falus A, Feher K, Walcz E, et al. Hormonal regulation of complement biosynthesis in human cell lines I. Androgens and gamma interferon stimulate the biosynthesis and gene expression of C1 inhibitor in human cell lines U937 and HepG2. Mol Immunol 1990;27:191–5. 92. Davis AE 3rd. The pathophysiology of hereditary angioedema. Clin Immunol 2005;114:3–9. 93. Frank MM. 8. Hereditary angioedema. J Allergy Clin Immunol 2008;121:S398–401. 94. Zuraw BL. Clinical practice. Hereditary angioedema. N Engl J Med 2008;359:1027–36. 95. Bouillet L, Boccon-Gibod I, Ponard D, et al. Bradykinin receptor 2 antagonist (icatibant) for hereditary angioedema type III attacks. Ann Allergy Asthma Immunol 2009;103:448. 96. Perricone R, De Carolis C, Giacomello F, et al. Impaired human ovarian follicular fluid complement function in hereditary angioedema. Scand J Immunol 2000;51:104–8. 97. Bork K, Barnstedt SE, Koch P, et al. Hereditary angioedema with normal C1-inhibitor activity in women. Lancet 2000;356:213–17. 98. Bork K, Gul D, Dewald G. Hereditary angio-oedema with normal C1 inhibitor in a family with affected women and men. Br J Dermatol 2006;154:542–5. 99. Nusinow SR, Zuraw BL, Curd JG. The hereditary and acquired deficiencies of complement. Med Clin North Am 1985;69: 487–504. 100. Trapp RG, Fletcher M, Forristal J, et al. C4 binding protein deficiency in a patient with atypical Behcet’s disease. J Rheumatol 1987;14:135–8. 101. Grumach AS, Leitao MF, Arruk VG, et al. Recurrent infections in partial complement factor I deficiency: evaluation of three generations of a Brazilian family. Clin Exp Immunol 2006;143:297–304. 102. Fremeaux-Bacchi V, Dragon-Durey MA, Blouin J, et al. Complement factor I: a susceptibility gene for atypical haemolytic uraemic syndrome. J Med Genet 2004;41:e84. 103. Genel F, Sjoholm AG, Skattum L, et al. Complement factor I deficiency associated with recurrent infections, vasculitis and immune complex glomerulonephritis. Scand J Infect Dis 2005;37:615–18. 104. Kavanagh D, Kemp EJ, Mayland E, et al. Mutations in complement factor I predispose to development of atypical hemolytic uremic syndrome. J Am Soc Nephrol 2005;16:2150–5. 105. Pangburn MK. Cutting edge: localization of the host recognition functions of complement factor H at the carboxyl-terminal: implications for hemolytic uremic syndrome. J Immunol 2002;169:4702–6. 106. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science 2005;308:419–21. 107. Caprioli J, Noris M, Brioschi S, et al. Genetics of HUS: the impact of MCP, CFH, and IF mutations on clinical presentation, response to treatment, and outcome. Blood 2006;108:1267–79. 108. Richards A, Kemp EJ, Liszewski MK, et al. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome. Proc Natl Acad Sci USA 2003;100:12966–71. 109. Zimmerhackl LB, Besbas N, Jungraithmayr T, et al. Epidemiology, clinical presentation, and pathophysiology of atypical and recurrent hemolytic uremic syndrome. Semin Thromb Hemost 2006;32:113–20.

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110. Yamashina M, Ueda E, Kinoshita T, et al. Inherited complete deficiency of 20-kilodalton homologous restriction factor (CD59) as a cause of paroxysmal nocturnal hemoglobinuria. N Engl J Med 1990;323:1184–9. 111. Shichishima T, Noji H. Heterogeneity in the molecular pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH) syndromes and expansion mechanism of a PNH clone. Int J Hematol 2006;84:97–103. 112. Cohen J, Caudwell V, Levi-Strauss M, et al. Genetic analysis of CR1 expression on erythrocytes of patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:393–7. 113. Sullivan KE, Jawad AF, Piliero LM, et al. Analysis of polymorphisms affecting immune complex handling in systemic lupus erythematosus. Rheumatology (Oxford) 2003;42:446–52.

Management of Complement Deficiencies 114. Fijen CA, Kuijper EJ, Drogari-Apiranthitou M, et al. Protection against meningococcal serogroup ACYW disease in complement-deficient individuals vaccinated with the tetravalent meningococcal capsular polysaccharide vaccine. Clin Exp Immunol 1998;114:362–9. 115. Schlesinger M, Kayhty H, Levy R, et al. Phagocytic killing and antibody response during the first year after tetravalent meningococcal vaccine in complement-deficient and in normal individuals. J Clin Immunol 2000;20:46–53. 116. Platonov AE, Beloborodov VB, Pavlova LI, et al. Vaccination of patients deficient in a late complement component with tetravalent meningococcal capsular polysaccharide vaccine. Clin Exp Immunol 1995;100:32–9. 117. Goodship TH. Factor H genotype-phenotype correlations: lessons from aHUS, MPGN II, and AMD. Kidney Int 2006;70:12–13. 118. Lapeyraque AL, Malina M, Fremeaux-Bacchi V, et al. Eculizumab in severe Shiga toxin-associated HUS. N Engl J Med 2011;364:2561–3. 119. Al-Akash SI, Almond PS, Savell VH Jr, et al. Eculizumab induces long-term remission in recurrent post-transplant HUS associated with C3 gene mutation. Pediatr Nephrol 2011;26:613–19.

Laboratory Assessment of Complement 120. Ekdahl K, Truedsson L, Sjoholm AG, et al. Complement analysis in adult patients with a history of bacteremic pneumococcal infections or recurrent pneumonia. Scand J Infect Dis 1995;27:111–17. 121. Ernst T, Spath PJ, Aebi C, et al. Screening for complement deficiency in bacterial meningitis. Acta Paediatr 1997;86:1009–10. 122. Fijen CA, Kuijper EJ, te Bulte MT, et al. Assessment of complement deficiency in patients with meningococcal disease in The Netherlands. Clin Infect Dis 1999;28:98–105. 123. Ellison RT, Kohler PH, Curd JG, et al. Prevalence of congenital and acquired complement deficiency in patients with sporadic meningococcal disease. N Engl J Med 1983;308:913–16. 124. Fijen CA, Juijper EJ, Hannema AJ, et al. Complement deficiencies in patients over ten years old with meningococcal disease due to uncommon serogroups. Lancet 1989;2(8663):585–8. 125. Merino J, Rodriguez-Valverde V, Lamelas JA. Prevalence of deficits of complement components in patients with recurrent meningococcal infections. J Infect Dis 1983;148:331–6. 126. Cremer R, Wahn V. Deficiency of late complement components in patients with severe and recurrent meningococcal infections. Eur J Pediatr 1996;155:723–4. 127. Sullivan KE, Wisnieski JJ, Winkelstein JA, et al. Serum complement determinations in patients with quiescent systemic lupus erythematosus. J Rheumatol 1996;23:2063–7. 128. Roumenina LT, Loirat C, Dragon-Durey MA, et al. Alternative complement pathway assessment in patients with atypical HUS. J Immunol Methods 2011;365:8–26. 129. Salmon JE, Heuser C, Triebwasser M, et al. Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort. PLoS Med 2011;8:e1001013.

CHAPTER 8  The Complement System

131.e1

SELF-ASSESSMENT QUESTIONS 1. Opsonization is a key function of the complement cascade. Which component represents the opsonin? a. C1 b. C2 c. C4 d. C3 e. C5 2. Increased susceptibility to neisserial disease is the hallmark of which set of complement deficiencies? a. Early classical pathway proteins b. Fluid phase regulatory proteins

c. Solid phase regulatory proteins d. Lectin activation components e. Terminal components 3. Systemic lupus erythematosus is associated with early classical pathway component deficiencies. Which test is the best screen for an early classical pathway component deficiency? a. CH50 b. AH50 c. Whole exome sequencing

9  Lipid Mediators of Hypersensitivity and Inflammation Tanya M. Laidlaw, Joshua A. Boyce

CONTENTS Generation of Lipid Mediator Precursors by Phospholipase A2, 132 Eicosanoid Formation, 133

SUMMARY OF IMPORTANT CONCEPTS • Lipid mediators can act to either propagate or suppress allergic inflammation, depending on the specific mediator and the receptor through which it signals. • Signaling through prostanoid receptors that increase cAMP generally restrain allergic inflammation. • LTB4 and the cysteinyl leukotrienes are important proinflammatory mediators. • Lipoxins are mediators involved in the active phase of resolution of inflammation. • Isoprostanes (IsoPs) are formed by the free radical-catalyzed peroxidation of arachidonic acid formation and are not only a dependable marker of oxidant injury both in vivo and in vitro but also are mediators that are biologically active and may regulate oxidant injury. • Sphingosine-1-phosphate is produced by mast cells and other cell types and is a major regulator of T lymphocyte function in preventing apoptosis, promoting CD4+CD25+ T regulatory cell activity, and enhancing chemotaxis. • Lipoxins (LXs) are produced at sites of either vascular or tissue injury, and abundant data suggest that these are involved in resolution of inflammation.

Lipid mediators were first recognized as important products of allergic reactions in the 1940 report by Kellaway and Trethewie, describing contraction of airway smooth muscle in the lungs of egg protein-sensitized guinea pigs with reexposure to the egg protein.1 In retrospect, the “slow reacting substance” responsible for the smooth muscle contraction they witnessed was cysteinyl leukotrienes (cys-LTs) generated in response to immunoglobulin E (IgE)-dependent stimulation of mast cells and basophils. Cys-LTs are now known to be generated by several cell types in response to both IgE-dependent and IgE-independent activation mechanisms and can regulate both immunologic and physiologic responses to allergen exposure. Moreover, recent evidence suggests that cys-LTs (and other lipid mediators) are also important as regulators of the initial dendritic cell response to antigens during primary sensitization, as well as having substantial effects on lymphocyte functions. Therefore our understanding of the scope of the mechanisms by which lipid mediators modulate allergic inflammation is more far reaching than was previously appreciated. In this chapter, we will review the pathways of lipid mediator generation, examine studies that confirm the presence of these products in allergic inflammatory states, and discuss in vivo

132

Individual Prostanoids, 135 Summary, 147

intervention studies in humans and recent murine studies that elucidate the activity of these mediators in the pathogenesis of allergic disease.

GENERATION OF LIPID MEDIATOR PRECURSORS BY PHOSPHOLIPASE A2 The phospholipases A2 (PLA2) are enzymes that hydrolyze fatty acids at the sn-2 position of membrane phospholipids, forming free fatty acids (including arachidonic acid) and lysoglycero-phospholipids.2 Arachidonic acid serves as the precursor for the synthesis of all prostaglandins and leukotrienes, collectively known as eicosanoids because the Greek word for twenty is “eikosi,” the number of carbon atoms in arachidonic acid. The lysoglycero-phospholipids are precursors for lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P).2 Both the generation and biologic function of the eicosanoids and the lysoglycero-phospholipids metabolites will be discussed in detail later in this chapter. The PLA2s are categorized into six major classes, secretory PLA2s (sPLA2), cytosolic PLA2 (cPLA2), Ca2+-independent PLA2 (iPLA2), platelet-activating factor acetylhydrolases (PAF-AH), lysosomal PLA2s, and adipose-specific PLA2.2 The current classification scheme of the PLA2s is based upon the catalytic mechanism of the individual PLA2, as well as their functional and structural properties, and consists of 16 groups (Table 9.1). Among these, the groups responsible for lipid mediator generation in vivo are limited to group IIA, group IID, group IVA, group V, group VI, and group X.2,3,4 The sPLA2 are small enzymes (14-18 kDa) that are secreted from their cellular source. They utilize an active site histidine and a His/Asp dyad and require µM levels of Ca2+ for their catalytic activity.2 After cellular release, the sPLA2s can participate in either paracrine or autocrine generation of arachidonic acid from the outer leaflet of plasma membranes. Group IIA sPLA2 is important in the generation of lysophosphatidyl choline for synthesis of LPA.2 Group IID sPLA2 expression increases with age and oxidative stress and is responsible for agedependent increases in PGD2 production in mouse lung.4 Group V sPLA2 is important for the development of allergic airway inflammation in mice. In a house dust mite model, group V sPLA2 -deficient mice had markedly reduced pulmonary inflammation and goblet cell metaplasia compared with wild type mice, perhaps through reduced antigen processing and maturation of antigen presenting cells, as well as impaired macrophage polarization.5 Mice lacking group X sPLA2 had decreased bronchial inflammation, airway remodeling, lung Th2 cytokine levels,

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation

TABLE 9.1  Phospholipase A2 (PLA2) Family

Thus the PLA2 enzymes, although critical for the generation of arachidonic acid, LPA, and S1P from membrane phospholipids also have many other important far-reaching biologic functions.

Group

Enzyme Type

IA-B

sPLA2

IIA-F

sPLA2

EICOSANOID FORMATION

III

sPLA2

IVA-F

cPLA2

Cyclooxygenase Pathway

V

sPLA2

VIA-F

iPLA2

VIIA-B

PAF-AH

VIIIA-B

PAF-AH

IX

sPLA2

X

sPLA2

XIA-B

sPLA2

XII

sPLA2

XIII

sPLA2

XIV

sPLA2

XV

LPLA2

XVI

AdPLA2

AdPLA2, Adipose-specific PLA2; cPLA2, cytosolic PLA2; iPLA2, Ca2+-independent PLA2; LPLA2, lysosomal PLA2; PAF-AH, plateletactivating factor acetylhydrolase [activity]; sPLA2, secreted PLA2.

and levels of multiple lipid mediators in a model of chicken ovalbumin (OVA)-induced airway disease, but this asthma-type phenotype can be restored with knock-in of human group X sPLA2.6 Group X sPLA2 is released in large quantities by the asthmatic airway epithelium and may play a particularly important role in asthma provoked by exercise and severe asthma in providing arachidonic acid for the rapid transformation of cysteinyl LTs.7-9 The cPLA2 are present in the cytosol and are larger than the sPLA2 (61-114 kDa).2 There are six subgroups (denoted A-F) of cPLA2 enzymes in group IV and these use a catalytic serine in a Ser/Asp dyad. The group IVA cPLA2 does not require Ca2+ for its catalytic activity, but Ca2+ is important for this enzyme’s translocation to intracellular membranes after binding to a C2-domain. Group IVA cPLA2 not only hydrolyzes glycerophospholipids at the sn-2 position to liberate arachidonic acid, but also has lysophospholipase and transacylase activities.2 A recent report suggests that this enzyme may have a role in asthma pathogenesis, because group IVA cPLA2 was overexpressed in patients with persistent asthma.10 The Ca2+-independent PLA2 are termed iPLA2 and are in group VI.2 Similar to the cPLA2, the iPLA2 uses a catalytic serine, and there are also six subgroups (denoted A-F) of iPLA2 enzymes in group VI. Group VIA and group VIB iPLA2 act to generate arachidonic acid release for eicosanoid production, whereas group VIA has roles in glycerophospholipid remodeling, protein expression, acetylcholine-modulated endotheliumdependent relaxation of vessels, apoptosis, and lymphocyte proliferation. The platelet-activating factor acetylhydrolases (PAF-AH) hydrolyze the acetyl group from the sn-2 position of PAF.2 There are two groups of PAF-AH, classified as groups VII and VIII. Although PAF-AH is not involved in eicosanoid formation, inactivation of PAF by PAF-AH may protect against anaphylaxis, because persons with lower levels of PAH-AH have more severe manifestations of anaphylaxis than those with higher levels of PAF-AH.11 However, early human studies of PAF antagonists failed to show significant benefit for the treatment of asthma or for the prevention of allergen-induced airway responsiveness.12

133

Arachidonic acid is oxidatively metabolized by the cyclooxygenase and lipoxygenase pathways.13 Cyclooxygenase catalyzes two reactions, first a cyclooxygenase reaction that inserts two molecules of oxygen into arachidonic acid to produce prostaglandin (PG)G2, followed by an endoperoxidase reaction that reduces PGG2 to PGH2 (Fig. 9.1). PGH2 is the precursor for the prostanoids PGD2, PGE2, PGF2α, and PGI2, and thromboxane A2 (TXA2). Each prostanoid is produced by tissue-specific enzymes and isomerases, as discussed later. There are two functional cyclooxygenase enzymes, COX-1 and COX-2. A third cyclooxygenase enzyme, COX-3, is encoded by the COX-1 gene but has an intron that is not retained in COX-1 and is thought not to be functional in humans. COX-1 and COX-2 are products of separate genes and have different biologic functions based on their different temporal and tissue-specific expression.13 The human COX-1 gene is located on chromosome 9, is constitutively expressed in most tissues, and although inducible in some contexts is presumed to be involved in homeostatic prostanoid synthesis.12 On the other hand, COX-2 expression is inducible and usually transient. The human COX-2 gene is present on chromosome 1. COX-2 expression can be induced by lipopolysaccharide (LPS) produced by gram-negative bacteria, in addition to interleukin (IL)-1, IL-2, and TNF.12 COX-2 expression can be induced in multiple cell types in response to inflammation or cellular stress.12 The multitude and diversity of stimuli that induce COX-2 expression, and the myriad of cells capable of expressing it, ensures that its function is a frequent concomitant of inflammatory diseases. There have been contradictory reports about the expression of COX-2 in the airway epithelium from persons with allergic diseases. One study reported a fourfold amplification in bronchial epithelial COX-2 immunostaining in asthmatic subjects compared with healthy controls14; however, another study discerned no difference in the level of immunostaining in asthmatics, chronic bronchitics, or controls who had no lung disease.15 COX-2 mRNA expression and immunoreactive protein was increased in the airway epithelium of asthmatics that have not been treated with corticosteroids compared with nonasthmatic controls, whereas corticosteroid-treated asthmatics had decreased COX-2 expression compared with their nontreated counterparts.16 The relationship between the cytokines implicated in the allergic response and COX-2 expression is complex. IL-4 and IL-13 suppress PGE2 production in bronchial epithelial cells by inhibiting expressions of both COX-2 and microsomal PGE synthase (mPGES) through JAK1 and STAT6 signaling.17 Thus, in asthmatic subjects IL-4 and IL-13 may inhibit the expression of COX-2 and the production of PGE2, a bronchoprotective prostanoid. In subjects with nasal polyps, prednisone increased COX-2 mRNA expression in polyp tissue 2 weeks after treatment was started, whereas there was no effect on COX-1 mRNA expression.18 It is possible corticosteroids modulate COX-2 expression by indirectly reducing IL-4 and IL-13, thus permitting induction of COX-2. This interpretation might help explain the apparently contradictory in vitro data whereby COX-2 immunoreactivity in airway epithelial cells is reduced by corticosteroid treatment.19 Corticosteroids decreased basal and bradykinin-induced levels of PGE2 production by airway epithelial cells, implying that COX-2 is the primary source of PGH2 for conversion to PGE2 in airway epithelium.19 COX-1 and COX-2 mRNA are expressed by resting human T lymphocytes.20 T cell activation does not affect COX-1 expression in T cells, whereas T cell stimulation upregulates

134

SECTION A  Basic Sciences Underlying Allergy and Immunology COOH

NSAIDs

Arachidonic acid

COX-1, COX- 2 O

COOH

PGG2

COOH

PGH2

O OOH

COX-1, COX- 2 O O OH

TxAS

PGDS

PGES

PGIS

PGFS

COOH

O

O

HO

HO COOH

COOH HO

OH

O

TXA2

OH

PGD2

HO

OH

PGE2

COOH

COOH

O OH

PGI2

HO

OH

PGF2α

Fig. 9.1  Prostanoid generation: Biosynthesis of prostaglandins. Arachidonic acid is metabolized by cyclooxygenases (COX-1 or COX-2) to the unstable endoperoxide prostaglandin H2 (PGH2), the common precursor for the five principal prostaglandins. Thromboxane A2 (TXA2), PGD2, PGE2, PGI2, and PGF2α are generated by individual prostaglandin synthase enzymes (TxAS, PGDS, PGES, PGIS, and PGFS) and elicit their biologic effects by activating cell surface G protein–coupled receptors. NSAIDs, Nonsteroidal antiinflammatory drugs. (From Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: multiple roles in inflammation and immune modulation. Pharmacol Ther 2004;103:147-66, with permission.)

COX-2 mRNA levels with increased COX-2 protein and cyclooxygenase activity.20 Therefore COX-2 is expressed both by resident airway cells and cells of the adaptive immune response.

Human Studies of the COX Pathway in Allergic Inflammation There are abundant data that COX activity is increased because of allergic inflammation. For instance, COX products in the bronchoalveolar lavage (BAL) fluid of allergic asthmatics are significantly increased compared with healthy nonasthmatic controls, and allergic antigen challenge of the airways further augments prostanoid production. BAL fluid levels of PGD2 and PGF2α were 12- to 22-fold greater in asthmatics than in nonallergic subjects, and 10 times greater in allergic asthmatics than in nonasthmatic subjects who had allergic rhinitis.21 Segmental allergen challenge led to a 17- to 208-fold increase in the levels of PGD2, thromboxane (Tx) B2, and 6-keto-PGF1α, a PGI2 metabolite in allergic asthmatics.22 Treatment of these subjects with prednisone for 3 days before segmental allergen challenge did not alter the BAL fluid prostanoid concentrations, revealing that corticosteroids do not inhibit activation of the COX pathway that occurs with an allergic inflammatory stimulus.23 Intervention studies examining the importance of the COX enzymes in allergic airway disease have been performed by treating subjects with indomethacin, which blocks both COX-1 and COX-2, before allergen challenge. Indomethacin did not affect lung function before allergen challenge in either allergic asthmatics or subjects with allergic rhinitis who did not have asthma.24 However, indomethacin treatment reduced the forced expiratory volume in one second (FEV1) and specific airway conductance in nonasthmatic subjects with allergic rhinitis in response to inhaled allergen challenge.24 Indomethacin administration before allergen challenge caused a small but significant decrease in specific airway conductance (sGaw) in the allergic asthmatic subjects compared

with placebo treatment, yet indomethacin had no effect on allergeninduced alterations in FEV1.24 Indomethacin treatment had no significant effect on airway responsiveness to histamine, nor did it change the immediate or late-phase pulmonary response to allergen challenge in allergic asthmatics.25,26 In subjects with exercise-induced asthma, indomethacin did not alter bronchoconstriction after exercise but did prevent refractoriness after exercise.27 However, studies using indomethacin for COX enzyme blockade need to be interpreted cautiously, because indomethacin has several other modes of action with clinical importance, including the inhibition of motility of polymorphonuclear leukocytes and the uncoupling of oxidative phosphorylation in mitochondria, and it is also a DP2 receptor agonist.28 Other nonselective COX inhibitors, such as the potent drug flurbiprofen, have shown attenuation of exercise-induced bronchoconstriction29 and allergen-induced bronchoconstriction,30 likely due to their effects on prostanoid generation. The apparent complex effect of COX inhibition on lung function reflects the diversity of the individual prostanoids and the receptors with which they interact (see below), some of which counteract one another’s actions.

Mouse Studies of the COX Pathway in Allergic Inflammation Mice with targeted deletions of COX-1 and COX-2 genes have been subjected to models of sensitization and challenge with OVA. COX-1 deficient mice that were sensitized and challenged with OVA showed heightened lung eosinophilia, serum IgE levels, augmented airway responsiveness to methacholine, increased numbers of lung CD4+ and CD8+ T cells, increased levels of Th2 cytokines, and increased concentrations of eotaxin and thymus- and activation-regulated chemokine (TARC, CCL17) compared with both mice deficient in COX-2 and wild type control mice.31,32 Thus, COX-1-derived prostanoids may be

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation homeostatic during allergen-induced pulmonary inflammation. One study reported that COX-2-deficient mice on a C57BL/6 genetic background had increased serum IgE levels, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) levels compared with control mice, but no difference in pulmonary eosinophilia or airway responsiveness.31,32 Another group reported that C57BL/6 COX-2 deficient mice had greater allergen-induced lung eosinophilia compared with wild type mice.33 Based on these findings that COX-1 inhibition augments allergic airway inflammation and airway responsiveness in mice, one would suspect that overexpression of COX-1 would have the opposite effect. However, COX-1 overexpression targeted to the airway epithelium decreased basal airway responsiveness and yet had no effect on the degree of allergic inflammation.34 Pharmacologic inhibition of COX enzymes profoundly alters the development of allergic inflammation in mice, though long-term studies to investigate this effect in humans have not been done. Mice treated with oral indomethacin during the induction of allergic airway disease had increased Th2-related cytokines in the lungs, increased pulmonary eosinophilia, and greater airway responsiveness to methacholine compared with vehicle-treated mice.35 Although BAL leukotriene levels were increased as a result of indomethacin treatment, 5-LO deficient mice also had increased allergen-induced inflammation with indomethacin treatment, effectively ruling out a causative role for enhanced leukotriene production in the exaggerated inflammatory response in this model.36 The heightened allergic inflammation with indomethacin depended on CD4+ cells but was independent of IL-4, IL-4 receptor alpha signaling, and STAT6.37 This augmented allergic phenotype is not specific to indomethacin, because both COX-1 and COX-2 inhibitors independently augmented lung levels of IL-13 and airway nonspecific responsiveness compared with vehicle-treated mice.38 COX-2 inhibition during epicutaneous sensitization with OVA in a mouse model of atopic dermatitis increased eosinophil skin infiltration, elevated total and antigen specific IgE, and resulted in a systemic Th2 response to antigen.39 As shown in several studies, COX inhibition during the development of allergic disease resulted in increased allergen-induced inflammation and airway responsiveness. These findings imply that one or more COX products restrain allergic inflammation and may potentially be a therapeutic target for the treatment of allergic diseases such as asthma and atopic dermatitis. It is critical to recognize that in these mouse studies COX was inhibited throughout the entire development of the allergic process, from the initial stage of antigen presentation and throughout all allergen challenges. In the human studies using indomethacin, COX inhibition

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occurred only at the time of an antigen challenge, long after the regulatory elements of allergic inflammation in the lung had been set in place. It is also important to note that prostanoids such as PGD2 that cause bronchoconstriction in humans fail to constrict mouse airways.40 Thus animal models of allergic pulmonary disease, whether COX functions are ablated pharmacologically or by gene deletion, are better suited to identify immunologic functions of prostanoids, rather than the direct effects on end organ physiology seen in human studies.

INDIVIDUAL PROSTANOIDS Prostaglandin D2 PGD2 is the major mast cell–derived prostanoid, being released in nanogram quantities by this cell type in response to IgE-mediated activation.13 Recent evidence suggests that eosinophils also synthesize PGD2.41 There are two distinct forms of PGD2-synthesizing enzymes, hematopoieticand lipocalin-PGD2 synthases (H-PGDS and L-PGDS, respectively); only the former is involved in PGD2 production by mast cells and other hematopoietic cell types. On a tissue level, H-PGDS is expressed to the greatest degree in humans in placenta, lung, adipose tissue, and fetal liver, whereas it is expressed in lower levels in the heart, lymph nodes, bone marrow, and appendix. On a cellular level, H-PGDS is expressed most abundantly in mast cells, CD8+ Tc2 cells, histiocytes, megakaryocytes, dendritic cells, and Kupffer cells. Recently two groups reported a population of antigen-specific human CD4+ Th2 cells with primed “innate-like” effector function that are notable for prominent H-PGDS expression.42,43 These cells also express the PGD2 receptor DP2 (also known as CRTH2), as well as COX-2, suggesting an autocrine regulatory role for PGD2 in Th2 effector responses. PGD2 can be metabolized to PGF2α, 9α,11β-PGF2 (the stereoisomer of PGF2α), and the J series of prostanoids which includes PGJ2, Δ12-PGJ2, and 15d-PGJ2.13 As is the case for all eicosanoids, PGD2 signals through distinct seven transmembrane, G-protein coupled receptors (GPCRs), termed DP1 and DP2 (Table 9.2).13 DP1 is expressed on mucus-secreting goblet cells in the nasal and colonic mucosa, nasal serous glands, vascular endothelium, Th2 cells, dendritic cells, basophils, and eosinophils. DP1 stimulation activates adenylate cyclase, resulting in an intracellular increase in cAMP levels and PKA activity. Signaling through DP1 has been reported to promote sleep, survival of eosinophils, secretion of mucus, vasodilation, and vascular permeability, while decreasing cytokine secretion and chemotaxis. DP2 is also known as chemoattractant receptorlike molecule expressed on Th2 cells (CRTH2). In addition to PGD2, other DP2 agonists include Δ12-PGJ2; 15-deoxy-Δ12,14PGJ2 (15d-PGJ2);

TABLE 9.2  Prostaglandin D2 Receptor Expression, Signaling, and Function Feature

DP1

DP2 (CRTH2)

Expression

Mucus-secreting goblet cells in nasal and colonic mucosa, nasal serous glands, vascular endothelium, Th2 cells, dendritic cells, eosinophils

Immune cells such as CD4+ Th2 and CD8+ Tc2 cells, basophils, and eosinophils

Receptor signaling

Stimulation activates adenyl cyclase, resulting in intracellular increase in cAMP levels and PKA activity

Decreases intracellular cAMP

Function

Promotes sleep, survival of eosinophils, secretion of mucus, vasodilation, and vascular permeability Decreases cytokine secretion and chemotaxis

Chemotaxis and activation of CD4+ Th2 and CD8+ Tc2 cells, basophils, eosinophils, and ILC2s Induces release of eosinophils from bone marrow, initiates their respiratory burst, and primes them for degranulation Increases microvascular permeability Depletes goblet cells Constricts coronary arteries

cAMP, Cyclic adenosine monophosphate; DP1, DP2, prostaglandin D receptor types 1 and 2; PKA, protein kinase A.

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13,15-dihydro-15-keto-PGD2; 11-dehydro-TXB2; and the COX inhibitor indomethacin. DP2 is expressed on immune cells such as CD4+ Th2 and CD8+ Tc2 cells, eosinophils, and basophils. These cells each respond chemotactically to PGD2 in a DP2-dependent manner. DP2 is expressed strongly by human group 2 innate lymphoid cells (ILC2s), as well as by allergen-specific Th2A cells. Notably, Th2A cells share many characteristics with ILC2s, including the ability to rapidly generate large quantities of Th2 cytokines in response to innate cytokines (IL-33, IL-25) as well as to PGD2.44 DP2 signaling can directly elicit the production of Th2 cytokines by both human ILC2s and Th2 cells.44,45 In the BAL fluid of asthmatic subjects DP2 is preferentially expressed by IL-4+/ IL-13+ T cells compared with IFN-γ+ T cells, potentially reflecting Th2A cells.46 DP2 signaling in eosinophils also induces their release from bone marrow, initiates their respiratory burst, increases the chemotactic response to other chemokines such as eotaxin, and primes them for degranulation. In addition, DP2 signaling is reported to increase microvascular permeability, deplete goblet cells, and constrict coronary arteries. In contrast to DP1 signaling, activation of DP2 results in decreased intracellular cAMP.13 Thus PGD2 signaling through DP2, via suppression of cAMP, would be predicted to facilitate allergic inflammation through its effect on chemotaxis and mediator release by effector cells. The smooth muscle contractile properties of PGD2 and its immediate metabolite, 9α, 11β-PGF2, are thought to be mainly mediated through the thromboxane TP receptor.47,48

Human Studies of PGD2 in Allergic Inflammation.  Inhalation challenge of human allergic asthmatic subjects with specific allergen increases the levels of PGD2 in the BAL fluid, reflecting IgE-dependent activation of mast cells.49 PGD2 is increased in the nasal lavage from subjects with allergic rhinitis relative to nonallergic controls,50 in tears from persons suffering from allergic conjunctivitis,51 and in the fluid from experimentally produced skin blisters in patients with late phase reactions of the skin.52 Treatment of asthmatic subjects with the COX-2 specific inhibitor celecoxib for 3 days failed to alter urinary levels of the stable PGD2 metabolite, 9α,11β-PGF2, suggesting that PGD2 is primarily synthesized via COX-1 in mild asthma.53 On the other hand, aspirin challenge of individuals with aspirin-exacerbated respiratory diseases does not suppress the levels of PGD2 in BAL fluid. Indeed, challenges with aspirin frequently induce marked increases in urinary and plasma PGD2 metabolites in subjects with aspirin-exacerbated respiratory disease (AERD), presumably reflecting the idiosyncratic activation of mast cells. These increases are highest among subjects who develop the most severe clinical reactions. A recent study reported that mast cells from nasal polyps of subjects both with and without AERD express substantially higher levels of mRNA encoding COX-2 than that encoding COX-1, suggesting a mechanism by which mast cell–derived PGD2 production could evade suppression by low-dose aspirin. Notably, subjects with AERD who are treated therapeutically with a high dose (650 mg) of aspirin (sufficient to suppress both COX-1 and COX-2) exhibit substantial (approximately 80%) reductions in basal urinary PGD2 metabolites. It is possible that suppression of PGD2 production accounts for some of the therapeutic benefit of aspirin in this context.54 PGD2 is a potent bronchoconstrictor (reflecting actions of 9α, 11β-PGF2) and vasodilator, and potentiates airway responsiveness.47 Intranasal administration of PGD2 increased nasal resistance 10 times more potently than histamine and 100 times greater than bradykinin.55 PGD2 induced vascular leakage in the conjunctiva and skin56 and led to eosinophil influx in the conjunctiva57 and trachea,58 suggesting that it may have a direct pathogenic role in allergic disease. The vascular effects of PGD2 are thought to largely reflect dilation mediated by DP1, whereas recruitment of effector cells is more likely to reflect chemotaxis via DP2.59,60 Additional effects reflect 9α,11β-PGF2 signaling at TP receptors.

There have been several recent trials of DP2 blockade in allergic diseases. For example, an orally available DP2 antagonist was shown to exert modest but significant beneficial clinical effects in adult patients with active eosinophilic esophagitis,61 and there have been a number of phase II trials of DP2 antagonists testing their efficacy in asthma and allergic rhinitis. Although some studies suggest that DP2 blockade may provide therapeutic benefit in patients with eosinophilic or allergic asthma,62-64 further studies are needed to confirm the clinical utility of this class of medication.

Mouse Studies of PGD2 in Allergic Inflammation.  Mouse studies reveal a complex role of PGD2 in experimental allergic disease.65 Transgenic mice that overexpress L-PGDS had greater BAL fluid levels of Th2 cytokines, eotaxin, eosinophils, and lymphocytes after allergen sensitization and challenge compared with nontransgenic littermates.66 Aerosolized PGD2 administered 1 day before inhalational challenge with low-dose antigen increased the numbers of eosinophils, lymphocytes, and macrophages, as well as IL-4 and IL-5, in BAL fluid of sensitized mice.67 These results suggest that PGD2 augments pulmonary Th2 responses. Rodent studies of DP1 function in allergic inflammation have been contradictory. Allergen sensitized and challenged DP1-deficient mice had significantly reduced BAL concentrations of IL-4, IL-5, and IL-13 compared with wild-type control mice, and diminished airways hyperresponsiveness (AHR), without a difference in the levels of IFN-γ in BAL fluid.65 These DP1-deficient mice had decreased BAL cellular influx with less eosinophils and lymphocytes compared with control mice, suggesting that DP1 signaling was important in the full expression of allergic inflammation.65 However, the DP1 agonist BW245C suppressed the function of lung dendritic cells, including lung migration and the ability of dendritic cells to stimulate T cell proliferation.68,69 Mice treated with BW245C or mice receiving adoptively transferred DP1-treated dendritic cells had increased numbers of Foxp3+ CD4+ regulatory T cells that suppressed inflammation in an interleukin 10–dependent mechanism.69 The reduction in allergic inflammation caused by the DP1 agonist on dendritic cell function was mediated by cyclic AMP– dependent protein kinase A.69 In addition, chimeric mice that lacked DP1 expression on hematopoietic cells had strongly enhanced airway inflammation when challenged with allergen, indicating an important homeostatic role of DP1 and endogenous PGD2.69 Collectively, these data suggest that DP1 signaling facilitates effector responses through structural cells but may dampen responses of dendritic cells so as to restrain the allergic inflammatory process at the sensitization phase. Several studies in different species support the notion that DP2 receptor promotes allergic inflammation. The DP2 receptor antagonist AM211 inhibited ovalbumin-induced airway eosinophil influx in a guinea pig model of allergic airway inflammation, while reducing the number of sneezes mice experienced after intranasal allergen challenge.70 Similarly, the DP2 antagonist MK-7246 inhibited antigen-induced late phase bronchoconstriction and airway responsiveness in sheep and antigeninduced eosinophilia in both sheep and monkeys.71 In addition, an oral, potently selective alkynylphenoxyacetic acid DP2 antagonist decreased ovalbumin-induced airway eosinophilia in mice.72 Thus, PGD2 signaling through DP2 increases allergic inflammation, and interference with this receptor attenuates such inflammatory responses in animals.

Prostaglandin E2 There are three distinct enzymes that can metabolize PGH2 to PGE2. These are microsomal PGE synthase-1 (mPGES-1), mPGES-2, and cytosolic PGE synthase (cPGES).13 mPGES-1 is membrane-associated, localized to the perinuclear area, and glutathione-dependent and has a trimeric structure. mPGES-1 is expressed inducibly (and concomitantly

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation with COX-2) in several cells and preferentially utilizes COX-2-derived PGH2. mPGES-1 can also metabolize PGH2 produced from COX-1, but requires exogenous administration of arachidonic acid for this function. For instance, mouse studies revealed that arachidonic acid generated by mast cell group IVA cPLA2 led to PGE2 production by fibroblast mPGES-1.73 The expression of cPGES is mostly constitutive and not induced by inflammatory stimuli. Compared with mPGES-1, cPGES coupled more efficiently with COX-1 than with COX-2 for PGE2 generation. Although this suggests that cPGE2 may provide PGE2 necessary for cellular homeostasis, mice lacking mPGES-1 show strikingly diminished levels of basal PGE2 production in most organs. In contrast, genetic deletion studies in mice have been unable to support a role of either cPGES or mPGES-2 as PGESs in vivo. cPGES is localized to the cytosol; there was evidence that it translocates from the cytosol to the nuclear membrane to assemble with COX-1 in PGE2 production, although it has a slight preference to interact with COX-2.74 Heat shock protein 90, casein kinase II, and bradykinin upregulated cPGE2 activity, whereas dexamethasone decreased cPGES activation.74 In contrast to mPGES-1, mPGES-2 is not dependent on glutathione.74 mPGES-2 is expressed constitutively in many cells and tissues, but can be induced in colorectal adenocarcinoma cells to high levels.74 In transfected cells, mPGES-2 uses PGH2 derived from COX-1 and COX-2 with equal efficiency. Local PGE2 concentrations are modulated by COX-2–driven synthesis and PGE2 degradation by 15-hydroxyprostaglandin dehydrogenase (15-PGDH).75 The effects of PGE2 both in vivo and in vitro are complex, relating to the fact that this prostanoid signals through four distinct GPCRs, termed EP receptors 1 through 4 (Table 9.3).13 Each EP receptor has a distinct G protein coupling preference and downstream signal activation, and some of these signals counteract one another. All four receptor subtypes are present in the lung and other organs associated with allergic responses.13 Signaling through the EP1 receptor increased inositol triphosphate and diacylglycerol, resulting in increased cell Ca2+ and smooth muscle contraction. Activation of the EP2 and EP4 receptors increased intracellular cAMP concentrations and relaxed smooth muscle.76 Stimulation of the EP2 receptor inhibits mast cell mediator release. 77 EP2 is expressed most abundantly in the uterus, lung, and spleen.78 EP4 receptor expression is greatest in the kidney and peripheral blood leukocytes, but there is high level of expression in the thymus, lung, and a number of other tissues.79 EP3 receptors caused smooth muscle contraction by decreasing the rate of cAMP synthesis.80 EP3 receptors are unique because of the diversity created by multiple splice variants that produce alternate sequences in the C-terminal tail of this receptor subtype; however, the functional importance of these alternative splice variants is not well understood.78 Usually these splice variants of EP3 decrease cAMP generation, in contrast to signaling through EP2 and EP4, which increase cAMP.78 Thus PGE2 activity can be diverse and possibly competing,

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dependent on the relative contributions of the receptors that are stimulated in a given context.

Human Studies of PGE2 in Allergic Inflammation.  PGE2 is a predominant COX product of the airway epithelium, macrophages, and smooth muscle.81,82 There is abundant evidence to support the proposition that endogenous PGE2 may be bronchoprotective in human asthma.83 PGE2 produced by epithelial cells inhibited vagal cholinergic contraction of airway smooth muscle.84 Bronchial epithelial cell–derived PGE2 also dampened dendritic cell migration and proinflammatory cytokine secretion.85 There was a negative correlation between the sputum levels of PGE2 from asthmatics and their sputum eosinophil count, consistent with antiinflammatory properties.86,87 PGE2 inhalation also inhibited the pulmonary immediate and late phase responses to inhaled allergen.88,89 Inhaled PGE2 decreased the change in methacholine airway reactivity and reduced the number of eosinophils after inhaled allergen challenge.88 In addition, PGE2 inhibited both exercise-induced and aspirin-induced bronchoconstriction in patients sensitive to these challenges.90,91 Interestingly, although PGE2 significantly protects against decrements in pulmonary function in challenge models, it does not alter baseline FEV1 or methacholine reactivity.89 The results from these studies suggest that PGE2 has greater immunomodulatory properties than directly affecting airway caliber. This is supported by the observation that PGE2 inhalation before segmental allergen challenge significantly reduced the BAL levels of PGD2, an important product of mast cell activation, and the concentrations of cys-LTs.92 Recent evidence suggests that the EP4 receptor in human, guinea pig, and rat airways mediates smooth muscle relaxation,93 whereas it is the EP3 receptor that mediates the cough properties of PGE2.94 PGE2 in combination with the β2adrenergic receptor agonist albuterol also inhibited human airway smooth muscle migration and mitogenesis,95,96 revealing that PGE has pleiotropic effects on airway function. PGE2 is rapidly metabolized and this has prompted investigators to utilize the more stable orally active PGE1 analog, misoprostol, in studies of allergen-induced airway inflammation and lung function in humans, but the results have largely been negative. Misoprostol did not have an impact on pulmonary function, β2 agonist use, or asthma severity score in aspirin-sensitive asthmatics, nor did it have any protective effect on the aspirin-induced reaction in patients with aspirin-exacerbated respiratory disease.97,98 These results contrast with the effects of inhaled PGE2, and may reflect a failure for orally administered misoprostol to reach the respiratory tract in pharmacologically active concentrations. Despite its capacity to suppress eosinophilia and allergic early and late-phase responses, in vitro studies demonstrate that PGE2 can either stimulate or suppress the function of immune cells. PGE2 in vitro inhibited lymphocyte production of the type 1 cytokines IL-2 and interferon-γ,

TABLE 9.3  Prostaglandin E2 Receptor Expression, Signaling, and Function Feature

EP1

EP2

EP3

EP4

Receptor expression

T cells, B cells, dendritic cells, smooth muscle

T cells, B cells, dendritic cells, mast cells, basophils, uterus, lung, spleen, smooth muscle

T cells, B cells, dendritic cells, smooth muscle

T cells, B cells, dendritic cells, kidney, lung, smooth muscle

Effect of receptor signaling

↑ intracellular Ca2+

↑ cAMP concentration

↓ cAMP synthesis

↑ cAMP concentration

Function

Smooth muscle contraction

Inhibition of dendritic cell differentiation and T cell stimulatory capacity; smooth muscle relaxation

Smooth muscle contraction

Enhances migration of antigen-stimulated dendritic cells to lymph nodes; inhibition of IL-12 production by monocytes and macrophages; promotes initiation of contact hypersensitivity reactions; smooth muscle relaxation

cAMP, Cyclic adenosine monophosphate; EP1 to EP4, prostaglandin E2 receptor types 1 to 4; IL-12, interleukin-12.

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thus promoting T cell differentiation toward a type 2 cytokine profile.99-102 These in vitro results suggest PGE2-promoted type 2 cytokine production may be regulated at the antigen presentation. Macrophages lacking either mPGES-1 or EP2 receptors display sharply reduced expression of IL-33 when stimulated by LPS, indicating a potential pro-Th2 effect of endogenous PGE2 in this cell type.103 Myeloid dendritic cells matured in the presence of IFN-γ produced type 1 CD4+ T lymphocyte responses, while dendritic cells matured in PGE2-elicited type 2 T cell responses.104 PGE2 induction of type 2 cytokine production mostly through its activity at the time of antigen presentation would not necessary contradict in vivo human studies that have suggested PGE2 is antiinflammatory. More recently, in combination with IL-23, PGE2 induced differentiation and expansion of CD4+ Th17, along with secretion of the signature cytokines by this lineage.105 Acute antigen challenge models probably more precisely reflect effector cell function, because allergic sensitization to an antigen occurs much earlier in life. Besides PGE2’s activity in the antigen-presenting cell function and Th1 and Th2 cell development, this prostanoid has important immunomodulatory effects on other inflammatory cells presumed to be pathogenic in asthma. In a cell culture system, both PGE2 and cAMP inhibited spontaneous eosinophil apoptosis, as did an EP2 agonist.106 Thus by prolonging eosinophil survival, PGE2 could potentially increase the inflammatory potential of these cells in asthma. However, PGE2 was also reported to decrease eosinophil chemotaxis, aggregation, degranulation, and IL-5-mediated survival.107,108 PGE2 inhibition of eosinophil trafficking was modulated through EP2 signaling, and its inhibition of eosinophil production of reactive oxygen species is modulated through EP4.109,110 Therefore the relevance of these in vitro findings to in vivo disease states is still to be determined. PGE2 also modulated the expression of granulocyte macrophagecolony stimulating factor (GM-CSF), from human airway smooth muscle cells.111 The COX inhibitor indomethacin upregulated GM-CSF production by cultured human airway smooth muscle cells; however, exogenous PGE2 decreased this indomethacin-induced GM-CSF production, suggesting that PGE2 restrained GM-CSF expression and the inflammation that is associated with this cytokine.111 In contrast, PGE2 increased IL-6 and GM-CSF production as a result of IgE-mediated mouse mast cell degranulation by signaling through EP1 and EP3 receptors.112 PGE2 has also been reported to have contrasting activities on the mast cell production of differing mediators. PGE2 has been reported to either reduce113-115 or enhance116,117 the release of histamine and other inflammatory mediators. These differences may relate to the relative dominance of EP3 (activating) versus EP2 (inhibitory) signaling in a given mast cell population. For instance PGE2 can activate human mast cells through EP3 but inhibit activation through the EP2-PKA signaling pathway.118 PGE2 is also thought to play a key role in aspirin-intolerant asthma with inhibition of COX-1, but not COX-2, being closely aligned with the ability of NSAIDs such as aspirin, and propionic acid derivatives (ibuprofen and naproxen) for precipitating this form of asthma (see Chapter 78).119 Additionally, PGE2 has recently been found to limit human ILC2 activation and reduce ILC2 production of IL-5 and IL-13, which was mediated through the combined action of the EP2 and EP4 receptors.120

Mouse Studies of PGE2 in Allergic Inflammation.  In the OVAsensitized and challenged model, mice that are deficient in the EP3 receptor had enhanced allergic inflammation compared with wild type (WT) mice, whereas there were no differences in the pulmonary allergic inflammation between WT, EP1-deficient, EP2-deficient, and EP4-deficient mice.121 Compared with WT mice, EP3-deficient mice had greater airway numbers of eosinophils, neutrophils, and lymphocytes in BAL fluid, as well as augmented BAL concentrations of IL-4, IL-5, and IL-13.121

Administration of the EP3 agonist AE-248 to OVA-sensitized and challenged WT mice significantly suppressed allergic airway cellularity and tended to decrease airway mucus expression and airways responsiveness to methacholine.121 In contrast, mice lacking EP2 receptors (and mice lacking mPGES-1) displayed sharply increased levels of lung inflammation in response to repetitive intranasal challenges with a house dust mite extract compared with wild type controls.122 There is evidence that PGE2 expression is reduced in chronic allergen exposure, possibly as a result of allergic inflammation, with a consequence being airway remodeling. In this model, there was an inverse relationship between the number of allergen challenges and both COX-2 and mPGES-1 expression in lung fibroblasts, resulting in decreased cytokine-induced PGE2 production.123 Interestingly, mPGES-1 derived PGE2 expression had no effect on allergen sensitization or effector T cell responses in a house dust mite mouse model comparing the phenotype of mPGES-1-deficient and wild type mice.124 However, mPGES1-deificent mice had significantly increased numbers of vascular smooth muscle cells and thickness of intrapulmonary vessels after allergen challenge.124 These results revealed that PGE2 produced by mPGES-1 protected the pulmonary vasculature from remodeling during allergeninduced lung inflammation.122,125 PGE2 also regulates airway tone in mice. Notably, dust mite allergen–primed mice develop sharp increases in airway resistance, lung cys-LT production, and mast cell activation products in BAL fluid after inhalation challenges with lysine-aspirin, a phenotype reminiscent of AERD. Immunologically naïve mice that lack 15-hydroxyprostaglandin dehydrogenase, the major catabolic enzyme of PGE2, and therefore which have elevated levels of PGE2, had a decreased bronchoconstrictor response to methacholine.126 Similarly, mice that had elevated PGE2 levels as a result of overexpression of PGE2 synthase in the lung had decreased methacholine-induced airway constriction.126 Thus PGE2 protects against lower airway bronchoconstriction, and other studies suggest that this effect is mediated through EP2. Pretreatment with aerosolized PGE2 blunted methacholine-induced bronchoconstriction in WT mice, but not in EP2-deficient animals.127 In addition, methacholine-induced bronchoconstriction was reversed by aerosolized PGE2 in WT mice, but not EP2-deficient mice.127 These findings were confirmed by another group that reported that PGE2-induced bronchodilation was a consequence of direct activation of EP2 receptors on airway smooth muscle, whereas PGE2 signaling through EP1 and EP3 led to bronchoconstriction.128 Collectively, these studies suggest that PGE2 regulates homeostasis of bronchomotor tone and pulmonary immune responses through different respective receptors.

Prostaglandin F2α PGF2α is produced by PGF synthase (PGFS).129 PGFS has two activities: (1) catalyzing the formation of PGF2α from PGH2 by PGH2 9,11-endoperoxide reductase in the presence of NADPH and (2) forming PGF2α from PGD2 by PGD2 11-ketoreductase.129 The PGFS binding sites for PGH2 and PGD2 are proposed to be distinct.129 PGFS is expressed in lung and peripheral blood lymphocytes, suggesting a possible role in allergic diseases such as asthma.130 PGFS may also have a role in the nervous system, because it has been identified in neurons and vascular endothelial cells in the rat spinal cord.131 PGFS is inhibited by nonsteroidal antiinflammatory drugs (NSAIDS) such as indomethacin, and this may partially explain the protective effect of this class of drugs in some gastrointestinal tumors in which PGFS activity is high.129 PGF2α binds a single receptor, termed FP (Table 9.4). FP binds PGF2 with high affinity, and also binds both PGD2 and PGE2 at nM range concentrations.132 Selective FP agonists such as fluprostenol and latanoprost have been produced that are used in clinical settings because of these agents’ ocular hypotensive properties.132 PGF2α plays a critical function in reproduction, renal physiology, and modulation of intraocular pressure. Tissue

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TABLE 9.4  Prostaglandin F2α, Prostaglandin I2, and Thromboxane A2 Receptor Expression,

Signaling, and Function Feature

FP

IP

TP

Receptor expression

Ovarian corpus luteum, kidney, eye, lung, stomach, heart

Th1 and Th2, dendritic cells, endothelium, platelets

Vascular tissues such as lung, heart, kidney smooth muscle

Effect of receptor signaling

↑ intracellular Ca2+

↑ cAMP concentration

Phospholipase C activation, calcium release, activation of protein kinase C TPα: activates adenylate cyclase TPβ: inhibits adenylate cyclase

Function

Smooth muscle contraction, cardiac hypertrophy

Inhibition of dendritic cell differentiation and T cell stimulatory capacity

Potentiates smooth muscle contraction, inhibits dendritic cell–T cell interaction

distribution of FP receptor mRNA expression is highest in the ovarian corpus luteum, followed by the kidney, with lower-level expression in the lung, stomach, and heart.133 FP expression has not been reported in the spleen, thymus, or on immune cells. As a result, in contrast to the other prostaglandins, there is very little evidence to suggest an important contribution of PGF2α-FP receptor signaling in inflammatory and immunologic processes.132

Human Studies of PGF2α.  PGF2α has not been studied to nearly the same extent as PGD2 or PGE2 in allergic disease and asthma. PGF2α inhalation leads to a dose-related decrease in sGaw in both control and asthmatic subjects.134-136 There has been a wide variation in the pulmonary function response to PGF2α reported in asthmatics, in contrast to the relatively small interindividual variation in healthy control subjects.136 Subjects who inhaled PGF2α, experienced wheezing, coughing, and chest irritation within 3 to 4 minutes, whereas watery sputum also occurred shortly thereafter.136 Maximal decrease in specific airway conductance after PGF2α occurred 6 minutes after inhalation, and recovery took place within 30 minutes.136 Asthmatic subjects experienced an approximate 150-times greater sensitivity to PGF2α than did healthy controls, yet asthmatics were only 8.5-times more sensitive to histamine than nonasthmatic subjects.136 There was decreased variation in individual responses to histamine compared with inhaled PGF2α challenge, but sensitivity to both mediators correlated with each other.136 In general, women had a diminished bronchoconstrictor response to PGF2α compared with men.136 Both PGE2 and isoprenaline shortened recovery from the decrease in pulmonary function caused by inhalation of PGF2α; however, neither atropine, sodium cromoglicate, nor flufenamic acid prevented PGF2αinduced bronchoconstriction.136 PGF2α (and PGE2 as well) decreased exhaled nitric oxide (NO) concentrations in both healthy controls and asthmatic subjects; however, the meaning of this outcome is unknown.137 Although FP is not expressed on immune cells, there is some evidence that PGF2α may have a role in airway inflammation. In subjects with asthma, the magnitude of sputum eosinophilia correlated with the log sputum PGF2α concentrations, whereas there was a negative correlation between sputum eosinophilia and PGE2 levels and no correlation between the number of sputum eosinophils and sputum levels of cys-LTs, thromboxane, and PGD2.87

Mouse Studies of PGF2α in Allergic Inflammation.  To our knowledge, there are no published studies examining the effect of PGF2α administration or signaling through the FP receptor in the mouse allergen challenge model. An FP-deficient mouse has been created, and these mice had attenuated bleomycin-induced pulmonary fibrosis independent of TGF-β expression.138 To date, no studies have examined whether FP-deficient mice are protected from collagen deposition and airway wall remodeling in a chronic allergen challenge model.

Prostaglandin I2 (Prostacyclin) PGI2 is converted from PGH2 by PGI synthase (PGIS). The gene encoding PGIS is located on chromosome 20q13.11-13.139 PGIS is strongly expressed in the heart, lung, smooth muscle, kidney, and ovary and expressed at moderate levels in the brain, pancreas, and prostate.139 There is low level PGIS expression in the placenta, spleen, and leukocytes.139 PGI2 signals through its receptor, IP, a GPCR (Table 9.4).78 Binding of PGI2 to its receptor activates adenylate cyclase via Gs in a dose-dependent manner, increasing the production of cAMP.140 This increase in intracellular cAMP mediates PGI2’s effect of inhibiting platelet aggregation and dispersing existing platelet aggregates both in vitro and in human circulation.140 Northern blot analysis reveals that IP mRNA is expressed to the greatest degree in the thymus, whereas a high level of IP mRNA expression is also found in spleen, heart, lung, and neurons in the dorsal root ganglia. IP is also expressed on mouse bone marrow– derived dendritic cells (BMDCs).141 The PGI2 analogs iloprost and cicaprost decreased BMDC production of proinflammatory cytokines (IL-12, TNF-α, IL-1α, IL-6) and chemokines (MIP-1α, MCP-1), whereas these analogs increased the production of the antiinflammatory cytokine IL-10 by BMDCs.141 The modulatory effect was associated with IPdependent upregulation of intracellular cAMP and downregulation of NF-κB activity.141 Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate antigen-specific CD4 T cell proliferation and production of IL-5 and IL-13.141 Iloprost also enhanced human dendritic cell production of IL-10 and in coculture experiments of iloprost-treated dendritic cells and naïve T cells, there was induction of T regulatory cells.142 IP is also expressed in T cells of mice, along with the PGE2 receptor (EP) subtypes and the thromboxane receptor (TP).143 IP has also been found in kidney smooth muscle and epithelial cells.144 Messenger RNA for IP is expressed in both CD4+ Th1 and Th2 cells.145 Recently, IP receptor signaling was shown to restrain proliferation and cytokine generation by both mouse and human ILC2s.146 Thus, IP has been located on several different cell types, including those critical to the immune response.

Human Studies of PGI2 in Allergic Inflammation.  PGI2 and PGD2 were the predominant COX products produced in antigen-induced anaphylactic reactions of human lung parenchyma, on the order of 3to 7-times greater concentrations than that of the other prostanoids.147 The PGI2 metabolite 6-keto-PGF1α was present in concentrations twoto three-fold higher than all the other prostanoids in both airway and subpleural lung fragments in an in vitro anaphylaxis assay of passively sensitized human lung.148 Plasma 6-keto-PGF1α was also increased after antigen challenge in which asthmatic subjects were pretreated with indomethacin.149 Thus PGI2 is produced in abundance in allergic inflammatory responses in the lung, presumably a reflection of

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activated endothelial cells that express almost all the PGIS in the human airway. Most of the published intervention studies examining the modulatory effect of PGI2 in human asthma were performed more than 15 years ago and were limited by the half-life of PGI2 (3 to 5 minutes). Therefore these studies may not accurately reflect the therapeutic capability of the currently available class of stable PGI2 agents. Pretreatment with PGI2 had no effect on allergen-induced immediate phase bronchoconstriction.150 In another study, PGI2 protected against both exercise and ultrasonic water-induced bronchospasm, yet again had no effect on allergen-induced airway reactivity.151 Inhaled PGI2 also had no effect on specific airway conductance but did result in consistent bronchodilation in two of the asthmatic subjects. In this study, there was a significant effect of PGI2 on the cardiovascular system. Inhaled PGI2 resulted in a fall in both diastolic (20 ± 3 mm Hg) and systolic (8 ± 2 mm Hg) blood pressure, as well as an increased pulse rate (29 ± 3 beats per minute).152 Intravenous PGI2 administration had no effect on the fall in airflow induced by aspirin in subjects with aspirin-induced asthma.153 Somewhat contradictory results of the effect of inhaled PGI2 in subjects with mild asthma have been reported.154 In these studies PGI2 did not alter specific airway conductance, but resulted in a concentration-dependent decrease in FEV1. In contrast, these same investigators found that PGI2 protected against bronchoconstriction induced by either PGD2 or methacholine. The authors proposed that these disparate findings might be explained by PGI2’s marked vasodilator effect, resulting in airway narrowing through mucosal blood engorgement, whereas this same phenomenon possibly reduced the spasmogenic properties of other inhaled mediators by increasing their clearance from the airways. An oral PGI2 analog (OP-41483) had no effect on FEV1 or airways responsiveness to methacholine in stable asthmatics.155 Since this last report which was published in 1991, to our knowledge, there have been no other published manuscripts examining PGI2 in human allergic inflammation in the lung or asthma. The therapeutic potential of newer, more stable PGI2 analogs in asthma, already approved for use in pulmonary hypertension, remains unexplored.

Mouse Studies of PGI2 in Allergic Inflammation.  Several studies using mouse models suggest that endogenous PGI2 signaling through IP is involved in homeostatic control of airway inflammation. In a model of short-term OVA challenge, IP-deficient mice had increased lung production of IL-4 and IL-5, serum antigen-specific and total IgE levels, and airway leukocyte accumulation compared with wild type mice.156 In another study, the period of allergen challenge was extended to generate signatures of “chronic” allergen exposure. In this study, IP-deficient mice had more airway eosinophils and lymphocytes, Th2 cytokine levels, and hydroxyproline concentrations compared with wild type mice.157 Mice that lacked the ability to signal through IP had augmented inflammatory and physiologic changes compared with WT mice in the model of bleomycin-induced fibrosis.158 In another bleomycin model of lung injury, mice that overexpressed PGIS in airway epithelial cells were protected against lung injury and had decreased production of F2-isoprostanes, marker of oxidant injury. In these experiments, PGI2 induced the expression of NAD(P)H:quinone oxidoreductase type I (NQO1), an enzyme that prevents generation of reactive oxidant species.159 Supporting the concept that PGI2 restrains airway inflammation, inhaled iloprost inhibited the maturation and migration of lung DCs to the mediastinal LNs after intranasal antigen administration, resulting in decreased induction of an allergen-specific Th2 response in these nodes.160 In this in vivo model, iloprost-treated DCs also downregulated Th2 differentiation from naïve T cells and were unable to boost effector cytokine production in primed Th2 cells.160 IP null mice also displayed increases in the numbers and activation of ILC2s in the

lung after challenge with an extract from the mold Alternaria alternata, reflecting the capacity of PGI2 to suppress ILC2 proliferation and cytokine generation.146 Although these results in animal models of allergic inflammation are encouraging for the use of PGI2 in the treatment of allergic airway inflammation, cost and difficulty in drug delivery are currently obstacles.161,162 However, the development of less expensive and longer acting agonists may make stable analogs of PGI2 a viable therapeutic option.

Thromboxane A2 Thromboxane A2 (TXA2) is the principal product of arachidonic acid metabolism formed by platelets and is a potent platelet aggregating agent.163 Thromboxane synthase (TXAS) is an endoplasmic reticulum membrane protein that catalyzes the conversion of prostaglandin H2 to thromboxane A2.164 TXAS is a member of the cytochrome P450 superfamily and is localized to band q33-q34 of the long arm of chromosome 7 in humans.164 TXAS is expressed abundantly in lung, liver, kidney, and blood cells, including megakaryocytes and monocytes.164 Lower, but significant, levels of TXAS mRNA are observed in kidney, placenta, and thymus.164 TXA2 is principally produced by platelets, monocytes, macrophages, neutrophils, and lung parenchyma.165 After it is formed, TXA2 is nonenzymatically hydrolyzed to thromboxane B2, which is further metabolized to the principle urinary metabolites 2,3-dinor-thromboxane B2 and 11-dehydro-thromboxane B2.166 The TXA2 receptor is termed TP (Table 9.4) and there are two isoforms, TPα and TPβ, which are produced by alternative splicing occurring in the carboxy-terminal region after the seventh transmembrane domain.167 Both of these isoforms functionally couple to a Gq protein, resulting in phospholipase C activation, calcium release, and activation of protein kinase C.168 However, these receptor isoforms couple oppositely to adenylate cyclase as TPα activates adenylate cyclase while TPβ inhibits this enzyme.169 The TP receptors are localized to both plasma membrane and cytosolic compartments and are mainly distributed in tissues rich in vasculature such as lung, heart, and kidney.132 These GPCRs are involved in a multitude of physiologic and pathologic processes such as vasoconstriction implicated in vascular diseases such as hypertension, stroke, atherosclerosis, and myocardial infarction.170 Notably, TP receptors are required for constriction of human airways to several prostanoids, including 9α,11β PGF2, PGF2, PGD2, and PGE2,171 and also mediate vascular responses to isoprostanes. Hence, some actions of TP receptor agonists may reflect functions unrelated to TXA2.

Human Studies of TXA2 in Allergic Inflammation.  TXA2 has a half-life of approximately 30 seconds,172 and because of this lability there is a paucity of in vivo studies examining the effect of TXA2 in the human airway. TXB2 did not cause bronchoconstriction of human airway in vivo.173 However, TXA2 was a potent stimulant of in vitro smooth muscle constriction.163 There are data that suggests that TXA2 might have a role in the physiology associated with acute asthma exacerbations. Levels of TXA2 metabolites were increased fourfold to sixfold in the urine of patients admitted to hospital with asthma compared with nonsmoking controls admitted for other diagnoses.173 Although one study reported a significant increase in urinary excretion of TXA2 products by atopic asthmatic subjects undergoing inhalation challenges with allergen,26,174 others have not found similar results.173 Inhibition of platelet COX by low dose aspirin inhibited the increase in urinary thromboxane metabolites, supporting that allergen inhalation causes platelet activation. Allergic asthmatics pretreated with indomethacin before inhaled allergen challenge resulted in a significant decline in urinary TXA2 metabolites; however, no change in pulmonary function was noted.26 Subjects that experience ozone-induced airway hyperresponsiveness had significant increases in BAL concentrations of TXA2 and airway

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation

Mouse Studies of TXA2 in Allergic Inflammation.  Both the TXA2

neutrophilia.175 Similarly, LTB4 inhalation also resulted in increased levels of TXA2 and neutrophils in BAL fluid.176 TXA2 antagonists have been used in challenge models and in shortterm studies in asthma to determine the effect of TXA2’s on pulmonary function and airway reactivity. In a nonrandomized, uncontrolled study the TP antagonist seratrodast (AA-2414) significantly reduced bronchial reactivity in asthmatic subjects after 4 weeks of once-daily therapy compared with a pretreatment baseline.177 In this study, seratrodast had no effect on either exhaled nitric oxide or on the percentage of eosinophils in sputum.177 In a follow-up double-blind, placebo-controlled study of asthmatics treated for 4 weeks, seratrodast treatment resulted in significant improvements in symptom score, peak expiratory flow (PEF) rates, diurnal variation of PEF, and bronchial responsiveness compared with the placebo group.178 These improvements were associated with a significant reduction in the number of submucosal eosinophils on bronchial biopsy.178 Seratrodast treatment resulted in a significant decrease in the number of cells in the epithelium expressing RANTES (CCL5) and macrophage inflammatory protein (MIP)-1α (CCL3), as well as a diminished number of cells in the submucosa expressing monocyte chemotactic protein-3, RANTES, MIP-1α, and eotaxin (CCL11).178 These findings suggest that TXA2 antagonism may reduce allergic inflammation in the lung, although the mechanisms are not well defined.

synthase inhibitor OKY-046 and the TP receptor antagonist S-1452 significantly decreased the number of total cells and eosinophils in BAL fluid in a dose-responsive relationship in OVA-sensitized and challenged mice.165 Treatment with either the TXA2 synthase inhibitor or the TP receptor antagonist significantly inhibited antigen-specific activation of splenic mononuclear cells from sensitized mice in ex vivo experiments as defined by proinflammatory cytokine production.165 A recent study revealed that genetic deletion of TP receptors from mice lacking mPGES-1 prevented the development of dust mite-induced pulmonary eosinophilia, airway hyperresponsiveness, Th2 cytokine generation, and vascular remodeling.179 Thus, the pathogenic contributions from TXA2 may be amplified when local concentrations of PGE2 are low.

Lipoxygenase Pathway As is the case for prostanoids, arachidonic acid is the precursor for lipoxygenase (LO) pathway products.180 Two major enzymes, 5-LO and 15-LO, metabolize arachidonate in the initial steps that form distinctive respective mediator classes (Fig. 9.2). The latter enzyme catalyzes the hydroperoxidation of arachidonic acid by the insertion of one molecule of oxygen at position 15 to form 15-HPETE, as well as the insertion of

Cell membrane

cPLA2 COOH

FLAP

Arachidonic acid

5-LO OH

OH COOH

COOH

5-HPETE

OH COOH

OH

OH COOH

6-trans-LTB4 LTC4S

OH COOH

S-Cys-Gly Glu LTC4

5-HETE

O

OH

COOH

LTA4H

LTA4 OH

LTB4 OH

COOH

COOH

S-Cys-Gly γ-GT, γ-GL

141

LTD4

S-Cys DiP

LTE4

Fig. 9.2  Leukotriene generation: Biosynthesis and molecular structures of cysteinyl leukotrienes (cys-LTs). The enzyme cPLA2 catalyzes the liberation of arachidonic acid from cell membranes. 5-LO translocates to the nuclear envelope, requiring the integral membrane protein FLAP to convert arachidonic acid to the precursor LTA4. LTA4 can spontaneously convert to the inactive metabolite 6-trans-LTB4, specifically hydrolyzed by LTA4H to LTB4, or conjugated to reduced glutathione by LTC4S, forming LTC4, the first committed molecule of the cys-LTs (red). After specific export, LTC4 is converted by the extracellular enzymes γ-GT and γ-GL to LTD4, and to LTE4 by dipeptidase (DiP). Enzymes essential for cys-LT synthesis are in bold. cPLA2, Cytosolic PLA2; FLAP, 5-LO–activating protein; γ-GL, gamma globulins; γ-GT, gamma-glutamyl transpeptidase; 5-HETE, 5-hydroxyeicosatetraenoic acid; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 5-LO, 5-lipoxygenase; LTA4, leukotriene A4; LTA4H, leukotriene A4 hydrolase; LTB4, leukotriene B4; LTC4, leukotriene C4; LTC4S, leukotriene C4 synthase; LTD4, leukotriene D4; LTE4, leukotriene E4. (From Kanaoka Y, Boyce JA. Cysteinyl leukotrienes and their receptors: cellular distribution and function in immune and inflammatory responses. J Immunol 2004;173:1503-10, with permission.)

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molecular oxygen into other polyunsaturated fatty acids and phospholipids. The 15-LO pathway is responsible for forming 15-hydroxyeicosa­ tetraenoic acid (15-HETE) and the dihydroxy acids 8,15-diHETE and 14,15-diHETE. 5-LO translocates in a Ca2+-dependent manner from either the cytoplasm or the nucleus to the perinuclear membrane and catalyzes the insertion of molecular oxygen into arachidonic acid to produce 5-hydroperoxyeicosatetraenoic acid (5-HPETE). 5-HPETE can then either be dehydrated to leukotriene (LT) A4 by 5-LO, or reduced to 5-hydroxyeicosatetraenoic acid (5-HETE) and further converted to the 5-oxo-ETEs.180,181 In intact cells, both of these catalytic functions require 5-LO activating protein (FLAP), an integral perinuclear membrane protein that transfers free arachidonic acid to 5-LO and is essential for the 5-LO function of generating LTA4.180

Leukotrienes Leukotrienes, named for their cells of origin (leukocytes) and three positionally conserved double bonds (trienes) are potent inflammatory mediators generated from the unstable precursor LTA4.182 There are two distinct classes of leukotrienes; LTB4 is a dihydroxyl compound formed by a cytosolic LTA4 hydrolase (LTA4H). LTA4H is expressed by mast cells, macrophages, and neutrophils, the major cellular sources of LTB4 in vivo. The human gene encoding LTA4H maps to chromosome 12q22.183 LTA4 can also be conjugated to reduced glutathione to form LTC4, the parent molecule of the cys-LTs by leukotriene C4 synthase (LTC4S).118,184 The major cellular sources of cys-LTs are eosinophils, basophils, mast cells, and macrophages, each of which express LTC4S. LTC4S expression is sharply upregulated in human mast cells by treatment with IL-4, potentially reflecting a mechanism for upregulating cys-LT production in allergic inflammation.185 The gene encoding LTC4S in humans maps to chromosome 5q35, distal to the Th2 cytokine gene cluster.186 Both LTB4 and LTC4 are exported by specific respective transporter proteins, members of the multidrug resistance proteins (MRPs). LTC4 is converted to LTD4 extracellularly by cleavage of glutamic acid from the glutathione moiety by γ-glutamyl-transpeptidase, or by a more specific γ-glutamyl-leukotrienease.187 A dipeptidase then cleaves glycine from LTD4 to form LTE4.187

Leukotrienes in Human Studies of Allergic Inflammation and Asthma.  Leukotriene B4 and the cys-LTs were increased in exhaled breath condensate from asthmatic subjects compared with healthy controls.188 After allergen challenge, there was a significant increase in leukotriene levels in the BAL fluid of allergic subjects, and this was associated with increased eosinophilic inflammation and bronchial responsiveness.189 Leukotriene levels in induced sputum from asthmatic subjects exceed those found in nonasthmatic controls and correlate with severity of disease.87 Urinary LTE4 is also increased in asthmatic subjects compared with controls and in proportion to disease severity and is especially high in subjects with AERD.190 Therefore leukotriene synthesis can reflect ongoing inflammation in the lung and the physiologic changes associated with asthma. Corticosteroid treatment had no impact on leukotriene levels in asthmatics, suggesting that leukotriene generation is relatively resistant to this class of antiinflammatory medication.188,191 As mentioned previously, both 5-LO and FLAP are critical to the generation of leukotrienes. Although there is abundant clinical experience with 5-LO inhibitors, no FLAP inhibitors have been approved for human use. Zileuton is a 5-LO inhibitor and its activity is presumed to be due to its ability to chelate iron at the active site of 5-LO, thus preventing its redox potential.192 In a study of asthmatics with mildto-moderate disease not receiving inhaled steroids, zileuton administration produced a 350 mL (15% from pretreatment baseline) increase in FEV1 within 1 hour and was statistically increased compared with placebo.

After a 4-week study period, there was also a significant improvement in peak expiratory flow rate in the zileuton-treated subjects (600 mg four times daily) over that in the placebo group. Of note, after the 4 weeks of treatment, zileuton reduced urinary LTE4 levels by only approximately 40% compared with the placebo group, indicating that even the highest clinically recommended dose of the 5-LO inhibitor did not fully block leukotriene generation.193 In another 13-week study of asthmatic subjects who had FEV1 between 40% and 80% predicted, zileuton significantly decreased the need for rescue β-agonists, reduced daytime and nighttime symptoms, and increased symptom-free days and nights compared with placebo.194 In this trial, zileuton also significantly reduced the number of subjects requiring corticosteroid therapy for an asthma exacerbation.194 In a trial to investigate the role of 5-LO inhibition on inflammation after segmental allergen challenge, zileuton inhibited urinary LTE4 production by 86% and prevented the increase in BAL eosinophils that was noted in the placebo-treated subjects.195 The fact that 5-LO inhibition blunts allergen-induced inflammation while improving lung function and symptoms in asthmatic subjects validates the role of leukotrienes in asthma pathogenesis. Disappointingly, no additional 5-LO inhibitors have made it to clinical use, and there are no clinically available FLAP inhibitors. There have been a number of studies over the past decades that collectively examine the genetic variants associated with both leukotrienerelated asthma pathogenesis and the differential regulation of responses to leukotriene modifiers. Indeed there are data to suggest that there are candidate genes in the cys-LT pathway that contribute to both the etiology of asthma itself, and regulate a person’s responsiveness to pharmacologic leukotriene modification.196

Murine Studies of Leukotriene Inhibition.  Unlike human airways, mouse airways are resistant to the bronchoconstrictor effects of the cys-LTs.40 Nonetheless, mouse models of allergen-induced pulmonary inflammation have uncovered key roles for these mediators in the induction and amplification of this process. Mice that lack 5-LO as a result of targeted gene disruption have decreased allergen-induced BAL eosinophilia, serum IgE, and airway responsiveness compared with wild type mice.197 A similar phenotype was described for mice lacking group IVA PLA2, which generates neither leukotrienes nor prostaglandins.198 Similarly, zileuton reduced allergen-induced leukotriene release in the BAL and eosinophil recruitment to the lungs, while dosedependently reducing AHR, mucus accumulation, and remodeling.199 Specific inhibitors of 5-LO and FLAP independently blocked airway mucus release and airway infiltration by eosinophils indicating a key role for leukotrienes in these features of allergic pulmonary inflammation; however, they had no effect on allergen-induced airway responsiveness.200 Zileuton treatment blocked airway responsiveness and inflammation that occurred as a result of intratracheal instillation of IL-13, monocyte chemoattractant protein (MCP)-1, MCP-5, and KC, the mouse homolog of IL-8.201 Thus the effects of pharmacologic and genetic deletion of 5-LO pathway products in mice are profound and reflect the composite loss of both cys-LT- and LTB4-mediated actions (see later discussion).

LTB4 LTB4 exerts its biologic effects by signaling through two distinct GPCRs, BLT1, and BLT2.202 LTB4 binds with much higher affinity to BLT1 compared with BLT2.202 Other eicosanoids, specifically 12(s)-HETE, 12(S)HPETE, and 15(S)-HETE can bind BLT2, but do not bind BLT1.202 BLT1 is predominantly expressed on activated leukocytes, with decreased expression in spleen, thymus, bone marrow, lymph nodes, heart, skeletal muscle, brain, and liver.202 BLT2 is expressed in most human tissues with the greatest degree in spleen, liver, ovary, and peripheral blood

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation white cells.202 Among the leukocytes, there are some significant differences in LTB4 receptor expression.202 For instance, neutrophils and eosinophils both express high levels of both BLT1 and BLT2, whereas mononuclear cells express high levels of BLT2, but very low levels of BLT1.202 BLT1 expression is upregulated on activated cells and expression is increased by both interferon-γ (IFN-γ) and glucocorticoids.202 The primary known function of LTB4 receptor signaling is leukocyte recruitment.202 LTB4 is a leukocyte chemoattractant and also changes leukocyte rolling to firm attachment through its upregulation of the integrin CD11b/CD18 on neutrophils.202 With these properties, exogenous LTB4 administration in both the skin and airways caused neutrophil migration to those sites.

Human Studies of LTB4 in Allergic Inflammation.  To date, there are no intervention studies using specific BLT1 or BLT2 antagonists or agonists to examine the role of signaling through these receptors in modulating allergic inflammation in humans. However, some studies suggest that signaling through these receptors may regulate the allergic phenotype. For instance, in healthy subjects, T cells that express BLT1 are rare in peripheral blood, but do express the activation markers CD38 and HLA-DR.203 Compared with T cells that do not express BLT1, a larger proportion of peripheral blood-expressing T cells also express the effector cytokines IFN-γ and IL-4, as well as inflammatory chemokine receptors, CCR1, CCR2, CCR6, and CXCR1.203 T cell BLT1 expression is tightly regulated by inflammation and only expressed transiently after naïve T lymphocytes are activated by dendritic cells. The number of peripheral blood T cells expressing BLT1 was increased in frequency in the airways of asymptomatic allergic asthmatics.203 LTB4, via BLT1, is strongly chemotactic for mast cell progenitors in vitro.204 Activated mature mast cells produced LTB4, which was highly chemotactic for 2-week-old mast cells that expressed high levels of mRNA for BLT1, whereas expression of this receptor was not present on mature mast cells.204 To date, there are no published studies in humans or human cells examining the effect of signaling through BLT2 on the regulation of allergic inflammation.

Mouse Studies of LTB4 in Allergic Inflammation.  BLT1 expression was weak in naïve murine CD4+ T cells, but strong in activated Th0, Th1, and Th2 cells, whereas BLT2 was not expressed in these cell populations.205 LTB4 induced CD4+ T cell chemotaxis in wild type mice, but not in BLT1-deficient mice, signifying the receptor specificity for this chemotaxis.205 BLT1-deficent mice had decreased numbers of both CD4+ and CD8+ T cells in BAL fluid after one and 2 days, but not after 3 days of allergen challenge in which mice had been sensitized first with an intraperitoneal injection of OVA and the adjuvant aluminum hydroxide, whereas there was no difference in the numbers of CD4+ and CD8+ cells in the lung parenchyma. BLT1-deficient and wild type mice did not differ in the expression of chemokines responsible for T cell recruitment, suggesting that reduced effector T cell trafficking into the airway in BLT1-deficient mice was a direct consequence of the absence of LTB4BLT1 signaling.205 In this model, there were no differences in serum IgE levels between the BLT1-deficient and wild type mice, and neither airway eosinophilia nor mucus expression was reported. However, adoptively transferred antigen-specific transgenic T cells did not require BLT1 for antigen-induced recruitment to the lungs of naïve mice.205 Thus the role of BLT1 signaling in the setting of allergen challenge is dependent on the model used. Another group using the OVA/aluminum hydroxide sensitization model found that BLT1-deficient mice were protected from AHR, eosinophilic inflammation, and hyperplasia of goblet cells.206 These BLT1-deficient mice also had reduced IgE production and levels of IL-5 and IL-13 in BAL fluid, suggesting BLT1 signaling was critical for the generation of a Th2-type immune response.206 Another group

143

found similar results in allergen-sensitized and challenged BLT1-deficient mice and was able to increase allergic inflammation by transfer of allergen-primed wild type T cells into the BLT1-deficient mice that were subsequently allergen challenged.207 Signaling through BLT1 has also been proposed to mediate CD8-dependent allergic airway inflammation and AHR. CD8-deficient mice that were adoptively transferred CD8 cells from allergen-sensitized wild type, but not BLT1-deficient, mice developed allergen inflammation and AHR.208,209 There have been no reports of murine studies examining the BLT2 receptor in regulating allergic inflammation, although dendritic cells from mice deficient in both BLT1 and BLT2 had a marked defect in the ability to migrate to draining lymph nodes compared with dendritic cells from wild type mice.210

Cysteinyl Leukotrienes Cys-LTs signal through three GPCRs; CysLT1, CysLT2, and the recently identified CysLT3 (also known as GPR99) (Table 9.5).182,211 In humans, CysLT1 maps to the X chromosome, whereas CysLT2 maps to chromosome 13q14.182 CysLT3/GPR99 maps to chromosome 13. CysLT1 binds LTD4 with high affinity and LTC4 and LTE4 equally with lower affinity.182 In contrast, CysLT2 binds LTC4 and LTD4 equally and with greater affinity than LTE4.182 CysLT3 exhibits a binding preference for LTE4. In humans, CysLT1 is expressed in the spleen, lung, placenta, and small intestine. 182 Specific cell types on which CysLT1 is expressed include bronchial smooth muscle, glandular epithelium, monocyte/macrophages, mast cells, basophils, eosinophils, dendritic cells, B and T lymphocytes, CD34+ hematopoietic progenitors, neutrophils, and human umbilical vein endothelial cells.182,212,213 CysLT1 expression can be upregulated on peripheral blood mononuclear cells by IL-4 and IL-13, or on an eosinophilic leukemic cell line by IL-5, with a resulting increase in expression of CysLT1 leading to enhanced chemotaxis to LTD4.182 CysLT2 is expressed in the lung, spleen, heart, lymph nodes, and brain. Cells expressing CysLT2 include monocyte/macrophages, mast cells, eosinophils, cardiac, Purkinje cells, bronchial smooth muscle, coronary smooth muscle, and human umbilical vein endothelial cells. CysLT3/GPR99 mRNA is expressed in the trachea and proximal convoluted tubule and, at least in mice, CysLT3/ GPR99 protein is richly expressed in nasal and tracheal epithelial cells.214 CysLT1 and CysLT2 have been shown to interact at least on human mast cells and mouse dendritic cells. CysLT2 inhibited CysLT1 mast cell surface expression and CysLT1-dependent proliferation of cord blood–derived mast cells.215 The specific CysLT1 receptor antagonists such as montelukast, zafirlukast, and pranlukast have been critical in determining the effects of signaling through this receptor in biological systems and in humans in vivo. Signaling through CysLT1 dilates blood vessels with a resultant increase in vascular permeability, amplified mucus expression, bronchial smooth muscle constriction, and inflammatory cell recruitment. Specifically, CysLT1 signaling augments transendothelial migration of CD34+ hematopoietic cells and eosinophil chemotaxis in vitro. In addition, cys-LTs increase IgE-mediated mast cell production of IL-5 and TNF via the CysLT1 receptor and regulate mast cell proliferation by inducing transactivation of the c-Kit receptor tyrosine kinase.216 The consequences of CysLT2-mediated signaling are far less well understood than that of CysLT1 signaling, because there are no specific CysLT2 receptor antagonists. A primary role of CysLT2 signaling may be activation of endothelial cells in vascular responses, as indicated by the CysLT2-dominant signaling reported in human umbilical vein endothelial cells.182 CysLT3 may play a role in the development of goblet cells.214

Human Studies of Cysteinyl Leukotrienes in Allergic Inflammation.  Site-directed allergen challenge increases the concentrations of cys-LTs in the skin, eye, nose, and lung, and these levels are strongly correlated with allergic symptoms.188,217,218 Direct administration of

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TABLE 9.5  Cysteinyl Leukotriene Receptor Expression, Signaling, and Function Feature

CysLT1

CysLT2

CysLT3/GPR99

Ligands

LTD4 > LTC4 = LTE4

LTC4 = LTD4 > LTE4

LTE4 > LTC4 > LTD4

Chromosome

Human: Xq13.2-21.1 Mouse: X-D

Human: 13q14.12-q21.1 Mouse: 14-D1

Human: 13q32.2 Mouse: 13q32.1

Receptor expression

Human Tissues: spleen, lung, placenta, small intestine

Human Tissues: spleen, lung, heart, lymph node, brain

Cell types: bronchial smooth muscle, monocytemacrophages, mast cells, eosinophils, CD34+ hematopoietic progenitor cells, neutrophils, HUVECs

Cell types: monocyte-macrophages, mast cells, eosinophils, cardiac Purkinje cells, pheochromocytes and ganglion cells in adrenal medulla, bronchial smooth muscle, HUVECs, coronary smooth muscle Mouse Tissues: spleen, lung, small intestine, kidney, brain, skin Cell types: monocyte-macrophages, fibroblasts, endothelial cells, cardiac Purkinje cells

Human Tissues: kidney, placenta, lung, nasal mucosa Cell types: eosinophils, mast cells, respiratory smooth muscle, epithelium

Mouse Tissues: lung, skin, small intestine Cell types: monocyte-macrophages, fibroblasts Function

Dilates blood vessels with increased vascular permeability Amplifies mucus expression, bronchial smooth muscle constriction Augments transendothelial migration of CD34+ hematopoietic cells and eosinophils Increases IgE-mediated mast cell production of IL-5 and TNF

Activation of endothelial cells, platelets

Mouse Tissues: kidneys, testes Cell types: smooth muscle, epithelium Goblet cell development, mucus secretion

CysLT1, CysLT2, CysLT3, Cysteinyl leukotriene receptor types 1 to 3; HUVECs, human umbilical vein endothelial cells; IgE, immunoglobulin E; IL-5, interleukin-5; LTC4, leukotriene C4; LTD4, leukotriene D4; LTE4, leukotriene E4; TNF, tumor necrosis factor.

cys-LTs into the human airways confirms that these lipids are the most potent known bronchoconstrictors, are proinflammatory, and that their effects are receptor mediated. In asthmatic subjects, inhaled LTE4 increased airway eosinophils. This increase is blocked by treatment with the CysLT1 receptor antagonist zafirlukast, albeit at higher dosages than are required to prevent allergen-induced bronchoconstriction.219 Notably, unlike LTE4, the CysLT1 agonist LTD4 does not elicit airway eosinophilia. Subjects with asthma demonstrate comparable degrees of airway reactivity to LTC4 or LTD4 as do nonasthmatic controls but display enhanced sensitivity to bronchoconstriction induced by inhalation of LTE4.220 Subjects with AERD display further enhancement of LTE4 reactivity over aspirin-tolerant controls.221 Whether these properties of LTE4 relate to the functions of CysLT3/GPR99 remains to be established. CysLT1 antagonists have been used in clinical trials of asthma, rhinitis, and urticaria. These studies reveal that signaling through this receptor is involved in the pathogenesis of these diseases. In a 12-week study of asthmatics with FEV1 50% to 85% predicted, montelukast significantly improved pulmonary function and symptoms compared with placebo, but was not as effective as inhaled corticosteroids for the same endpoints.222 CysLT1 receptor antagonists have also been shown to inhibit both early- and late-phase pulmonary reactions to allergen challenge as well as airway obstruction induced by exercise, SO2, hyperpnea, adenosine 5’-monophosphate, aspirin, and mannitol.223-228 In clinical studies of rhinitis, CysLT1 receptor antagonists have reduced rhinorrhea, nasal congestion, and sneezing, while improving daytime and nighttime symptoms, although there is variability between studies in the effectiveness of this medication class on these individual endpoints.213 CysLT1 antagonists have also been shown to reduce the symptoms of chronic urticaria compared with placebo.229-231 Several of these clinical trials identified significant suppression of blood and/or tissue eosinophil counts by the administration of CysLT1 antagonists, implying a role for

cys-LTs in regulating eosinophil homeostasis.213 Presently, the lack of antagonists selective for CysLT2 or CysLT3/GPR99 preclude direct examination of the contributions potentially made by these receptors to human allergic disease.

Mouse Studies of cys-LTs in Allergic Inflammation.  In a mast cell–dependent model of OVA-induced pulmonary inflammation, BALB/c mice lacking LTC4S showed strikingly diminished AHR, goblet cell metaplasia, OVA-specific IgE, and cytokine production by restimulated lymph node cells compared with wild type, allergen-treated control mice.232 These mice also showed strikingly decreased mast cell numbers in the bronchial epithelium compared with wild type controls.232 In another model of OVA-induced allergic pulmonary inflammation, wild type BALB/c mice were treated with a long (76 day) period of allergen challenge to induce changes of airway remodeling.233 In this study, the administration of the CysLT1 receptor-selective antagonist montelukast during the challenge phase decreased lung expression of IL-4 and IL-5 and decreased bronchial eosinophil numbers while inhibiting smooth muscle hypertrophy and collagen deposition.233 In this model, inflammation subsided after cessation of the allergen challenge, but the smooth muscle hypertrophy and collagen deposition persisted to 3 months.233 Interestingly, montelukast reversed the remodeling signatures when administered from days 73 to 163 (after the challenge phase of the experiment), whereas dexamethasone did not.233 Cys-LTs signaling through the CysLT2 receptor can mediate tissue fibrosis. In a model of atopic dermatitis induced by epicutaneous sensitization with OVA, WT mice had increased skin thickening and collagen deposition compared with either LTC4 synthase-deficient or CysLT2 receptor-deficient mice.234 In this model, eosinophils were the primary source of cys-LTs. Thus cys-LTs orchestrate both sensitization and remodeling events in models of allergic pulmonary inflammation in mice, consistent with their abundant generation by mast cells and eosinophils, as well as the broad

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation distribution of their receptors on both hematopoietic and structural cells in the lung. When pulsed ex vivo with dust mite antigen, mouse myeloid dendritic cells exposed to exogenous LTD4 show augmented production of IL-10 and attenuated IL-12 generation compared with antigen pulsing alone.235 Treatment of these DCs with CysLT1 receptor-selective antagonists during antigen pulsing attenuates IL-10 generation and augments IL-12 production.235 Myeloid dendritic cells treated with CysLT1 antagonists during stimulation with dust mite allergen in vitro were substantially less able to support an eosinophil-dominated inflammatory response to inhaled allergen when adoptively transferred into the tracheas of naïve recipient mice.235 Dust mite allergen–pulsed dendritic cells from mice lacking either CysLT1 or LTC4 synthase show markedly impaired ability to prime recipient naïve mice for Th2 responses to dust mite allergens, whereas CysLT2 receptor null dendritic cells exhibit markedly enhanced Th2 priming function due to loss of CysLT2 receptor– dependent interference with CysLT1 function.236 Thus as is the case for PGD2, cys-LTs participate in the regulation of dendritic cell maturation responses. Recently, several studies have linked cysLTs to the effector arm of type 2 immunity in mice. LTC4, LTD4, and LTE4 can all induce increases in the numbers of ILC2s in the lungs of mice that undergo challenges in Alternaria alternata extract and can elicit the production of IL-5 and IL-13 from these cells.237 The responses of ILC2s to LTC4 and LTD4, but not to LTE4, depend exclusively on CysLT1.238 LTC4 potently activates mouse platelets exclusively through CysLT2 and strongly induces the recruitment of eosinophils to the lungs of mice that are sensitized and challenged with low-dose OVA. The latter response depends on CysLT2mediated platelet activation and thromboxane-mediated endothelial activation.239 These observations reflect additional mechanisms by which cys-LTs may facilitate allergic inflammation.

Lipoxins Lipoxins (LXs) are produced at sites of either vascular or tissue injury, and abundant data suggest that these are involved in resolution of inflammation (reviewed in reference 240). LX can be formed by several pathways. In the vasculature, LTA4 produced by activated leukocytes can be converted to LX by platelet 12-LO. In the lung parenchyma, LTA4 can be converted to LX by epithelial cell 15-LO. LXs can also be formed by transformation of 15-LO–derived 15-hydroperoxy-eicosatetraenoic acid (15-H(p)ETE) by 5-LO. 15-epimer-LXs are produced by 5-LO–mediated conversion of 15(R)-hydroxy-eicosatetraenoic acid (15(R)-HETE) to 15-epi-LXA4 and 15-epi-LXB4. Interestingly, statins also demonstrate the capability of inducing 15-epi-LXA4 formation. In addition, cell-cell interactions between neutrophils and airway epithelial cells in the presence of statins leads to 15-epi-LXA4 biosynthesis. LXA4 and 15-epi-LXA4 are both agonists for a LXA4 receptor termed ALX/FPR2, a GPCR that binds these lipid products with high affinity. ALX is expressed on both human airway epithelial cells and leukocytes, and can be induced by specific inflammatory mediators. In addition to LXs signaling through ALX, LXs can also act as antagonists at CysLT1 receptors and can also signal via the aryl hydrocarbon receptor. LXs can inhibit granulocyte locomotion, shape change, transmigration, and degranulation. Through these actions, LXs are both antiinflammatory for neutrophils and eosinophils, and appear to have an important role in the clearance of inflamed tissue by monocytes and macrophages. In addition to these leukocyte-specific actions, LXs promote restoration of injured airway epithelium by stimulating bronchial basal epithelial cell proliferation, blocking the release of the proinflammatory cytokines IL-6 and IL-8, and inhibiting neutrophil transmigration across differentiated human bronchial cells. Injury upregulates ALX receptor expression in both the proximal and distal airway. In line with promoting resolution of

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injury, LXs inhibit inflammation-induced angiogenesis and endothelial cell migration in response to proinflammatory mediators. Animal models reveal that LXs block inflammation and its consequences in several different models of lung injury. Allergic airway inflammation is significantly reduced by LXs in mouse models, and resolution occurs more quickly compared with vehicle treatment. Similar findings have been published for a rat model of allergic pleurisy, bleomycin-induced fibrosis in mice, acid-induced acute lung injury, and pneumonia. LXs are decreased in the whole blood, sputum, and BAL fluids of persons with severe asthma, suggesting that decreased generation of LXs, and therefore inability to resolve inflammation, may be associated with the severe phenotype. There is also a decrease in LXs in subjects with aspirin-intolerant asthma, exercise-induced asthma, scleroderma lung disease, and cystic fibrosis compared with healthy persons. Further research in the LX field is necessary to determine whether these lipids may serve a therapeutic purpose.

Resolvins and Protectins Resolvins and protectins are mediators that are proposed to be critically involved in the active phase of inflammation resolution (reviewed in reference 241). Resolvins and protectins are enzymatically generated from the n-3 polyunsaturated fatty acids eicosapentaenoic acid (EPA, 20 : 5n-3) and docosahexanoic acid (DHA, 22 : 6n-3) that are enriched in fish oils. Resolvins are divided into the EPA-derived E-series resolvins, RvE1 and RvE2, and the DHA-derived D-series resolvins, RvD1-4. In vitro, RvE1 blocked transendothelial migration and superoxide generation, thus revealing potent inhibitory effects of this lipid on neutrophil function. RvE1 also increased the clearance of neutrophils from the apical surface of mucosal epithelial cells and phagocytosis of apoptotic neutrophils by macrophages. In vivo, RvE1 promoted resolution of periodontal, colonic, and allergic airway inflammation in mice. RvE1 was log-order more potent than either dexamethasone or aspirin. ReV2 is structurally distinct from RvE1, and these two mediators have additive effects at low concentrations, suggesting that they signal through separate receptors. The D-series resolvins also inhibit neutrophil tissue infiltration. Protectin D1 (PD1) is the lead member of the protectin family and is produced from DHA. PD1 has been isolated from mouse lung, human blood, and exhaled breath condensate from asthmatic and healthy humans. In mice, PD1 prevented allergen-induced airway eosinophilia, T cell influx, mucus metaplasia, and methacholine-induced hyper­ responsiveness. In addition, PD1 administration accelerated the resolution of established allergic airway inflammation. Clinical trials are now underway to determine the utility of resolvins and protectins in human inflammatory diseases.

Isoprostanes Isoprostanes (IsoPs) are prostaglandin-like molecules that are not formed by the action of the COX enzymes, but instead are formed by the free radical-catalyzed peroxidation of arachidonic acid.242 IsoP formation is not only a dependable marker of oxidant injury both in vivo and in vitro, but these mediators also are biologically active and may regulate oxidant injury.242 There are several classes of IsoPs that differ based on the functional groups present on the prostane ring.242 The classes include the F2-IsoPs, the D2/E2-IsoPs, the A2/J2-IsoPs, the isothromboxanes, and the isoketals (IsoKs).242 These classes are distinguished by the type of isoprostane ring that each contains.242 A2/J2-IsoPs are formed from the dehydration of E2/D2-IsoPs, respectively. F2-IsoP can be detected in all normal biologic fluids in both humans and animals, whereas the D2/E2-IsoPs, the A2/J2-IsoPs, and the isothromboxanes cannot.242 However, levels of all of these classes are substantially increased in vivo after oxidant injury such as carbon tetrachloride administration,

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with the exception of the A2/J2-IsoPs and the IsoKs, which still cannot be detected.242 The neuroprostanes are similar to the IsoPs in that they contain various prostane ring functional groups. However, the neuroprostanes are formed from docosahexaenoic acid that is present in high levels of neural tissue.242 IsoPs have been shown to have potent biologic activity and perhaps regulate adverse effects of oxidant injury. The biologic activities of 15-F2tIsoP (also known as 8-isoprostane or 8-iso-PGF2α) and 15-E2t-IsoP have been particularly well studied, and these were potent vasoconstrictors in the kidney, lung, heart, retina, portal vein, and lymphatics.243-249 At least part of the vasoconstrictive properties of 15-F2t-IsoP resulted from its interaction with the thromboxane receptor TP.250 15-F2t-IsoP also promoted the release of endothelin and vascular smooth muscle cell proliferation.251,252 Other activities ascribed to 15-F2t-IsoP include osteoclastic differentiation resulting in bone resorption and augmented resistance to aspirin-induced blockade of platelet aggregation.253-255 Because the F2-IsoPs can be easily detected in human biologic fluid at baseline, changes in the levels of these compounds have been used as an in vivo index of endogenous lipid peroxidation or oxidant stress. The fact that the F2-IsoPs can be measured in urine further increases the usefulness of these products as a noninvasive marker of oxidant stress activity and the activity of antioxidant compounds in vivo. Similar to the prostaglandins, measurement of F2-IsoPs in the urine is the most dependable method to assess total endogenous levels of these products, because blood levels may reflect only recent trends because the biologic half-life of these mediators is very short, on the order of 16 minutes. The preferred method of measurement of the IsoPs is gas chromatography/ negative ion chemical ionization mass spectroscopy. Immunoassays are commercially available; however, the presence of substances in biologic fluids that interfere with these assays may confound results obtained with this technique. It is important to note that biologic material in which IsoPs are to be measured should be stored at −70° C or with the addition of antioxidants to prevent production of IsoPs ex vivo.

Human Studies of Isoprostanes in Allergic Inflammation.  Levels of IsoPs have been studied in asthma and other allergic diseases. Compared with healthy nonasthmatic controls, exhaled breath condensates of the F2-IsoP series member 15-F2t-IsoP were greater in children with asthma, whether they had either mild persistent asthma, stable mildto-moderate persistent asthma that was treated with inhaled corticosteroids, or unstable asthma.256 Similarly, steroid-naïve asthmatic children and children in stable condition with mild-to-moderate persistent asthma who were being treated with inhaled corticosteroids had greater levels of 15-F2t-IsoP compared with healthy nonasthmatic children.257,258 In children experiencing an asthma exacerbation, oral corticosteroids significantly reduced exhaled breath condensate levels of 15-F2t-IsoP, although not to the same concentrations as measured in nonasthmatics, suggesting that corticosteroids may not be fully effective in reducing oxidative stress in children with an exacerbation of asthma.259 IsoPs have also been measured in exhaled breath condensates in adults. In women with mild allergic asthma, the levels of 15-F2t-IsoP were inversely correlated with the percent predicted FEV1.260 Exhaled breath condensate levels of 15-F2t-IsoP were also elevated in steroid-naïve subjects with aspirin-induced asthma compared with both subjects with aspirininduced asthma treated with steroids and healthy control subjects.261 Allergen challenge increased 15-F2t-IsoP in exhaled breath condensate, indicating that allergen challenge increases airway oxidative stress in allergic asthma.262 Induced sputum has also been used as a biologic fluid in which IsoPs may be measured. Adults who either had asthma or bronchiectasis had greater levels of sputum 15-F2t-IsoP than in healthy control subjects.263 In addition, 15-F2t-IsoP levels decreased in the induced sputum of asthmatics as they recovered from an exacerbation.263 Urinary

levels of IsoPs were also elevated in asthmatic subjects experiencing allergen challenge. 15-F2t-IsoP was elevated from as early as 2 hours, and as long as 8 hours, after inhaled allergen challenge in subjects with mild allergic asthma.264 In contrast, there was no increase in the urinary excretion of 8-isoprostane after methacholine challenge.264 Therefore, IsoP levels are increased during ongoing allergic inflammatory responses and decrease with treatment.

Mouse Studies of Isoprostanes in Allergic Inflammation.  IsoP measurements have also been performed in mouse models of allergic lung inflammation. In the OVA-model, F2-isoprostanes in whole lung were increased on the ninth day of daily aerosol allergen challenge.265 Increased immunoreactivity to 15-F2t-IsoP or to isoketal protein adducts was found in epithelial cells 24 hours after the first aerosol challenge and after 5 days of allergen exposure in macrophages.265 Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased 15-F2t-IsoP immunoreactivity after OVA challenge.265 Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes, whereas supplemental vitamin E suppressed their formation.265 Similar changes in immunoreactivity to F2-isoprostanes were seen.265 Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant.265 Therefore, IsoPs are also increased in the mouse model of allergic inflammation and these are reduced with antioxidant treatment.

Sphingosine-1-Phosphate There are two primary members of lysosphingolipids (LPLs) that have immunomodulatory functions. These include the lysoglycerophospholipids, such as lysophosphatidic acid (LPA), and the lysosphingolipids, of which sphingosine-1-phosphate (S1P) is a key member.266 S1P is synthesized intracellularly with the primary sources among immune cells being mast cells, platelets, and macrophages; however, a variety of nonimmune cells can also produce this mediator.267 The first step in S1P production is the sphingomyelinase conversion of endogenous membrane-derived sphingomyelin to ceramide, then ceramidase converts ceremide to sphingosine.266 Sphingosine is then converted to S1P through phosphorylation by either of two Sph kinases, SphK1 or SphK2.266 In mouse bone marrow–derived mast cells SphK2 was the principal regulator of intracellular signaling events such as calcium flux and downstream calcium-dependent activation of protein kinase C, NF-κB, eicosanoid production, and cytokine secretion.268 SphK1 had no apparent role in these intracellular activities, but instead regulated the concentration of extracellular S1P and the sensitivity of mast cells to antigen-driven degranulation.268 S1P can be transformed back into sphingosine by S1P phosphatase or removed from the production pathway by a S1P lyase.267 S1P functions within cells as a modulator of calcium homeostasis and as a regulator of cellular survival and proliferation.267 The receptors responsible for these intracellular functions are not clearly defined. S1P can be transported extracellularly and functions to activate cell motility.267 This function is mediated by S1P signaling through five GPCRs S1P1-5.267 The principal functional receptor for leukocyte chemotaxis to S1P is S1P1, which is expressed on T and B lymphocytes, mononuclear phagocytes, dendritic cells, mast cells, and NK cells.269 S1P signaling through S1P1 is a major regulator of T lymphocyte function in preventing apoptosis, promoting CD4+CD25+ T regulatory cell activity, and enhancing chemotaxis.269 S1P1 signaling promotes thymocyte emigration and movement of lymphocytes from lymph nodes, but not from the spleen, into efferent lymph and subsequently to blood.269 S1P1 receptors also regulate the chemotaxis of mast cells toward antigen, whereas S1P2 receptors facilitate IgE-dependent mast cell degranulation. It is thus likely that S1P generated by mast cells plays an important autocrine role in their function.270

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation

Human Studies of S1P in Allergic Inflammation.  Segmental allergen challenge in allergic asthmatics induced a twofold increase in S1P levels in BAL fluid 24 hours after the challenge; however, there was no change in BAL S1P levels in nonallergic nonasthmatic controls after challenge.271 In in vitro experiments using human airway smooth muscle cells, S1P administration resulted in a dose dependent increase in both DNA synthesis and cell proliferation.271 S1P also induced IL-6 production by human smooth muscle airway cells that was further increased by treatment of the cells with both S1P and TNF. However, S1P inhibited TNFinduced RANTES expression. Therefore further work will need to be performed to conclusively define the role of S1P in the human allergic inflammatory response.

Mouse Studies of S1P in Allergic Inflammation.  The pharmacologic agent FTY720 downregulates the activity of S1P1 in addition to S1P2 and S1P5, but not S1P3 or S1P4, and has been used in animal models to test the activity of signaling through the receptors in which it exerts inhibitory activity.269 In a model in which OVA-specific polarized Th2 cells were adoptively transferred into naïve mice, orally administered FTY720 inhibited the influx of T lymphocytes and eosinophils into the lungs when these mice were subsequently challenged with aerosolized OVA.272 In mice that were sensitized with an intraperitoneal injection of OVA formulated with aluminum hydroxide, oral FTY720 at the time of OVA challenge inhibited the airway accumulation of lymphocytes and eosinophils, prevented the induction of bronchial hyperresponsiveness, and reduced goblet cell hyperplasia.272 Intratracheal administration of FTY720 significantly reduced the number of BAL macrophages, neutrophils, lymphocytes, and eosinophils when it was given 30 minutes before OVA aerosolization in mice that had been previously sensitized to OVA.273 In addition, intratracheal FTY720 inhibited allergen-induced AHR and parenchymal lung inflammation.273 Although FTY720 had no effect on the number of blood-circulating lymphocytes or lymphocytes in peripheral lymph nodes, the number of T cells in lung-draining lymph nodes was significantly reduced.273 The effect of FTY720 seemed to be predominantly mediated by its inhibition of the migration of lung dendritic cells to the mediastinal lymph nodes, which in turn blunted the formation of allergen-specific Th2 cells in lymph nodes.273 In addition, FTY720-treated dendritic cells were intrinsically less effective in activating naïve and effector Th2 cells because of an inhibited capacity to form stable interactions with T cells and thus to produce an immunologic synapse.273 Thus inhibition of signaling through several S1P receptors downregulates allergic inflammation in the mouse. Very recently, FTY720 was reported to potently and selectively inhibit group IVA PLA2.274 It is thus possible that some of its immunosuppressive effects may be mediated by interference with eicosanoid generation.

SUMMARY The lipid mediators are a diverse array of potent molecules that can be rapidly generated by structural cells as well as leukocytes in response to environmental perturbations. The spectrum of homeostatic, immunologic, and inflammatory functions served by these mediators can now be better understood because of the identification of the GPCRs and enzyme systems responsible for the actions and synthesis of each and the development of receptor-deficient mice and receptor-selective agonists. Although the development of specific enzyme inhibitors and receptor antagonists for therapeutic use is still in its infancy, the success of the COX and 5-LO inhibitors and the CysLT1 antagonists strongly support the role of eicosanoids in human disease. It is likely that additional reagents under development, such as FTY720, will both provide efficacious treatment for human disease and new insights

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into the biologic importance of lipid mediators in both health and disease. Furthermore, the increasing recognition that lipid mediator receptors can form heterodimers and even trimers raises the level of complexity of eicosanoid signaling275 but at the same time identifies new opportunities for selective interventions on causal pathways of allergic disease.

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213. Peters-Golden M, Gleason MM, Togias A. Cysteinyl leukotrienes: multi-functional mediators in allergic rhinitis. Clin Exp Allergy 2006;36(6):689–703. 214. Bankova LG, Lai J, Yoshimoto E, et al. Leukotriene E4 elicits respiratory epithelial cell mucin release through the G-protein-coupled receptor, GPR99. Proc Natl Acad Sci USA 2016;113(22):6242–7. 215. Jiang Y, Borrelli LA, Kanaoka Y, et al. CysLT2 receptors interact with CysLT1 receptors and down-modulate cysteinyl leukotriene dependent mitogenic responses of mast cells. Blood 2007;110(9):3263–70. 216. Jiang Y, Kanaoka Y, Feng C, et al. Cutting edge: interleukin 4-dependent mast cell proliferation requires autocrine/intracrine cysteinyl leukotriene-induced signaling. J Immunol 2006;177(5):2755–9. 217. Bisgaard H, Ford-Hutchinson AW, Charleson S, et al. Production of leukotrienes in human skin and conjunctival mucosa after specific allergen challenge. Allergy 1985;40(6):417–23. 218. Creticos PS, Peters SP, Adkinson NF Jr, et al. Peptide leukotriene release after antigen challenge in patients sensitive to ragweed. N Engl J Med 1984;310(25):1626–30. 219. Laitinen A, Lindqvist A, Halme M, et al. Leukotriene E(4)-induced persistent eosinophilia and airway obstruction are reversed by zafirlukast in patients with asthma. J Allergy Clin Immunol 2005;115(2):259–65. 220. Arm JP, O’Hickey SP, Spur BW, et al. Airway responsiveness to histamine and leukotriene E4 in subjects with aspirin-induced asthma. Am Rev Respir Dis 1989;140(1):148–53. 221. Christie PE, Schmitz-Schumann M, Spur BW, et al. Airway responsiveness to leukotriene C4 (LTC4), leukotriene E4 (LTE4) and histamine in aspirin-sensitive asthmatic subjects. Eur Respir J 1993;6(10):1468–73. 222. 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 1999;130(6):487–95. 223. Brannan JD, Anderson SD, Gomes K, et al. Fexofenadine decreases sensitivity to and montelukast improves recovery from inhaled mannitol. Am J Respir Crit Care Med 2001;163(6):1420–5. 224. Lazarus SC, Wong HH, Watts MJ, et al. The leukotriene receptor antagonist zafirlukast inhibits sulfur dioxide-induced bronchoconstriction in patients with asthma. Am J Respir Crit Care Med 1997;156(6):1725–30. 225. Leigh R, Vethanayagam D, Yoshida M, et al. Effects of montelukast and budesonide on airway responses and airway inflammation in asthma. Am J Respir Crit Care Med 2002;166(9):1212–17. 226. Pearlman DS, van Adelsberg J, Philip G, et al. Onset and duration of protection against exercise-induced bronchoconstriction by a single oral dose of montelukast. Ann Allergy Asthma Immunol 2006;97(1):98–104. 227. Rorke S, Jennison S, Jeffs JA, et al. Role of cysteinyl leukotrienes in adenosine 5’-monophosphate induced bronchoconstriction in asthma. Thorax 2002;57(4):323–7. 228. Rundell KW, Spiering BA, Baumann JM, et al. Effects of montelukast on airway narrowing from eucapnic voluntary hyperventilation and cold air exercise. Br J Sports Med 2005;39(4):232–6. 229. Di Lorenzo G, Pacor ML, Mansueto P, et al. Randomized placebo-controlled trial comparing desloratadine and montelukast in monotherapy and desloratadine plus montelukast in combined therapy for chronic idiopathic urticaria. J Allergy Clin Immunol 2004;114(3):619–25. 230. Erbagci Z. The leukotriene receptor antagonist montelukast in the treatment of chronic idiopathic urticaria: a single-blind, placebo-controlled, crossover clinical study. J Allergy Clin Immunol 2002;110(3):484–8. 231. Pacor ML, Di Lorenzo G, Corrocher R. Efficacy of leukotriene receptor antagonist in chronic urticaria. A double-blind, placebo-controlled comparison of treatment with montelukast and cetirizine in patients with chronic urticaria with intolerance to food additive and/or acetylsalicylic acid. Clin Exp Allergy 2001;31(10):1607–14. 232. Kim DC, Hsu FI, Barrett NA, et al. Cysteinyl leukotrienes regulate Th2 cell-dependent pulmonary inflammation. J Immunol 2006;176(7):4440–8.

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation 233. Henderson WR Jr, Tang LO, Chu SJ, et al. A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002;165(1):108–16. 234. Oyoshi MK, He R, Kanaoka Y, et al. Eosinophil-derived leukotriene C4 signals via type 2 cysteinyl leukotriene receptor to promote skin fibrosis in a mouse model of atopic dermatitis. Proc Natl Acad Sci USA 2012;109(13):4992–7. 235. Machida I, Matsuse H, Kondo Y, et al. Cysteinyl leukotrienes regulate dendritic cell functions in a murine model of asthma. J Immunol 2004;172(3):1833–8. 236. Barrett NA, Rahman OM, Fernandez JM, et al. Dectin-2 mediates Th2 immunity through the generation of cysteinyl leukotrienes. J Exp Med 2011;208(3):593–604. 237. Doherty TA, Khorram N, Lund S, et al. Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production. J Allergy Clin Immunol 2013;132(1):205–13. 238. von Moltke J, O’Leary CE, Barrett NA, et al. Leukotrienes provide an NFAT-dependent signal that synergizes with IL-33 to activate ILC2s. J Exp Med 2017;214(1):27–37. 239. Liu T, Garofalo D, Feng C, et al. Platelet-driven leukotriene C4-mediated airway inflammation in mice is aspirin-sensitive and depends on T prostanoid receptors. J Immunol 2015;194(11):5061–8. 240. Carlo T, Levy BD. Molecular circuits of resolution in airway inflammation. ScientificWorldJournal 2010;10:1386–99. 241. Levy BD. Resolvins and protectins: natural pharmacophores for resolution biology. Prostaglandins Leukot Essent Fatty Acids 2010;82(4–6):327–32. 242. Fam SS, Morrow JD. The isoprostanes: unique products of arachidonic acid oxidation-a review. Curr Med Chem 2003;10(17):1723–40. 243. Fukunaga M, Takahashi K, Badr KF. Vascular smooth muscle actions and receptor interactions of 8-iso-prostaglandin E2, an E2-isoprostane. Biochem Biophys Res Commun 1993;195(2):507–15. 244. Hoffman SW, Moore S, Ellis EF. Isoprostanes: free radical-generated prostaglandins with constrictor effects on cerebral arterioles. Stroke 1997;28(4):844–9. 245. Kang KH, Morrow JD, Roberts LJ, et al. Airway and vascular effects of 8-epi-prostaglandin F2 alpha in isolated perfused rat lung. J Appl Physiol 1993;74(1):460–5. 246. Lahaie I, Hardy P, Hou X, et al. A novel mechanism for vasoconstrictor action of 8-isoprostaglandin F2 alpha on retinal vessels. Am J Physiol 1998;274(5 Pt 2):R1406–16. 247. Marley R, Harry D, Anand R, et al. 8-Isoprostaglandin F2 alpha, a product of lipid peroxidation, increases portal pressure in normal and cirrhotic rats. Gastroenterology 1997;112(1):208–13. 248. Mobert J, Becker BF, Zahler S, et al. Hemodynamic effects of isoprostanes (8-iso-prostaglandin F2alpha and E2) in isolated guinea pig hearts. J Cardiovasc Pharmacol 1997;29(6):789–94. 249. Sinzinger H, Oguogho A, Kaliman J. Isoprostane 8-epi-prostaglandin F2 alpha is a potent contractor of human peripheral lymphatics. Lymphology 1997;30(3):155–9. 250. Takahashi K, Nammour TM, Fukunaga M, et al. Glomerular actions of a free radical-generated novel prostaglandin, 8-epi-prostaglandin F2 alpha, in the rat. Evidence for interaction with thromboxane A2 receptors. J Clin Invest 1992;90(1):136–41. 251. Fukunaga M, Makita N, Roberts LJ, et al. Evidence for the existence of F2-isoprostane receptors on rat vascular smooth muscle cells. Am J Physiol 1993;264(6 Pt 1):C1619–24. 252. Fukunaga M, Yura T, Badr KF. Stimulatory effect of 8-Epi-PGF2 alpha, an F2-isoprostane, on endothelin-1 release. J Cardiovasc Pharmacol 1995;26(Suppl. 3):S51–2. 253. Cranshaw JH, Evans TW, Mitchell JA. Characterization of the effects of isoprostanes on platelet aggregation in human whole blood. Br J Pharmacol 2001;132(8):1699–706.

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254. Csiszar A, Stef G, Pacher P, et al. Oxidative stress-induced isoprostane formation may contribute to aspirin resistance in platelets. Prostaglandins Leukot Essent Fatty Acids 2002;66(5–6):557–8. 255. Tintut Y, Parhami F, Tsingotjidou A, et al. 8-Isoprostaglandin E2 enhances receptor-activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway. J Biol Chem 2002;277(16):14221–6. 256. Zanconato S, Carraro S, Corradi M, et al. Leukotrienes and 8-isoprostane in exhaled breath condensate of children with stable and unstable asthma. J Allergy Clin Immunol 2004;113(2):257–63. 257. Shahid SK, Kharitonov SA, Wilson NM, et al. Exhaled 8-isoprostane in childhood asthma. Respir Res 2005;6:79. 258. Baraldi E, Ghiro L, Piovan V, et al. Increased exhaled 8-isoprostane in childhood asthma. Chest 2003;124(1):25–31. 259. Baraldi E, Carraro S, Alinovi R, et al. Cysteinyl leukotrienes and 8-isoprostane in exhaled breath condensate of children with asthma exacerbations. Thorax 2003;58(6):505–9. 260. Battaglia S, den Hertog H, Timmers MC, et al. Small airways function and molecular markers in exhaled air in mild asthma. Thorax 2005;60(8):639–44. 261. Antczak A, Montuschi P, Kharitonov S, et al. Increased exhaled cysteinyl-leukotrienes and 8-isoprostane in aspirin-induced asthma. Am J Respir Crit Care Med 2002;166(3):301–6. 262. Brussino L, Badiu I, Sciascia S, et al. Oxidative stress and airway inflammation after allergen challenge evaluated by exhaled breath condensate analysis. Clin Exp Allergy 2010;40(11):1642–7. 263. Wood LG, Garg ML, Simpson JL, et al. Induced sputum 8-isoprostane concentrations in inflammatory airway diseases. Am J Respir Crit Care Med 2005;171(5):426–30. 264. Dworski R, Roberts LJ, Murray JJ, et al. Assessment of oxidant stress in allergic asthma by measurement of the major urinary metabolite of F2-isoprostane, 15-F2t-IsoP (8-Iso-PGF2alpha). Clin Exp Allergy 2001;31(3):387–90. 265. Talati M, Meyrick B, Peebles RS Jr, et al. Oxidant stress modulates murine allergic airway responses. Free Radic Biol Med 2006;40(7):1210–19. 266. Lin DA, Boyce JA. Lysophospholipids as mediators of immunity. Adv Immunol 2006;89:141–67. 267. Beaven MA. Division of labor: specialization of sphingosine kinases in mast cells. Immunity 2007;26(3):271–3. 268. Olivera A, Mizugishi K, Tikhonova A, et al. The sphingosine kinase-sphingosine-1-phosphate axis is a determinant of mast cell function and anaphylaxis. Immunity 2007;26(3):287–97. 269. Goetzl EJ, Rosen H. Regulation of immunity by lysosphingolipids and their G protein-coupled receptors. J Clin Invest 2004;114(11):1531–7. 270. Jolly PS, Bektas M, Olivera A, et al. Transactivation of sphingosine-1phosphate receptors by FcepsilonRI triggering is required for normal mast cell degranulation and chemotaxis. J Exp Med 2004;199(7):959–70. 271. Ammit AJ, Hastie AT, Edsall LC, et al. Sphingosine 1-phosphate modulates human airway smooth muscle cell functions that promote inflammation and airway remodeling in asthma. FASEB J 2001;15(7):1212–14. 272. Sawicka E, Zuany-Amorim C, Manlius C, et al. Inhibition of Th1- and Th2-mediated airway inflammation by the sphingosine 1-phosphate receptor agonist FTY720. J Immunol 2003;171(11):6206–14. 273. Idzko M, Laut M, Panther E, et al. Lysophosphatidic acid induces chemotaxis, oxygen radical production, CD11b up-regulation, Ca2 + mobilization, and actin reorganization in human eosinophils via pertussis toxin-sensitive G proteins. J Immunol 2004;172(7):4480–5. 274. Payne SG, Oskeritzian CA, Griffiths R, et al. The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors. Blood 2007;109(3):1077–85. 275. Woodward DF, Jones RL, Narumiya S. International Union of Basic and Clinical Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress. Pharmacol Rev 2011;63(3):471–538.

CHAPTER 9  Lipid Mediators of Hypersensitivity and Inflammation

153.e1

SELF-ASSESSMENT QUESTIONS 1. Prostaglandin D2 can be generated through the sequential actions of which of the following 2 enzymes: a. Cyclooxygenase-1 and prostaglandin E synthase b. Cyclooxygenase-1 and prostaglandin D synthase c. 5-Lipoxygenase and prostaglandin D synthase d. 12-Lipoxygenase and prostaglandin E synthase 2. Leukotriene C4 can be generated through the sequential actions of which of the following two enzymes: a. Cyclooxygenase-2 and leukotriene C4 synthase b. 5-Lipoxygenase and leukotriene A4 hydrolase

c. 5-Lipoxygenase and leukotriene C4 synthase d. Cyclooxygenase-1 and 12-lipoxygenase 3. The cysteinyl leukotrienes signal through the following three G-protein-coupled receptors: a. BLT1, BLT2, and CysLT3/GPR99 b. EP1, EP2, and EP3 c. DP1, DP2, and CysLT1R d. CysLT1, CysLT2, and CysLT3/GPR99

10  Molecular Biology and Genetic Engineering Sudhir Gupta, Leman Yel

CONTENTS Anatomy of the Gene, 154 RNA and Protein Synthesis, 155 DNA Repair, 157 DNA Replication, 157 Control of Gene Expression, 158

SUMMARY OF IMPORTANT CONCEPTS • In the 21st century, the advent of novel molecular biology and genetic engineering approaches after the completion of the landmark Human Genome Project, continues to expand our understanding of human biology, resulting in accurate disease diagnosis, discovery of genetic defects and predispositions, and targeted treatment strategies toward precision medicine. • Genomics and proteomics, by means of microarray technology, help to translate the human genetic blueprint into knowledge of gene location, expression profiling, and function. Microarray technology has further enabled next-generation sequencing (i.e., whole exome sequencing, whole genome sequencing, and targeted gene panel [TGP] sequencing) to genetically diagnose rare Mendelian disorders. • Pharmacogenetics, the study of interindividual genomic variations related to the effect of drugs, is an area of relevant research to personalize pharmacologic treatment. • RNA interference/RNA silencing, a Nobel prize–winning discovery, is a powerful reverse genetics tool to clarify gene function and to modify expression of the gene of interest. MicroRNAs, endogenous short RNAs, have recently been found to be an evolutionarily significant component of genetic regulation and to have links with certain disease processes. • DNA methylation and histone modification are two prominent epigenetic mechanisms for formation and storage of cellular information in response to transient environmental signals.

Since the landmark discovery of the double helix structure of DNA in the 1950s, a series of accelerating advances in genetics has taken place. Classically, the function of a gene was studied by examining the effect of its mutation in the whole organism or in cultured cells. The technology of gene cloning, in vitro mutagenesis and gene expression in heterologous cells, along with the development of transgenic and knockout animals, genomics, and proteomics have revolutionized genetic analysis. The Human Genome Project (HGP), an international research effort to sequence and map all of the genes of our species, Homo sapiens, emerged as one of the great achievements in human biology. Completed in April 2003, the HGP has started the “biology century” with an impact

154

DNA Rearrangement: Genetic Recombination, 160 Recombinant DNA Technology, 160 Epigenetics, 171 Summary, 172

on numerous areas of molecular medicine, including disease diagnosis, genetic predisposition, pharmacogenetics, and 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 has 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 of the sugar molecules are numbered 1′ to 5′ proceeding clockwise from the oxygen atom (Fig. 10.1). 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 present in equal amounts, but the amount of adenine in a DNA molecule is always equal to the amount of thymine, and the amount of guanine always equals the amount of cytosine (as expressed in nucleotide symbols, 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 diffraction pattern suggested that the DNA molecule had the shape of a helix or corkscrew, with a diameter of 2 nm and a complete helical turn every 3.4 nm. James Watson and Francis Crick1 built models of nucleotides and tried to assemble the nucleotides into a molecule. After exploring various possibilities, they proposed a “double helix” structure

CHAPTER 10  Molecular Biology and Genetic Engineering

Central axis

H A G

T

C 5′

O

4′

1′

H

C

A

T

H

–O G H

T

H G

P

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O

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T

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4′

1′

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which polypeptides are assembled. Transfer RNA (tRNA) transports the amino acids to the ribosome for the synthesis of polypeptide.4,5 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) consists of 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 formed from multiple polypeptide chains with a molecular weight of 500,000.6,7 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.

Base

H

A

H

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3′ O

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–O

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Transcription

H

H Base

O

O H H

G

C T

C 5′

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A G

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1′ H 3′ O

H 2′

155

H

H

Fig. 10.1  Structure of DNA. Left, Model of Watson-Crick DNA double helix. A, Adenine; C, cytosine; G, guanine; T, thymine. Right, Chain-linked deoxyribose and phosphate residues forming the sugar-phosphate bond. (Courtesy Baback Roshanravan.)

of the DNA molecule, in which the bases of two strands pointed inward toward one another (base pairing). In this model, the base pairing is 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 paired bases: adenine makes double hydrogen bonds with thymine, and guanine forms three hydrogen bonds with cytosine (Fig. 10.1).

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 (Fig. 10.2). 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 in codon designations) instead of thymine.2 In addition, RNA does not have a regular helical structure. The class of RNA present in ribosomes is called ribosomal RNA (rRNA).3 rRNA and ribosomal proteins provide sites at

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 transcription.6–9 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 this process to begin. 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 guanine, cytosine, thymine, and adenine in the DNA would signal the addition of cytosine, guanine, adenine, and uracil, 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 of 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 heterogeneous nuclear RNA) vary greatly in size because of the presence of long noncoding intron sequences. This is in contrast with mature, more uniform, smallsize RNA sequences that are needed for encoding proteins. The primary RNA transcript is then capped by the addition of a methylated guanine 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 accounts for approximately 3% of the quantity of cellular RNA.

156

SECTION A  Basic Sciences Underlying Allergy and Immunology

Loaded tRNA binds to Amino acids

ribosome, amino acids are assembled in the order Completed

directed by the mRNA

protein Growing peptide chain

ADP ATP

tRNA binds amino acid tRNA

Nucleus

Ribosomal

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subunit

subunit

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polymerase

pore

RNA transcribed mRNA

from DNA Primary transcript

Introns

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RNA transcript spliced to produce mRNA

Cytoplasm

Fig. 10.2  Steps in protein synthesis. ADP, Adenosine diphosphate; ATP, adenosine triphosphate; mRNA, messenger RNA; tRNA, transfer RNA. (Courtesy Baback Roshanravan.)

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 a time, termed a codon (Table 10.1).10 Because RNA is made up of four types of nucleotides, there are 64 possible sequences composed of three nucleotides each—the familiar three-letter codon designations. Of the 64 sequences, 3 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. Translation is mediated by tRNA, also termed adapter molecule, which has two important properties.11–15 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, 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 the 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

CHAPTER 10  Molecular Biology and Genetic Engineering

TABLE 10.1  The Genetic Code, With 20 a

Specified Amino Acids First Position (5′ End)

Second Position U

C

A

G

Third Position (3′ End)

Phe Phe Leu Leu

Ser Ser Ser Ser

Tyr Tyr Stop Stop

Cys Cys Stop Trp

U C A G

C

Leu Leu Leu Leu

Pro Pro Pro Pro

His His Gln Gln

Arg Arg Arg Arg

U C A G

A

Ile Ile Ile Met

Thr Thr Thr Thr

Asn Asn Lys Lys

Ser Ser Arg Arg

U C A G

G

Val Val Val Val

Ala Ala Ala Ala

Asp Asp Glu Glu

Gly Gly Gly Gly

U C A G

U

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

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 process. The eukaryotic releasing factor (eRF), a cytoplasmic protein, binds directly to any stop codon that reaches the A-site on the ribosome, thereby altering the activity of peptidyl transferase. As a result, instead of an amino acid, a water molecule is added to the peptidyl-tRNA. This change in configuration 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. Additional factors are required at each stage of protein synthesis,11,12 characterized by their cyclic association with, and dissociation from, the ribosome. During the initiation phase of protein synthesis, the two subunits of ribosomes are 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 in turn determines how the ribosome assembles. The initiation process involves a number of steps catalyzed by a set of proteins, the initiation factors. To start a new protein chain, the ribosome must bind an aminoacyltRNA 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

157

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 an 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; most of these spontaneous changes are temporary, however, because they are immediately corrected by a mechanism termed DNA repair.16,17 Only rarely are these changes in the DNA permanent; such a change is 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 complimentary 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 also are 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 in that 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 are 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 complementary nucleic acid sequence.19,20 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 DNA-binding proteins.21,22 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 (helixdestabilizing 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 conformation. Several classes of eukaryotic DNA polymerase have been identified. DNA polymerase a is a nuclear replicase; however, it synthesizes only one of the daughter strands. DNA polymerase δ also is involved in replication and probably synthesizes the other daughter strand. DNA

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SECTION A  Basic Sciences Underlying Allergy and Immunology DNA polymerase

3′ 5′

5′ 3′

Most recently synthesized DNA strands 5′

3′

5′ Okazaki fragment

3′ DNA polymerase

3′

5′

Fig. 10.3  Structure of DNA replication fork. (Courtesy Baback Roshanravan.)

polymerases β and ε probably are involved in DNA repair reactions, and DNA polymerase γ is responsible for replication of mitochondrial DNA. The actual process of replication occurs at the DNA replication fork,23–25 which is asymmetric (Fig. 10.3). 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 polymerase molecules work at the DNA fork, one (polymerase S) 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 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. By 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 (bp) 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 rotates 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 to a DNA phosphate, thereby breaking a phosphodiester bond in a DNA strand.26,27 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 double-strand break in the DNA helix. Topoisomerase II enzymes are activated where two double helixes cross over each other. When topo­ isomerase 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 processing, RNA transport, mRNA degradation, translational, and posttranslational by protein phosphorylation.28–32 The most common and important gene regulation is at the transcription level, which is mediated by binding of proteins to regulatory sequences within the DNA.

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, 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) composed of several subunits binds to a short TATA sequence (Fig. 10.4). 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, is likely to inhibit transcription. Regulatory proteins bind to the edges of base pairs exposed in the major grooves of DNA. Most of these regulatory proteins contain

CHAPTER 10  Molecular Biology and Genetic Engineering

159

Repressor Enhancer Silencer Enhancer

Enhancer

Activator

Basal factors 30

Activator

Activator 40 110 60

250

150

80

E F

B

H

RNA polymerase

A

Coactivators

TATA box

TATA-binding protein

Coding region

Core promoter Fig. 10.4  Structure of human transcription complex, consisting of four types of proteins: basal factors, coactivators, activators, and repressors. (The numbered proteins are the names of subunits of RNA polymerase II. Each subunit is named according to its molecular mass in kilodaltons. The letters [A,B,F,E,H] indicate the basal transcription factors.) (Courtesy Baback Roshanravan.)

structural motifs, such as zinc finger or leucine zipper. The regulatory proteins are composed of two distinct 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 configuration 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, binding of translational repressor proteins, and selective degradation of mRNA transcripts. Most eukaryotic genes are made up of 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 (accounting for approximately 90% of the primary transcript) are removed by enzymes in a process of RNA splicing or RNA processing. This is a point at which 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 adenine and uracil 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

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3′ end that are recognition sites for endonucleases, causing these transcripts to be digested quickly.

DNA REARRANGEMENT: GENETIC RECOMBINATION To adapt to an ever-changing environment, DNA undergoes rearrangement, which is caused by genetic recombination.33–40 The mechanisms of genetic recombination allow large sections of DNA helix to move from one chromosome to another. There are two classes of genetic recombination: 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 chromosome.36,37 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 that they have extensive sequence homology. The homologous recombination does not normally change the rearrangement of the genes in a chromosome. By contrast, site-specific recombination alters the relative positions of the nucleotide sequence in a chromosome, because DNA homology between the recombining DNA molecules 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.33,38–40 There are two types of site-specific recombination: conservative and transpositional. The conservative site-specific recombination was first demonstrated in bacteriophage lambda (λ).38 This applies to many viruses. DNA sequences in the virus code 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 then, by using a short region of sequence homology, reseals the reaction. Finally, the integrase dissociates from the DNA and is ready to be used for the next recombination reaction. In transpositional sitespecific 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 • Next Generation Sequencing • RNA interference/RNA silencing/microRNA

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 sequenced.41,42 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 10.2). Certain restriction nucleases produce staggered cuts, leaving short, single-stranded tails at the two ends (cohesive ends) of each DNA fragment (Fig. 10.5). Any two DNA fragments can be joined together, provided that both DNA fragments have the same cohesive ends (generated either by the same restriction nuclease or

Result of cleavage

Recognition sequence

Enzyme

A G

A

A

T

T

C

G

C

T

T

A

A

G

C

A

T

T

C G

T

T

A

A

G

A

T

C

EcoRI

G

G

A

T

C

C

G

C

C

T

A

G

G

C

C G

C

T

A

G

BamHI Fig. 10.5  Cleavage sites for commonly used restriction nucleases. (Courtesy Baback Roshanravan.)

CHAPTER 10  Molecular Biology and Genetic Engineering

161

TABLE 10.2  Selected Restriction Endonucleases and Their Recognition Sequences and

Cleavage Sites

Cleavage Site Enzyme

5′

Tetranucleotides TaqI

3′

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

Heptanucleotides MstII

C

C

*

T

N

A

G

G

A, Adenine; C, cytosine; G, guanine; N, any base; T, thymine; *, cleavage site.

using another restriction nuclease, as long as the DNA fragments have 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 also are used in DNA cloning and DNA 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.  Gel electrophoresis techniques separate DNA molecules by size.43–45 Polyacrylamide gel with a small pore size is used to separate single-stranded DNA fragments less than 500 nucleotides long (with a size range of 10 to 500 nucleotides) that differ in size by as little as a single nucleotide. Agarose gel with a medium pore size is used to fractionate the double-stranded DNA molecule (with a size range of 300 to 10,000 nucleotide pairs). The DNA bands in polyacrylamide gel and agarose gel electrophoresis are invisible, unless DNA is stained with ethidium bromide or labeled with radioisotope 32P before performing electrophoresis. A variation of agarose gel electrophoresis, the pulse-field agarose gel electrophoresis, separates extremely long DNA molecules. This technique has been used to separate 16 different Saccharomyces cerevisiae chromosomes that range in size from 220,000 to 2.5 million nucleotide pairs.

Labeling of Purified DNA Molecules.  Isolated DNA molecules can be labeled by either of two methods.46 In one method, DNA is copied by Escherichia coli DNA polymerase I in the presence of nucleotides that have either been labeled with 32P or chemically labeled. These labeled nucleotides are then used as “DNA probes” for nucleic acid hybridization. In the second method, bacteriophage polynucleotide kinase is used to transfer a single 32P-labeled phosphate from ATP to the 5′ end of each chain of DNA. This method has both advantages and disadvantages. DNA molecules labeled by this technique have low radioactivity and therefore cannot be used as DNA probes; however, they are extremely useful for DNA sequencing and DNA footprinting. Sequencing of DNA Fragments.  It is now possible to determine the complete DNA sequence of genes by either a chemical or an enzymatic method.47–50 In the chemical method a set of identical end-labeled double-stranded DNA molecules are dissociated and exposed to a chemical that destroys one of the four bases (e.g., cytosine residue) in the DNA. This generates a family of DNA fragments of different lengths. To determine the full sequence, a similar procedure can be done on four separate samples of identical end-labeled double-stranded DNA, using four different chemicals that cleave DNA preferentially at thymine, cytosine, guanine, or adenine residues. The fragments of DNA produced by mild chemical treatments are then separated on gel and detected by autoradiography. Because the chemical method is less specific than the enzymatic method, it is no longer used. The enzymatic method is the standard procedure currently used for DNA sequencing. In this procedure, in vitro DNA synthesis is carried out in the presence of chain-terminating nucleoside triphosphate. The DNA to be sequenced is used as a template to synthesize in vitro a set of replicas, using DNA polymerase. All replicas begin at the same place but terminate at different points along the DNA. The most important step of this method is the use of the dideoxynucleoside dideoxyadenosine triphosphate (ddATP), in which the deoxyribose 3′-OH group present

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in normal nucleotides is missing. Therefore, when incorporated into a DNA chain, ddATP blocks the addition of the next nucleotide. This reaction generates a ladder of DNA fragments, which can be detected by a chemical or radioactive label that is incorporated either into oligonucleotide primers or into one of the deoxyribonucleoside triphosphates. To determine the full sequence of the desired DNA, four different chain-terminating nucleoside triphosphates are used in separate DNA synthesis reactions on the same primed single-stranded DNA template. Products of these reactions are analyzed in polyacrylamide gel electrophoresis.

Recognizing DNA.  DNA recognition is one of the central control points in the regulation of cellular processes. The nuclear proteins scan the surface of DNA molecules with extraordinary sensitivity and specificity, using them as a map to find the correct position for assembling the transcriptional apparatus. The ability of these regulatory factors to recognize specific DNA sequences underlies the selective expression of genes in a particular cell type. Important sequences in a regulatory region are identified by directly examining an interaction between protein and DNA. Several methods have been used to identify such proteinDNA interactions.51 Electrophoretic mobility-shift assay.  The electrophoretic mobilityshift assay, or gel-shift assay or retardation assay, relies on gel electrophoresis to determine whether a radioactive DNA fragment binds nuclear protein and to what extent the binding is sequence specific.52 The radiolabeled DNA fragment that contains a protein-binding site runs through the gel more slowly than the DNA fragment alone, and therefore the band corresponding to protein-DNA complex shifts upward relative to the band corresponding to DNA alone. To distinguish sequencespecific from sequence-nonspecific interactions between protein and DNA, competitive assays are performed with two competitor DNA, one nonspecific competitor (unrelated sequence) and one specific competitor (an exact sequence of the probe). The unlabeled specific competitor competes with the labeled DNA fragment for nuclear protein binding, resulting in the disappearance of the shifted band. A further variation of the gel-shift assay uses antibodies against specific nuclear proteins to confirm that the shifted protein-DNA complexes contain the corresponding protein factor. The antibody binds to the protein, forming a supercomplex of protein-DNA, and on autoradiograph is visualized as a “supershifted” band. The gel-shift assay cannot identify the position at which the protein binds along the DNA. Furthermore, this assay cannot be used to determine whether the shifted band is caused by two proteins binding to two different sites on the same DNA fragment. The DNA footprinting technique addresses these limitations of gel-shift assay. DNA footprinting.  Some proteins play a crucial role in determining which genes in a particular cell type are active by binding to regulatory DNA sequences that are located outside the coding region of a particular gene.53,54 To determine the function of a protein, it is important to identify the specific sequences to which it binds. DNA footprinting reveals the site where proteins bind on a DNA molecule. In this technique, first a pure DNA fragment that is labeled at one end with 32P is isolated; then a trace amount of a DNA endonuclease is added to the mixture of nuclear protein and radiolabeled DNA. The endonuclease cuts double-stranded DNA at various sites, except where a protein is bound to the DNA (protein prevents endonuclease cleavage). The DNA fragments are subjected to electrophoresis on a gel containing denaturing agents. Only the protein-labeled DNA complex is visualized on autoradiograph. The labeled fragments that terminate with binding sites are missing, leaving a gap in the gel pattern called a “footprint.” Binding site selection assay.  In the binding site selection assay, specificity of DNA-protein interaction is complemented with the

amplification capacity of the polymerase chain reaction (PCR) to identify a protein’s DNA binding site without prior knowledge of the gene it may control.55 A set of synthetic potential DNA-binding sites are required for this assay. Nuclear protein is incubated with DNA fragments that allow protein to bind to its preferred binding site among the random collection of DNA fragments. The protein-DNA complexes are separated from unbound DNA fragments by gel-shift or an antibody to protein. The protein is then removed from the DNA by heating, and the selected DNA fragments are amplified by PCR, using primers complementary to the sequences flanking the random oligonucleotides. It is possible to obtain a homogeneous DNA sequence containing the preferred DNAbinding site by repeating the process of protein binding, purification, and amplification.

Nucleic Acid Hybridization Two strands of the double-helix DNA dissociate when an aqueous solution of DNA is exposed to very high pH (= 13) or heated at 100° C, a process called DNA denaturation. However, if the solution is kept at 65° C for a prolonged period, the complementary single strands of DNA will re-form double helixes, a process called DNA hybridization. Similar hybridization will occur between single strands of DNA-DNA, RNA-RNA, or RNA-DNA, provided that they have complementary nucleotide sequences.56,57 The rate of nucleic acid hybridization depends on the rate at which two complementary nucleic acid chains collide, which in turn is proportional to the concentration of the chains. Therefore the rate of hybridization may represent the concentration of any desired DNA or RNA sequence in a mixture of other sequences. Nucleic hybridization assay requires a pure single-stranded DNA fragment that is complementary in sequence to the desired DNA or RNA. Such a single-stranded DNA fragment can be obtained by cloning or can be chemically synthesized if its sequence is short. The DNA fragment is labeled either with radioisotope or with a chemical and used as an indicator to follow its incorporation during hybridization. Such an indicator DNA is called a DNA probe. Nucleic acid hybridization reactions using DNA probes are so sensitive and selective that one molecule of complementary sequence present in one cell can be detected. Therefore it is possible to determine the number of copies of a particular DNA sequence in a cell’s genome. DNA probes also can be used to hybridize with RNA rather than DNA to determine whether a particular gene is expressed in a cell. In this case, DNA probe is hybridized with purified cellular RNA to determine whether the RNA includes molecules matching the probe DNA. In more extensive analysis, DNA probe, after hybridization has completed, is treated with specific nucleases to determine the exact region of DNA probe that has hybridized with cellular RNA. The start and stop sites for RNA transcription can thus be determined. The hybridization of DNA probes to cellular RNA also allows determination of whether change in gene expression is caused by controls that act on the transcription of DNA, splicing of the RNA of the gene, or translation of mature mRNA into protein.

Northern and Southern Blotting.  Northern and Southern blotting are gel transfer hybridization techniques to analyze RNA and DNA, respectively.58,59 In Southern blotting (named for the inventor of the procedure), isolated genomic DNA is cut into fragments of manageable size (which can be readily separated) with usually more than one restriction endonuclease.43 In general, maximum lengths of DNA that can be directly manipulated are 15 to 20 kilobases (kb). The double-stranded DNA fragments are then separated according to their size by gel electrophoresis. Double-stranded DNA molecules are separated into singlestranded DNA by alkaline denaturation after the gel has been run. To identify DNA fragments, DNA is transferred from agarose gel to a nitrocellulose filter paper (nylon), on which they become immobilized.

CHAPTER 10  Molecular Biology and Genetic Engineering This process of DNA transfer from agarose gel to nitrocellulose paper is similar to blotting, hence the term blotting. DNA fragments on nitrocellulose paper now can be hybridized with radiolabeled DNA probe. Those fragments that are complementary to DNA probe will be hybridized and can be visualized by autoradiography. The size of the DNA molecule in each band that binds to the probe can be determined by reference to bands of DNA standard that are electrophoresed side-byside with the experimental samples. The usefulness of this technique depends on the specificity of the available probes. In Northern blotting, instead of DNA, RNA containing the gene of interest is analyzed with DNA probe. Analogous to Southern blotting, in Northern blotting, purified RNA is separated by agarose gel electrophoresis, transferred to nitrocellulose membrane, hybridized with labeled DNA probe, and visualized by autoradiography.

Polymerase Chain Reaction.  PCR is an extremely sensitive and rapid technique to detect the presence of a specific gene and is extremely useful as a diagnostic tool for detecting a large number of diseasecausing or associated genes.60–62 However, the most significant contribution of PCR technology has been in gene cloning. It is possible to isolate a gene from a single cell. This procedure involves in vitro amplification of specific pieces of DNA (Fig. 10.6). Two oligonucleotide primers, which are homologous to some 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 (cDNA). 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 run can be inserted into a vector, cloned, and sequenced.

Denature target strands and anneal primers

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Quantitative real-time polymerase chain reaction assay.  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 real-time PCR assay is based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product.63–65 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 6-carboxyfluorescein [6-FAM]), and a quencher fluorochrome (6-carboxy-tetramethylrhodamine [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 degrades the probe, releasing the reporter fluorochrome. The amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle (Fig. 10.7). Compared with other quantitative PCR methods, the real-time PCR technique is more accurate and sensitive and less labor-intensive, does not require post-PCR sample handling, and allows a faster and higher throughput. Therefore real-time PCR analysis can be performed on very small samples, for example, to quantify cytokine profiles in cells of the immune system. T cell receptor rearrangement excision circle (TREC) measurement is another application of real-time PCR assay in immunology. TREC offers a novel tool to identify recent thymic emigrants in peripheral blood and T cell production by the thymus.

Fluorescence in Situ Hybridization.  Fluorescence in situ hybridization (FISH) is a nonradioactive technique used by cytogeneticists and molecular biologists to identify chromosomal aberrations and for gene mapping. Chromosomal aberrations indicate clinical abnormalities and therefore are important in prenatal diagnosis of several diseases. In this technique, DNA probes detect segments of the human genome

Extend primers to make copies of targets

Cycle 1

Cycle 2

Ad infinitum Fig. 10.6  Polymerase chain reaction, a cyclic process in which the number of DNA targets doubles with each cycle. (Courtesy Baback Roshanravan.)

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R

Q

Q

A

Relative fluorescence

10

1

0.1

Ct

0

B

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle

Fig. 10.7  Quantitative real-time polymerase chain reaction (PCR). (A) Primers are extended as in traditional PCR. 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.

by DNA-DNA hybridization of samples of lysed metaphase cells prepared under conditions that preserve the morphology of condensed human chromosomes.66,67 Attachment of the fluorescent molecule to the DNA probe allows the visualization, by light microscopy, of the position on a chromosome. FISH is an improvement on in situ hybridization methods that depend on labeling of probes with radioactive isotopes. FISH has played an important role in the HGP and subsequent studies.

Gene Cloning One of the most important developments in the field of recombinant DNA technology has been the technique of gene cloning. The first step in the cloning of a specific gene is the construction of a comprehensive collection of cloned DNA fragments, the DNA library or gene library, which includes at least a fragment that contains a gene of interest (see later). The cloning of genetic material begins with the insertion of a DNA fragment that contains a gene of interest into the purified DNA genome of a self-replicating element, generally a virus or a plasmid, and the propagation of this chimeric DNA molecule in a host organism. The process of gene cloning leads to the amplification of specific DNA fragments more than 1012-fold. This allows the isolation and chemical characterization of specific DNA sequences. A virus or plasmid used in this way is known as a cloning vector. A cloning vector is a DNA molecule that has the following characteristics: (1) it is capable of replicating independently of the host chromosome; (2) an organism containing the vector can be grown preferentially; and (3) additional DNA can be inserted into the vector. There are two classes of vectors: the plasmid vectors and the phage vectors.

Plasmid Vectors.  Plasmids are small, circular molecules of doublestranded DNA derived from larger plasmids that occur naturally in bacteria.68 Most plasmid-cloning vectors are designed to replicate in E. coli.69 All of the enzymes required for replication of the plasmid DNA are produced by a host bacterium. The classic example of plasmid vector is pBR322, which was one of the first such vectors to be recognized. The three important features of plasmid vectors are as follows: • Origin of replication. This origin permits the efficient replication of plasmid to a large number of copies of cells, by the plasmid’s replicon, a region of approximately 1000 bp encoding the site at which DNA replication is initiated. • Presence of selectable marker. Most plasmid vectors encode a gene that confers bacterial resistance to antibiotic. This allows selection of clones carrying the plasmid in the medium containing antibiotic. • Cloning, or restriction enzyme, cleavage site. All cloning vectors must have at least one cloning site (a specific DNA sequence that is recognized and cut by a restriction endonuclease), where the foreign DNA is inserted. Three classes of restriction enzymes bind to DNA at the recognition sequence and hydrolyze the phosphodiester bond on both strands of DNA. Such restriction sites usually have twofold symmetry; that is, the restriction sites are palindromic. Class II restriction endonucleases, which recognize a DNA sequence of four to eight nucleotides, are preferred for DNA technology. The restriction enzyme EcoRI, isolated from E. coli, cleaves DNA at the sequence 5′-GAATTC.69 The EcoRI scans the plasmid until it finds the GAATTC sequence, where it hydrolyzes the phosphodiester bond between deoxyguanosine and deoxyadenosine on both strands of the DNA, creating a 4-bp (AATT) single-stranded overhang. Because EcoRI is palindromic, the overhanging single-stranded ends (sticky ends) are complementary to each other and can hybridize or anneal to each other by base pairing. Now the DNA to be cloned (cleaved from its source by EcoRI) is inserted into a plasmid vector whose DNA sequence has been cut by restriction endonuclease. The DNA fragment anneals to the vector through DNA ligase, which catalyzes the covalent joining of the vector DNA to the new piece of DNA (chimeric DNA). The gene (DNA fragment to be cloned) now becomes a passenger on the vector molecule, ready to be introduced into bacteria (DNA transformation). Electroporation is the most efficient of the several techniques used to achieve DNA transformation.70 The chimeric DNA is mixed with bacteria in a cuvette, and an electric potential is created across the wall of the container, allowing the DNA to enter the bacteria (transfection). The bacteria are then grown in the presence of antibiotic (e.g., ampicillin, neomycin) for which the resistant gene is present in the chimeric DNA.71 This will allow the bacteria with recombinant plasmid to proliferate, whereas any bacteria that were not transformed with the recombinant plasmid will die. The clones or colonies of bacteria containing cloning vector can be isolated for further characterization. There are several ways to characterize clones, but the most common technique is to culture individual bacterial clones, isolate their DNA, and analyze the clones. Once the plasmid DNA has been purified, its structure can be analyzed by digesting the DNA with restriction endonuclease (e.g. EcoRI) and then subjecting it to agarose gel electrophoresis. DNA is visualized by staining the gel with ethidium bromide. Phage Vectors.  Practically all phage vectors are based on bacterio-

phage λ, a bacterial virus that infects Escherichia coli.72 Phage vectors have several advantages over plasmid vectors. First, phage vectors induce their genes into bacteria with high efficiency. Second, they can be exploited to clone larger pieces of DNA, because many phages have large genomes. The bacteriophage λ DNA (approximately 50 kb) encodes approximately 60 genes that are arranged in three groups:

CHAPTER 10  Molecular Biology and Genetic Engineering the immediate early genes, the delayed early genes, and the late genes. After entry of λ phage DNA into a bacterium, its 12-base cohesive (cos) ends are joined by DNA ligase, resulting in the formation of a circular molecule. The virus then follows a lysogenic (dormant) or lytic pathway, depending on a balance between host and the viral factors. If immediate early genes are not expressed, the viral genome integrates into bacterial chromosome; however, transcription of almost all viral genes is suppressed, and therefore virus is maintained in a dormant or lysogenic state. By contrast, if the immediate early genes are transcribed, their proteins, through gene transcription, induce expression of delayed early genes. The delayed early genes are responsible for induction of the lytic pathway through their involvement in replication of viral DNA. The late genes, whose expression follows the expression of delayed early genes, encode proteins that are involved in the synthesis of viral capsid, packaging of the viral RNA into the capsid, and lysis of the infected bacterium. The middle third genome of the phage vector, which is essential to the lysogenic pathway, is not critical for the lytic pathway. Therefore this segment can be replaced with a new piece of DNA. In summary, phage λ cloning vector can be considered as having two pieces of DNA. One piece (approximately 20 kb) encodes the late genes that are involved in capsid assembly. The other piece (approximately 10 kb) encodes delayed early genes required for replication of phage DNA and late genes, which regulate the lysis of bacterium. The terminal portions of both pieces are cos sites, which signal the packaging of the viral DNA into capsid. One of the most versatile phage λ vectors is λ zeta-associated protein (λZAP), which has several important features. First, λZAP has a polyclonal site that has multiple restriction enzyme recognition sequences that are large (six to eight bases). The enzymes cut λZAP only in the polyclonal site. Second, λZAP can make fusion proteins that are useful in purifying the protein encoded by the cloned gene. The fusion proteins also can be used to raise antibodies to a cloned gene’s product by injecting them in the animal. Third, the promoters of two specific RNA polymerases flank λZAP’s polyclonal site. One side has a promoter that is recognized by the RNA polymerase of bacteriophage T3, and on the other side is a promoter that is recognized by the RNA polymerase of bacteriophage 7. These RNA polymerases transcribe complementary strands of the cloned insert. The synthetic RNA can be translated in vitro or in vivo to make the protein so that the structure and function of the encoded protein can be studied. Fourth, λZAP encodes a phagemid vector that can be excised in vivo from the λ vector. This is helpful to prepare large amounts of cloned DNA fragments from phagemid vector rather than from a λ vector. The process of ligating a DNA insert into a λ phage vector is the same as with a plasmid vector. However, a DNA insert must have terminal ends that are compatible with ligation into the λ cloning site. After ligation, the DNA is packaged into phage λ in vitro as protein capsid, using a mixture of structural proteins and enzymes. The recombinant bacteriophages are then allowed to infect the bacterium. The infected bacteria are mixed with agarose and plated. In about 5 hours the recombinant bacteriophage undergoes many rounds of replication and lyses bacteria within a 1-mm diameter. As a result, a clear area, the plaque, develops that corresponds to a clone generated by phage λ vector. Each plaque contains 1 million copies of a single viral clone. Individual plaques can be purified, and the phages from the plaque can be grown in large quantities to isolate the DNA. Some phage particles stick to a piece of nitrocellulose paper when it is placed over a plaque. The phage particles can be disrupted and the DNA irreversibly attached to nitrocellulose membrane. This DNA can be screened by hybridization with a radiolabeled nucleic acid probe. Labeled probes are then visualized by radiography. In this way, labeled nucleic acid probes can be used to identify a gene within a λ gene library (see later).

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Cosmid Vectors.  Cosmid vectors are hybrids between plasmid and

phage λ vectors. The classic example of cosmid vector is c2RB, which carries an origin of replication and a cloning site and has antibioticresistant genes. As with the phage λ vector, the cosmid vector encodes the cos sequences required for packaging of DNA into λ capsid. Cosmid vectors are designed to clone large fragments of DNA and to grow their DNA as a virus or as a plasmid. Cosmid vectors are used in homologous recombination between two different plasmids in the same cell and grown in both bacteria and animal cells. Cosmid vectors, along with λ vectors, are used as standard vectors for the cloning of genomic DNA.73

Phagemid Vectors.  Phagemid vectors are hybrids between plasmids and a gene from a filamentous DNA bacteriophage M13 that infects but does not lyse E. coli. These vectors can convert a double-stranded plasmid into a single-stranded plasmid; the latter is useful for DNA sequencing, site-directed mutagenesis, and subtraction hybridization.74,75

Yeast Plasmid Vectors.  Although bacterial plasmid vectors are used most often, yeast plasmid vectors are preferred in special circumstances,76 most notably the development of artificial chromosomes for gene mapping. Yeast plasmid vectors can carry DNA fragments that are 20 times (106 bases) as big as the DNA that even the cosmid vectors can propagate. Yeast artificial chromosomes (YACs), the most efficient vectors for cloning large pieces of DNA, encode a bacterial and yeast origin of replication, an antibiotic-resistant gene, a cloning site, a yeast centromere, two telomerases, and a selectable gene for growth in yeast. Because these vectors clone large fragments of DNA, yeast plasmid vectors are particularly useful in demonstrating linkage between two genes that may be more than 50 kb apart; other cloning techniques will not be able to show such physical linkage. This technique has already resulted in the isolation of several disease-causing genes, including genes for neurofibromatosis type 1, cystic fibrosis, and Duchenne muscular dystrophy. Eukaryotic Plasmid Vectors.  Eukaryotic plasmid vectors, also known as shuttle vectors, can express genes in both bacteria and eukaryotic cells.77 These vectors carry a bacterial origin of replication (replicon) and an antibiotic-resistant gene, which allow them to grow in bacteria. In addition, shuttle vectors carry a eukaryotic enhancer and a promoter at 5′ to the coding sequence of a gene and poly-A site located at 3′ to the gene. Furthermore, many of the eukaryotic vectors also have introns, either before or after the coding sequence. Plasmid pSV2gpt was one of the first eukaryotic vectors described. A number of genes have been used as selectable markers in animal cells, including gpt (expressed by pSV2gpt), which codes for xanthine-guanine phosphoribosyl transferase; apt, encoding aminoglycoside phosphotransferase; tk, encoding thymidine kinase; gdfr, encoding dihydrofolate reductase; hpt, encoding hygromycin B phosphotransferase; and ad, the adenosine deaminase gene. Eukaryotic vectors have been used to isolate genes on the basis of their function. For this purpose, cDNA is inserted into the vector between the promoter and the poly-A site. The pCD vector was the first eukaryotic vector designed to express cDNA library in human cells.78 The function of a eukaryotic gene can be evaluated by introducing the plasmid DNA into eukaryotic cells by electroporation, transfection of precipitable DNA, or microinjection. Eukaryotic expression vectors also can be used to make a transgenic animal that carries a new, artificially introduced gene in the embryo.79,80 A cloned gene in a eukaryotic expression vector is introduced into the female pronucleus of a single-cell embryo by microinjection. This cloned DNA integrates into the host chromosome and becomes a part of the host genome. Transgenic animals have been extensively used to study the effects of mutation and aberrant expression of a gene. Genes also can be introduced into the embryo with eukaryotic viral vectors.

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Eukaryotic Viral Vectors.  Viruses that infect eukaryotic cells can be used to introduce DNA into animal cells not as a primary vector for cloning a gene, but to demonstrate the expression of a cloned gene in eukaryotic cells. First, eukaryotic viral vectors were based on the SV40 papovavirus, which is a double-stranded DNA virus of 5400 bp that carries only a small amount (2000 bp) of new DNA.81 Two types of eukaryotic viral vector systems have been used: retroviral vectors and herpesvirus vectors.82,83 The advantage of retroviral vectors is that all genes can be replaced with new DNA. Retroviruses contain a singlestranded RNA genome that is converted into double-stranded DNA by reverse transcriptase (RT) inside the infected cell.84 The double-stranded DNA integrates into the chromosome of host cells. This provirus transcribes its entire genome, which encodes all of the proteins that are required for the synthesis of new virions. Most retroviruses do not kill the cells they infect. Therefore infected cells continue to make new viruses. In the retroviral genome, tandem long terminal repeats (LTRs) flank the coding sequences of the virus at either end. LTRs play an important role in the integration of provirus into the infected host cell chromosome and in regulating transcription. The sequences just internal to LTRs are involved in replication of viral genome and packaging of viral RNA (packaging sequence [Psi]) into the virus. Most of the retroviral genome is composed of three structural genes: gag, pol, and env. The gag gene codes for several core proteins, the pol gene encodes RT and integrase, and the env gene codes for viral envelope glycoprotein, which is required for viral entry into host cell. The essential elements of retroviral vectors are a cloning site between two LTRs, the packaging signal, and the sequence necessary for DNA replication. After insertion and ligation of a gene into the cloning site, the vector is grown in bacteria to produce large amounts of DNA. The DNA is purified and introduced into an animal cell line.85 The recombinant RNA is integrated into a host chromosome and transcribed into recombinant DNA, which carries the packaging signal. The RNA transcript is then packaged into capsid. In addition to their use in making transgenic mice,86–89 eukaryotic retroviral vectors have been used in gene therapy.90,91 Retroviral vectors require cell division, however, so efficient transduction of quiescent stem cells has been difficult to achieve. Currently, more efficient vector systems such as lentivirus vectors are being developed to transduce nondividing cells (e.g., quiescent hematopoietic stem cells).

is packaged into phage capsids. The phage capsid is allowed to infect bacteria (e.g., E. coli). The recombinant phage multiplies as the bacteria multiply, generating millions of genomic DNA clones. The phage can be purified, and the collection of virions is called a phage genomic library. Genomic libraries usually are stored as phage particles in solution. A genomic library can be prepared in cosmid vectors provided that the size of the partially digested DNA fragments is adjusted to approximately 40 to 45 kb. A cosmid library has the advantage of a larger insert size; however, rearrangements may occur. More recently, YAC libraries have been constructed for mapping large regions of human genome. Yeasts have advantages over bacteria because they are eukaryotes and therefore more like animal and human cells. cDNA libraries can be prepared from selected populations of mRNA molecules.94,95 When the gene of interest is expressed at high levels, most cDNA clones are likely to contain the gene sequence, so cDNAs can be selected from these cells with minimal effort. However, various methods can be used to enrich particular mRNAs before making the cDNA library from cells in which genes of interest are less abundantly transcribed. One example is the use of antibody against the protein to precipitate selectively those polyribosomes to which the mRNA coding for the protein is attached. The precipitate may enrich the desired mRNA by as much as 1000-fold. mRNA from the cells is isolated by dissolving the cells in a solution that inactivates ribonucleases, and RNA is then separated by cesium chloride density-gradient centrifugation. mRNA (contains poly-A tail) is separated from rRNA and tRNA by passing through chromatographic column containing cellulose to which short polymer of thymidine (oligodeoxythymidine [dT], or poly-dT) are covalently attached. Because of the adenines at the 3′ end of mRNA, mRNA hybridizes to oligo-dT and is retained by the column, whereas the poly-A RNA (e.g., rRNA and tRNA) passes through the column. The pure poly-A plus mRNA is eluted from the column by washing it with water. mRNA is now converted into double-stranded DNA by means of a series of enzymatic reactions. A double-stranded hybrid molecule containing one strand of RNA and one strand of DNA is made with the help of RT. The RNA strand is then converted into DNA by DNA polymerase, DNA ligase, and RNase H, resulting in a cDNA molecule. The cDNA molecule is ligated into one of several cloning vectors (e.g., phagemid, eukaryotic vector, λ phage). Then DNA is introduced into bacteria to create the cDNA library.

Gene Libraries.  To clone a specific gene by plasmid or viral vector,

Gene Isolation

one must construct a DNA library, which is a collection of cloned DNA fragments that includes the gene of interest. A DNA library generally is stored in a population of bacterial cells. There are two types of DNA libraries: genomic and cDNA. A genomic library is a collection of DNA fragments contained within self-replicating vectors that represent the entire genome of the individual from which the DNA was made. The cDNA library represents a collection of only those DNA fragments that were transcribed into mRNA in the cell from which the mRNA was isolated.92,93 A genomic library is constructed from chromosomal DNA. The DNA of the cell is digested with a specific restriction nuclease (e.g., EcoRI) into a large number of DNA fragments. The digestion reaction can be controlled so that the average size of the DNA fragments is approximately 20,000 bps (DNA is partially digested). The advantage of the partial-digest library is that it contains a series of overlapping DNA fragments (approximately 18 to 20 kb) covering the genome. The DNA is then fractionated by size. Because the size of genomic DNA fragments tends to be relatively large (more than 10,000 bases), phage or cosmid vectors (more often phage vectors) are used to generate genomic DNA clones. The bacteriophage λ DNA is cleaved with the same restriction nuclease that is used to cleave genomic DNA, and the two are then mixed; DNA ligase is added; and the chimeric DNA

Once a gene library has been generated, the gene of interest is isolated from millions of clones in the library by any of a number of screening methods, based on the particular properties of the genes and the proteins they encode. In general, these methods can be categorized as follows: (1) screening by homology to nucleic acid probes; (2) screening for an immunoreactive product with antibodies to the gene’s product; (3) screening with a functional assay for the gene product; (4) differential screening; (5) subtraction hybridization; and (6) PCR.

Homology to Nucleic Acid Probes.  Many genes have homologies to other genes that have been previously cloned. In this method, one constructs an oligonucleotide probe based on the structure of the known gene, which is suspected to have homology to the gene in question, and then probes to detect a cDNA clone within a cDNA library. For example, to isolate immunoglobulin G subclass 1 (IgG1) gene expressed by cloned B cells, a cDNA library can be made from the B cells’ mRNA and the library screened with a probe homologous to a highly conserved domain in all IgG genes, the IgG constant region domain.96

Screening With Antibodies to Gene Product.  In this screening technique, antibodies against a particular protein encoded by the gene

CHAPTER 10  Molecular Biology and Genetic Engineering of interest are used to screen cDNA libraries for a particular gene cloned in an expression vector. The antibody binds to the replica of phage plaques containing the protein encoded by the gene of interest and detected by one of several methods already described. The recombinant phage that bound the antibody is purified and can be sequenced and functionally assayed to confirm that it is encoded by the gene of interest.97

Expression Systems to Screen for Functional Gene Product.  To verify that the isolated gene codes for a protein that has a functional role, in vitro and in vivo expression systems are used. One of the methods for analyzing the function of a cloned gene is the oocyte expression system. In this in vitro expression assay, synthetic mRNA made from the cloned cDNA is injected into Xenopus oocyte, where the transcript is then translated in vitro.98 After translation, the presence of the desired protein can be assayed by a number of methods. For example, if an antibody to a protein is available, the protein can be immunoprecipitated, purified, and characterized for its function, or if the gene encodes an enzyme, the enzyme activity can be measured. In the in vivo expression system, the cloned gene is expressed in cells, as in the oocyte expression system. Additional in vivo expression assays depend on eukaryotic expression vector pCD and eukaryotic viral vector systems. Expression systems have been used to isolate a gene. The expression of a cDNA library in tissue culture cells has been used to isolate cDNA clones encoding lymphokines99,100 and CD28, a T cell receptor cell surface protein.101

Differential and Subtraction Hybridization.  The differential and subtraction hybridization methods are used to identify genes that are expressed in one type of cell but not in another, for example, a gene expressed in terminally differentiated cells but not in undifferentiated precursor cells, or genes encoding cell surface proteins present in T cells but absent in B cells. In differential hybridization, mRNA is extracted from two cell types from the same organism, and a cDNA library of a large number of recombinant clones is made from the target tissue from which the gene of interest is to be isolated.102 Next, a selected library is grown on agar plates as discrete colonies. Bacterial colonies are picked individually and replated in a matrix. Two replicas of the matrix are made on nitrocellulose paper; bacteria on the filter paper are chemically lysed, and their DNA is fixed. The mRNAs from both cell types or tissues are converted into cDNAs, which are now radiolabeled. Two separate hybridizations are performed on two nitrocellulose filters carrying the target bacterial matrix. Each filter is probed with cDNA made from each cell type or tissue. In this reaction, bacterial colonies that contain cDNA homologous to the probe mRNA will hybridize to the radiolabeled probe, which then can be identified by autoradiography. The differential hybridization is a simple procedure; however, it is less sensitive than subtraction hybridization because it requires abundant mRNA (greater than 0.1%). Subtraction hybridization is a powerful tool for enriching particular nucleotide sequences before cDNA cloning.103–105 This procedure can be used to identify any differentially expressed gene. Subtraction hybridization was first used to isolate cDNA coding for the T cell antigen receptor (TCR),106 a gene that is expressed only in T cells and is lacking in B cells. In the case of lymphocytes (or in any other cell type), poly-A plus RNA is prepared from both T and B lymphocytes. Then cDNA strands from T cells are synthesized using oligo-dT primers. The RNA is removed from DNA-RNA hybrids by alkaline hydrolysis. These cDNAs are then hybridized with a large excess of mRNA from B cells. All T cell cDNAs for genes that also are expressed in B cells hybridize to B cell RNA, and the T cell–specific cDNAs remain single-stranded and

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are further purified by passing the mixture on a hydroxyapatite column, which retains double-stranded hybridized molecules and allows the single-stranded T cell–specific cDNA to pass through. These singlestranded cDNAs are converted into double-stranded cDNAs and cloned. The library is screened with a subtracted 32P-labeled cDNA probe.

Polymerase Chain Reaction Cloning.  PCR can clone selected DNA fragments. The availability of purified DNA polymerases and chemically synthesized DNA oligonucleotides has made it possible to clone specific DNA sequences rapidly without the need for living cells. PCR cloning technique is rapidly replacing Southern blotting for the diagnosis of genetic disease and for the detection of low levels of viral particles, including human immunodeficiency virus (HIV).

Gene Mapping The power of gene mapping for the study of human diseases is extra­ ordinary. Restriction fragment length polymorphism (RFLP) has been successful in pinpointing the chromosomal location of a number of genes for different diseases, the cause of which is unknown. More than 100 human diseases have been mapped to both X loci and autosomal loci using RFLP.107–109

Restriction Fragment Length Polymorphism.  Gene mapping, or restriction endonuclease mapping, has become an important technique to analyze a large number of genetic disorders; large genomes can be mapped by either physical or genetic techniques. Physical mapping, which includes restriction maps and a library of genomic clones, directly analyzes the DNA molecules that constitute each chromosome. A restriction map identifies a linear series of sites in the DNA that are separated from one another by actual distance along the nucleic acid. A genetic map is based on the frequency of coinheritance of two or more features of an organism that serve as a genetic marker. If the difference in DNA sequence in a given population is rare, it is termed mutation; if it is common, it is called polymorphism. RFLP is a difference in the size of DNA restriction fragment (restriction map) between individuals. It can serve as a useful genetic marker for the analysis and mapping of a large genome. RFLP is based on the principle that small differences in the DNA sequence can alter restriction enzyme cutting patterns. For example, a single base-pair difference in a particular chromosome, or short deletions or insertions of a base pair, may eliminate a site for restriction enzyme action, resulting in a large size difference in DNA restriction fragments. The inherited difference in the size of RFLPs provides a large number of linkage markers for following mutant genes through families.

DNA Engineering To determine the role of genes, RNA molecules, and proteins in an intact organism, scientists have relied on naturally occurring mutations and on a number of techniques to generate mutations. DNA engineering has transformed this area of investigation. A specific mutation now can be generated in selected genes, and stable strains of mutants can be produced in cells or mice to study the function of a desired gene.

Gene Editing.  Genome editing is the deliberate alteration of a selected DNA sequence in a living cell. Genome editing techniques can be used to alter how a gene functions, for example, by changing a variant of a gene that may give rise to disease to one that does not. As well as modifying the genome itself, the technique can be used to modify the epigenome—a set of chemical modifications associated with the genome that can control gene activity, such as changing gene expression without changing the DNA sequence.110 CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–CRISPR associated protein 9) is an

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example of a new method of genome editing that is now widely used in research. It is the simplest, most versatile and precise method of genetic manipulation. The CRISPR-Cas9 system consists of two key molecules that introduce a change or mutation into the DNA. Cas9 acts as a pair of “molecular scissors” that cut the two strands of DNA at a specific location in the genome so that a portion of DNA can then be added or removed. The location of the cut is determined by a piece of RNA (guide RNA; about 20 bases) located within a longer RNA scaffold. The scaffold part binds to DNA and the predesigned sequence directs Cas9 to the right part of the genome to cut it at the predetermined point. The guide RNA has a base sequence that is complementary to those of the target DNA sequence in the genome so that it will only bind to the target sequence and no other; thus Cas9 makes a cut across both strands of the DNA.

Study of the Gene’s Function.  In vitro, a mutation in a gene of interest can be produced by two different techniques. In the first, or classic technique, cells in which the gene is known to be expressed are treated with mutagen, and mutant cells are isolated by selecting against expression of gene product. This approach has not been very successful, because the mutations can only be detected if both copies of a gene in the diploid cell are mutated, which occurs rarely when using conventional mutagenesis. Therefore the more reliable technique of homologous recombination (discussed previously) is used. Cloned copies of the desired gene are altered to render them nonfunctional and then introduced into the cell’s chromosome, replacing the normal gene with a nonfunctional copy. Because of the high frequency of homologous recombination, both copies of the gene in a diploid cell can be mutated, resulting in homozygous mutant cells. A defect then can be ascribed to the mutated gene if the mutant phenotype is reverted by a copy of the wild type of gene transferred into the mutant cell by transfection. Several approaches have been used to study the function of an isolated gene by inserting it in “foreign” cells. First, using transient expression systems, it is possible to insert genes into cells and study both the quantity and the structure of their transcript. Second, genes can be introduced into established cultures of cells of the appropriate lineage. Third, it is possible to insert chromosomes containing the gene of interest into a foreign cell. Fourth, genes can be introduced into fertilized eggs and their patterns of integration and expression studied over the next several generations; several variations on this approach have developed. For example, the application of homologous recombination to embryonic stem (ES) cells has allowed the manipulation of mouse genome. ES cells are derived from the inner cell mass of a blastocyst and can be kept indefinitely in culture without affecting their totipotent characteristics. New genes can be inserted into the mouse genome by transgenesis, creating transgenic mice.111 In transgenesis, the desired DNA is injected into the male pronucleus of a fertilized ovum, which is then implanted into the uterus of a pseudopregnant female mouse. The injected DNA is randomly integrated into the genome of some of the eggs. This results in a mouse that has extra genetic material of a known structure (transgene). Therefore it is possible to study the effect of a transgene on development, to localize the region of the gene required for its expression in normal tissues, and to study the effect of overexpression and mutation on gene function. Transgenic mice have been especially useful in studying the role of T cell and B cell receptors in lymphocyte development. In contrast with transgenic animals, in certain circumstances, the function of a particular gene can be understood if a mutant animal that does not express the gene of interest can be obtained, using a technique of gene knockout by homologous recombination. Such mice are called knockout mice.112 A common approach to disrupt gene function by homologous recombination in ES cells is to construct a target or

knockout vector. A standard knockout vector contains a positive selection marker (neomycin gene) within the coding sequence of a genomic DNA fragment that leads to disruption of the target gene. However, to improve the recombination events, a knockout vector containing a positive selection marker that lacks either its own promoter or poly-A site and knockout vectors that contain a negative selection gene in addition to a positive marker have been developed. Furthermore, the frequency of homologous recombination has been increased by using a syngeneic DNA. These approaches have been used to generate a large number of null-mutant mice strains, including major histocompatibility complex (MHC) class I–deficient113 and MHC class II–deficient mice.114 The two techniques for generating transgenic mice and knockout mice are now well established. These animals have been instrumental in the in vivo study of the effect of point mutation. As a result, functions of various molecules and the molecular basis of various genetic diseases have been delineated.

Genomics and Proteomics

Gene Arrays.  The HGP, started in 1990 and officially completed in 2003, initiated a new era in genetics.115–118 At present, a huge amount of DNA sequence data that make up the entire human genetic blueprint is available. The challenge is to translate the genome into knowledge, making the focus of research to analyze the interaction and regulation of the identified genes. Advances in functional genomic technology help to determine the location and function and the orchestrated expression of these genes. DNA array technology has become a powerful, high-throughput, versatile tool that can be applied to the study of functional genomics. DNA arrays are collections of large sets of DNA sequences immobilized onto solid substrates as individual spots. The principle of DNA array technology is based on the highly sensitive and specific hybridization of complementary strands of nucleic acids. cDNA is tagged with a radioactive or fluorescent label during reverse transcription from sample RNA and hybridized to an array. This single-stranded tagged cDNA, or probe, binds to corresponding DNA immobilized to an array. Excess and nonspecific tagged cDNA is washed away, and the pattern of tagged cDNA binding is imaged and analyzed for the intensity of hybridization to each spot. The intensity of the signal is proportional to the quantity of the hybridized cDNA. The level of expression relative to another cDNA or another DNA sequence spot on the array is calculated (Fig. 10.8). The data processing enables analysis of thousands of transcripts at the same time and profiling of the relationship between the many genes in the arrays, termed expression profiling. The substrates used to immobilize the target DNA can be either porous, such as nylon membrane, or nonporous, such as glass slides. When DNAs are dotted onto nylon membranes, the gene arrays are referred as macroarrays. In this case, the detection is based on radioactivity of 32P or 33P and does not rely on competition between an experimental and a control set of cDNAs. By contrast, in glass micro­ arrays, also called chips, the detection depends on fluorescence labeling of the probe. Although fluorescence-based detection is less sensitive than the radioactive system, glass has many advantages over nylon as a support: Because of its low autofluorescence, glass does not contribute significantly to background “noise,” which can mask data in nylon arrays. More important, however, two different probes can be labeled with different fluorochromes and simultaneously incubated with one glass array. The DNA sequences immobilized onto substrate can be either full-length or short oligonucleotides of 10 to 50 bp. Short oligonucleotides may be more efficient in hybridization.119,120 Recently, microarrays have been designed using oligonucleotide probes synthesized in situ by applying a resequencing array tiling strategy to achieve a high-throughput sequencing that allows for the detection of both known and novel

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CHAPTER 10  Molecular Biology and Genetic Engineering Test RNA

Reference RNA Reverse transcription

Cy3-labeled test cDNA

Reference protein

Cy3

Test cDNA

Reference cDNA

Cy3-dUTP

Test protein

Cy5

Cy3-labeled test protein

Cy5-labeled reference protein

Cy5-dUTP Cy5-labeled reference cDNA

Mix and expose to antibody array

Mix and expose to DNA array

Glass antibody array Glass DNA array

Fluorescence reading Fluorescence reading

Fig. 10.8  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 to Cy5, in which significant deviations from 1 show increased (>1) or decreased ( ILC2s

High fat diet

• AHR dependent on ILC3s, IL-1β and NLRP3.

Dermatitis Models MC903 (vitamin D analog)

• ILC2 responses and skin inflammation dependent on TSLP, not IL-33 signaling • Ear swelling reduced in Rorasg/sg mice and CD90.1-depleted RAG1-/- mice • Ear swelling and skin ILC2 number dependent on IL-25 and IL-33, and less so on TSLP • ILC2 activation by basophil IL-4

AHR, Aryl hydrocarbon receptor; BMT, bone marrow transplant; HDM, house dust mite; IL, interleukin; ILC, innate lymphoid cell; OVA, ovalbumin; RSV, respiratory syncytial virus; TSLP, thymic stromal lymphopoietin. Adapted from Doherty TA. At the bench: understanding group 2 innate lymphoid cells in disease. J Leukoc Biol 2015;97(3):455–67.

and DCs that could either indirectly activate ILC2s or directly promote inflammatory responses through other cell types. Overall, it is clear that IL-33 and IL-25 are critical initiators of type 2 inflammation, and future studies will define whether these cytokines will be effective targets of therapy for allergic diseases.

Thymic Stromal Lymphopoietin TSLP is an IL-7-like cytokine that is found increased in epithelial tissues from patients with chronic rhinosinusitis (CRS), atopic dermatitis, and asthma. Genome-wide association (GWAS) studies have also linked TSLP single nucleotide polymorphisms with increased susceptibility to asthma.8,22 TSLP binds to a heterodimer consisting of TSLP receptor (TSLPR) and IL-7Rα, expressed on DCs, mast cells, and ILC2s. In addition to epithelial cells, TSLP is produced by DCs, mast cells, and basophils. Human TSLP exists in long and short isoforms that have varying levels of proinflammatory capabilities. TSLP has important roles in innate and adaptive type-2 responses, and initial work showed that TSLP primes DCs to induce CD4+ Th2 cell differentiation.19 Subsequent work demonstrated that TSLP also potently activates human ILC2s.18 Peripheral blood and nasal polyp ILC2s stimulated with TSLP show upregulation of GATA3 and increases

in IL-4, IL-5, IL-13 production, which are enhanced in the presence of IL-33. Interestingly, TSLP further induces corticosteroid resistance of mouse and human ILC2s suggesting that corticosteroids (CSs), which are used as a mainstay of treatment for allergic diseases, may not reduce ILC2 responses driven by TSLP.25 Importantly, early studies using an anti-TSLP antibody in allergen-challenged asthmatics demonstrated reductions in hyperresponsiveness and airway eosinophils, suggesting that targeting the TSLP/ILC2 axis may be a promising therapeutic strategy for allergic diseases.

Lipid Mediators A critical link between ILC2s and allergic inflammation was further established as eicosanoids known to promote or inhibit type 2 inflammation were found to directly modulate ILC2 responses.8 Human peripheral blood ILC2s stimulated with PGD2 showed increased IL-13 production in the presence of IL-33 and IL-25, whereas lipoxin A4 (LXA4) inhibited ILC2 activation by PGD2.26 CysLTs that include LTC4, LTD4, and LTE4 also promote ILC2 activation in mice and humans.8,17 Additional lipid inhibitors of ILC2 function include prostaglandin E2 (PGE2), prostaglandin I2 (PGI2), and the omega-3 derivative Maresin-1.8,27 Interestingly, sex hormones also regulate ILC2s; androgens inhibit

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Epithelial damage (viruses, allergens, pollutants)

Mucus AHR

Tissue repair

Mast cell TSLP, IL-33, IL-25

IL-13

Mucus

AREG IL-9

Dendritic cell

CysLTs PGD2

TL1A ILC2

IL-4

Macrophage IL-5

CD4+ Th2 cell B cell

IgE

Eosinophil

Fig. 12.4  Innate lymphoid cell (ILC) 2–driven type 2 inflammation. Viruses, allergens, and pollutants damage intact epithelium to release “alarmin” cytokines TSLP, IL-33, and IL-25 that in turn activate ILC2s. Lipid mediators prostaglandin D2 (PGD2) and cysteinyl leukotrienes (CysLTs) produced by mast cells, eosinophils, and macrophages also promote ILC2 responses. The tumor necrosis factor (TNF) family member TL1A produced by dendritic binds to DR3 expressed on ILC2s, resulting in ILC2 proliferation and cytokine production. Activated ILC2s produce IL-4, IL-5, IL-9, IL-13, and amphiregulin. ILC2 IL-4 secretion promotes Th2 cell differentiation and memory responses in addition to IgE production by B cells. IL-5 promotes tissue infiltration of eosinophils, and IL-13 induces airway hyperresponsiveness, remodeling features, and dendritic cell migration to lymph nodes for Th2 cell responses. IL-9 promotes mast cell accumulation, and along with IL-13, induces mucus metaplasia. Amphiregulin binds to epidermal growth factor (EGFR) on structural cells to induce repair responses. (Adapted from Doherty TA. At the bench: understanding group 2 innate lymphoid cells in disease. J Leukoc Biol 2015;97(3):455–67.)

ILC2 function and mouse uterine ILC2s are controlled by estrogen levels.28 The effects of sex hormones on ILC2s may provide clues to gender differences in the development and natural history of allergic diseases, including asthma. Overall, lipid mediators appear to be critical modulators of ILC2 function, and lipid-ILC2 interactions likely contribute critically to human allergic diseases. The central mediators and cell types that activate ILC2s in tissue inflammation are shown in Fig. 12.4.

Costimulatory Molecules and Neuropeptides Additional regulators of ILC2 function deserve mention and include costimulatory molecules (ICOS/DR3) as well as neuromedin U1 receptor (NMUR1).8,29 Costimulatory interactions between ICOS and ICOS ligand as well as DR3 and TL1A provide critical immunomodulatory signals between many immune cell types, including antigen-presenting cells (APCs) and T cells. In addition to adaptive immune cell responses, ICOS and DR3 have been detected on ILC2s and positively regulate

Th2 cytokine production in mouse and human ILC2s. NMUR1 has also been detected on mouse and human ILC2s and regulates Th2 cytokine production in response to the secreted neuropeptide neuromedin U (NMU). Furthermore, ILC2s colocalized with NMU-expressing nerves in the mouse GI tract. The discovery of the NMU/NMUR1/ ILC2 axis suggests the presence of a critical pathway by which ILC2s may contribute to neuroinflammation, or reciprocally, nerve control of peripheral type 2 inflammation.

ILC2s AND TYPE 2 INFLAMMATION IN PRECLINICAL DISEASE MODELS Viral-Induced Airway Inflammation Mouse models of asthma recapitulate some of the cardinal features of human asthma including peribronchial inflammation, epithelial mucus production, airway hyperresponsiveness, and airway smooth muscle

CHAPTER 12  Innate Lymphoid Cells increases and fibrosis. The first reports to demonstrate the involvement of ILC2s in such models used influenza virus to induce airway inflammation and hyperresponsiveness.20,30,31 IL-13 produced by ILC2s was shown to be critical to the development of AHR during viral infection. In contrast, appropriate airway repair after influenza virus infection in mice required ILC2 production of the growth factor amphiregulin. Thus ILC2s appear to have dual roles in both pathogenic and normal repair responses in the airway after viral infection. In addition to influenza, rhinovirus infection is a major cause of asthma morbidity, including triggering asthma exacerbations. Interestingly, rhinovirus infection in neonatal, but not adult, mice led to expansion of IL-13+ lung ILC2s, mucus production, and AHR that were dependent on IL-25.16,31 Importantly, the expanded ILC2s persisted for weeks in the lung, suggesting that increased ILC2 numbers could be primed for greater responses upon reexposure to viruses or allergens. Further work has shown that TSLP, IL-33, and IL-25 participate in ILC2 responses in mice during rhinovirus infection highlighting the redundancy and complexity of innate responses during upper respiratory tract infections. Human studies have also demonstrated ex vivo ILC2 activation after rhinovirus exposure, supporting the translational aspect of the preclinical models. Respiratory syncytial virus (RSV) is also tied to airway disease in humans, and mice challenged with RSV have increased IL-13, producing ILC2 expansion that is dependent on TSLP.32

Allergen-Induced Airway Inflammation In addition to responding to different viruses associated with asthma, ILC2s are also activated in response to multiple allergens including Alternaria alternata, house dust mite (HDM), papain, and ovalbumin (OVA, Table 12.1).16,31 The initial lung disease mouse models used RAG knockout mice (that lack B and T cells but have ILCs) or used short-term (few days) allergen challenge models to skew toward an innate response before the onset of adaptive immunity. Multiple studies revealed that ILC2s are strongly activated after exposure of mice to the fungal allergen Alternaria alternata that induces rapid eosinophilic lung inflammation in previously unsensitized mice. This finding is intriguing because of the association between Alternaria sensitization and exposure with severe asthma, including fatal exacerbations. Alternaria induces high levels of lung IL-33 and ILC2 activation that is dependent on the IL-33 receptor (T1/ST2). During longer-term models in HDM- and OVA-challenged mice, ILC2s continue to be activated, and in some cases, numbers are similar to Th2 cytokine-producing CD4+ cells at later stages during adaptive responses. It is important to note that methods to detect cytokine production by various cell types in vivo do not account for the levels of cytokine produced by individual cells. Importantly, ILC2s produce significantly more IL-5 and IL-13 in vitro (µg amounts per 5000 cells) compared with mast cells, basophils, NKT cells, and Th2 cells when stimulated with IL-33, suggesting that total numbers of ILC2s may not reflect their impressive contribution to tissue cytokine levels.6,16 Given the limitations of interpreting responses from RAG-deficient mice and lack of reagents to specifically deplete ILC2s, a mouse model was needed that specifically lacked ILC2s but had intact adaptive Th2 cell responses. Multiple investigators have since used RORα bone marrow transplant (BMT) to better isolate the effects of ILC2s during adaptive responses to allergens.10,16,31 Because RORα is required for ILC2 development but has a minor effect on adaptive CD4+ cell development or responses, RORα-deficient bone marrow transferred into wild type hosts limits the effect of RORα to the hematopoietic cells. Interestingly, these “ILC2-deficient mice” fail to mount an adaptive Th2 cell response to dust mite and papain despite having an intact adaptive immune system and mounting a normal Th2 response to conventional systemic sensitization with OVA. The mechanism whereby ILC2s promote adaptive Th2 responses appears to partially be related to the effects of ILC2

197

IL-13 production on DC licensing and migration for Th2 cell priming and memory responses. A similar requirement for development of CD4+ Th2 responses by ILC2s was found in helminth-infected mice through ILC2 production of IL-4 induced by LTD4. Thus, in addition to providing a rapid and robust source of innate Th2 cytokine production, ILC2s also appear to control development and possibly persistence of adaptive Th2 responses. Resistance to CS is a feature of severe asthma, and ILC2s are insensitive to CS under specific conditions. Some models using allergenchallenged mice have shown ILC2 responses that are CS resistant but develop sensitivity after TSLP blockade or in the absence of TSLP.8,31 Furthermore, TSLP signals through STAT5 and pharmacologic inhibition of phospho-STAT5 also reverses steroid resistance of ILC2s.25 These studies have therapeutic implications; anti-TSLP treatment has shown efficacy in early studies of allergen-challenged human asthmatics and could potentially be used as an adjunct in CS-resistant asthmatics.

Other Models of ILC-Induced Airway Inflammation Obesity as well as exposure to heightened levels of pollution, cigarette smoke, and ozone levels have been associated with asthma morbidity. In mice, ozone exposure results in a mixed response with neutrophilia and type 2 inflammation that is dependent on ILCs.33 In contrast, HDMchallenged mice receiving diesel exhaust have enhanced type 2 lung inflammation that appears to be largely dependent on adaptive immunity and not ILC2s.34 Differences in models may reflect the duration and presence of adaptive Th2 cells in response to allergen (HDM) versus lack of allergen (ozone without allergen). Cigarette smoke appears to have a silencing role in ILC2 responses to viral infections through downregulation of the IL-33 receptor ST2, leading to an exaggerated type 1 response.31 In a mouse model of obesity-associated asthma, a high-fat diet administered to mice induces AHR that is dependent on IL-17, ILC3s, and the NLRP3 inflammasome that processes IL-1β and IL-18.35 Mouse models of lung inflammation have also suggested that ILC2s contribute to bleomycin-induced pulmonary fibrosis and after hyperoxia.31 Taken together, ILC2s, and in some cases ILC3s, appear to be contributors to lung inflammation in multiple models.

Preclinical Atopic Dermatitis Models Atopic dermatitis is a common skin inflammatory skin disease that clinically manifests as itchy scaly rashes, and pathologically as skin barrier disruption, eosinophilic infiltration, high serum IgE, and predisposition to other atopic disorders including asthma and food allergy. Similar to asthma models in mice, models of atopic dermatitis exist that show some features of the disease, including eosinophilic infiltration, epidermal hyperplasia, dermal thickening, and systemic IgE production.16,31 Studies of skin ILC2s in mice have revealed that ILC2s are present, and the majority of IL-13–positive cells at baseline in IL-13 reporter mice are non-T cells. C57BL/6 mice administered the vitamin D analog MC903 that induces dermal thickening and inflammation and skews toward TSLP-mediated responses, require ILC2s as well as TSLP for the pathologic skin changes.16,31 However, Balb/c mice administered the same topical compound show dependence of inflammation and ILC2 responses on IL-25 and IL-33 and, to a lesser extent, TSLP. In another model, skin-specific overexpression of IL-33 induces dermal eosinophilia and skin Th2 cytokine increases along with expansion of ILC2s. Additionally, administering IL-2 to RAG1–/– mice (that lack T or B cells but have ILC2s) also induces epidermal hyperplasia, dermal thickening, eosinophilic infiltration, skin Th2 cytokine production, and ILC2 activation. This is somewhat surprising, because IL-2 is largely regarded as a homeostatic cytokine for ILC2s but appears, in certain contexts, to be proinflammatory.

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SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 12.2  ILC2s Isolated from Human Fluid and Tissues Tissue Compartment

Findings

Reference

Blood

ILC2s higher in asthma patients than healthy controls.

Bartemes KR, J Allergy Clin Immunol 2014;134:671

BAL fluid

Number of IL-13-producing ILC2s increased in asthmatics.

Christianson CA, J Allergy Clin Immunol 2015;136:59–68

Esophagus

Percentage of ILC2s higher in eosinophilic esophagitis.

Doherty TA, J Allergy Clin Immunol 2015;136:792–794

Nasal polyps

Increased ILC2 frequency compared with turbinates.

Bal SM, Nat Immunol 2016;17:636–645

Skin

ILC2s are markedly increased in atopic dermatitis.

Bruggen MC, J Invest Dermatol 2016;136:2396–2405

Sputum

ILC2s greater in patients with eosinophilic asthma.

Smith SG, J Allergy Clin Immunol 2016;137:75–86

Blood and BAL fluid

Allergen challenge increased ILC2s.

Chen R, Am J Respir Crit Care Med 2017;196:700–712

Blood

ILC2s greater in females than males with severe asthma.

Cephus JY, Cell Rep 2017;21:2487–2499

Nasal scrapings

ILC2s greater in AERD patients after aspirin challenge.

Eastman JJ, J Allergy Clin Immunol 2017;140:101–108

AERD, Aspirin exacerbated respiratory disease; BAL, bronchoalveolar lavage fluid.

ILC2 IN HUMAN ALLERGIC DISEASES Increases in the number and function of ILC2s have been reported in humans who have allergic diseases, including asthma, allergic rhinitis, and atopic dermatitis (Table 12.2). There were twice as many ILC2s in the peripheral blood of patients with allergic asthma compared with patients with allergic rhinitis or healthy controls, and there was no difference in the number of peripheral blood ILC2s between the patients with allergic rhinitis and the healthy controls.36 The ILC2s from subjects with allergic asthma were significantly more responsive to IL-33 and IL-25 ex vivo, secreting higher levels of IL-5 and IL-13 compared with patients either with allergic rhinitis or healthy controls.36 Another study confirmed that peripheral blood ILC2s from subjects with asthma had increased type 2 cytokine secretion in response to IL-33 and IL-25 stimulation ex vivo compared with persons without asthma.37 In this study, patients with asthma had a greater number of peripheral blood IL-13, but not IL-5, producing ILC2s, compared to healthy controls. Interestingly, the number of IL-13–expressing ILC2 was dramatically decreased in those patients whose asthma was well controlled. It is important to note that one study did not find a difference in the number of peripheral blood ILC2s between healthy controls or persons with either mild or severe asthma.26

Airway Diseases Including Asthma The relationship between the number of peripheral blood ILC2s and eosinophilic airway inflammation has been investigated in two studies. In the first, the proportion of ILC2s in the peripheral blood was significantly threefold greater in patients with eosinophilic asthma compared with those with non-eosinophilic asthma.38 The percent of ILC2s in peripheral blood significantly correlated with sputum eosinophil counts and those subjects with the greatest percentage of peripheral blood ILC2s were those with the greatest degree of airway eosinophilic inflammation. In the second study, a cross-sectional analysis of patients with steroid-naïve mild atopic asthma and nonatopic control subjects, significantly greater numbers of total and type 2 cytokine-producing ILC2s were detected in blood and sputum of patients with severe asthma compared with mild asthmatics, whereas intracellular cytokine expression by CD4 cells within the airways did not differ between the asthmatic groups.39 In patients with severe asthma, ILC2s were the predominant source of type 2 cytokines, and there were significantly greater numbers of sputum type 2 cytokine-producing ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers. These results suggested that ILC2s may promote the persistence of airway eosinophilia in patients

with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy. Similar observations were made within the airways, with both the total number of ILC2s and the number of IL-13–producing ILC2s increased in the BAL fluid of asthmatics compared with nonasthmatic controls. In a study of adults, the frequency of ILC2s in BAL was significantly elevated threefold in asthmatic patients compared with healthy controls.40 In the patients with asthma, IL-33 in BAL fluid was also significantly increased compared with healthy controls, and elevated BAL IL-33 predicted increased ILC2s in the airways. The BAL IL-33 levels negatively correlated with FEV1 percent predicted and asthma control test (ACT) scores, which reflect symptoms. In a study of children older than 6 years, there were significantly more pulmonary ILC2s in BAL and induced sputum than in peripheral blood, and a significantly higher proportion of ILC2s was present in patients with severe therapy-resistant asthma than in patients without asthma.41 In this study, ILC2s were not identified in children with lower respiratory tract infections, suggesting that ILC2s may have a specific role in allergic airway diseases. The presence of pulmonary, rather than systemic, ILC2s, which corresponded with elevated IL-33 in children with severe therapy-resistant asthma, suggests that therapies designed to target these cells locally should be pursued. Allergen challenge increased the number of ILC2s in the airway and peripheral blood. In one report where all subjects were selected based on their ability to develop allergen-induced early and late responses, airway eosinophilia, and increased methacholine airway responsiveness, there was a significant increase in total, IL-5–, and IL-13–expressing ILC2s in sputum, 24 hours after allergen, coincident with a significant decrease in blood ILC2.42 Airway eosinophilia correlated with IL-5– expressing ILC2s at all time points and allergen-induced changes in IL-5–expressing CD4 cells at 48 hours after allergen. Sex hormones are important regulators of ILC2 number and function in the setting of asthma. Women with moderate to severe asthma have an increased frequency of peripheral blood ILC2s compared with men who have the same severity of disease.43 In contrast, there was no difference in the peripheral blood frequency of ILC2s between women and men who did not have asthma. Studies in mice provided support for the possible mechanism by which sex hormones regulated ILC2 function. ILC2s from adult female mice had increased IL-2–mediated proliferation compared with ILC2s from adult male mice, as well as prepubescent female and male mice. 5α-dihydroxytestosterone, a hormone downstream of testosterone, decreased lung ILC2 numbers and ILC expression of IL-5 and IL-13.

CHAPTER 12  Innate Lymphoid Cells ILC2s may have an important role in regulating airway epithelial cell barrier function in asthma. In an in vitro system in which human bronchial epithelial cells (HBEC) at the air–liquid interface were cocultured with human ILC2s isolated from peripheral blood, the presence of ILC2s significantly reduced transepithelial electrical resistance.44 Coculture of HBEC with ILC2s for 24 hours significantly increased the passage of large molecules, signifying greater epithelial cell permeability, which was further increased by IL-33 stimulation of the ILC2s. Neutralization of IL-13 restored the HBEC integrity that had been disrupted by the ILC2s. There is evidence that the epithelial cell–derived cytokine TSLP regulates steroid resistance of airway ILC2s from patients with severe asthma. ILC2s were isolated from the BAL fluid from patients with poorly controlled asthma and then stimulated with IL-2, IL-7, and IL-33.25 Intracellular cytokine staining by flow cytometry revealed that there was negligible inhibition of IL-5 expression with dexamethasone treatment of the airway ILC2s, whereas similarly cultured and treated ILC2s from peripheral blood had a significant decrease in IL-5 expression. TSLP was elevated in the BAL fluid from the subjects with asthma, and there was consideration that the airway TSLP may have increased ILC2 resistance to corticosteroid treatment. TLSP treatment of blood ILC2s made them more resistant to corticosteroid treatment, and this was reversed by inhibition of STAT5, through which TSLP signals. TSLP treatment of ILC2s increased IL-7 receptor expression, a possible mechanism by which TSLP enhances resistance to corticosteroid treatment.

Chronic Rhinosinusitis ILC2s have been identified in CRS with polyposis (wNP). ILC2s were present at an increased frequency in nasal polyps from patients with CRSwNP.45 A comparison of the expression of transcripts encoding IL-4 or IL-12 in the nasal polyps with that in healthy turbinate tissue revealed that the amount of IL4 mRNA was elevated in polyp tissue relative to that in healthy turbinate tissue, whereas IL12 mRNA was undetectable in polyp tissue. This suggested that IL-4 may have a role in promoting ILC2s in nasal polyp tissue, because IL-12 transdifferentiates ILC2 into ILC1. To determine whether IL-4 was involved in inducing or maintaining expression of the PGD2 receptor DP2, also known as chemoattractant receptor-homologous molecule expressed on Th2 (CRTH2), which is a defining characteristic of human ILC2s, peripheralblood-derived ILC2s were first cultured under ILC1-polarizing conditions and then restimulated in the presence of IL-4. ILC2 expression of DP2 was restored in the presence of IL-4. Additionally, IL-4 acted in synergy with IL-33 to induce ILC2 proliferation, in addition to promoting ILC2 production of IL-5 and IL-13 to a level comparable with that induced by IL-33 and TSLP. IL-4, by itself, was unable to induce IL-5 or IL-13 production by ILC2s. Thus IL-4 is an important factor in promoting ILC2 function in nasal polyps by working in concert with IL-33. Another group similarly found that ILC2s were the dominant ILC subset in nasal polyps (NPs). These investigators found that ILC2s isolated from NPs spontaneously released the type 2 cytokines IL-5 and IL-13, whereas peripheral blood ILC2s did not.46 This suggests that the threshold for cytokine production in ILC2s from NPs might be lower, resulting in greater inflammation. NPs vary in the degree of eosinophilia that exists within them. Eosinophilic nasal polyps contained double the number of ILC2s versus noneosinophilic polyps.47 Polyp ILC2s were also reduced by 50% in patients treated with systemic corticosteroids. These results strongly suggest that ILC2s may have a contributing role to the presence of eosinophils in NPs.

Skin Diseases Including Atopic Dermatitis Atopic dermatitis in humans is also linked to dysregulation of ILC2s. ILC2s are markedly increased in the skin of patients with atopic dermatitis compared with skin samples collected from healthy controls.48–51

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In one study, there were significantly more ILC2s detected in lesional skin biopsies from atopic patients relative to healthy individuals, yet there was a similar frequency of circulating ILC2s in the peripheral blood of both groups.51 These human atopic skin-derived ILC2s had significantly greater surface expression of IL-17RB (IL-25R), ST2 (IL-33R), and TSLP receptors compared with healthy controls. Gene expression analysis on skin samples isolated from either noninvolved skin or AD-involved skin clearly demonstrated the upregulation of genes encoding the IL-17RB (IL-25R), ST2 (IL-33R), and TSLP receptors in the AD skin samples. Analysis for cytokine gene expression also detected increased levels of IL33 and IL25 in the AD samples. These authors also assessed the ability of ILC2s from AD skin to produce IL-13 and IL-5 in response to stimulation with IL-25 and IL-33. IL-33 stimulation elicited significant levels of IL-13, IL-6, and IL-5 production, but induced little IL-4. TSLP and IL-25 did not induce significant IL-4, IL-13, IL-6, or IL-5 production in isolation, but interestingly were able to enhance cytokine production when present in combination. Using transmigration assays, IL-33, in contrast to IL-25, elicited significant migration of skin-derived in vitro–cultured ILC2s. Although migration toward TSLP was also observed, this occurred only at high concentrations. In this study, IL-33 was a potent stimulus for the activation of human skin–derived ILC2s, induced the upregulation of its own receptor, enhanced the expression of type 2 cytokine expression, and increased the migratory capacity of these cells. Within the dermis of inflamed atopic skin, ILC2s were found in close proximity to basophils or T cells, suggesting possible coordination between these proinflammatory cell types.50 Another group confirmed that the frequencies of ILC2s, basophils, and Th2 cells, but not mast cells, were significantly elevated in the skin, but not peripheral blood, of patients with atopic dermatitis, but not psoriasis.52 Skin basophils were only detected in the skin from atopic dermatitis patients and expressed IL-4 after stimulation. In atopic dermatitis, basophils, and ILC2s were positively correlated in the skin, whereas basophils were inversely correlated with blood ILC2s. A third study further confirmed an increased number of ILC2s in the lesional skin of patients with atopic dermatitis compared with healthy controls.48 Collectively, these data strongly suggest a pathologic role for ILC2s in the setting of atopic dermatitis. There is new evidence that ILC2s have a role in surveillance in the skin. A subset of ILC2s express MHC class II molecules and have the capability of presenting peptide antigens to T lymphocytes.53 ILC2s also express the nonclassical MHC molecule CD1a display lipids and small molecules to T lymphocytes. In a human skin challenge model, ILC2 obtained from human skin presented endogenous antigens to T cells.54 Expression of CD1a on the skin ILC2s was upregulated by TSLP, when the levels of TSLP were similar to those of patients with atopic dermatitis, and this response was PLA2G4a-dependent. Additionally, the CD1a pathway was used to sense Staphylococcus aureus via a toll-like receptor–dependent CD1a-reactive T cell response to endogenous ligands. These results reveal a previously undefined role for ILC2s in limit surveillance.

Allergic Rhinitis There are conflicting data about whether ILC2s are increased in the setting of allergic rhinitis. As mentioned earlier, one study reported that there was no difference in the number of peripheral blood ILC2s between the patients with allergic rhinitis and the healthy controls.36 In cat-allergic subjects, nasal challenge with cat antigen significantly increased the frequency of ILC2s in the peripheral blood by twofold 4 hours after challenge, as compared with no change in the ILC2 frequency after diluent challenge.55 ILC2s are hypothesized to play a key role, because proinflammatory cells in allergic rhinitis and patients challenged with intranasal exposure to allergen have increases in the number of ILC2s in nasal secretions that strongly and positively correlated with

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SECTION A  Basic Sciences Underlying Allergy and Immunology

airway eosinophilia.56 Interestingly, the magnitude of ILC2 induction and cytokine production in allergic rhinitis patients varies by antigen, with house dust mite–monosensitized patients having significantly increased frequencies of circulating ILC2s compared with mugwort monosensitized patients.57 Pollen grass subcutaneous immunotherapy (SCIT) was effective in preventing ILC2 accumulation in the blood of seasonal allergic rhinitis patients, demonstrating the potential for therapeutic intervention in attenuating ILC2 responses.58 These data broadly support a role of ILC2s in the pathogenesis of allergic rhinitis, though factors such as specificity of the antigen may influence ILC2 reactivity.

Chronic Obstructive Pulmonary Disease Although ILC2s are increased in allergic diseases, there was a decrease in ILC2s relative to ILC1 in persons with chronic obstructive pulmonary disease, as examined in stable patients in the chronic obstructive pulmonary disease (COPD) gene study that investigated underlying genetic factors of COPD.59 In healthy control subjects, approximately 40% of peripheral blood ILCs expressed the ILC2 marker DP2, whereas approximately 5% of the circulating ILCs were ILC1. The frequency of peripheral blood ILC1 was significantly higher in patients with COPD, and this corresponded with a lower frequency of peripheral blood ILC2s. Patients with severe COPD, as defined by Global Initiative for Chronic Obstructive Lung Disease (GOLD) classification III–IV, had significantly higher frequency of ILC1 compared with patients with milder COPD (GOLD classification I–II). Smokers, who were otherwise healthy, had a significantly greater frequency of peripheral blood ILC1s than was present in nonsmokers who were healthy. There was an inverse correlation of ILC1s and lung function, as measured by percent FEV1 predicted and the FEV1/forced vital capacity (FVC) ratio. In contrast, there was a positive correlation between ILC2s and lung function.

Aspirin-Exacerbated Respiratory Disease and Eosinophilic Esophagitis Emerging evidence supports a potential role for ILC2s in the context of several other allergic diseases. Patients with aspirin-exacerbated respiratory disease (AERD) challenged intranasally with nonsteroidal antiinflammatory drugs had increased ILC2 burdens in nasal scrapings collected during challenge compared with ILC2 levels in nasal scrapings at baseline.60 This increase in ILC2 burden in the nasal scrapings was associated with a simultaneous decrease in ILC2 frequency in the blood, suggesting potential tissue infiltration of ILC2s from the blood to the nasal mucosa. Furthermore, this increase in ILC2 burden in the nasal scrapings positively correlated with severity of symptoms.60 These results revealed that ILC2s are recruited to the nasal mucosa during COX-1 inhibitor–induced reactions in patients with AERD. Additionally, several eosinophilic disorders have been associated with ILC2 activation. In active eosinophilic esophagitis (EoE) there was an increase in the percentage of ILC2s in the esophagus compared with patients with inactive EoE, proton pump inhibitor–responsive EoE (PPI-REE), or healthy controls.61 Esophageal ILC2s correlated strongly with eosinophils in the esophageal biopsies. Finally, eosinophilic pleural effusion (EPE) induces a marked increase in the number of ILC2s in the pleural fluid.62 We are currently in the very early stages of defining associations between the number and function of ILC2s with specific disease states. Once specific ILC2 inhibitors are developed and used for clinical trials, then we will have a much better understanding of the role of these cells in disease pathogenesis.

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2. Spits H, Artis D, Colonna M, et al. Innate lymphoid cells–a proposal for uniform nomenclature. Nat Rev Immunol 2013;13(2):145–9. 3. Busse WW, Lemanske RF Jr. Asthma. N Engl J Med 2001;344(5):350–62. 4. Neill DR, Wong SH, Bellosi A, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010;464(7293):1367–70. 5. Price AE, Liang HE, Sullivan BM, et al. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc Natl Acad Sci USA 2010;107(25):11489–94. 6. Moro K, Yamada T, Tanabe M, et al. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells. Nature 2010;463(7280):540–4. 7. Peebles RS Jr. At the bedside: the emergence of group 2 innate lymphoid cells in human disease. J Leukoc Biol 2015;97(3):469–75. 8. Cavagnero K, Doherty TA. Cytokine and lipid mediator regulation of group 2 innate lymphoid cells (ILC2s) in human allergic airway disease. J Cytokine Biol 2017;2(2). 9. Zook EC, Kee BL. Development of innate lymphoid cells. Nat Immunol 2016;17(7):775–82. 10. Halim TY, MacLaren A, Romanish MT, et al. Retinoic-acid-receptor-re lated orphan nuclear receptor alpha is required for natural helper cell development and allergic inflammation. Immunity 2012;37(3):463–74. 11. Wong SH, Walker JA, Jolin HE, et al. Transcription factor RORalpha is critical for nuocyte development. Nat Immunol 2012;13(3):229–36. 12. Lim AI, Verrier T, Vosshenrich CA, et al. Developmental options and functional plasticity of innate lymphoid cells. Curr Opin Immunol 2017;44:61–8. 13. Mjosberg J, Spits H. Human innate lymphoid cells. J Allergy Clin Immunol 2016;138(5):1265–76. 14. Mjosberg JM, Trifari S, Crellin NK, et al. Human IL-25- and IL-33responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011;12(11):1055–62. 15. Mjosberg JM, Trifari S, Crellin NK, et al. Human IL-25- and IL-33responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol 2011;12(11):1055–62. 16. Doherty TA. At the bench: understanding group 2 innate lymphoid cells in disease. J Leukoc Biol 2015;97(3):455–67. 17. Doherty TA, Khorram N, Lund S, et al. Lung type 2 innate lymphoid cells express cysteinyl leukotriene receptor 1, which regulates TH2 cytokine production. J Allergy Clin Immunol 2013;132(1):205–13. 18. Mjosberg J, Bernink J, Golebski K, et al. The transcription factor GATA3 is essential for the function of human type 2 innate lymphoid cells. Immunity 2012;37(4):649–59. 19. Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol 2015;16(1):45–56. 20. Monticelli LA, Sonnenberg GF, Abt MC, et al. Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus. Nat Immunol 2011;12(11):1045–54. 21. Xue L, Salimi M, Panse I, et al. Prostaglandin D activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on T2 cells. J Allergy Clin Immunol 2014;133(4):1184–94. 22. Mitchell PD, O’Byrne PM. Epithelial-derived cytokines in asthma. Chest 2017;151(6):1338–44. 23. Drake LY, Kita H. IL-33: biological properties, functions, and roles in airway disease. Immunol Rev 2017;278(1):173–84. 24. Stier MT, Zhang J, Goleniewska K, et al. IL-33 promotes the egress of group 2 innate lymphoid cells from the bone marrow. J Exp Med 2018;215(1):263–81. 25. Liu S, Verma M, Michalec L, et al. Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: the role of thymic stromal lymphopoietin. J Allergy Clin Immunol 2018;141(1):257–68. 26. Barnig C, Cernadas M, Dutile S, et al. Lipoxin A4 regulates natural killer cell and type 2 innate lymphoid cell activation in asthma. Sci Transl Med 2013;5(174):174ra26. 27. Zhou W, Toki S, Zhang J, et al. Prostaglandin I2 signaling and inhibition of group 2 innate lymphoid cell responses. Am J Respir Crit Care Med 2016;193(1):31–42.

CHAPTER 12  Innate Lymphoid Cells 28. Laffont S, Blanquart E, Guery JC. Sex differences in asthma: a key role of androgen-signaling in group 2 innate lymphoid cells. Front Immunol 2017;8:1069. 29. Klose CSN, Mahlakoiv T, Moeller JB, et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 2017;549(7671):282–6. 30. Chang YJ, Kim HY, Albacker LA, et al. Innate lymphoid cells mediate influenza-induced airway hyper-reactivity independently of adaptive immunity. Nat Immunol 2011;12(7):631–8. 31. Halim TY. Group 2 innate lymphoid cells in disease. Int Immunol 2016;28(1):13–22. 32. Stier MT, Bloodworth MH, Toki S, et al. Respiratory syncytial virus infection activates IL-13-producing group 2 innate lymphoid cells through thymic stromal lymphopoietin. J Allergy Clin Immunol 2016;138(3):814–24.e11. 33. Yang Q, Ge MQ, Kokalari B, et al. Group 2 innate lymphoid cells mediate ozone-induced airway inflammation and hyperresponsiveness in mice. J Allergy Clin Immunol 2016;137(2):571–8. 34. De Grove KC, Provoost S, Hendriks RW, et al. Dysregulation of type 2 innate lymphoid cells and TH2 cells impairs pollutant-induced allergic airway responses. J Allergy Clin Immunol 2017;139(1):246–57.e4. 35. Kim HY, Lee HJ, Chang YJ, et al. Interleukin-17-producing innate lymphoid cells and the NLRP3 inflammasome facilitate obesity-associated airway hyperreactivity. Nat Med 2014;20(1):54–61. 36. Bartemes KR, Kephart GM, Fox SJ, et al. Enhanced innate type 2 immune response in peripheral blood from patients with asthma. J Allergy Clin Immunol 2014;134(3):671–8.e4. 37. Jia Y, Fang X, Zhu X, et al. IL-13(+) type 2 innate lymphoid cells correlate with asthma control status and treatment response. Am J Respir Cell Mol Biol 2016;55(5):675–83. 38. Liu T, Wu J, Zhao J, et al. Type 2 innate lymphoid cells: a novel biomarker of eosinophilic airway inflammation in patients with mild to moderate asthma. Respir Med 2015;109(11):1391–6. 39. Smith SG, Chen R, Kjarsgaard M, et al. Increased numbers of activated group 2 innate lymphoid cells in the airways of patients with severe asthma and persistent airway eosinophilia. J Allergy Clin Immunol 2016;137(1):75–86. 40. Christianson CA, Goplen NP, Zafar I, et al. Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33. J Allergy Clin Immunol 2015;136(1):59–68.e14. 41. Nagakumar P, Denney L, Fleming L, et al. Type 2 innate lymphoid cells in induced sputum from children with severe asthma. J Allergy Clin Immunol 2016;137(2):624–6.e6. 42. Chen R, Smith SG, Saslter B, et al. Allergen-induced increases in sputum levels of group 2 innate lymphoid cells in subjects with asthma. Am J Respir Crit Care Med 2017;196(6):700–12. 43. Cephus JY, Stier MT, Fuseini H, et al. Testosterone attenuates group 2 innate lymphoid cell-mediated airway inflammation. Cell Rep 2017;21(9):2487–99. 44. Sugita K, Steer CA, Martinez-Gonzalez I, et al. Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions through IL-13 in asthmatic patients. J Allergy Clin Immunol 2018;141(1):300–10. 45. Bal SM, Bernink JH, Nagasawa M, et al. IL-1beta, IL-4 and IL-12 control the fate of group 2 innate lymphoid cells in human airway inflammation in the lungs. Nat Immunol 2016;17(6):636–45.

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46. Poposki JA, Klingler AI, Tan BK, et al. Group 2 innate lymphoid cells are elevated and activated in chronic rhinosinusitis with nasal polyps. Immun Inflamm Dis 2017;5(3):233–43. 47. Walford HH, Lund SJ, Baum RE, et al. Increased ILC2s in the eosinophilic nasal polyp endotype are associated with corticosteroid responsiveness. Clin Immunol 2014;155(1):126–35. 48. Bruggen MC, Bauer WM, Reininger B, et al. In situ mapping of innate lymphoid cells in human skin: evidence for remarkable differences between normal and inflamed skin. J Invest Dermatol 2016;136(12):2396–405. 49. Kim BS, Siracusa MC, Saenz SA, et al. TSLP elicits IL-33-independent innate lymphoid cell responses to promote skin inflammation. Sci Transl Med 2013;5(170):170ra16. 50. Kim BS, Wang K, Siracusa MC, et al. Basophils promote innate lymphoid cell responses in inflamed skin. J Immunol 2014;193(7):3717–25. 51. Salimi M, Barlow JL, Saunders SP, et al. A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med 2013;210(13):2939–50. 52. Mashiko S, Mehta H, Bissonnette R, et al. Increased frequencies of basophils, type 2 innate lymphoid cells and Th2 cells in skin of patients with atopic dermatitis but not psoriasis. J Dermatol Sci 2017;88(2):167–74. 53. Oliphant CJ, Hwang YY, Walker JA, et al. MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion. Immunity 2014;41(2):283–95. 54. Hardman CS, Chen YL, Salimi M, et al. CD1a presentation of endogenous antigens by group 2 innate lymphoid cells. Sci Immunol 2017;2(18). 55. Doherty TA, Scott D, Walford HH, et al. Allergen challenge in allergic rhinitis rapidly induces increased peripheral blood type 2 innate lymphoid cells that express CD84. J Allergy Clin Immunol 2014;133(4):1203–5. 56. Dhariwal J, Cameron A, Trujillo-Torralbo MB, et al. Mucosal type 2 innate lymphoid cells are a key component of the allergic response to aeroallergens. Am J Respir Crit Care Med 2017;195(12):1586–96. 57. Fan D, Wang X, Wang M, et al. Allergen-dependent differences in ILC2s frequencies in patients with allergic rhinitis. Allergy Asthma Immunol Res 2016;8(3):216–22. 58. Lao-Araya M, Steveling E, Scadding GW, et al. Seasonal increases in peripheral innate lymphoid type 2 cells are inhibited by subcutaneous grass pollen immunotherapy. J Allergy Clin Immunol 2014;134(5):1193–5.e4. 59. Silver JS, Kearley J, Copenhaver AM, et al. Inflammatory triggers associated with exacerbations of COPD orchestrate plasticity of group 2 innate lymphoid cells in the lungs. Nat Immunol 2016;17(6):626–35. 60. Eastman JJ, Cavagnero KJ, Deconde AS, et al. Group 2 innate lymphoid cells are recruited to the nasal mucosa in patients with aspirin-exacerbated respiratory disease. J Allergy Clin Immunol 2017;140(1):101–8.e3. 61. Doherty TA, Baum R, Newbury RO, et al. Group 2 innate lymphocytes (ILC2) are enriched in active eosinophilic esophagitis. J Allergy Clin Immunol 2015;136(3):792–4.e3. 62. Kwon BI, Hong S, Shin K, et al. Innate type 2 immunity is associated with eosinophilic pleural effusion in primary spontaneous pneumothorax. Am J Respir Crit Care Med 2013;188(5):577–85.

CHAPTER 12  Innate Lymphoid Cells

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SELF-ASSESSMENT QUESTIONS 1. Which of the following is required for innate lymphoid cell (ILC) 2 development? a. Aryl hydrocarbon receptor (AHR) b. Inhibitor of DNA binding 2 (Id2) c. Eomesodermin (EOMES) d. Recombination-activating genes (RAG) 2. Which of the following is present on human innate lymphoid cell (ILC) 2s? a. CD3 b. CD20 c. CD45 d. CD56 3. Which prostaglandin functions in chemotaxis and cytokine production of innate lymphoid cell (ILC) 2s? a. PGD2 b. PGE2 c. PGI2 d. PGF2α

4. Which cytokine activates innate lymphoid cell (ILC) 2s? a. Interleukin (IL)-6 b. IL-12 c. IL-21 d. IL-33 5. For which cytokine is there evidence that it might induce steroid resistance in patients with asthma? a. Interleukin (IL)-25 b. IL-33 c. Transforming growth factor (TGF)-β d. Thymic stromal lymphopoietin (TSLP)

13  Antigen-Presenting Dendritic Cells Bart N. Lambrecht, Hamida Hammad

CONTENTS Introduction, 202 Dendritic Cell Terminology and Heterogeneity, 202 Antigen Uptake, 203 Antigen Presentation, 204 Integrated Function of Dendritic Cells in the Immune Response, 205

SUMMARY OF IMPORTANT CONCEPTS • Dendritic cells are the most important antigen-presenting cells that are responsible for allergic Th2 sensitization to inhaled allergens. • Allergens, viruses and bacteria are not recognized directly by T lymphocytes, but rather are digested into immunogenic peptides for presentation onto major histocompatibility (MHC) I and MHC II to CD8+ and CD4+ T cells, respectively. • Tolerance is the usual outcome of allergen exposure. Sensitization occurs when dendritic cells become activated by adjuvants like cigarette smoke, diesel exhaust particles, or enzymatically active allergens. • Dendritic cells determine the T cell polarization process into Th1 (producing mainly interferon [IFN]γ), Th2 (producing mainly interleukin [IL]-4, IL-5, and IL-13), Th17 (producing mainly IFN-γ and IL-17), and Treg (producing mainly IL-10 and/or transforming growth factor [TGF]β). • Dendritic cells are recruited into inflamed sites in allergic disease. They are crucial in mounting a Th2 effector response in already established disease. • Dendritic cells are ideal targets for developing new treatments against allergic disease, because they control so many aspects of the allergic cascade.

INTRODUCTION The prevalence of sensitization to allergens and allergic diseases has reached epidemic proportions in Western societies.1 Allergic sensitization is the presence of immunoglobulin E (IgE) antibody to common environmental and food allergens and is controlled by interleukin (IL)-4 producing T follicular helper (Tfh) cells that provide help for IgE synthesis by B cells.2,3 In addition, many of the inflammatory cell types found within sites of allergic inflammation, such as eosinophils and mast cells, depend on IL-4-, IL-5-, and IL-13-producing Th2 cells for their development and function.4 We also realize now that many of the features of asthma can also be controlled by type 2 innate lymphocytes (ILC2) that produce the same set of cytokines, but lack a specific antigenrecognition T cell receptor (TCR).4,5 Th2 cells will only react to allergen when it is presented in the context of major histocompatibility complex (MHC) molecules by professional antigen-presenting cells (APCs) such as dendritic cells (DCs), macrophages, and B cells. The ability to capture

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Role for Dendritic Cells in Allergic Sensitization in Humans, 208 Dendritic Cells in Allergic Asthma, 208 Concluding Remarks, 211

and process foreign antigens and to deliver the three signals for T cell activation (peptide MHC-TCR, costimulation, and polarizing cytokines) is the defining characteristic of professional APCs. Additionally, for induction of primary immune responses in a naïve host, the professional APCs should be able to migrate to the draining lymph nodes, to efficiently activate those few recirculating naïve T cell clones that express “useful” cognate antigen receptors.6,7 DCs are the most important APC found throughout the body and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to allergens. Increasingly, these cells are also recognized for their potential to maintain ongoing effector responses and therefore they might be crucial in maintaining allergic inflammation.8 B cells present allergen to T cells mainly in the context of immunoglobulin synthesis, for which they need TFH cell help.9,10 Macrophages are important regulators of tissue homeostasis but are also seen as scavenger cells that can control pathogen clearance and tissue remodeling. They derive from a variety of circulating mononuclear precursors or in steady state often from self-replenishing embryonic progenitors.11–16

DENDRITIC CELL TERMINOLOGY AND HETEROGENEITY Dendritic Cell Subsets in the Mouse in Steady State DCs were originally described by their capacity to efficiently process and present antigens and to prime naïve T cells. In peripheral tissues, they make up a network of highly DCs, hence their name (Fig. 13.1 for lung DCs). Initially considered a single population of cells, it is now clear that there are many subsets of DCs, in most species studied. The most simple division is in conventional or classical DCs (cDCs) and plasmacytoid (p)DCs (for detailed discussion on mouse lung DCs, see references 8 and 17, see Fig. 13.2 for a detailed schematic on mouse DC subsets). There are two major subsets of cDCs that all express MHC II and CD11c; cDC1 additionally expresses the transcription factors interferon regulatory factor (IRF) 8 and basic leucine zipper ATF-like transcription factor (Batf) and can be best detected by expression of XCR1, CADM1, and DNGR1; cDC2 expresses the transcription factor IRF4 and ZEB2 and can be best distinguished by staining for CD172 (signal regulatory protein [SIRP] α) or CD11b.18–20 The conducting airways of the lung contain

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Some cDC2 can express langerin.31 These populations correspond to equivalent populations in the mouse.20,23,32,33 In lung tissue as well as lung draining lymph nodes, obtained through mechanical or enzymatic dispersion of resected lung specimens, the same cDC subsets are present, along with up to four subpopulations of mononuclear phagocytes (MP), which are also CD11c+ and HLA-DR+. Compared with mice, there are considerably less CD141+ cDC1 and CD303+ pDC in human steady state lung, whereas a fair amount of CD14+ MPs express CD141.12,34,35 In the skin, an elaborate network of DCs, including LCs is now precisely defined functionally and molecularly.20,28,36

Origin and Turnover of Steady State and Inflammatory Lung Dendritic Cells

Fig. 13.1  Major histocompatibility class II–positive mucosal dendritic cell network visualized by immunohistochemical staining (brown) on a murine tracheal wholemount. The trachea was taken from a naïve unimmunized mouse.

Human DCs originate in the bone marrow from a CD34+ precursor and circulate in the human bloodstream as a CD123+CD33+CD45RA+ preDC before entering peripheral tissues.37 Both in mice and humans, some of these preDC precursors are already committed to become cDC1 (pre-cDC1) or cDC2 (pre-cDC2), and some become pDC.37–39 There has been great progress in understanding the transcriptional control over development of DCs over the last years, and these concepts have led to a better understanding of the development of lung DCs, particularly derived from studies in the mouse.40 In broad terms, there seems to be a dedicated progenitor expressing the hematopoietic cytokine receptor Flt3 and giving rise to macrophages and all subsets of cDCs and pDCs.29 In addition, classical monocytes can also give rise to cells strongly resembling DCs under conditions of inflammation, a system modelled in vitro by growing monocytes in GM-CSF and IL-4.41,42

ANTIGEN UPTAKE all subsets of DCs in the epithelial layer (see Fig. 13.1 for mouse lung epithelial DCs) as well as the lamina propria. Similarly, the lung interstitium contains all DC subsets.21 Some DCs also patrol the vessel wall of the pulmonary arterial vasculature and can capture injected embolic material.22 When the lung is challenged with any foreign substance that has the potential to trigger an inflammatory event, there is recruitment to the conducting airways and lung parenchyma of additional monocytederived cells (MC), which very much resemble a subset of the steady state cDCs and are therefore easily confused with them. Although these cells are often referred to as moDCs, they resemble macrophages in many ways and can best be identified using markers like CD64, MER protooncogene tyrosine kinase (MerTK), and pro­resolving lipid mediator maresin (MAR) 1 in the mouse.20,23,24 In the lung interstitium around mouse bronchi, there are also many other subsets of MerTK+ interstitial macrophages that can be easily confused with DCs.25 In the skin, a similar network of cDCs is found, moDCs can be recruited, and additionally Langerhans cells are found.20,26–28

Human Dendritic Cell Subsets Paul Langerhans first described DCs (first known as Langerhans cells [LCs]) in the skin of humans in the late 19th century but originally thought these cells to belong to the nervous system. We know now that LCs are probably much more related to macrophages and derive from an early embryonic progenitor that self-replenishes in the skin.24 Human DCs were first described in 1975 and found to reside in the T cell area of lymph nodes and spleen as so-called interdigitating DCs. Since these initial studies it is clear that also in humans, many subsets of DCs can be found.20,29,30 More detailed identification and phenotyping of human pulmonary DCs was performed since the advent of blood DC markers became widely available. In BAL, three types of CD11c+ DC populations were detected. These include cDC type 1 (CD141+, XCR1+, CADM1+), cDC2 (CD1c+, CD172+), and plasmacytoid DCs (CD123+, CD303+).

There are various ways by which an APC can acquire foreign antigen.43 A first mechanism is via receptor-mediated endocytosis involving clathrincoated pits. Immature DCs express a plethora of specialized cell receptors for patterns associated with foreign antigens, such as the C-type lectin receptors (langerin, DC-SIGN, dectin, BDCA-2, macrophage mannose receptor, and the unique carbohydrate receptor DEC-205). Lectin-receptor mediated uptake by DCs results in an approximately 100-fold more efficient antigen presentation to T cells, compared with antigens internalized via fluid phase. Pollen starch granules were shown to bind to C-type lectin receptors on alveolar macrophages (AMs) and DCs, although internalization occurred only in macrophages.44 DC-SIGN appears to be a major receptor for house dust mite (HDM) allergens.45 The major cat allergen Fel d 1 is captured by DCs through the mannose receptor.46 In allergic individuals, DCs are furthermore loaded with allergen-specific IgE binding to the high-affinity IgE receptor (FcεRI), thus leading to efficient receptor mediated endocytosis of the allergen, a mechanism that also occurs in vivo.47–49 A second mechanism of antigen uptake is constitutive macropinocytosis that involves the actin skeleton-driven engulfment of large amounts of fluid and solutes (approximately one cell volume per hour) by the ruffling membrane of the DC followed by concentration of soluble antigen in the endocytic compartment. Macropinocytosis seems to be a dominant mechanism involved in the uptake of recombinant Bet v 1 and Phl p 1 pollen allergens by LCs and of Der p 1 by cultured DCs and can be inhibited by cytochalasin D and amiloride.50 Third, immature LCs, cultured DCs, plasmacytoid DCs, and macrophages have been shown to phagocytose particulate antigens such as latex beads and even whole bacteria, as well as apoptotic cells, and this could be the dominant mechanism of uptake of particulate allergens.51 Increasingly, it is shown that differential uptake of allergens or allergoids (chemically modified allergens with hypoallergenic properties) might affect the functions of DCs, and this could be achieved by conjugating

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Steady state

Inflammation

cDC1 CD11chi CD11b– XCR1+ CD103+

Inflammatory moDC CD11b+ CD11c+ Ly6C+ SIRP1α+ CD64+ MerTK+ MAR1+

cDC2 CD11chi CD11b+ SIRP-1α pDCs CD11cdim CD11b– Siglec H+ PDCA1+

Allergens

Alveolar DC CD11b+ CD103+ Non-autofluorescent

Alv. Mac CD11b– F4/80+ Siglec F+ CD2+ CD64 Autofluorescent Fig. 13.2  Different subsets of mouse lung dendritic cells (DCs). In steady-state conditions (left), CD11chi conventional DCs (subdivided into cDC1 and cDC2 subsets) line the conducting airways. They can also be found back in the deeper interstitial compartments and obtained by enzymatic digestion of peripheral lung. CD11cdim plasmacytoid DCs (pDCs) are also found in both compartments with a slight preference for the interstitial compartment, and they express cell surface markers SIGLEC-H and PDCA1 (plasmacytoid DC antigen -1). Finally, the alveolar space contains DCs that can be easily confused with CD11chi alveolar macrophages if one does not take autofluorescence of the latter into account. Specific markers that can be used to discriminate alveolar macrophages from DCs are SiglecF and CD2. Under inflammatory conditions, there is recruitment of CD11b+ monocytes to the lungs, and these rapidly become DCs. They can still express Ly6C as part of their monocytic lineage, but this is so for only a limited amount of time. These moDCs are known to express the macrophage markers CD64 and MerTK, and they crossreact with the antibody Mar1 directed against the high-affinity FcεRI receptor.

allergoids to nonoxidized mannan.52,53 The extracellular antigens that are taken up by any of these mechanisms accumulate in the endocytic compartment, where they are loaded on newly synthesized and recycling MHC class II molecules but may also be transported into the cytosol, where they become accessible to the class I antigen presentation pathway, a process called cross-presentation (see later).

ANTIGEN PRESENTATION Presentation of Exogenous Antigens on Major Histocompatibility Class II to CD4+ T Cells Allergens are extracellular antigens, and like most extracellular antigens they are processed for presentation onto MHC class II molecules. The T cell receptor of CD4+ T lymphocytes will respond only to processed antigen in the context of MHC class II, a process called MHC restriction. In contrast to MHC class I, which is expressed on all nucleated cells types, MHC class II is mainly expressed by professional APCs, but also to a lesser extent by epithelial cells, mast cells, and possibly eosinophils. Within the endocytic compartment, antigen is cleaved into short immunogenic peptides by proteolytic enzymes of the cathepsin family (for review see reference 54). Antigen loading on MHC class II molecules occurs in an acidic cellular compartment rich in newly synthesized

MHC class II molecules, called the MIIC compartment. This multivesicular complex is located at the intersection of the biosynthetic (ER, Golgi complex, secretory granules) and endocytic pathway of vesicle transport within the cell and contains the MHC II–related HLA-DM peptide exchanger that is essential for loading high-affinity antigenic peptides on MHC II. Alternatively, there is a pathway of peptide loading onto preformed MHC II molecules that have been internalized into mildly acidic endosomal vesicles after being expressed on the cell surface. At present, it is unclear how allergens are loaded onto MHC class II molecules by DCs. In sensitized individuals, internalization of allergens via receptor-mediated endocytosis by multivalent crosslinking FcεRI on immature DCs targets the antigen to the MIIC compartment.55 In contrast, the generation of peptide–MHC complexes derived from macropinocytosis of Bet v 1 and Phl p 1 pollen allergens was only partly inhibited when the pH of the endosomes was altered, suggesting that part of the molecules were not metabolized in the lysosomal MIIC compartment.

Antigen Presentation on Major Histocompatibility Class I to CD8+ T Cells After a virus enters a host and infects cells, the major adaptive immune response that clears the infection is mediated by CD8+ cytotoxic T

CHAPTER 13  Antigen-Presenting Dendritic Cells lymphocytes (CTLs). These cells also provide the major defense against cancers. CD8+ lymphocytes recognize infected cells that display on their surface MHC class I molecules presenting antigenic peptides derived from viral proteins or tumor antigens expressed in the cytoplasm. All nucleated cells have the capacity to present peptides derived from the cytoplasm onto MHC class I molecules. A second, less well-defined, approach to load peptides on MHC class I molecules is for the CD141+ XCR1+ cDC1 DCs to capture extracellular antigens and to process these captured exogenous antigens into the MHC class I pathway, a process called cross-presentation (for review see reference 56).

INTEGRATED FUNCTION OF DENDRITIC CELLS IN THE IMMUNE RESPONSE Dendritic Cell Activation Like the gut, the lung and skin are constantly and highly exposed to the external environment, and therefore they must cope with a number of challenges such as allergens, particles, toxic gases, or invading microorganisms, and lung DCs play an important role in tuning the optimal immune response to these challenges. Like other cells of the innate immune system, DCs have evolved to recognize conserved pathogenassociated molecular patterns (PAMPs) contained in these antigens. PAMPs are recognized by cells through specific pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I like receptors (RLRs), and C-type lectin receptors (CLRs) (Fig. 13.3). DCs reside in an immature state in the periphery of the lung, where they are located strategically to detect inhaled particulate and soluble antigen (Fig. 13.4). Within the DC population, cDCs and pDCs differ in their TLR expression pattern.57 The expression profile of TLRs on DCs seems however to be organ-specific. In human lung, cDC type 1 and BDCA3+ cDC type 2 express mRNA transcripts for TLR1, TLR2, TLR3, TLR4, TLR6, and TLR8. In response to TLR2 and TLR4 ligands, cDC type 1 and cDC type 2 release proinflammatory

cytokines (TNF-α, IL-1β, IL-6, and IL-8). Human lung pDCs express TLR7 and TLR9 and release proinflammatory cytokines in response to the TLR7 ligand imiquimod, and IFN-α in response to TLR9 ligand CpG oligonucleotides.58 Although direct recognition of foreign PAMPs by PRRs is the most likely explanation of how DCs respond to foreign antigen, it is now clear that recognition of PAMPs by nearby epithelial cells is at least as important in activating the lung DC network (Fig. 13.4) (for review see references 59 and 60). This conclusion was reached by studying the in vivo response of lung DCs to the toll-like receptor (TLR)-4 agonist endotoxin (lipopolysaccharide [LPS]), in bone marrow chimeric mice that lacked TLR4 exclusively on either radiosensitive hematopoietic cells or radioresistant epithelial cells. In the absence of TLR ligation, lung DCs demonstrated a sessile behavior. Provision of LPS led to a dramatic increase in motility and antigen sampling behavior that led to crawling of DCs in between basal epithelial cells. Strikingly, instruction for this pattern of motility required TLR4 triggering of epithelial cells and not on DCs directly.61 Lung epithelial cells also produce the essential chemokines that attract immature cDCs and inflammatory monocytes to the site of antigen exposure. For lung DC recruitment to inflammatory stimuli, several chemokines and cytokines have been implicated (Fig. 13.4). The chemokine CCL20 and epithelial β-defensin are ligands for CCR6 expressed by immature (lung) DCs, and bronchial epithelial cells produce these factors in response to TLR ligation, C-type lectin triggering, allergen inhalation, virus infection, and exposure to environmental pollutants.62

Dendritic Cell Migration to the Draining Lymph Nodes Intratracheal administration of fluorescently labeled large MW antigens leads to uptake in different subsets of mediastinal LN DCs, as early as 12 hours later (for review see reference 63). Several mechanisms explain how inhaled antigen could reach the mediastinal LN. Most of the antigen transport is DC-mediated, because it can be strongly reduced by toxinmediated depletion of lung DCs and occurs in a CC-chemokine receptor

Recognition of microbial motifs (PAMPs)

Recognition of inflammation and tissue damage (DAMPs)

Toll like receptors 1–10

Protease activated receptors (PAR2) Complement receptors (C1aR, C5aR) Prostanoid receptors DP1, EP4, IP Neuropeptide receptors NK1, CGRPR Purinergic receptors P2X, P2Y Receptors for Uric acid HMGB1 Heat shock proteins

Intracellular receptors NLR (NOD1/2, NLRP3) TLR 3, 7, 9 PKB C-type lectin receptors Dectins Langerin Macrophage mannose receptor DEC205 BDCA-2 Thrombomodulin (BDCA-3)

205

Fig. 13.3  Expression of “danger” receptors by dendritic cells. Dendritic cells express the ancient receptors of the innate immune system also expressed by macrophages, such as the toll-like receptors (TLRs) and C-type lectin receptors. These receptors react to foreign pathogen-associated molecular patterns (PAMPs). In addition, DCs express numerous receptors for inflammatory mediators and necrotic cell debris, the socalled damage-associated molecular patterns (DAMPs). The exact receptors for uric acid, high-mobility group box 1 (HMGB1) protein, and heat shock proteins are not yet known. Nod-like receptors (NLRs) are specialized in recognizing endosomal or cytoplasmic motifs of pathogens. Protein kinase B (PKB) responds to virus infection. Receptors for prostanoids include the prostaglandin D2 type 1 receptor (DP1) and the prostaglandin E2 type 4 receptor (EP4) as well as the PGI2 receptor IP. Receptors for neurokinins include the neurokinin-1 receptor (NK1) for substance P, and the calcitonin gene–related protein receptor (CGRPR). Purinergic receptors are for ATP and metabolites and consist of a P2X and P2Y family, each made up of several members. High-mobility group box 1 is a danger signal released from damaged cells.

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Primary response

Direct DC activation

TLR, CLR, PAR ROS production Indirect DC activation CD80, CD86, OX40L CD11b+ DCs

TSLP, GM-CSF, IL-33, IL-25, IL-1 ATP, uric acid

CCL2 CCL20 β-defensins

Th2

IL-13 cDC2 migration CCR7-dependent Recruitment of monocytic precursors

Secondary response

Activation of innate immune cells

ILC2

Th2

CCL17 CCL22

Th2

Basophils CD4 OX40L, Jagged1, IL-6, IL-23, LTC4 Lack of IL-12

PGD2 Histamine

Th2

Mediastinal lymph node

IgE-mediated allergen presentation

Th2

Mast cell

Recruitment and reactivation of Th2 cells

IgE synthesis Th2

Th2/Th17 polarization Fig. 13.4  Induction of Th2 immunity by lung dendritic cells (DCs). Both lung DCs and epithelial cells express pattern recognition receptors (PRRs) and can be activated directly by allergens. In response to allergens, lung epithelial cells produce chemokines that attract immature conventional (c)DCs and inflammatory monocytes (CCL2, CCL20). Activated epithelial cells produce instructing cytokines (e.g., interleukin 1 [IL-1], granulocyte macrophage colony–stimulating factor [GM-CSF], and thymic stromal lymphopoietin [TSLP]) and danger signals (ATP, uric acid) that favor DC maturation. Activated lung DCs then migrate to the draining mediastinal lymph nodes. At the same time, DC maturation will be fully induced. When mature DCs arrive in the mediastinal lymph nodes (MLNs), they select specific T cells from the polyclonal repertoire of cells that migrate through the high endothelial venules and T cell area. Within 4 days, this will lead to clonal expansion of antigenspecific T cells. T cell polarization then ensues, and, in response to allergen presentation, a mixed Th2 and Th17 response is induced. DCs receive help from basophils to sustain Th2 responses. (Right) DCs also play a predominant role during the Th2 effector phase of asthma, when the lung is repeatedly exposed to allergens. During allergen challenge, DCs could locally restimulate effector function in lung-resident lymphocytes or they could recruit effector Th2 cells through CCL17 and CCL22 production. IgE-mediated allergen recognition enhances Th2 responses to inhaled allergens.

7 (CCR7)- and CCR8-dependent manner. Also in neonatal mice, where the lung epithelium is much more leaky, the transport of inhaled antigen to the draining nodes is CCR7 dependent and mediated by DCs.64 Lipid mediators like prostaglandins, leukotrienes, and sphingosine lipids have been implicated in controlling the emigration of DCs from the lung (for review see reference 65).

Dendritic Cells Control T Effector Responses When Properly Triggered DCs are crucial in regulating the immune response by bridging innate and adaptive immunity. Signals from the type of antigen and the response of the innate immune system to it are translated by DCs into a signal that can be read by the cells of the adaptive immune response leading to an optimal response for a particular insult. Lung DCs that have seen inhaled antigen migrate to the T cell paracortex of the draining mediastinal nodes. When antigen is being recognized, there is formation of

a long-term immunologic synapse, leading to maximal T cell activation. After a few hours, the T cell detaches, divides, and differentiates into an effector and possibly memory T cell. In response to pathogen inhalation, or in the case of concomitant exposure to inhaled environmental adjuvants like cigarette smoke, fine particulate matter, or bacterial contaminants, the outcome of DC-driven antigen presentation in the lung is dramatically altered. Under these conditions, lung DCs get proper maturation stimuli and release cytokines and express costimulatory molecules that determine the outcome of the adaptive cellular immune response (Figs. 13.4 and 13.5). Many groups have shown that lung DCs are biased to promote Th2 responses, most likely via production of IL-6 in the absence of Th1-prone IL-12p70 production, or through production of leukotriene C4 (LTC4), but it is not yet clear whether this Th2 proneness is linked to a particular cDC subset. Recently, however, different markers like c-KIT or FcεRI identified a Th2-prone subset of lung DCs.66,67 Age also determines the Th2 potential of cDC2. In the

CHAPTER 13  Antigen-Presenting Dendritic Cells

Dendritic cell surface ligand

Secreted mediator

OX40L CD86 Jagged

ICOSL dim CD86 dim CD80 Jagged?

CD80 Delta ICAM-1

Th0

207

Cytokines produced by T cell types

High IL-6 Low IL-2 T1/ST2L/IL-33 CCL2?

Th2

IL-4 IL-5 IL-13 TNF-α

Low IL-6 Low IL-12 TGF-β PGD2

Treg

IL-10 TGF-β

High IL-6 High TGF-β

Th17

IL-17

High IL-6 High IL-12 High IL-13 CCL3 High IFN-γ

Th1

IFN-γ TNF-α

Fig. 13.5  T helper cell polarization by dendritic cells (DCs). Depending on the type of antigen, the dose, the genetic background, and the tissue environment where antigen is first introduced, DCs can induce various types of Th responses (Th2, Th1, Th17, and Treg), tailor-made to protect the host, while avoiding autoimmunity. Often the response is extremely well balanced to avoid tissue damage while allowing clearance of the threat. The various cytokines and costimulatory molecules that favor a particular direction are indicated.

neonatal period, mouse cDC2s express OX40L and produce little polarizing IL-12, which may help explain the bias toward type 2 immunity in this early period of life.64 In models of HDM-driven asthma and immunity to the helminth H. polygyrus, generation of TFH responses in the lung-draining lymph nodes depend on IRF4-expressing cDC2 that also express CXCR5 and migrate to the T-B cell border of lymph nodes.68,69 In models of N. brasiliensis helminth infection and immunization with the Th2 adjuvant alum, IL-4+ TFH development was also shown to depend on Notch ligand expression by conventional DCs and T cell intrinsic Notch signalling, whereas Th2 development did not.2 A key question in the field is whether subsets of cDC2s exist that would preferentially induce TFH over effector Th2 immune responses. Through production of IL-23, IL-6, and IL-1β, lung DCs can also induce Th17 responses, and this pathway seems to be regulated by the complement system.70 Recent data also suggest that the potential of cDC2s to induce pure Th2 immunity or a mixed Th2/Th17 type of response is tightly regulated by cell intrinsic mechanisms, including fine tuning of TLR signalling pathways and metabolic programming.71,72 Understanding induction of such mixed Th2/Th17 responses is important, because this profile is often seen in steroid-resistant difficult-totreat allergic diseases. In our view, any lung DC can induce CTL responses, and Th1, Th2, or Th17 Th effector responses depending on the type and strength of inciting stimulus, with only minor bias related to lineage or surface markers. Others have proposed that cDC1s are particularly prone to inducing CD8 T cell immunity to apoptotic self-antigens in a process of cross-presentation.57,73

Dendritic Cells Control Inhalational Tolerance DCs can also transport antigen without inducing T effector responses. Inhalation of harmless antigen devoid of strong activating TLR ligands

induces T cell unresponsiveness via a mechanism involving abortive T cell proliferation, deletional tolerance, and/or differentiation of regulatory T cells (Tregs), a process also influenced by age and presence of a proper microbiome.1,74–76 The induction of respiratory tolerance is also related to the basic mechanisms of the hygiene hypothesis, explaining how allergy risk is increasing in an environment devoid of microbial instruction (for review see reference 1). The precise subset of DCs presenting inhaled antigen in a tolerogenic manner is currently unknown. The IRF8-expressing cDC1 as well as plasmacytoid DCs seem to control tolerance to allergens and actively suppress Th2 responses, sometimes via induction of Tregs.77–82 Some groups indeed showed that tolerance induction and formation of a Treg response depends on antigen presentation by pDCs.83,84 Removal of lung pDCs was able to turn inhalation of a harmless protein antigen from a tolerogenic to a fully immunogenic event, with ensuing development of respiratory Th2 immunity.78,85 Others found that tolerance required cDCs that migrated to the mediastinal LNs in a CCR7-dependent way, and IRF-8 dependent cDC1 might be the cell type doing this.86 When antigens are administered chronically to mice or rats, a specific form of inhalation tolerance ensues, and animals no longer mount inflammatory responses to inhaled antigen, whereas they initially did. This form of tolerance is explained by the fact that there is reduced antigen uptake by lung DCs, or induction of long-lived Tregs.87

Dendritic Cells Control Aspects of Humoral Immunity There is accumulating evidence that lung DCs might also induce humoral immunity by directly communicating with B cells or by providing crucial factors involved in B cell immunoglobulin class switching or promoting the development of IL-4 producing TFH cells, and this is mainly a function of migratory cDC2 that might express NOTCH receptor ligands.2,68 Lung DCs can promote the production of IgA in a process dependent

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on TGF-β.88,89 Intratracheal injection of the mucosal adjuvant cholera toxin B subunit also induces DC-dependent IgA class switching.90 In contrast to gut epithelial DCs, a recent study on lung DCs identified a role for both RALDH1 and RALDH2 (enzymes that promote retinoic acid production) in IgA class switching. Both subsets of lung cDCs had equal levels of RALDH activity.91 The potential of lung DCs to induce IgA responses is also related to microbial instruction of these cells.89 Dendritic cells inside tertiary lymphoid follicles that are formed at sites of chronic lung inflammation were also shown to maintain local (antiviral) IgA synthesis and contribute to the maintenance of the organized lymphoid structures, most likely through secretion of chemokines that attract and retain naïve T and B cells (for review see references 92 and 93).

ROLE FOR DENDRITIC CELLS IN ALLERGIC SENSITIZATION IN HUMANS Although it has not been proven directly in humans that DCs are responsible for the Th2 sensitization process, some in vitro findings strongly imply these cells. The way in which allergens are handled by DCs is fundamentally different between atopic and nonatopic individuals.52,94 When DCs obtained from HDM-sensitive asthmatic subjects were exposed to the endotoxin-free major allergen component Der p1 in vitro, they mainly produced IL-10, but little IL-12. They expressed the costimulatory molecules CD86 and PDL1.95 When monocyte-derived DCs from non-HDM-allergic donors or nonallergic donors were exposed to Der p1, they produced mainly IL-12, expressed CD80, and generated the TH1 cell–specific chemokine CXCL10. Not surprisingly, monocytederived DCs from allergic patients induced TH2 cell responses of naive alloreactive T cells in vitro, whereas those DCs from nonallergic individuals induced Th1 responses. Therefore the way HDM is handled by DCs is crucial to the generation of Th2-cell sensitization and is clearly different in patients with allergy to HDM. The cysteine protease activity of Der p1 induced these changes in the DCs of allergic individuals, indicating that the activation of a protease-activated receptor on DCs leads to aberrant cellular activation in patients with allergies.95 However, allergens without enzymatic activity can also directly activate DCs to induce Th2 priming. For instance, phytoprostane lipids contained in pollen allergens can induce DC maturation and inhibit IL-12 production by LPS-activated DCs. When cocultured with allogeneic naïve T cells, pollen-treated DCs polarized the immune response toward Th2.96

DENDRITIC CELLS IN ALLERGIC ASTHMA Dendritic Cells Are Prime Inducers of Th2 Immunity in the Lung Lung DCs or GM-CSF–cultured bone marrow–derived DCs injected into the lung97 are able to induce the typical Th2 response to inhaled allergen. In particular, IRF4-expressing CD11b+ CD172+ type 2 conventional DCs (cDC2s) are necessary and sufficient to drive Th2 polarization in the skin, gut, and lung.23,26,67,68,98–103 Type 2 immunity induced by cDC2s is heavily instructed by barrier epithelial cells. By their release of Th2 instructive cytokines including TSLP, GM-CSF, IL-33, IL-1, and IL-25, and via induction of a type 1 interferon response, the barrier epithelial cells actively determine whether DCs respond or tolerate antigenic challenges and what type of immune response is induced.27,59,64,102,104 This paradigm that DCs can induce Th2 immunity was however challenged by the proposition that basophils, and not DCs, were the inducers of Th2 immunity by acting as APCs and an early source of Th2 polarizing cytokine IL-4, based on studies using the model allergen papain, and helminth infection.105 It was however shown that the conclusions of these studies could be questioned because the depleting antibodies used also targeted conventional lung DCs.67 Recently, however, the idea of

the basophil as an APC has been reignited. Although it was shown that basophils in mice and humans express little MHC II on their surface,67 bone marrow–cultured murine basophils generated in vitro using IL-3 and GM-CSF, have been reported to substantially upregulate MHC II on their surface, although these cells still showed little transcription of the corresponding MHC II gene, and these cultures also lead to expansion of MHC II–positive DCs.106 More in-depth cell biologic analysis using coculture experiments of wild type DCs and Mhc2-deficient basophils revealed transfer of MHC II molecules from DCs to basophils in a contact-dependent manner.106 In vivo basophils were also found to display low levels of MHC II on their cell surface, possibly acquired from DCs through trogocytosis—a process whereby lymphocytes (B, T, and NK cells) conjugated to antigen-presenting cells extract surface molecules from these cells and express them on their own surface.107,108 Even in the absence of antigen processing, the cell surface display of peptide-loaded MHC II in these basophils was able to stimulate T cells.108 Together, these data provide evidence that although basophils may not be directly involved in primary antigen presentation, they can modulate type 2 immune responses in a variety of ways, although the mechanisms by which this happens and the different outcomes need further in-depth study.

Induction of Th2 Immunity to Allergens Depends on PRR Signalling Epidemiologic studies as well as animal experimental work has shown that many environmental and genetic factors control the process of allergic sensitization. Common known risk factors are the level of exposure to inhaled allergens (like HDM, cat dander, pollen, cockroach allergen, and fungal spores), concurrent or prior viral or bacterial infections, and concurrent exposure to environmental air pollution (like diesel or other fine dust particles, cigarette smoke, ozone, and other noxious gases). Together with allergen exposure, these factors have to integrate into a signal that can be interpreted by the cells of the adaptive immune system, and accumulating evidence suggests that this occurs through close communication between airway epithelial cells and DCs (reviewed in references 4 and 59). It has become clear that PRRs expressed by both epithelial cells and DCs not only mediate innate responses to microbes, but that they can also play a dominant role in innate responses to noninfectious environmental triggers. Most allergens are known to contain motifs capable of activating different classes of PRRs, either directly or via release of endogenous mediators like fibrinogen cleavage products or complement.43,109 Some allergens such as cockroach or HDM extracts require TLR2 and TLR4 signalling, respectively, to induce allergic asthma in mice.61,110

Molecular Crosstalk Between Epithelial Cells and Lung Dendritic Cells Leading to Th2 Immunity In the recent years, there has been more focus on trying to dissect the molecular mechanisms underlying allergenicity and Th2 sensitization. Certainly, the triggering of TLRs on DCs by allergens and environmental triggers seems to be essential. However, one major caveat is that most of the studies performed have used BMDCs in mice or monocytederived DCs in humans to mimic lung DC responses. The direct effect of allergens on purified lung DCs remains unclear. Although there is evidence that allergens administered in the airways of mice or humans can recruit and induce lung DC maturation, the activation of DCs by allergens is not necessarily direct. An emerging theme is that the stromal cells of the airways, consisting mainly of epithelial cells, endothelial cells, and fibroblasts, can also react to allergens and strongly influence the behavior of DCs. In experiments using bone marrow chimeric mice without TLR4 on either immune cells or radioresistant epithelial cells, several groups have shown that the response to allergens is substantially

CHAPTER 13  Antigen-Presenting Dendritic Cells altered when epithelial cells cannot detect the endotoxin in the allergen. From these studies, a model emerged (Fig. 13.4) in which bronchial epithelial cells first attract immature DCs or their precursors through the release of CCL2 or CCL20 chemokines and subsequently instruct them to induce Th2 immunity to allergens via the release of innate pro-Th2 cytokines such as IL-1, GM-CSF, TSLP, and IL-25 and IL-33.61,109,111–114 All of these innate cytokines have pleiotropic effects, yet all share the propensity to drive DCs into a Th2-activating mode, via suppression of IL-12 and/or upregulation of Ox40L.115 Particularly in the neonatal period and in early life, the epithelium is poised to produce IL-33 and in this way promote type 2 immunity to allergens via effects on DCs.64,116 Although contamination of allergens with motifs that stimulate PRRs provides a good explanation for how immunity can be induced to harmless allergens, another could be that allergens trigger release of damage-associated molecular patterns (DAMPs) that occur intracellularly in homeostasis but are released extracellularly upon physical or metabolic stress. We have previously reported that a single administration of HDM allergen in the airways of mice induced the release of both ATP and uric acid (UA).117 Others have shown that protease allergens like papain and fungal proteases also trigger release of UA.118 HDM-induced UA promotes Th2 cell sensitization by amplifying the production of epithelial-derived innate pro-Th2 cytokines that instruct DCs like TSLP, GM-CSF, and IL-25, and by activating lung DCs.117,118 We found airway epithelial cells to be a source of UA, and the release of UA was TLR4-dependent. Others have also found that type 2 immunity to inhaled antigens promoted by particulate matter airway pollution was associated with mucosal production of UA.119 The effects of UA in promoting Th2 immune responses did not require activation of the NLRP3 inflammasome or IL-1R axis, yet required signalling through spleen tyrosine kinase (Syk) and the PI3 kinase delta isoform in DCs. Uric acid–driven Th2 immunity resembled that induced by the prototypical adjuvant alum, which has been widely used to induce asthmatic airway inflammation in mice in a pathway requiring UA and production of PGE2 in an NLRP3-independent manner.120,121 It is also possible that some type 2 triggers like rhinovirus infection or RS virus infection trigger the release of extracellular DNA through a process called NETosis (formation of neutrophil extracellular DNA traps).122 Epithelial and neutrophil cell death is closely regulated and lung epithelial cells capturing dying neighboring cells regulate epithelial production of IL-33.123 Another trigger for DC activation could be the release and catabolism of low molecular weight extracellular matrix polysaccharide, hyaluronan.124

Collaboration Between Innate Immune Cells and Dendritic Cells Promote Type 2 Immunity Allergens often trigger an innate immune response made up of innate lymphoid cells type 2 (ILC2), eosinophils, and basophils. These cells accumulate in the lungs at a time when DCs also get activated by allergens, and it has been proposed that these innate immune cells can extensively collaborate with DCs to promote type 2 adaptive immunity. Indeed, in some mouse models, DCs alone were unable to skew T cells to a Th2 phenotype ex vivo and required additional help of basophils.125,126 In a model of spontaneous development of type 2 immunity by skinspecific overexpression of TSLP in mice, dermal DCs induced IL-3 production by recently activated unpolarized CD+ T cells in an OX40Ldependent manner, which led to the recruitment of IL-4 producing basophils and the subsequent polarization of Th2 T cells, suggesting that the collaboration between basophils and DCs requires a responding cognate T cell, but not necessarily pin-pointing the basophil as the presenting cell type.127 Recently, genetic models to deplete basophils have become available, and these allowed for a more definitive

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assessment whether basophils are really required as APCs to generate adaptive Th2 immunity. Th2 cell polarization was found to be unaffected in genetically basophil-depleted mast cell protease 8 (Mcpt8)Cre conditional knockout mice during primary infection with different helminths.128,129 Sensitization and challenge of Mcpt8Cre mice with ovalbumin (OVA)-alum and papain-OVA also showed normal expansion of Th2 responses, whereas genetically DC-depleted animals showed severely impaired responses.128,130 Genetic depletion of basophils using Basoph8 x Rosa-DTA mice followed by footpad immunization with the parasite Schistosoma mansoni also resulted in normal Th2 priming.131 Not only do these experiments question the role of the basophil as an APC for type 2 immunity, they even cast some doubts on the dogma that the basophil is an important bystander provider of the crucial polarizing IL-4 cytokine during initiation of type 2 immunity.130,132 Just like basophils cooperate with DCs during the antigen presentation process, MCs and DCs join forces in type 2 immunity by forming immunologic synapses with each other. These dynamic interactions facilitate transfer of endosomal contents, including the transfer of antigen and membrane of activated MCs to DCs, resulting in the processing and presentation of transferred antigens and ultimately the activation of T cell responses.133,134 MCs certainly differ from professional APCs in being able to use different mast cell granule proteolytic pathways to process antigen, and it would be favorable for DCs to exploit these additional pathways to generate peptide ligands for MHC, because the peptides generated might lead to diversification of the TCR repertoire.135 Finally, MCs can also indirectly help shape the nature of the adaptive immune response by promoting the migration of DCs to draining nodes in mice.136,137 In this regard MC-deficient mice might demonstrate defects in antigen presentation that are not intrinsic to mast cells. Similar to basophils and mast cells, ILC2s seem to also collaborate with DCs to regulate antigen presentation. ILC2s are a predominant source of OX40L, and ILC2-specific loss of OX40L, has been shown to lead to deficient induction of Th2 priming to allergens and helminths in mice, leading to failure to clear helminths.138 Additionally, adoptive transfer of ILC2s can potentiate Th2 responses in recipient mice, including an IL-13-driven enhanced production of Th2 selective chemokine CCL17 by cDC2s relative to controls, and an increased migration of DCs to the draining lymph node T cell area.139–141

Th2 Adjuvant Effects of Environmental Pollutants Increasingly, it is reported that other inhaled environmental factors such as ambient particulate matter including diesel exhaust particles (DEP), ozone, or cigarette smoke are also able to trigger TLR signalling, release of DAMPs, and activation of innate immune cells. The concept of epithelial control of DC functions by cytokines has also been shown for ambient particulate matter that also induces GM-CSF and TSLP and thus could activate DCs or ILC2s indirectly.142 Reactive oxygen species (ROS) are produced by epithelial cells and DCs in response to allergens or particulate matter. Reactive oxygen species can alter epithelial cell barrier integrity, enhance DC-attracting chemokine production by epithelial cells, and ultimately participate in the induction of Th2 responses through several cellular and molecular mechanisms.126 Remarkably, allergens found in ragweed extracts can themselves produce ROS because they contain a NADPH oxidase-like activity. Pollen-derived ROS can activate NF-κB in DC in a process that is regulated by Nrf-2.143

Dendritic Cells in Ongoing Allergic Airway Inflammation After a priming and proliferation phase in the draining lymph nodes, Th2 effector cells migrate back to the exposed tissues to enforce ongoing innate immunity.113,144–146 Given the nature of the type of antigens that induce it (e.g., environmental ubiquitous allergens and tissue dwelling

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parasites that evade immune recognition), type 2 immunity often becomes chronic and localized to tissues, because the antigen is persistently present.147 Some of the Th2 cells will also adopt a phenotype of long-lived tissue resident TRM cells, which can be restimulated months later and produce immediate cytokines in the periphery.148,149 For immediate CD4+ effector T cell reactivation,150 as well as for setting up a niche for long-term survival of CD4+ T cells, interactions with MHC II+ APCs are needed, although the precise nature of these MHC II expressing cells has not been elucidated. Given the fact that effector and memory T cells are less dependent on costimulatory molecules, are kept alive by low-level TCR signaling, and reside in tissues rather than lymph nodes, any nonprofessional nonmigratory APC that expresses MHC II might suffice. Another major change that occurs in the antigen-experienced host is the persistence of antigen-specific immunoglobulins that can significantly alter the activation state of professional and nonprofessional APCs by binding to various activating or inhibitory Fc receptors.151 Antigen-specific IgE and IgG1 can bind to low- and high-affinity Fc receptors on mast cells, basophils, eosinophils, macrophages, and DCs

and can be used to much more efficiently capture allergens for presentation to tissue-dwelling effector or resident memory T cells. Despite these changes that take place in the immune host, DCs still play a predominant role during the Th2 effector phase of asthma, when the lung is repeatedly exposed to allergens and effector T cells migrate back to the lung (Fig. 13.4).152 The number and activation state of lung and airway CD11b+ cDC2s is enhanced during the acute phase after allergen challenge, and these DCs form adhesion clusters with activated T lymphocytes. Depletion of these DCs in CD11cDTR mice at the time of lung allergen challenge reduces the salient features of asthma, like airway eosinophilia, metaplasia of goblet cells, and bronchial hyperreactivity.150,153,154 The function of DCs during allergen challenge could be the local restimulation of effector function in lung resident lymphocytes, or alternatively could be the recruitment of these effector Th2 cells (Fig. 13.6). In allergen-challenged mice, CD11b+ DCs are a prominent source of the chemokines CCL17 and CCL22, thus recruiting CCR4+ Th2 cells to the lungs.155 A study using selective depletion of lung CD11b+ myeloid cells (most likely CD11b+ moDCs) in CD11bDTR mice indeed identified these cells, and not bronchial epithelial

Allergens (e.g. Derp1) break down epithelial tight junctions

Allergens

Fig. 13.6  Interaction between epithelial cells and dendritic cells during established inflammation. Allergens stimulate epithelial cells to release chemokines and growth factors for dendritic cells (DCs), Th2 cells, and eosinophils. Thymic stromal lymphopoietin (TSLP) and granulocyte macrophage colony–stimulating factor (GMCSF) are instrumental in inducing a Th2-prone phenotype in lung DCs. Epithelial cell tight junctions are opened up by protease activity of certain allergens like Der p 1 from house dust mites. In this way, allergens gain access to the DC extensions. The recruited DCs are also stimulated directly by allergen and produce even more chemokines for Th2 cell recruitments (CCL17/TARC and CCL22/MDC). Locally attracted Th2 cells interact with DCs in the airways, leading to local DC maturation and T cell costimulation of effector cytokine production. In addition, locally activated DCs can also activate T resident memory T cells (Trm). These activated Th2 cells and Trm cells eventually control the inflammatory process by activating eosinophils and mast cells and by feeding back on the epithelium and DCs. At the same time, DCs also migrate to the draining lymph nodes where they restimulate recirculating memory Th2 cells to become effector cells, and they recruit new cells into the response. In this way, effector cells are continuously replenished. DCs are also crucial for maintaining IgE synthesis, through their stimulation of interleukin 4–producing TFH cells.

C

GM-CSF, IL33 enhances CCL5, DC survival attracts Eos, Th2, DCs DC

TSLP PGE2 IL10

CCL20, attracts DCs

CCL17/CCL22 chemokines attract Th2 cells

IL-4 IL-13

Th2

DC

CCR4 CCR8

Th2

Th2

Trm

Local costimulation to Th2 cells or Trm cells

New effector cells migrate to lung Ag loaded DCs migrate to draining lymph nodes CCR7 Th0

TFH IL-4

Stimulation of recirculating memory Th2 cells and naïve T cells Local stimulation of Tfh cells

IgE synthesis

CHAPTER 13  Antigen-Presenting Dendritic Cells cells, as the critical source of CCL17 and CCL22.156 We have also shown that monocyte-derived DCs are the predominant source of chemokines that attract inflammatory cells to the airways.23,64 Several publications report the presence of FcεRI on lung DCs in humans and mice,67,157 but only recently have we fully appreciated the importance of this finding. In a very elegant manner, it was shown that mice that express the human FcεRI on DCs had greatly enhanced Th2 responses to inhaled allergens.48 The reason for this is the lowering of the threshold for allergen recognition by DCs armed with allergenspecific IgE on their surface. It has long been shown in humans that anti-IgE therapies indeed lead to lowering of the threshold of allergen recognition, and eventually to a reduction in Th2 immune responses to inhaled allergens. Work in the same humanized FcεRI transgenic strain of mice has shown that FcεRI might also provide a feedback mechanism on DC activation, however, by suppression of chemokine production.47 An alternative explanation why DCs are so important during ongoing allergen challenge is that they migrate to the lymph nodes and restimulate resting memory T cells, to feed the effector response with new effector cells (Fig. 13.6).4,144

CONCLUDING REMARKS DCs that communicate with epithelial cells and innate immune cells are crucial in determining the functional outcome of an allergen encounter in the lung, nose, and skin. Antigen presentation by cDC2s leads to Th2 sensitization typical of allergic disease. It is increasingly clear that DCs have an antigen-presenting function beyond sensitization. Therefore DCs constitute a novel target for the development of antiallergic therapy aimed at suppressing the origin of the allergic inflammatory cascade.

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CHAPTER 13  Antigen-Presenting Dendritic Cells not plasmacytoid dendritic cells to induce Foxp3 de novo in CD4+ T cells and promote airway tolerance. J Immunol 2013;191(1):25–9. 80. Kuipers H, Lambrecht BN. The interplay of dendritic cells, Th2 cells and regulatory T cells in asthma. Curr Opin Immunol 2004;16(6): 702–8. 81. Lombardi V, Speak AO, Kerzerho J, et al. CD8alpha(+)beta(-) and CD8alpha(+)beta(+) plasmacytoid dendritic cells induce Foxp3(+) regulatory T cells and prevent the induction of airway hyper-reactivity. Mucosal Immunol 2012;5(4):432–43. 82. Everts B, Tussiwand R, Dreesen L, et al. Migratory CD103+ dendritic cells suppress helminth-driven type 2 immunity through constitutive expression of IL-12. J Exp Med 2016;213(1):35–51. 83. Xanthou G, Alissafi T, Semitekolou M, et al. Osteopontin has a crucial role in allergic airway disease through regulation of dendritic cell subsets. Nat Med 2007;13(5):570–8. 84. Alissafi T, Kourepini E, Simoes DCM, et al. Osteopontin promotes protective antigenic tolerance against experimental allergic airway disease. J Immunol 2018;200(4):1270–82. 85. Kool M, van Nimwegen M, Willart MA, et al. An anti-inflammatory role for plasmacytoid dendritic cells in allergic airway inflammation. J Immunol 2009;183(2):1074–82. 86. Akbari O, DeKruyff RH, Umetsu DT. Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat Immunol 2001;2:725–31. 87. Fear VS, Burchell JT, Lai SP, et al. Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance. J Immunol 2011;187(9):4561–70. 88. Naito T, Suda T, Suzuki K, et al. Lung dendritic cells have a potent capability to induce production of immunoglobulin A. Am J Respir Cell Mol Biol 2008;38(2):161–7. 89. Ruane D, Chorny A, Lee H, et al. Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses. J Exp Med 2016;213(1):53–73. 90. Smits HH, Gloudemans AK, van Nimwegen M, et al. Cholera toxin B suppresses allergic inflammation through induction of secretory IgA. Mucosal Immunol 2009;2(4):331–9. 91. Guilliams M, Crozat K, Henri S, et al. Skin-draining lymph nodes contain dermis-derived CD103(-) dendritic cells that constitutively produce retinoic acid and induce Foxp3(+) regulatory T cells. Blood 2010;115(10):1958–68. 92. Neyt K, GeurtsvanKessel CH, Deswarte K, et al. 1 signaling promotes iBALT induction after influenza virus infection. Front Immunol 2016;7:312. 93. Neyt K, Perros F, GeurtsvanKessel CH, et al. Tertiary lymphoid organs in infection and autoimmunity. Trends Immunol 2012;33(6): 297–305. 94. Hammad H, Lambrecht BN, Pochard P, et al. Monocyte-derived dendritic cells induce a house dust mite-specific Th2 allergic inflammation in the lung of humanized SCID mice: involvement of CCR7. J Immunol 2002;169(3):1524–34. 95. Hammad H, Charbonnier AS, Duez C, et al. Th2 polarization by Der p 1–pulsed monocyte-derived dendritic cells is due to the allergic status of the donors. Blood 2001;98(4):1135–41. 96. Traidl-Hoffmann C, Mariani V, Hochrein H, et al. Pollen-associated phytoprostanes inhibit dendritic cell interleukin-12 production and augment T helper type 2 cell polarization. J Exp Med 2005;201(4):627–36. 97. Lambrecht BN, De Veerman M, Coyle AJ, et al. Myeloid dendritic cells induce Th2 responses to inhaled antigen, leading to eosinophilic airway inflammation. J Clin Invest 2000;106(4):551–9. 98. Phythian-Adams AT, Cook PC, Lundie RJ, et al. CD11c depletion severely disrupts Th2 induction and development in vivo. J Exp Med 2010;207(10):2089–96. 99. Kumamoto Y, Linehan M, Weinstein JS, et al. CD301b+ dermal dendritic cells drive T helper 2 cell-mediated immunity. Immunity 2013;39:733–43.

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100. Gao Y, Nish SA, Jiang R, et al. Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells. Immunity 2013;39(4):722–32. 101. Williams JW, Tjota MY, Clay BS, et al. Transcription factor IRF4 drives dendritic cells to promote Th2 differentiation. Nat Commun 2013;4:2990. 102. Connor LM, Tang SC, Cognard E, et al. Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles. J Exp Med 2017;214(1):125–42. 103. Tussiwand R, Everts B, Grajales-Reyes GE, et al. KLF4 expression in conventional dendritic cells is required for T helper 2 responses. Immunity 2015;42(5):916–28. 104. Lambrecht BN, Hammad H. Allergens and the airway epithelium response: gateway to allergic sensitization. J Allergy Clin Immunol 2014;134(3):499–507. 105. Sokol CL, Chu NQ, Yu S, et al. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response. Nat Immunol 2009;10(7):713–20. 106. Yamanishi Y, Miyake K, Iki M, et al. Recent advances in understanding basophil-mediated Th2 immune responses. Immunol Rev 2017;278(1):237–45. 107. Yoshimoto T, Yasuda K, Tanaka H, et al. Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. Nat Immunol 2009;10(7):706–12. 108. Miyake K, Shiozawa N, Nagao T, et al. Trogocytosis of peptide-MHC class II complexes from dendritic cells confers antigen-presenting ability on basophils. Proc Natl Acad Sci USA 2017;114(5):1111–16. 109. Millien VO, Lu W, Shaw J, et al. Cleavage of fibrinogen by proteinases elicits allergic responses through Toll-like receptor 4. Science 2013;341(6147):792–6. 110. Trompette A, Divanovic S, Visintin A, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 2009;457(7229):585–8. 111. Willart MA, Deswarte K, Pouliot P, et al. Interleukin-1alpha controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33. J Exp Med 2012;209(8):1505–17. 112. Verma M, Liu S, Michalec L, et al. Experimental asthma persists in IL-33 receptor knockout mice because of the emergence of thymic stromal lymphopoietin-driven IL-9(+) and IL-13(+) type 2 innate lymphoid cell subpopulations. J Allergy Clin Immunol 2018;142(3):793–803.e8. 113. Van Dyken SJ, Nussbaum JC, Lee J, et al. A tissue checkpoint regulates type 2 immunity. Nat Immunol 2016;17(12):1381–7. 114. Rajavelu P, Chen G, Xu Y, et al. Airway epithelial SPDEF integrates goblet cell differentiation and pulmonary Th2 inflammation. J Clin Invest 2015;125(5):2021–31. 115. Eiwegger T, Akdis CA. IL-33 links tissue cells, dendritic cells and Th2 cell development in a mouse model of asthma. Eur J Immunol 2011;41(6):1535–8. 116. Saluzzo S, Gorki AD, Rana BM, et al. First-breath-induced type 2 pathways shape the lung immune environment. Cell Rep 2017;18(8):1893–905. 117. Kool M, Willart MA, van Nimwegen M, et al. An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma. Immunity 2011;34(4):527–40. 118. Hara K, Iijima K, Elias MK, et al. Airway uric acid is a sensor of inhaled protease allergens and initiates type 2 immune responses in respiratory mucosa. J Immunol 2014;192(9):4032–42. 119. Gold MJ, Hiebert PR, Park HY, et al. Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization. Mucosal Immunol 2016;9(3):809–20. 120. Kool M, Soullie T, van Nimwegen M, et al. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 2008;205(4):869–82. 121. Kuroda E, Ishii KJ, Uematsu S, et al. Silica crystals and aluminum salts regulate the production of prostaglandin in macrophages via NALP3 inflammasome-independent mechanisms. Immunity 2011;34(4):514–26.

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122. Toussaint M, Jackson DJ, Swieboda D, et al. Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation. Nat Med 2017;23(6):681–91. 123. Juncadella I, Kadl A, Sharma AK, et al. Apoptotic cell clearance by bronchial epithelial cells critically influences airway inflammation. Nature 2013;493(7433):547–51. 124. Gebe JA, Yadava K, Ruppert SM, et al. Modified high-molecular-weight hyaluronan promotes allergen-specific immune tolerance. Am J Respir Cell Mol Biol 2017;56(1):109–20. 125. Allenspach EJ, Lemos MP, Porrett PM, et al. Migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive CD4 T cells. Immunity 2008;29(5):795–806. 126. Tang H, Cao W, Kasturi SP, et al. The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling. Nat Immunol 2010;11(7):608–17. 127. Leyva-Castillo JM, Hener P, Michea P, et al. Skin thymic stromal lymphopoietin initiates Th2 responses through an orchestrated immune cascade. Nat Commun 2013;4:2847. 128. Ohnmacht C, Schwartz C, Panzer M, et al. Basophils orchestrate chronic allergic dermatitis and protective immunity against helminths. Immunity 2010;33(3):364–74. 129. Schwartz C, Oeser K, Prazeres da Costa C, et al. T cell-derived IL-4/ IL-13 protects mice against fatal Schistosoma mansoni infection independently of basophils. J Immunol 2014;193(7):3590–9. 130. Kim S, Karasuyama H, Lopez AF, et al. IL-4 derived from non-T cells induces basophil- and IL-3-independent Th2 immune responses. Immune Netw. 2013;13(6):249–56. 131. Sullivan BM, Liang HE, Bando JK, et al. Genetic analysis of basophil function in vivo. Nat Immunol 2011;12(6):527–35. 132. Sokol CL, Barton GM, Farr AG, et al. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat Immunol 2008;9(3):310–18. 133. Carroll-Portillo A, Cannon JL, te Riet J, et al. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation. J Cell Biol 2015;210(5):851–64. 134. Kambayashi T, Baranski JD, Baker RG, et al. Indirect involvement of allergen-captured mast cells in antigen presentation. Blood 2008;111(3):1489–96. 135. Lotfi-Emran S, Ward BR, Le QT, et al. Human mast cells present antigen to autologous CD4(+) T cells. J Allergy Clin Immunol 2018;141(1):311–21.e10. 136. Galli SJ, Gaudenzio N. Human mast cells as antigen-presenting cells: when is this role important in vivo? J Allergy Clin Immunol 2018;141(1):92–3. 137. Suto H, Nakae S, Kakurai M, et al. Mast cell-associated TNF promotes dendritic cell migration. J Immunol 2006;176(7):4102–12. 138. Halim TYF, Rana BMJ, Walker JA, et al. Tissue-restricted adaptive type 2 immunity is orchestrated by expression of the costimulatory molecule OX40L on group 2 innate lymphoid cells. Immunity 2018;48(6):1195–207.e6. 139. Neill DR, Wong SH, Bellosi A, et al. Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 2010;464(7293):1367–70.

140. Kim BS, Wojno ED, Artis D. Innate lymphoid cells and allergic inflammation. Curr Opin Immunol 2013;25(6):738–44. 141. Halim TY, Hwang YY, Scanlon ST, et al. Group 2 innate lymphoid cells license dendritic cells to potentiate memory TH2 cell responses. Nat Immunol 2016;17(1):57–64. 142. Gold MJ, Hiebert PR, Park HY, et al. Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization. Mucosal Immunol 2016;9(3):809–20. 143. Rangasamy T, Williams MA, Bauer S, et al. Nuclear erythroid 2 p45-related factor 2 inhibits the maturation of murine dendritic cells by ragweed extract. Am J Respir Cell Mol Biol 2010;43(3):276–85. 144. Harris NL, Watt V, Ronchese F, et al. Differential T cell function and fate in lymph node and nonlymphoid tissues. J Exp Med 2002;195(3): 317–26. 145. Shinkai K, Mohrs M, Locksley RM. Helper T cells regulate type-2 innate immunity in vivo. Nature 2002;420(6917):825–9. 146. Coquet JM, Schuijs MJ, Smyth MJ, et al. Interleukin-21-producing CD4(+) T cells promote type 2 immunity to house dust mites. Immunity 2015;43(2):318–30. 147. Li BW, de Bruijn MJ, Tindemans I, et al. T cells are necessary for ILC2 activation in house dust mite-induced allergic airway inflammation in mice. Eur J Immunol 2016;46(6):1392–403. 148. Lloyd CM, Harker JA. Location, location, location: localized memory cells take residence in the allergic lung. Immunity 2016;44(1):13–15. 149. Hondowicz BD, An D, Schenkel JM, et al. Interleukin-2-dependent allergen-specific tissue-resident memory cells drive asthma. Immunity 2016;44(1):155–66. 150. van Rijt LS, Jung S, Kleinjan A, et al. In vivo depletion of lung CD11c+ dendritic cells during allergen challenge abrogates the characteristic features of asthma. J Exp Med 2005;201(6):981–91. 151. Guilliams M, Bruhns P, Saeys Y, et al. The function of Fcgamma receptors in dendritic cells and macrophages. Nat Rev Immunol 2014;14(2):94–108. 152. Lambrecht BN, Hammad H. Taking our breath away: dendritic cells in the pathogenesis of asthma. Nat Rev Immunol 2003;3(12):994–1003. 153. Lambrecht BN, Salomon B, Klatzmann D, et al. Dendritic cells are required for the development of chronic eosinophilic airway inflammation in response to inhaled antigen in sensitized mice. J Immunol 1998;160(8):4090–7. 154. van Rijt LS, Vos N, Willart M, et al. Persistent activation of dendritic cells after resolution of allergic airway inflammation breaks tolerance to inhaled allergens in mice. Am J Respir Crit Care Med 2011;184(3):303–11. 155. Beaty SR, Rose CE Jr, Sung SS. Diverse and potent chemokine production by lung dendritic cells in homeostasis and in allergic lung inflammation. J Immunol 2007;178(3):1882–95. 156. Medoff BD, Seung E, Hong S, et al. CD11b+ myeloid cells are the key mediators of Th2 cell homing into the airway in allergic inflammation. J Immunol 2009;182(1):623–35. 157. Tunon-De-Lara JM, Redington AE, Bradding P, et al. Dendritic cells in normal and asthmatic airways: expression of the alpha subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI -alpha). Clin Exp Allergy 1996;26(6):648–55.

CHAPTER 13  Antigen-Presenting Dendritic Cells

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SELF-ASSESSMENT QUESTIONS 1. Induction of CD4 Th2 immunity is a specific function of: a. Alveolar macrophages. b. Type 2 dendritic cells. c. Plasmacytoid dendritic cells. d. Epithelial cells. 2. Dendritic cells and epithelial cells communicate with each other via: a. Thymic stromal lymphopoietin (TSLP) and granulocyte macrophage colony–stimulating factor (GM-CSF). b. GM-CSF and interleukin 8 (IL-8). c. Neuropeptides. d. Gap junctions.

3. Dendritic cells are drug targets for allergic inflammation because: a. They are short lived. b. They take up drugs avidly. c. They control effector cell influx in tissues. d. They metabolize drugs. 4. Dendritic cells will induce Th2 immune responses when: a They communicate with basophils. b They express interferon regulatory factor (IRF) 4. c They fail to produce interleukin 12 (IL-12). e. All of the above. f. None of the above.

14  Biology of Mast Cells and Their Mediators Peter Bradding, Hirohisa Saito

CONTENTS Introduction, 215 Basic Mast Cell Biology, 215 Mast Cell Homing to Tissue, 217 Mast Cell Heterogeneity, 218

SUMMARY OF IMPORTANT CONCEPTS • Mast cells secrete a wide range of biologic mediators of direct relevance to the pathophysiology of allergic diseases and asthma. • There is evidence of acute mast cell activation in isolated allergic responses such as anaphylaxis and allergen challenge and chronic ongoing activation in diseases such as asthma. • Mast cells infiltrate key airway structures in asthma, placing activated mast cells within the dysfunctional airway elements (airway smooth muscle, submucosal glands, airway epithelium). • Mast cells can be activated by numerous immunologic (IgE) and nonimmunologic pathways; many are likely to be operative in chronic disease. • Mast cells have many roles beyond allergic disease and asthma such as pathogen recognition, protection against envenomation, wound healing, and promotion of tissue fibrosis. • Stem cell factor is an obligate growth factor for human mast cells and signals through the receptor tyrosine kinase Kit. Gain-of-function mutations in Kit contribute to the development of mastocytosis.

INTRODUCTION Mast cells (MC) were discovered by Paul Ehrlich more than a century ago on account of the characteristic metachromatic staining of their secretory granules,1 but their role in human pathophysiology is still not fully understood. They are present in all classes of vertebrates, including amphibians, reptiles, birds, and mammals, and it has been estimated that the storage of histamine in vertebrate MCs and its use as an inflammatory mediator was established in primitive reptiles (Lepidosauria) approximately 276 million years ago.2 This suggests that they mediate functions essential for life rather than representing a vestigial remnant of the immune system. No MC-deficient human has so far been identified. In healthy humans, MCs are present throughout connective tissues and mucosal surfaces and are especially prominent at the interface with the external environment such as the skin, respiratory tract, conjunctiva, and gastrointestinal tract. MCs are armed with a vast array of biologic mediators and respond rapidly to a variety of tissue insults. Their primary role is to initiate an appropriate program of inflammation and repair in response to tissue damage initiated by a

Mast Cell Ultrastructure and Mediators, 219 Mechanisms of Mast Cell Activation, 221 Mast Cells in Allergic Diseases and Asthma, 227 Conclusion, 235

variety of diverse stimuli. MCs contribute to the maintenance of tissue homeostasis with examples including roles in wound repair,3 revasculari­ zation,4 and protective roles in the response to bacterial infection5 and envenomation.6 Their “misguided” activation by allergens contributes to the development of allergic symptoms. In situations of an ongoing tissue insult, their sustained release of numerous proinflammatory mediators, proteases, and cytokines within specific tissue structures contributes to the pathophysiology of various chronic diseases such as asthma, pulmonary fibrosis, rheumatoid arthritis, and atherosclerosis. A key goal is the development of novel treatments that attenuate adverse MC function when administered chronically to humans in vivo. Such therapies do not currently exist but may offer a novel approach to the treatment of many life-threatening diseases. This chapter focuses on the biologic properties of MCs and how these remarkable cells contribute to the development and propagation of asthma and related allergic diseases.

BASIC MAST CELL BIOLOGY Mast Cell Development and Survival MCs develop from pluripotent hematopoietic bone marrow stem cells (Fig. 14.1) that are released into the systemic circulation as undifferentiated, CD34+, receptor tyrosine kinase Kit-positive (CD117+) mononuclear cells. These subsequently migrate into tissue, potentially proliferate,7 and then differentiate (mature) under the influence of the local cytokine milieu, the tissue matrix, and resident cells such as fibroblasts that profoundly influence their phenotype.8 Stem cell factor (SCF, Kit ligand, Steel factor) is the ligand for Kit, encoded by the protooncogene c-Kit, and is an obligate human MC growth factor that is derived from many cellular sources including epithelial and mesenchymal cells in both soluble and membrane-bound forms. Removal of SCF leads to rapid MC apoptosis, although several factors such as IL-6 are able to delay this. Human MCs differentiate from bone marrow or circulating peripheral blood progenitors in vitro with SCF as the only exogenous factor but remain immature and predominantly contain secretory granules of the “tryptase-only” type (designated MCT phenotype).9 However, MCs grown in suspension culture in medium supplemented with SCF and IL-6 are more mature in terms of their nuclear morphology and granular structure but remain as the MCT phenotype. In contrast, cells grown on a fibroblast or endothelial cell monolayer develop a tryptase+

215

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Tissue

Fig. 14.1  An overview of mast cell (MC) development. SCF, Stem cell factor.

chymase+ (MCTC phenotype) and resemble skin MCs, whereas IL-4 dramatically increases chymase expression in human cord blood–derived MCs (HCBMC).10 In addition, human bone marrow–derived MC (HBMMC) and HCBMC grown in SCF plus conditioned medium from a cell line derived from a patient with systemic mastocytosis yielded cells containing chymase only (MCC phenotype). These observations indicate that factors other than SCF are required for complete MC maturation. Gain-of-function mutations in Kit, most commonly the D816V type, are found in mastocytosis and MC leukemia (see Chapter 74). The vast majority of patients with systemic mastocytosis carry the somatic D816V mutation in the KIT gene. Although such mutations can contribute to neoplastic transformation, their presence is not adequate for this in isolation. Several cofactors enhance or inhibit the effects of SCF depending on the MC origin and its level of maturity.11 Nerve growth factor (NGF), IL-3, IL-6, IL-9, and IL-10 potentially enhance SCF-dependent MC growth and/or promote survival. Conversely, granulocyte macrophage– colony stimulating factor (GM-CSF), retinoids, and transforming growth factor β (TGF-β) inhibit MC growth and differentiation. IL-4 inhibits proliferation of immature human peripheral blood–derived MCs (HPBMC) but potentiates proliferation of more mature HPBMCs, whereas IL-5 and interferon γ (IFN-γ) prolong HCBMC survival on SCF withdrawal but inhibit immature HPBMC proliferation. IL-6 is important for the maintenance of human lung mast cell (HLMC) MCT viability through inhibition of apoptosis but is without effect on HLMC MCTC or human skin MCs. IL-33 inhibits human skin MC apoptosis via upregulation of the antiapoptotic protein BCL-XL12 and prolongs human cord blood–derived MC survival after the removal of SCF.13 Lysophosphatidic acid (LPA) is a lipid mediator that exerts comitogenic effects with SCF in HCBMC.14 Experiments from adoptive transfer in MC-deficient mice indicate that tissue MCs may survive for up to several months. In keeping with this, primary human MCs isolated from skin, gut, or lung can be maintained in cell culture for several months in the presence of SCF and IL-6. The β chain of FcεRI, MS4A2, amplifies FcεRI-dependent MC activation.15 However, two truncations of MS4A2 have been described with distinct functions. A truncation arising from inclusion of intron 5 results in an in-frame stop codon (MS4A2 variant 2) and has been detected in HCBMC and basophils.16 This truncation decreases the expression of FcεRI because it chaperones the α chain to endosomes for degradation; its effects on cell function are not known. A truncation of MS4A2

(MS4A2trunc) from HLMCs that lacks exon 317 MS4A2trunc gene expression was negatively regulated by SCF, and its expression was not detected in the Kit gain-of-function human MC line HMC-1. Unlike MS4A2, MS4A2trunc did not traffic to the cytoplasmic membrane but instead was associated with the nuclear membrane. Overexpression of MS4A2trunc induced HLMC death and profoundly inhibited HMC-1 cell proliferation by inducing G2-phase cell cycle arrest and apoptosis.17 This extends the roles of the MS4A2 gene beyond the regulation of acute allergic responses and suggests that its silencing may contribute to pathologic MC proliferation. Another MS4A family member, MS4A4, is expressed by HLMCs and HCBMCs, and the HMC-1 and LAD2 human MCs lines.18 In HCBMCs, MS4A4 expression increases as the cells mature over 8 weeks of culture. MS4A4 influences MC function by promoting the surface expression of Kit by directing Kit trafficking through endocytic recycling rather than degradation pathways, via a mechanism that involves recruitment of Kit to caveolin-1-enriched microdomains. MS4A4 also regulates signaling through the phospholipase (PL)C γ1 pathway for Kit, possibly by recruitment of receptors to lipid rafts. Altered Kit trafficking and signaling with MS4A4 silencing resulted in an increase in AKT signaling, LAD2 cell proliferation, and human cord blood–derived MC migration. These observations suggest that MS4A4 dysfunction in MCs may contribute to aberrant cellular responses and may explain the observations that aberrant genetic expression of MS4A family members is associated with a range of disease processes, including Alzheimer’ disease.19 MCs require the tight control of ion flux to ensure their survival and optimal functioning. The widely expressed transient receptor potential (TRP) melastatin (M) family member TRPM7 is expressed by HMC-1 and HLMC.20 This channel is involved in Mg2+ homeostasis, and it also carries numerous trace divalent cations essential to normal functioning and survival. Thus knockdown of TRPM7 in HMC-1 cells and HLMC leads to rapid cell death.20

Regulation of Mast Cell Survival by Cell Adhesion Molecule-1 Cell adhesion is a fundamental mechanism through which cells communicate, permitting the delivery of specific cell-cell signals. In recent years, cell adhesion molecule-1 (CADM1; also known previously as TSLC1, IGSF4, SgIgSF, Necl2, Syncam1, RA175) has emerged as a particularly important adhesion protein regulating MC function and signaling through Kit.

CHAPTER 14  Biology of Mast Cells and Their Mediators CADM1 is a member of the immunoglobulin (Ig) superfamily and is implicated in several diseases including cancer (due to its roles in tumor suppression), autism spectrum disorder, pulmonary fibrosis, and venous thromboembolism. CADM1 binds to itself (homophilic adhesion) and to heterophilic binding partners including CADM2, CADM3, Nectin-3, and cytotoxic and regulatory T cell molecule (CRTAM), all of which are members of the same Ig superfamily. The intracellular domain of CADM1 contains two important sites required for protein binding; a juxtamembrane protein 4.1/ezrin/radixin/moesin (FERM) binding motif, which interacts with DAL-1 (protein 4.1B) and anchors CADM1 to the f-actin cytoskeleton,21 and a PDZ-binding motif located within the C-terminus. This motif mediates interactions with the PDZ domains of several other proteins, including members of the membraneassociated guanylate kinases (MAGUK) family.22–24 FERM- and PDZbinding domains are particularly important components of CADM1; if either the FERM-binding domain or PDZ-binding domain is deleted, CADM1 is no longer able to mediate cell-cell adhesion, and its tumor suppressor activity in epithelial cells is lost. This suggests that there is an important cooperative interaction between the PDZ- and FERMinteracting proteins in tumor suppression and inside-out signaling mediating cell adhesion. CADM1 also plays an important role in cell motility.25 CADM1 is expressed in epithelial cells, nerves, endothelial cells, fibroblasts, and MCs, but not other leukocytes, with the exception of malignant T cells in adult T cell leukemia/lymphoma (ATLL).26 Several isoforms of CADM1 exist because of variable splicing between exons 7 through 11 in the juxtamembranous extracellular region.27,28 Several functional CADM1 isoforms and several cryptic nonfunctional isoforms are detectable in the immature HMC-1 human MC line derived from a patient with MC leukemia, the differentiated LAD2 human MC line derived from a patient with MC sarcoma, and in primary human lung MCs.28,29 The SP4 CADM1 isoform, encoded by exons 1 to 8 and 11 to 12, is the only functional isoform present in the MC line HMC-1, whereas LAD2 cells and human lung MCs express approximately 80% of SP4, 16% to 18% of the longer SP1 isoform (exons 1 to 9 and 11 to 12) and approximately 2% to 5% of the longest SP6 (exons 1 to 12) isoforms. Real-time PCR has also detected the SP3 (exons 1 to 7 and 11 to 12) isoform in human lung MCs, but not SP2, soluble SP5 (exon 1 to intron 7) or CADM1b (exons 1 to 7, 9, and 11 to 12).29 The failure to detect SP5 in human MCs is significant; SP5 has been cloned from mouse MCs, where it is proposed to function as a negative regulator of CADM1 function.30 The activities of each CADM1 isoform in MCs differ. For instance, the SP4 isoform mediates homotypic (MC-MC) adhesion through a homophilic (CADM1-CADM1) trans-interaction, and also promotes human lung MC and HMC-1 survival in monoculture, although these two processes are not entirely interdependent.28 In contrast, although the SP1 isoform also supports homotypic MC adhesion, it is less efficient than SP4, and SP1 overexpression acts in a dominant-negative manner to reduce HMC-1 and human lung MC survival. CADM1 downregulation results in reduced viability and decreased expression of the prosurvival protein Mcl-1L, but not Blc-2 or Bcl-XL, and increases caspase-3/7 activity in both HMC-1 cells and HLMCs.28 This coincides with decreased basal Kit levels in HLMCs. Interestingly, SP4 expression, which promotes survival, is approximately fourfold higher in rapidly proliferating neoplastic HMC-1 cells than human lung MCs. Consequently, it is plausible that high SP4 expression and absent SP1 expression contributes to the neoplastic transformation of MCs in a manner similar to that evident in ATL.26 CADM1 expression not only promotes homotypic adhesion between MCs but also facilitates the formation of heterotypic interactions between human MCs and other cell types. For example, CADM1 mediates

217

adhesion of human lung MCs to primary human parenchymal lung fibroblasts31 and, of relevance to asthma, primary human airway smooth muscle (ASM) cells.32 This CADM1-dependent adhesive process promotes MC survival and proliferation in the absence of serum and exogenous SCF or IL-6.31,33 Thus CADM1 has a key role in MC biology. More extensive experiments with human ASM cells have suggested that CADM1 and Kit have an intimate relationship in MCs. For example, CADM1 and Kit cooperate and contribute to human lung MC survival and proliferation.33 In addition, both CADM1 and Kit exist under the transcriptional control of microphthalmia transcription factor (MITF),34,35 coimmunoprecipitate from HMC-1 cells,33 and colocalize in human lung MCs at points of adhesion to human ASM muscle cells, as determined by confocal imaging.33 Downregulation or overexpression of CADM1 also reduces or increases, respectively, surface Kit expression in both human lung MCs and HMC-1 cells.28,36 Furthermore, CADM1 downregulation leads to increased caspase-3 and 7 activity in both HMC-1 cells and human lung MCs,28 and this coincides with decreased basal Kit levels in human lung MCs. Thus it is thought that CADM1 plays an important role localizing Kit to the surface of MCs at points of cell-cell adhesion. This will facilitate the interaction between Kit and membrane-bound SCF expressed on cells such as fibroblasts and ASM cells, hence promoting MC survival. CADM1 is also required for the efficient function of integrindependent adhesion to extracellular matrix through heterophilic interactions. CADM1 regulates both E-cadherin and α6β4 integrin function in other cell types, and downregulation of CADM1 in HMC-1 cells results not only in reduced adhesion to human ASM cells, but also reduced adhesion to their extracellular matrix.36 Collectively these findings suggest that CADM1 is a key adhesion receptor which regulates net MC adhesion, both directly, through CADM1-dependent adhesion, and indirectly, through the regulation of other adhesion receptors. Thus CADM1 is a key adhesion molecule that interacts with Kit in a cooperative manner to promote MC survival. Targeting MC-specific CADM1 signal transduction pathways or inhibiting the function of its counterreceptors on fibroblasts and ASM cells has the potential to become a novel strategy for the inhibition of MC function in disease.

MAST CELL HOMING TO TISSUE Many MC chemoattractants have been identified and are summarized in Table 14.1. The mechanisms through which MC progenitors are mobilized from the bone marrow and migrate into tissue are still poorly understood. In mice, MC homing to the gut requires α4β7 integrin and the CXCR2 chemokine receptor expression on the MC progenitors and expression of the adhesion molecules MAdCAM-1 and VCAM-1 on the intestinal endothelium.8 Unlike human lungs, healthy mouse lungs do not contain many MCs, but numbers increase markedly with induction of experimental “allergic” inflammation. This inflammatory recruitment of committed MC progenitors to mouse lung requires the expression of MC progenitor α4β7 and α4β1 integrins. In addition, there is involvement of the CCR2/CCL2 pathway in these murine models.37 Leukotriene B4 is a chemoattractant for mouse and human MC progenitors, and mouse MCs activated with anti-IgE release this lipid, suggesting that resident MCs might regulate progenitor recruitment.38 PGE2 is also chemotactic for mouse MC progenitors and is active in the conditioned medium from processed nasal mucosa that has been challenged with OVA in sensitized animals.39 How these mechanisms relate to human MC progenitor recruitment is unclear, and there may be some important differences. In particular, HMC-1 cells, HBMMC, and HLMC do not express CXCR2,40 although it is possible that early progenitors might. Prostaglandin E2 inhibits HLMC migration through the Gs-coupled EP2 receptor but is chemotactic if

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TABLE 14.1  Chemoattractants for

Mast Cells Mast Cell Chemotactic Factor

Receptor(s) Demonstrated

Chemotaxis Shown In Vitro Nonchemokine GPCR Ligands C3a, C5a PTX-sensitive pathway

Cell Type(s)

HMC-1

Serum amyloid A

PTX-sensitive pathway

HMC-1, human CBMCs

Platelet-activating factor (PAF)

PAF receptor

HMC-1, human CBMCs

5-HT (serotonin)

5-HT(1A) receptor

Mouse BMMCs, human PBMCs

LTB4

BLT1, BLT2

Mouse BMMCs, HMC-1

C1q

C1q receptor

Mouse BMMCs

PGE2

EP3 receptor

Mouse BMMCs, HLMCs

Cytokines/Growth Factors TGF-β TGF-β serine-threonine type I and II receptors

HMC-1, human CBMCs

Stem cell factor (SCF)

Kit

HMC-1, human CBMCs

Chemokines CCL2 (MCP-1)

CCR2

Mouse BMMCs

CCL3 (MIP-1α)

CCR1

Mouse BMMCs

CCL4 (MIP-1β)

CCR5

Mouse BMMCs

CCL5 (RANTES)

CCR1, CCR3, CCR4

HLMCs

CCL11 (eotaxin)

CCR3, CCR5

Mouse BMMCs, human CBMCs

CXCL8

CXCR1

HLMCs, human CBMCs

CXCL9 (MIG), CXCL10 (IP-10), CXCL11 (I-TAC)

CXCR3

HLMCs (but not human BMMCs)

CXCL12

CXCR4

HLMC, human CBMCs

CX3CL1 (fractalkine)

CX3CR1

HMC-1

Chemotactic Role Demonstrated In Vivo CCL2 (MCP-1) Increase in human wounds precedes mast cell infiltrate. CCL5 (RANTES)

Injection into rat paw, but not human skin, caused accumulation of mast cells

BMMCs, Bone marrow–derived mast cells; CBMCs, cord blood–derived mast cells; GPCRs, G protein–coupled receptors; HLMCs, human lung mast cell; HMC-1, human mast cell line; 5-HT, 5-hydroxytryptamine; IP-10, interferon gamma–induced protein 10; I-TAC, interferon-inducible T-cell alpha chemoattractant protein; LTB4, leukotriene B4; MCP-1, macrophage chemoattractant protein 1; MIP-1α, MIP-1β, macrophage inflammatory proteins 1α, 1β; PBMCs, peripheral blood–derived mast cells; PTX, pertussis toxin; TGF-β, transforming growth factor β.

the PGE2 EP2 receptor is blocked.41 The relative dominance of the EP2 receptor in less mature HCBMC suggests that PGE2-dependent recruitment of progenitors in humans is perhaps unlikely.

MAST CELL HETEROGENEITY There is marked heterogeneity of MCs with respect to ultrastructure, receptor expression, mediator content, immunologic and nonimmunologic activation, and pharmacologic responsiveness. This is evident across species, between different organs, and even within the same organ (summarized in Table 14.2).42–45 The factors leading to this heterogeneity are multifactorial and include interactions with the tissue matrix and resident cells such as fibroblasts. Although it is also possible that progenitors are committed to a particular phenotype early on in their development,46 marked plasticity in protease expression is also evident.47,48 Human MCs are classified according to their protease content. Traditionally there have been those that contain tryptase only (MCT) and others containing tryptase, chymase, carboxypeptidase A, and cathepsin G (MCTC) (Table 14.2).45 The MCT phenotype is typically found at mucosal surfaces such as the nasal and lower airway epithelium in rhinitis and mild asthma, and the bronchial lamina propria in health and disease.43,49 In contrast, the MCTC phenotype favors connective tissues such as healthy skin,49 the ASM bundles in asthma,50 and atherosclerotic lesions. Recently, a further phenotype expressing tryptase and carboxypeptidase A, but not chymase, has been described in the airway epithelium in asthmatic airways48 (discussed further later). There are also reports of rare MCs containing chymase and carboxypeptidase without tryptase (MCC) in the lung, nose, gut, and kidney whose function is unknown.51 The heterogeneity of MCs also extends to their cytokine content (Fig. 14.2). IL-4 and IL-13 are predominantly found in MCTC, whereas IL-5 and IL-6 are almost exclusively present in MCT,51 suggesting distinct roles for these phenotypes. Marked intratissue heterogeneity is highlighted further by evidence demonstrating marked differences in size and shape, and expression of tryptase, chymase, FcεRIα, IL-9R, histidine decarboxylase, 5-lipoxygenase, LTC4 synthase, renin, VEGF, and basic FGF in MCs located in the large airways, small airways, alveoli, pulmonary vessels, and pleura.44 Recently, a new multifunctional IL-9-producing mucosal MC (MMC9s) was described in mice that secretes large amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, and this was associated with decreased food allergy development, which was restored by adoptively transferred MMC9s. Atopic individuals that developed food allergy similarly displayed increased intestinal expression of Il9 and MC-specific transcripts, suggesting that the induction of MMC9s is a pivotal step to acquire IgE-mediated food allergy.52 It is perhaps not surprising that such extensive heterogeneity exists as an MC interacting with an ASM cell, for example, is likely to have different functions compared with one interacting with an epithelial cell. The differential expression of receptors and mediators makes it clear that different MC phenotypes will have distinct roles, but what these are is largely unclear. It is not known whether the phenotype of an individual MC is “plastic,” and as such, whether it changes in disease. Both seem likely, because the activation-dependent pattern of gene expression in fairly homogeneous MC populations such as those from cord blood is highly specific to the stimulus,53 and MCs in lung parenchyma in idiopathic pulmonary fibrosis are the dominant cells expressing basic FGF, although this growth factor was notably absent in alveolar MCs in nonfibrotic lung parenchyma.54 Furthermore, remarkably rapid conversion of MCTC cells to MCT has been reported in coculture with human airway epithelium.47

CHAPTER 14  Biology of Mast Cells and Their Mediators

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TABLE 14.2  Functional Mast Cell Heterogeneity RODENT MAST CELLS

HUMAN MAST CELLS

Characteristic

MMC

CTMC

MCT

MCTC

Protease content

Rat mast cell protease 2 Mouse mast cell protease-1, -2

Rat mast cell protease 1 Mouse mast cell protease-3, -4, -5, -6, -7

Tryptase

Tryptase Chymase

Proteoglycan content

Chondroitin sulfate

Heparin

Predominant granule patterning evident on electron microscopy

Heparin

Heparin

Scroll

Lattice

Common location

Mucosa

Submucosal tissues

Epithelium

Lamina propria, connective tissue, skin, airway smooth muscle

Putative primary role

Host defense

Tissue repair

Host defense

Tissue repair

Relative LTC4 release

High

Absent

High

Skin: low

Relative PGD2 release

Low

Low

Cytokine profile

Activated by antigen

Yes

Yes

Activated by substance P

No

Yes

Responds to C5a

High

Skin: high

IL-4: low IL-5: high IL-6: high IL-13: low

IL-4: high IL-13: high

Yes

Yes

No

Yes

No

Yes

Responds to PAF

Yes

No

Responds to opiates

No

Yes

Yes (weak effect)

No

Inhibited by sodium cromoglycate

No

Yes

Mast cell types: CTMC, rodent connective tissue mast cell; MCT, human tryptase-only mast cell; MCTC, human tryptase and chymase–containing mast cell; MMC, rodent mucosal mast cell. IL, Interleukin; LTC4, leukotriene C4; PAF, platelet-activating factor; PGD2, prostaglandin D2.

A

B Chymase

C IL-6

Tryptase

Fig. 14.2  Heterogeneity of human mast cells within the nasal lamina propria in a patient with perennial rhinitis. An MCT mast cell (arrows) contains tryptase and interleukin (IL)-6 (B and C) but not chymase (A), whereas a nearby MCTC mast cell (arrowheads) contains chymase and tryptase (A and C) but not IL-6 (B). (Reproduced with permission from Bradding P, Okayama Y, Howarth PH, et al. Heterogeneity of human mast cells based on cytokine content. J Immunol 1995;155:297-307.)

MAST CELL ULTRASTRUCTURE AND MEDIATORS MC membranes contain finger-like projections (microplicae), and while immature MCs may have a multi-lobed nucleus, mature cells have a mono-lobed nucleus with no apparent nucleoli and little condensed chromatin (Fig. 14.3).55 The prominent cytoplasmic structures are the

electron dense granules that are membrane-bound and contain preformed mediators (Fig. 14.3A). Dense lipid bodies contain arachidonic acid. The membrane-bound secretory granules contain crystalline structures that resemble scrolls, lattices, crystals, or whorls (Fig. 14.3B–D).55 Electron microscopy demonstrates that MCT granules contain predominantly scroll patterns, and MCTC granules contain predominantly lattice patterns.56

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A 2 µm

B

C

D 1 µm

E

F Fig. 14.3  Ultrastructural appearance of human mast cells on electron microscopy. (A) Transmission electron micrograph showing a skin mast cell containing many prominent electron-dense granules. The 1- to 2-µm processes emanating from the plasma membrane (arrow) are termed microplicae. (B) through (D) At higher magnification, electron-dense granules show three structural arrangements: scrolls (B), gratings (C), and lattices (D). (E) Anaphylactic mast cell degranulation with fusion of mast cell granules. (F) Piecemeal mast cell degranulation in asthmatic bronchial mucosa. Scale bar = 3 µm. (A through D, Courtesy Daniel S. Friend, MD, Harvard Medical School; E and F, courtesy Susan Wilson, PhD, University of Southampton.)

CHAPTER 14  Biology of Mast Cells and Their Mediators MCs can appear round or spindle-shaped in tissues and in humans; the most effective way to identify the location and subtype of MCs histologically is to use immunohistochemistry with antibodies raised against MC-specific proteases. The backbone of the granule matrix is formed from proteoglycans, long core proteins with glycosaminoglycan (GAG) side chains. In human MCs, the proteoglycan content of the granules is predominantly heparin, which stabilizes the β-tryptase tetramer, with some chondroitin E also present. Neutral proteases, acid hydrolases, and histamine molecules are attached to heparin by ionic linkage to the sulfate groups on the GAGs. The sulfate groups generate an acidic environment within the granules that maintains the mediators in an inactive state. IgE-dependent activation of the MC induces granule swelling, crystal dissolution, and granule fusion, both with surrounding granules and the cell membrane. This is followed by exocytosis with release of mediators into the extracellular space. This process is termed compound exocytosis or anaphylactic degranulation (Fig. 14.3E). Once in the extracellular space, the neutral pH mobilizes and activates the mediators. MC activation through this pathway is not a cytotoxic event but is energy-dependent; even after almost complete degranulation, HLMCs are able to survive and regranulate over a period of 48 hours.57 However, in many diseased tissues including asthmatic bronchial mucosa, the ultrastructural appearance of MCs typically demonstrates piecemeal degranulation,58,59 where there is variable loss of granule contents, although the granules and their membranes remain intact (Fig. 14.3F). The mechanisms leading to piecemeal degranulation in MCs are poorly understood and require further research. The effects of preformed mediators often remain localized, because histamine is short-lived in vivo, being broken down by diamine oxidase (histaminase) and histamine-N-methyltransferase (HMT), and the active tetramer of tryptase rapidly dissociates into inactive monomers in the absence of heparin. In addition to the stored granule-derived mediators, newly formed metabolites of arachidonic acid are also released from MCs after IgEdependent activation (Table 14.3). This phospholipid is liberated from the cell membrane, nuclear envelope, or lipid bodies during immunologic activation and is rapidly oxidized by either the cyclooxygenase or the lipoxygenase pathways to form the eicosanoids prostaglandin D2 (PGD2) and leukotriene C4 (LTC4), respectively (see Chapter 9). MCs also synthesize and secrete numerous cytokines and chemokines depending upon the stimulus (see later). The principal biologic properties of MC autacoids (histamine, PGD2, LTC4), proteases, and cytokines are summarized in Tables 14.3 and 14.4. A list of MC-derived chemokines is provided in Table 14.5. Further specific points of interest in relation to mediator function in disease are discussed later.

MECHANISMS OF MAST CELL ACTIVATION IgE-Dependent Activation The best-studied mechanism of MC activation, and the one considered most relevant to allergic disease, is activation through the high-affinity IgE receptor FcεRI (for reviews see reference 60 and Chapter 24). FcεRI is a tetrameric structure composed of an α chain (FcεRIα) that binds IgE, a β chain signaling subunit (FcεRIβ or MS4A2), and two γ subunits that exist as an immunoreceptor tyrosine-based activation motif (ITAM)– containing homodimer signaling subunit (FcεRIγ). The tetramer is stabilized on the cell surface by the binding of IgE. Thus therapeutic anti-IgE, which binds to the receptor-binding domain of IgE and thus prevents IgE from binding to the α chain, reduces cell-surface FcεRI expression. FcεRI expression is also increased further by IL-4 and IL-13, which enhance FcεRI-dependent secretion.61 Most of the downstream signaling events identified after FcεRI engagement have been defined

221

TABLE 14.3  Classic Preformed and Newly

Generated Human Mast Cell Autacoid Mediators and Proteases With Examples of Their Biologic Effects Mediator

Activity

Histamine (stored)

Bronchoconstriction; tissue edema; ↑ vascular permeability; ↑ mucus secretion; ↑ fibroblast proliferation; ↑ collagen synthesis; ↑ endothelial cell proliferation, dendritic cell differentiation and activation

Heparin (stored)

Anticoagulant; mediator storage matrix; sequesters growth factors; fibroblast activation; endothelial cell migration

Tryptase (stored)

Degrades respiratory allergens and cross-linked IgE; generates C3a and bradykinin; degrades neuropeptides; TGF-β activation; increases basal heart rate and ASM contractility; ↑ fibroblast proliferation and collagen synthesis; epithelial ICAM-1 expression and CXCL8 release; potentiation of mast cell histamine release; neutrophil recruitment

Chymase (stored)

↑ Mucus secretion; ECM degradation, type I procollagen processing; converts angiotensin I to angiotensin II; ↓ T cell adhesion to ASM; activates IL-1β, degrades IL-4, releases membrane-bound SCF

PGD2 (synthesized)

Bronchoconstriction; tissue edema; ↑ mucus secretion; dendritic cell activation; chemotaxis of eosinophils, Th2 cells, and basophils via the CRTH2 (CD294) receptor

LTC4/LTD4 (synthesized)

Bronchoconstriction; tissue edema; ↑ mucus secretion; enhances IL-13-dependent airway smooth muscle proliferation; dendritic cell maturation and recruitment; eosinophil IL-4 secretion; mast cell IL-5, IL-8, and TNF-α secretion; tissue fibrosis

ASM, Airway smooth muscle; CRTH2, chemoattractant receptor of Th2 cells; ECM, extracellular matrix; ICAM-1, intercellular adhesion molecule 1; IgE, immunoglobulin E; IL, interleukin; LTC4, leukotriene C4; LTD4, leukotriene D4; PAF, platelet-activating factor; PGD2, prostaglandin D2; SCF, stem cell factor; TGF-β, transforming growth factor β; TNF-α, tumor necrosis factor α.

in rodent models. Where human cells have been investigated, some important differences in signaling have been observed.62 A detailed description of the multiple signaling cascades activated after receptor activation can be found in Chapter 24. For IgE-dependent processes, influx of extracellular Ca2+ is a critical requirement for the release of both preformed and newly generated mediators and several Ca2+-dependent cytokines.63 IgE-dependent Ca2+ influx in both mouse and human MCs is carried predominantly by the store-operated Ca2+-selective channel Orai1 (also known as CRACM1), with minor contributions from Orai2 and Orai3.64,65 In an Orai1-/mouse, MC degranulation, LTC4 release, and tumor necrosis factor α (TNF-α) production after IgE-dependent activation were markedly impaired.66 HLMCs express mRNA for Orai1-3, and protein for Orai1 and 2.64 The use of specific blockers of Orai channels reduced HLMC IgE-dependent Ca2+ influx, and histamine, LTC4, and cytokine (IL-5, IL-13, TNF-α, CXCL8) release by approximately 50%.64

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TABLE 14.4  Biologic Activity of Human Mast Cell–Derived Cytokines In Vitro Cytokine

Target Cells

Biologic Effects

IL-4

B cells T cells Monocyte-macrophages Eosinophils Fibroblasts Endothelial cells Mast cells ASM cells

Proliferation; ↑ MHC class II, ↑ CD40, ↑ CD25; ↑ IgE production, ↑ IL-6 Proliferation, differentiation to Th2 phenotype ↓ H2O2 and O2-, ↓ parasite killing, ↓ tumoricidal activity; monocyte → macrophage differentiation; ↓ IL-1, IL-6, IL-8, and TNF-α, ↑ MHC class II and CD23; 15-lipoxygenase expression Transendothelial migration Proliferation, chemotaxis, ↑ ECM; ↑ ICAM-1 Proliferation, ↑ VCAM-1, ↓ ICAM-1 ↑ FcεRI, ↑ ICAM-1 Bronchial hyperresponsiveness

IL-3, IL-5, GM-CSF

Eosinophils

Growth, adhesion, transendothelial migration, chemotaxis, activation, and prolonged survival

IL-6

B cells T cells Mast cells

↑ Immunoglobulin secretion including IgE Growth, differentiation Survival of HLMC MCT

IL-13

B cells Monocyte-macrophages Eosinophils Endothelial cells Fibroblasts ASM cells Epithelial cells

IgE synthesis Same as for IL-4 Activation, ↑ survival ↑ VCAM-1 ↑ CCL11 secretion Bronchial hyperresponsiveness ↑ TSLP expression; mucus hypersecretion

TNF-α

Monocyte-macrophages T cells Neutrophils Eosinophils Endothelial cells Fibroblasts Mast cells

↑ Cytotoxicity, chemotaxis, prolonged survival MHC class II and IL-2R expression, proliferation ↑ Cytotoxicity, chemotaxis ↑ Cytotoxicity, oxidant production ↑ E-selectin, ↑ ICAM-1, ↑ VCAM-1; ↑ recruitment of most leukocytes Growth, chemotaxis, ↓ collagen, ↑collagenase, ↑ IL-6, ↑ IL-8 Histamine and tryptase secretion

SCF

Mast cells

Growth, differentiation, survival; chemotaxis

NGF

B cells T cells Eosinophils Basophils Neutrophils Monocyte-macrophages Fibroblasts Smooth muscle cells Mast cells

Differentiation, proliferation, ↑ immunoglobulin secretion Differentiation, proliferation Proliferation Activation, mediator release Chemotaxis, survival, mediator release Proliferation, mediator release Migration, contraction Migration, contraction, proliferation Differentiation, survival, activation, mediator release

TGF-β1

ASM cells Epithelial cells Endothelial cells

Differentiation, activation, increased α-smooth muscle actin, increased contractility Inhibition of proliferation ↑ Angiogenesis

FGF-2

Fibroblasts Endothelial cells NK cells Macrophages Dendritic cells T cells

Proliferation ↑ Angiogenesis ↑ Cytotoxicity Development, maturation Activation, maturation, ↑ IFN-γ ↑ Survival of activated T cells, ↑ Th1 phenotype

TSLP

Dendritic cells Mast cells

Induction of Th2 immunity ↑ IL-13 expression

ASM, Airway smooth muscle; FGF-2, fibroblast growth factor-2; GM-CSF, granulocyte-macrophage colony-stimulating factor; ICAM-1, intercellular adhesion molecule 1; IFN-γ, interferon γ; IgE, immunoglobulin E; IL, interleukin; MHC, major histocompatibility complex; NGF, nerve growth factor; NK, natural killer; SCF, stem cell factor; TGF-β1, transforming growth factor β1; TNF-α, tumor necrosis factor-α; TSLP, thymic stromal lymphopoietin; VCAM-1, vascular cell adhesion molecule 1.

K+ channels have the potential to modulate Ca2+ influx and hence mediator release because of their profound effects on the cell membrane potential. In both RBL cells and rat IL-3-dependent BMMCs, an inwardlyrectifying K+ channel (Kir2.1) is open when the cells are at rest. However, currents carried by these Kir channels have never been seen in human

skin MCs, HLMCs, HBMMCs, HPBMCs, LAD2 cells, or HMC-1 cells.20,62,67,68 After IgE-dependent activation, human MCs rapidly open the Ca2+-activated K+ channel KCa3.1.62,68 This channel has also been identified in murine MCs.69 Orai1 and KCa3.1 interact in the HLMC plasma membrane with the result that KCa3.1 indirectly enhances Ca2+

CHAPTER 14  Biology of Mast Cells and Their Mediators

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TABLE 14.5  Human Mast Cell-Derived Chemokines and Their Biologic Effects In Vitro Chemokine and Known Stimuli

Target Cells

Biologic Effects

T cells

T cell recruitment

CCL2 - Constitutive - SCF - FcεRI - Dengue virus - IL-1β - Catestatin

Mast cells Epithelial cells Fibrocytes T cells Eosinophils Monocytes Basophils

Chemotaxis Chemotaxis, proliferation Chemotaxis Induces Th2 phenotype Chemotaxis Chemotaxis Activation, mediator release

CCL3 - FcεRI - Nod1 ligand - Dengue virus - Catestatin - IgE

Mast cells T cells Macrophages Neutrophils Eosinophils Monocytes Basophils

Activation, mediator release Chemotaxis (selective for Th1), induces Th1 phenotype Differentiation Chemotaxis (in vivo), cytotoxicity Chemotaxis Chemotaxis Activation, mediator release

CCL4 - FcεRI - Nod1 ligand - Dengue virus - Catestatin

T cells Eosinophils Neutrophils

Chemotaxis (selective for Th1), induces Th1 phenotype Chemotaxis Chemotaxis (in vivo)

CCL5 - FcεRI - Dengue virus

Mast cells T cells Eosinophils Monocytes

Chemotaxis Chemotaxis (selective for Th1), induces Th1 phenotype Chemotaxis Chemotaxis

CCL7 - FcεRI

Eosinophils Monocytes Basophils

Chemotaxis Chemotaxis Activation, mediator release

CCL8 - Constitutive - Dengue virus

T cells Basophils B cells Eosinophils

Chemotaxis Chemotaxis, histamine release Chemotaxis Chemotaxis

CCL13 - Constitutive

Eosinophils Basophils Monocytes Immature dendritic cells T cells Endothelial cells Fibroblasts

Chemotaxis Histamine release, chemotaxis Chemotaxis Chemotaxis Chemotaxis Chemotaxis Inhibits apoptosis

CCL17 - Constitutive

T cells

Chemotaxis (selective for Th2)

CCL18 - FcεRI

Epithelial cells T cells B cells Immature dendritic cells Basophils Macrophages

Epithelial mesenchymal transition Chemotaxis Modulates activation and chemokine-induced responses Chemotaxis Chemotaxis, histamine release Chemotaxis, activation

CCL19 - Constitutive

Airway smooth muscle cells

Chemotaxis

CCL20 - FcεRI - Pseudomonas

Dendritic cells T cells

Chemotaxis Chemotaxis

CCL22 - Constitutive

T cells

Chemotaxis (selective for Th2)

-CCCCL1 - FcεRI - IgE

Continued

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TABLE 14.5  Human Mast Cell-Derived Chemokines and Their Biologic Effects In Vitro—cont’d Chemokine and Known Stimuli

Target Cells

Biologic Effects

CCL23 - FcεRI

Monocytes T cells Neutrophils Myeloid progenitor cells Dendritic cells

Chemotaxis, adhesion, mediator release Chemotaxis Chemotaxis Suppressed development Chemotaxis

CCL25 - Constitutive

Periosteal progenitor cells Lymphocytes Mesenchymal stem cells

Chemotaxis Adhesion Chemotaxis

CCL28 - Constitutive

Lymphocytes T cells Hematopoietic progenitor cells

Adhesion Migration Growth and survival

Neutrophils Airway smooth muscle cells

Chemotaxis, adhesion Migration

CXCL3 - FcεRI

Airway smooth muscle cells

Migration

CXCL4 - Constitutive

T cells Endothelial cells Monocytes Neutrophils

Chemotaxis Angiogenesis Impairs chemotaxis Adhesion

CXCL5 - Constitutive

Neutrophils

Chemotaxis

CXCL7 - Constitutive - Dengue virus

Neutrophils

Chemotaxis, degranulation

CXCL8 - Constitutive - IgE - FcεRI - CXCL12 - Substance P - TSLP + IL-1β - IL-33 - Nod1 ligand - Dengue virus

Mast cells Endothelial cells Neutrophils Eosinophils

Reduces chemotaxis and mediator release Chemotaxis, proliferation, survival, angiogenesis Chemotaxis Chemotaxis following priming with IL-3, IL-5, or GM-CSF

CXCL10 - Constitutive - Dengue virus

Mast cells

Chemotaxis

CXCL14 - Constitutive

Bacteria Monocytes Dendritic cells

Antimicrobial action Chemotaxis Chemotaxis

CXCL16 - Constitutive

T cells Monocytes

Chemotaxis Chemotaxis

CXCL17 - Constitutive

Bacteria Dendritic cells Monocytes

Antimicrobial action Chemotaxis Chemotaxis

T cells

Chemotaxis, activation of apoptosis

Mast cells Leukocytes Fibroblasts

Chemotaxis Chemotaxis, adhesion Increased MMP2 production

-CXCCXCL2 - FcεRI

-CXCL1 - Constitutive -CX3CCX3CL1 - Constitutive

IL, Interleukin; GM-CSF, granulocyte macrophage–colony stimulating factor; MMP, matrix metalloproteinase; TSLP, thymic stromal lymphopoietin.

CHAPTER 14  Biology of Mast Cells and Their Mediators influx and histamine release but is not critical for secretion, and can thus be considered as increasing the signal strength of an immunologic stimulus.62,68–70 This channel is closed in a membrane-delimited manner by compounds that inhibit MC secretion and migration via Gs-coupled receptors, including β2-adrenoceptor agonists,71 adenosine72 (via the A2a adenosine73 receptor), and PGE2, providing a mechanism for the coupling of receptor activation to impaired secretion. The truncation of FcεRIβ (MS4A2trunc) described previously is not only important for regulating human MC survival and proliferation but is also important for microtubule formation and degranulation in the LAD2 human MC line. In this respect, MS4A2trunc appears to function by trafficking adaptor molecules and kinases to the pericentrosomal and Golgi region in response to Ca2+ signals.74 MS4A2trunc contains a putative calmodulin-binding domain, and mutagenesis studies suggest that calmodulin binding to MS4A2trunc in the presence of Ca2+ is critical for MS4A2trunc function. Thus gene targeting of MS4A2trunc in LAD2 cells attenuated microtubule formation, degranulation, and IL-8 production downstream of Ca2+ signals.74 Thus the function of the MS4A2 gene extends beyond the production of FcεRIβ and FcεRI expression and potentially plays a crucial role in the regulation of both human MC survival and activation.

Monomeric IgE-Dependent Mast Cell Activation In addition to the cross-linking of FcεRI by allergen, the binding of monomeric IgE alone to FcεRI initiates intracellular signaling events and Ca2+ influx.75–77 In rodents this results in the release of granulederived mediators and the secretion of cytokines including IL-6. This IL-6 acts in an autocrine manner and prolongs MC survival after growth factor withdrawal.78 In HCBMCs, monomeric IgE binding alone induces the release of CCL1, CCL3, and GM-CSF without histamine release. However, in HLMCs IgE binding induces the secretion of histamine, LTC4 and CXCL8, which is markedly enhanced in the presence of SCF.75 Interestingly, in both rodent MCs and HLMCs, on-going signaling is dependent on the presence of “free” IgE, and this ceases immediately when free IgE is removed, suggesting that these findings are physiologically relevant.75,79 The mechanisms behind this are uncertain but in part appear to involve FcεRI aggregation. These observations appear important biologically because both SCF and free IgE concentrations are elevated in asthmatic airways,80 and there is a good correlation between total serum IgE and the presence of asthma and bronchial hyperresponsiveness. This provides a mechanism for the ongoing activation of MCs through FcεRI in the absence of acute allergen exposure and could explain, in part, the efficacy of anti-IgE therapy in chronic allergic disease.

Mast Cell Activation by Superantigens Human lung and/or heart MCs can be activated through FcεRI by bacterial, viral, and endogenous superantigens including several staphylococcal enterotoxins, Peptostreptococcus protein L,81 protein Fv (released from the liver during infection with hepatitis viruses A, B, C, and E),82 and the HIV-1 protein gp12083 (and reviewed in reference 84). These superantigens bind and cross-link IgE bound to FcεRI, leading to classical FcεRI-dependent mediator release (reviewed in reference 84). Increased exposure to staphylococci has been documented in patients with atopic eczema, asthma, and nasal polyposis and has been linked to asthma severity.85–87 Staphylococcal superantigens may therefore contribute to both the pathogenesis and severity of these diseases and engage MCs in the process.

Activation of Mast Cells Independently of FcεRI Human MCs are also activated through a plethora of non-IgE-dependent stimuli that are relevant to many disease processes.88 These include

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proteases (including tryptase), cytokines (e.g. SCF, TNF-α, IFN-γ, IL-33), complement, adenosine, toll-like receptor (TLR) ligands, neuropeptides (particularly skin MC), Ig-free light chains (mouse), pollutants, occupational agents (e.g., plicatic acid in western red cedar), cell-cell contact, and hyperosmolarity (summarized in Fig. 14.4). It is therefore evident that the numerous mast cells resident within human airways are able to respond to all of the stimuli implicated in both the development of asthma and the ongoing symptomatology and exacerbations. The signaling pathways used by many of the receptors involved in the responses to these stimuli are poorly understood in human mast cells. Nevertheless there is evidence of important receptor cross-talk. For example, SCF potentiates IgE-dependent MC mediator and cytokine release at 10 ng/mL, whereas at 100 ng/mL it directly activates the cells. SCF enhances LTD4-induced Ca2+ mobilization and expression of inflammatory genes such as TNF-α in the LAD2 human MC line,89 and LTD4 and SCF together induce the synergistic activation of MC Kit.89 SCF is also interesting because it inhibits β2-adrenoceptor (β2-AR) signaling in HLMC and HMC-1 within minutes of exposure through the rapid phosphorylation of the β2-AR on Tyr350 followed by internalization.90 This leads to impaired β2-AR-dependent inhibition of both histamine and LTC4 release and inhibition of β2-AR-dependent ion channel modulation.90 Remarkably, in the presence of IgE and SCF together, salbutamol counterintuitively increases histamine release in a dose-dependent manner.90 If this occurs in vivo, it might explain why regular use of β2-AR agonists sometimes reduces asthma control91 and actually enhances MC mediator release and the early asthmatic response after allergen challenge.92,93 Because MCs within the ASM in asthma are exposed to membrane SCF present on the surface of ASM cells, such a mechanism is highly plausible. SCF and Kit expression are increased in asthma, and so targeting SCF/Kit might not only exert antiinflammatory/ remodeling effects through the inhibition of MC function but remove the potentially detrimental effects of β2-AR agonists. In vitro, TNF-α stimulates MC degranulation directly and enhances the effects of other stimuli.94,95 After IgE-dependent activation the release of preformed MC-associated TNF-α exerts a positive autocrine feedback signal to augment NF-κB activation and further production of TNF-α and other cytokines such as GM-CSF and IL-8.96 However, large studies of anti-TNF-α treatment in unselected patients with asthma have failed to show clinical benefit97,98 and have significant adverse effects.99 IL-33 expression by ASM100 and airway epithelium101 is increased in asthmatic airways. Human cord blood–derived MCs express the receptor for IL-33 known as ST213 and respond to IL-33 exposure by releasing several cytokines and chemokines including IL-5, IL-8, IL-13, and TNFα.13,102 In the presence of IgE, IL-33 may induce human cord blood MC degranulation and PGD2 generation,102 but this is not a consistent finding.13 In HMC-1 cells and mouse bone marrow–derived MCs, IL33R-dependent signaling induced and required cross-activation with Kit, with evidence of a physical association between the two receptors.103 It has been suggested that ASM-derived IL-33 may play a key role in the activation of ASM-infiltrating MCs and the propagation of ASM dysfunction in asthma. However, chronic exposure (>72 hours) of human cord blood MCs to IL-33 resulted in substantially reduced Ca2+ mobilization and degranulation in response to FcεRI-dependent activation.104 These findings suggest that IL-33 may play a protective rather than a causative role in MC activation under chronic conditions. The potential outcomes of targeting IL-33 in asthma remain uncertain. Thymic stromal lymphopoietin (TSLP) is another epithelial- and ASM-derived cytokine, which works cooperatively with IL-1β to induce the release of numerous cytokines and chemokines from human MCs, including IL-5 and IL-13,105–107 but it does not induce degranulation. Tryptase induces histamine release from human MCs in several tissues, including the lung.108,109 In keeping with this, tryptase inhibitors

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Allergens Superantigens Autoantibodies IgE

PAF

Ca2+ influx

+

Orai1

Adenosine

SCF

FcεRI PAF receptor A2B, A3 receptors

C5a

Drugs (including aspirin)

PI3K PLCγ

Neurotensin (psychological CRH stress)

IP3

CRH-R

Flagellin

TLR-5

PAR-2

+

CADM1

↑ Ca2+

CD88

Proteases

Kit

TLR-4

Ca2+ store release

LPS

TLR-2

PGN

CD48

Degranulation, arachidonic acid metabolism, cytokine generation, gene transcription

FimH

TLR-3

dsRNA ST2

Physical stimuli Hyperosmolarity ADDS

Air pollutants (NO2, O3–, PM10)

FcR

FcγRI

Bacterial products

Viral products

Cytokines (e.g. TNFα, TSLP)

Integrin β1

IL-33

Occupational agents (plicatic acid)

Cell-cell signals IgG

Parasite IgG

IgE

Ig free light chains

MBP-1 (with membrane SCF priming)

(e.g. airway smooth muscle cell, fibroblasts)

Fig. 14.4  Many non–IgE-dependent pathways exist for the activation of human mast cells and are likely to contribute to mast cell activation in chronic diseases such as asthma, rheumatoid arthritis, and pulmonary fibrosis.

reduce FcεRI-dependent human lung MC degranulation, suggesting that tryptase amplifies this response in an autocrine fashion.108 In animal studies, exogenously administered tryptase induced bronchoconstriction and AHR,110,111 whereas pretreatment with antihistamines blocked these effects, suggesting that MC activation is likely to play a role. Tryptase also potentiated the contractile response of sensitized human bronchi to histamine.112

Directional Mast Cell Mediator Release Mast cell degranulation is undoubtedly a highly regulated process, highlighted by the directional degranulation described by Joulia et al.113 In this study, human peripheral blood–derived MCs were examined with live cell imaging, using fluorochrome-labeled avidin as a probe that binds to heparin during degranulation. Of note, granule contents including tryptase and chymase were retained on the MC surface for several hours after FcεRI-dependent activation. This is in keeping with observations that MCs express IL-4, a heparin/heparan-binding cytokine on their cell surface,114,115 and that they are capable of presenting functionally active proteoglycan-bound molecules such as IFN-γ on their cell surface when added exogenously.116 IgE bound to CD20 on B cells was able to activate FcεRI on human MCs in coculture leading to MC degranulation, and this degranulation

localized predominantly at points of MC-B cell contact. Similar results were seen with IgG-bound B cells through activation of FcγRIIA on human MCs, and also in the presence of IgG opsonized parasites. This process was termed the antibody-dependent degranulatory synapse (ADDS) and demonstrates that MCs establish discrete directional cell-cell cross-talk.

Toll-Like Receptors Human progenitor-derived MC and mouse MC express TLRs 1-7 and -9. These play an important role in the innate host response to pathogens, activating diverse programs of gene expression depending on the stimulus. For example, in murine MC functional responses to TLR2 (the receptor for bacterial peptidoglycan) results in production of TNFα, IL-4/5/6/13 and IL-1β, whereas activation of TLR4 (the receptor for LPS) induces production of TNF-α, IL-1β, and IL-6/13, but not IL-4/5. In addition, activation of TLR-2 but not TLR-4 induces Ca2+ mobilization, degranulation, and LTC4 production.117,118 Examination of the gene expression profile from HCBMC after activation with LPS compared with anti-IgE demonstrates that both induce a core response, plus an LPS or anti-IgE–specific program of gene expression.53 Perhaps of more relevance to asthma is MC activation via TLR3, the ligand for which is double-stranded viral RNA.119 Because viruses are a common cause

CHAPTER 14  Biology of Mast Cells and Their Mediators for asthma exacerbations, a viral-mediated MC activation response may be an important contributor to the deteriorating airway physiology.

MAST CELLS IN ALLERGIC DISEASES AND ASTHMA MCs are involved in biologic processes in health and the pathophysiology of many diverse diseases. However, they are best known with respect to their role in asthma and related allergic diseases. IgE-mediated allergic diseases include asthma, allergic rhinoconjunctivitis, eczema, urticaria, and systemic anaphylaxis. It is evident from Tables 14.3 through 14.5 that MC secrete a plethora of autacoids, proteases, and cytokines, which are relevant to the pathophysiology of allergy. Depending on the site of mediator release, acute signs and symptoms manifest clinically as rhinitis, conjunctivitis, urticaria, angioedema, erythema, bronchospasm, diarrhea, vomiting, and hypotension that can be fatal in severe reactions (such as anaphylactic shock). The potential role of MCs in the process of allergen sensitization will be discussed, followed by a description of their role in anaphylaxis and seasonal allergic rhinoconjunctivitis where an IgE-dependent role appears relatively clear cut. Some consideration will then be given to atopic dermatitis (eczema), in which the evidence for MC involvement is less apparent, followed by an in-depth review of the evidence implicating MC in the pathophysiology of chronic asthma where the involvement of allergen and IgE is probably one of many factors driving MC activation. The role of the MC in urticaria is discussed in detail in Chapter 35.

Mast Cells and Allergen Sensitization Both rodent and human MCs express class II MHC antigens and are capable of presenting soluble antigens to T cells with subsequent T cell proliferation.120 Rat MCs are capable of skewing the differentiation of T cells toward the Th2 phenotype through the release of IL-4,121 and it is therefore plausible that MCs could contribute to Th2 differentiation at the onset of an immune response. This could occur in the absence of IgE, because a number of diverse allergens including bee venom phospholipase (PL)A2 and Der p I induce the release of histamine and IL-4 from HLMC in the absence of cell-bound IgE.122,123 Phospholipases and proteases are associated with further allergens including those originating from cockroaches, fungal spores, pollens, and cats. The molecular mechanism by which proteases release mediators from MCs involves in part the protease-activated receptor (PAR)2. The reason that allergen sensitization does not occur in everyone may be explained by environmental factors (level of allergen exposure), genetic factors (MC releasability, epithelial integrity/permeability, local antiprotease activity, and regulation of cytokine production), and immunologic tolerance. MCs also influence dendritic cell development and their ability to activate T cells. For example, histamine increases IL-10 and decreases IL-12 production by mature dendritic cells, with the result that naïve T cells become polarized toward a Th2 phenotype. Similar effects have been observed with PGD2,124 and MC-dependence for the generation of Th2-promoting dendritic cells is evident in mice in vivo.125 MC exosomes induce immature dendritic cells (DCs) to become mature plasmacytoid DCs capable of antigen presentation to T-cells by upregulating MHC class II, CD80, CD86, and CD40 molecules.126 MC-derived TNF-α is important for dendritic cell migration during immune responses.127

Mast Cells in Anaphylaxis Systemic anaphylaxis is the most striking and immediately life-threatening IgE-dependent reaction. Food allergies are the most common cause, but it can also result from drug allergies, in particular penicillin, insect venom such as bee stings, or physical stimuli such as exercise or may

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be idiopathic. Reactions that are clinically indistinguishable from anaphylactic reactions but that are not IgE-dependent are sometimes termed anaphylactoid reactions. Anaphylaxis is a syndrome with varied triggers and clinical presentations, which is mediated predominantly by MCs and perhaps basophils. In exercise-induced anaphylaxis, MC degranulation has been demonstrated in the skin using electron microscopy.128 However, the best marker of systemic MC activation in anaphylaxis is an acute rise in the concentration of β-tryptase in the peripheral circulation.129 Unlike α-tryptase, which is released by MC constitutively, β-tryptase is stored in MC granules and released after IgE-dependent activation, and therefore represents a more specific marker of acute activation than total tryptase. Histamine and β-tryptase are released from MC together, but whereas histamine concentrations in blood peak within 5 minutes, the peak of tryptase is maximal between 15 and 120 minutes after the onset of symptoms.130 This is because tryptase is a larger molecule than histamine but also remains bound to heparin longer, delaying its diffusion from the tissue. Tryptase is therefore not only a more specific marker for MC activation than histamine, which is also released by basophils, but more convenient to measure after a suspected anaphylactic event. Interestingly, patients with systemic mastocytosis are at increased risk of anaphylactic and anaphylactoid reactions and often demonstrate increased baseline serum concentrations of tryptase. This is predominantly α-tryptase which is released constitutively and reflects the increased MC mass.131 The key difference between anaphylaxis and other MC-associated diseases is that anaphylaxis involves the systemic activation of MCs leading to gastrointestinal symptoms, urticaria, cardiovascular collapse, and respiratory embarrassment due to bronchospasm and/or laryngeal edema. The reason for the systemic spread of MC activation has been assumed to relate to the systemic spread of allergen, but this is perhaps implausible, and amplification mechanisms need to be considered, such as neurologic reflexes. Recent work has also identified platelet-activating factor (PAF) as a potential amplifier. PAF is implicated in the pathogenesis of anaphylaxis in humans and can activate human MCs. In the skin, neural reflexes activate MCs after the administration of PAF, but isolated human skin MCs do not degranulate in response to PAF directly. HLMC and HPBMC release histamine and PGD2 rapidly in response to concentrations of PAF similar to those measured in human blood during anaphylactic reactions.132 This response is partially dependent on Ca2+ influx. PAF also induces the release of CXCL8 and transiently upregulates mRNA expression for several other chemokines.132 PAF signals via the PAF receptor coupled to Gαi with activation of phospholipase (PL)-Cγ1 and PLCβ2 and also enhances IgE-dependent mediator release. The observations that PAF can directly activate HLMC and HPBMC and potentiate IgE-dependent secretion provides a mechanism whereby PAF amplifies the effects of allergen exposure. This provides a biologically plausible mechanism that links elevated PAF concentrations in human anaphylaxis to an amplified airway MC response. The source of PAF in human anaphylaxis is uncertain but in part might come from MCs.133 Here it is suggested that PAF generated locally by MCs, and perhaps other cells in response to MC activation at the point of allergen contact, may lead to an exaggerated MC response at more distal sites, such as the airway. In turn, skin MCs may be activated indirectly by PAF through neural reflexes.134 Such a PAF amplification loop may be a key factor in the generation of systemic anaphylaxis.

Mast Cells in Allergic Rhinitis Allergic rhinitis is characterized by the symptoms of nasal and palatal itch, rhinorrhea, sneezing, nasal blockage, and, in severe cases, anosmia. Symptoms may be either seasonal (SAR) due to sensitivity to seasonal

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SECTION A  Basic Sciences Underlying Allergy and Immunology

allergens such as grass, or perennial (PAR), often due to house dust mite sensitivity.

Seasonal and Perennial Allergic Rhinitis (see Chapter 40).  Allergic rhinitis is characterized by the presence of a mucosal inflammatory response, similar in many respects to that seen in asthma. In both SAR and PAR there are increased numbers of eosinophils in the lamina propria and epithelium, increased MC numbers in the epithelium, and increased expression of Th2 cytokines in MCs, eosinophils, and T lymphocytes.135–138 Increased numbers of CD34+, tryptase-negative cells have been documented in the nasal epithelium, suggesting that there is recruitment of MC progenitors to this site.139 These intraepithelial MCs are predominantly of the MCT phenotype, whereas those in the lamina propria are about 60% MCTC.135,140 MCs in the airway epithelium and lamina propria are activated with ultrastructural evidence of MC degranulation,141 and increased concentrations of LTC4 but not histamine are present in nasal lavage.142 In addition, MCs express IL-4, IL-5, IL-6, and TNF-α in the nasal mucosa and demonstrate increased expression of IL-4 in both SAR and PAR, a feature that is reversed by the application of topical corticosteroids.137 Although histamine concentrations are not elevated, antihistamine therapy is highly effective at ameliorating symptoms. Anti-IgE therapy is also effective at treating both SAR and PAR when serum IgE levels are suppressed effectively, supporting the view that these are IgE-driven MC-dependent diseases.143 MCs recovered from the nasal mucosa of patients with allergic rhinitis induce plasma cell IgE synthesis through the IgE-dependent release of IL-4 and IL-13 and the interaction of CD40 (expressed by B cells) with its ligand (CD40L expressed by MCs), suggesting that MCs might contribute to the local production of IgE in the nasal mucosa.144 This is relevant because increased numbers of IgE-secreting plasma cells are present in the allergic nasal mucosa.145 In addition, nasal exposure to allergen and diesel exhaust particles induces a rapid upregulation of IL-4 within human nasal MCs,146 and diesel exhaust particles potentiate allergen-induced histamine release.147 In summary, ongoing MC activation within the allergic nasal mucosa coupled with the biologic effects of MC products (Tables 14.3 through 14.5) can explain much of the symptomatology and pathology of allergic rhinitis.

of the disease can give rise to more severe symptoms including pain, corneal scarring, cataract, or glaucoma with the potential to threaten sight. The most common form is seasonal allergic conjunctivitis (SAC), with perennial allergic conjunctivitis (PAC), atopic keratoconjunctivitis (AKC), atopic blepharoconjunctivitis (ABC), and vernal conjunctivitis (VC) less common (see Chapter 38, and for a detailed review reference 155). MCs are recognized as central effector cells in all types of allergic eye disease, demonstrating increased numbers and morphologic evidence of degranulation.156–158 After conjunctival allergen challenge, elevated levels of histamine, tryptase, and LTC4 are present in tears.159,160 The MCTC phenotype predominates in the normal conjunctiva, but in PAC, SAC, and VKC the number of MCT cells increases in both the conjunctival epithelium and subepithelial layers.161,162 In contrast, in AKC and ABC, the numbers of MCTC increase.163 It has been proposed that MCTC contribute to tissue fibrosis in various diseases, and their increased number may therefore contribute to the excess fibrosis evident in AKC and ABC. As in the nose and bronchi, the MCs in the conjunctiva in SAC demonstrate heterogeneity in terms of cytokine expression, with the MCTC subset expressing predominantly IL-4 and IL-13, whereas the MCT subset expresses IL-5 and IL-6.164 Allergen challenge again produces early- and late-phase responses, suggesting similar mechanisms to those in the nose and lower airway. Antihistamines are useful for the treatment of symptoms suggesting that MC-derived histamine contributes to the symptomatology.

Mast Cells in Atopic Dermatitis (Eczema) and Urticaria

Experimental Allergen-Induced Rhinitis.  Nasal allergen challenge provides further evidence that MCs are important effectors and orchestrators of rhinitis. Most patients with allergic rhinitis develop an early phase response (EPR) after appropriate allergen challenge, with the acute development of rhinitic symptoms. In about 40% of subjects, a late phase response (LPR) follows after about 6 hours with a recurrence of symptoms. During the EPR there is the release of a spectrum of inflammatory mediators including histamine,148,149 tryptase,148,149 PGD2,150 and LTC4,151,152 indicative of MC activation. These symptoms are attenuated by antihistamines and markedly inhibited by anti-IgE therapy.153 During the LPR there is tissue infiltration by eosinophils and CD4+ T cells, but this is also markedly attenuated by anti-IgE therapy, suggesting that MC-driven events at the time of challenge are key. A model using low-dose repeat allergen challenge more closely mimics events during the pollen season.154 After repeated daily challenges, MC numbers start to increase in the nasal epithelium at day 6, indicating that if this involves recruitment and differentiation of progenitors, then this occurs relatively quickly.

Although atopic dermatitis (Chapter 33) is strongly associated with atopy, as indicated by its name, the role of IgE in its pathogenesis is poorly defined. There are no controlled trials examining the effects of omalizumab in atopic dermatitis that might support or refute a pathogenic role for IgE, and unlike the other “allergic” diseases described previously, evidence of MC involvement in atopic dermatitis is relatively sparse.165 However, bearing in mind their wide distribution in the skin, and the multiple modes MCs exhibit for sensing the surrounding environment, they are likely to be involved in the immunopathology. Whereas healthy skin contains MCs that are approximately 90% MCTC,49 the number of MCT increases in the skin of patients with atopic dermatitis.166 In addition, skin MCs demonstrate increased expression of IL-4 in atopic dermatitis.167 It is evident from Tables 14.3 to 14.5 that a number of MC mediators have the potential to contribute to disease pathogenesis, but evidence for this is currently lacking. Urticaria occurs in acute and chronic forms, with a complex classification and multiple etiologies. This is covered in more detail in Chapter 35. In various forms of acute urticaria, MC degranulation is evident ultrastructurally,168 and antihistamines provide a useful means of treatment, suggesting that the skin lesions occur predominantly as a result of MC activation. In chronic idiopathic urticaria (CIU), MC activation is also a factor. Interestingly, cultures of HPBMC derived from patients with CIU exhibit increased constitutive histamine release compared with controls,169 and in 30% of patients with CIU, circulating autoantibodies to FcεRI or IgE have been identified, suggesting a pathogenic role involving MCs.170,171 However, these patients do not usually have evidence of MC activation elsewhere in the body, which is unexplained. Nevertheless, in CIU refractory to treatment with antihistamines, administration of omalizumab is highly effective and brings rapid relief, further supporting a role for IgE-dependent MC activation.172

Mast Cells in Allergic Conjunctivitis

Mast Cells in Asthma

Allergic conjunctivitis commonly accompanies allergic rhinitis. In mild allergic eye disease, patients complain of variable itch, tearing, and swelling that is uncomfortable but does not threaten sight. Chronic forms

Asthma is a complex disease characterized by the presence of airway obstruction. The pathophysiology differs markedly from the diseases described previously because of the presence of smooth muscle, which

CHAPTER 14  Biology of Mast Cells and Their Mediators

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surrounds the airways. In asthma this ASM is hyperresponsive to bronchoconstrictor stimuli and is a major factor contributing to airflow obstruction. This obstruction is potentially reversible, at least in part, either spontaneously or with pharmacologic intervention, and is characterized by the symptoms of wheeze, dyspnea, cough, and chest tightness. Exacerbations may be triggered by a number of different stimuli, one or more of which may predominate in any individual. The major pathologic processes through which airflow obstruction occurs are ASM contraction, mucosal edema related to increased vascular permeability, excessive mucus secretion, airway inflammation, and various structural changes referred to as airway wall remodeling. This section concentrates on the evidence that places MCs as central effector cells in asthma pathophysiology as determined by the profile and biology of the mediators they release, evidence of their ongoing activation and release of these mediators, and their infiltration of key structures within the airways.

concentrations of histamine, PGD2, and LTC4 are present in the LAR, but in different ratios than during the EAR, raising the possibility that a contribution comes from non-MC sources such as macrophages and eosinophils.177,180 Tryptase levels fall during the LAR,177 which might indicate an absence of MC activity. However, MC degranulation is not “all or nothing,” with clear examples available of differential mediator release under various conditions.181–184 Interestingly, GM-CSF, which is released after allergen provocation, inhibits expression of tryptase in HMC-1 cells, but does not attenuate histamine release, and in HLMC may in fact potentiate IgE-dependent histamine release.185 Thus plausible mechanisms exist to explain the disparity between tryptase and histamine levels during the LAR. The LAR is also attenuated markedly by omalizumab therapy,179 indicating that MC activation during the EAR initiates events leading to the LAR. It is therefore likely that the secretion of MC mediators and cytokines orchestrate the development of the late inflammatory response.

Evidence of Mast Cell Activation in Asthma

Chronic Allergic Asthma.  Asthma is a heterogeneous and chronic disease. In chronic allergic-type “Th2 high” asthma (the most common form), MCs present in the bronchial mucosa are in an activated state. Morphologic assessment using electron microscopy indicates that in “stable” atopic asthma there is continuous ongoing degranulation within the airway epithelium, submucosa, and ASM58,59,186 (see Fig. 14.3). Several studies have also shown increased numbers of MCs in BAL from stable allergic asthmatics compared with healthy controls,187–189 together with increased concentrations of histamine and tryptase.174,187,188 As referred to earlier, MCs within the bronchial mucosa in asthma express several cytokines including IL-4, IL-5, IL-6, IL-13, and TNFα.114,115,190 When asthmatic airways are compared with healthy airways, there is evidence of increased expression of both IL-4 and IL-5 mRNA in MCs191 and increased expression of MC-associated IL-4 and TNF-α protein.192 Strong correlations have been described between eosinophil numbers and IL-4+, IL-5+, and TNF-α+ MC densities, and it is likely that MC-derived cytokines make a major contribution to asthma pathophysiology as an ongoing source of preformed and newly generated cytokines. MCs in the BAL of steroid-naïve symptomatic asthmatic subjects appear to be both primed and activated. They exhibit greater spontaneous histamine release than healthy MCs,189,193 and in several studies demonstrated enhanced IgE-dependent release.194,195 Both features might perhaps be related to the higher IgE concentrations in atopic subjects with consequent upregulation of FcεRI leading to enhanced IgE-related signaling75 and allergen-dependent mediator release.61 Others found that IgE-dependent activation of MCs from symptomatic asthmatics did not produce any significant increase in histamine release compared with the already high spontaneous release, in contrast to the findings in asymptomatic asthmatic subjects, perhaps suggesting that enhanced spontaneous release in the presence of symptoms is related to IgE-dependent activation in vivo.193 Enhanced mediator releasability in asthma may also stem from in vivo activation by other inflammatory stimuli but could also represent a fundamental functional difference predisposing to asthma, perhaps as a result of genetic factors. MCs are therefore in a state of continuous activation in chronic allergic asthma, with evidence to suggest that they may exhibit increased mediator releasability, both spontaneously and in response to IgEdependent activation. When this information is coupled with the epidemiologic data implicating allergen exposure in the development of asthma and the efficacy of omalizumab, it is easy to envisage a scenario where the atopic asthmatic phenotype results in part from the everyday interaction between allergens, IgE, and hyperreactive MCs.

Experimental allergen-induced asthma.  Because of the evidence that asthma is at least in part an IgE-dependent disease for many patients, the method of acute bronchial challenge with a relatively large dose of allergen in the laboratory has been used as a model for studying asthma pathophysiology. After bronchial allergen challenge there is a rapid fall in pulmonary function (e.g., FEV1) at 10 to 20 minutes in nearly all atopic asthmatics and many atopic nonasthmatics; this gradually recovers over the next 2 hours and is termed the early asthmatic reaction (EAR). In about 50% of subjects, there is a further fall in FEV1 between 4 and 6 hours, the late asthmatic reaction (LAR). For reasons not understood, this is more severe after house dust mite allergen challenge than grass pollen challenge. This LAR may last up to 12 hours and in some individuals may be followed by recurring airway obstruction for several days or even weeks. The early asthmatic reaction.  During the EAR several vasoactive and spasmogenic mediators are released from MCs resident within the airway mucosa. The relative rate of mediator release from HLMC in vitro is histamine > PGD2 > LTC4 with one half maximal release occurring at 2, 5, and 10 minutes respectively. This is reflected in vivo by the recovery of these mediators in bronchoalveolar lavage fluid (BAL) within 5 to 10 minutes after local bronchial allergen challenge.173–176 Histamine, PGD2, and LTC4/LTD4 induce bronchoconstriction, mucosal edema, and mucus secretion. Evidence for their direct role in the EAR is provided by studies using potent and selective synthesis inhibitors and receptor antagonists, which demonstrate that the EAR is markedly attenuated by inhibitors of histamine (H1 receptor), LTC4/ LTD4 (cysteinyl LTRl), and to a lesser extent PGD2 (thromboxane TP receptor). The MC origin of these mediators is supported by many pieces of evidence. Firstly, the kinetics of IgE-dependent mediator release in vivo parallels that of purified HLMC in vitro. Second, there is a rapid increase in the concentration of MC-specific tryptase in BAL within minutes after local bronchial allergen challenge.174,177 Third, β-agonists such as salbutamol, known inhibitors of MC degranulation when applied acutely in vitro,178 completely abolish the early reaction and the associated increase in plasma histamine levels. Last, the EAR is almost completely ablated after 12 to 16 weeks pretreatment with omalizumab,179 confirming that IgE-dependent signaling is critical. The Late Asthmatic Reaction.  The LAR is associated with inflammatory cell accumulation and activation. This situation has been considered to be analogous to that seen with chronic airway inflammation, although caution is needed with respect to this extrapolation. The role of the MC as a source of bronchospastic mediators during the LAR is more difficult to define than during the EAR. Increased

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Nonallergic (“Intrinsic”) Asthma.  Many asthmatic patients, particularly those with late-onset disease, do not have evidence of sensitization to common aeroallergens. These patients have traditionally been described as having so-called “intrinsic” or nonallergic asthma. This form of the disease is often of later onset, more severe and persistent, and more often associated with nasal polyposis and aspirin sensitivity. However, despite the different clinical picture, a pattern of inflammation more or less identical to that found in extrinsic asthma is present in the bronchial mucosa of nonallergic asthmatics.196 This suggests that a common mechanism may in fact be operating in the development of both phenotypes of asthma. In support of this, MC FcεRI+ expression is increased in the bronchial mucosa of both atopic and nonatopic asthmatic subjects compared with healthy controls.197 Epsilon germline gene (Iε) and mature epsilon heavy chain (Cε) mRNA+ B cells in the bronchial mucosa in both atopic and nonatopic asthma are increased, suggesting that there is local IgE synthesis,198 which may account for this increase in MC FcεRI+ expression. Secondly, and of equal interest, there is increased expression of the Th2 cytokines IL-4 and IL-5 at both the mRNA and protein level in nonallergic asthma.199 As in atopic asthma, MCs account for a significant proportion of the cells expressing mRNA and protein for both of these cytokines.190,200 Both IL-4 and IgE are powerful inducers of FcεRI expression on MCs,61 and IL-4 is a key cytokine involved in the promotion of IgE synthesis.201 Because human MCs can induce IgE synthesis by B cells in an IL-4 and IL-13 dependent manner,144 lung MCs might contribute to local bronchial IgE production, supported by the observation that the concentration of IgE in BAL is increased 24 hours after bronchial allergen challenge.202 Taking the above observations together, it seems likely that in nonallergic asthma, activated MCs producing Th2 cytokines support local B cell IgE production, which together with IL-4 and IL-13 upregulates FcεRI expression on MCs. Anti-IgE therapy might, therefore, be very effective in some patients with nonallergic asthma, and this is supported by a recent clinical study.203 In patients with severe asthma, high concentrations of MC proteases (tryptase, chymase, CPA3) are present in BAL and sputum, irrespective of whether they are atopic or nonatopic, indicating that ongoing MC activation is a common feature across asthma phenotypes.204 Occupational Asthma (see Chapter 56).  Occupational asthma is defined as asthma that develops, or is exacerbated, after specific exposure in the workplace and should exclude nonspecific stimuli that will produce bronchoconstriction in any asthmatic subject. More than 300 agents are recognized as inducers of occupational asthma, and common to all of these compounds is that they are inhaled (which probably informs us about the induction of atopic and intrinsic asthma). They fall into three main groups: (1) those associated with the synthesis of specific IgE antibodies; (2) those that are thought to produce sensitization through as yet undefined immunologic mechanisms, with specific IgE antibodies usually absent (examples from this group include the lowmolecular-weight chemicals plicatic acid [present in the dust of western red cedar] and the isocyanates; and (3) agents that are mostly irritant gases, fumes, or chemicals and that are capable of producing asthma after a single large exposure (reactive airways dysfunction syndrome or irritant-induced asthma). Interestingly, the pathology of occupational asthma (with the exception of irritant-induced asthma) is virtually identical to that seen in atopic and intrinsic asthma, including those cases where IgE-independent mechanisms have been advocated such as with western red cedar asthma (WRCA) and toluene diisocyanate (TDI) asthma.205,206 Thus in TDI asthma, numbers of MCs are increased in the bronchial epithelium compared with healthy controls, and electron microscopy demonstrates that the majority of MCs are degranulated.206 Subjects who develop

occupational asthma after a short period of exposure to TDI (2 years) have more MCs in their airway mucosa than subjects who develop asthma after a long period of exposure (22 years).207 Bronchial provocation with plicatic acid results in the rapid release of histamine into the BAL of patients with WRCA but not healthy subjects.208 Similarly, in vitro, plicatic acid releases histamine from MC in both BAL and bronchial mucosal biopsies obtained from patients with WRCA but not those with atopic asthma.209 This occurs via an undefined IgE-independent mechanism, which is in keeping with the observations that patients with WRCA do not usually have specific IgE to plicatic acid.209 In summary, MCs are strongly implicated in the pathogenesis of occupational asthma.

Exercise-Induced Asthma (see Chapter 54).  Exercise-induced asthma is not a distinct disease entity but a marker of poor asthma control and ongoing airway inflammation, indicating the need for more intensive therapy. Typically bronchoconstriction occurs 5 to 10 minutes after exercise and usually recovers within 30 minutes. The mechanisms behind exercise-induced asthma probably relate to the effects of airway cooling and water loss during exercise and may be mimicked by hyperventilation of cold dry air (reviewed in reference 210). A few but not all studies have identified increased concentrations of histamine in the serum of asthmatic subjects after exercise, and analysis of induced sputum after exercise has demonstrated increased concentrations of histamine, cysteinyl leukotrienes, and tryptase indicative of MC activation.211,212 In support of this, histamine H1 receptor antagonists, cyclooxygenase inhibitors, and leukotriene receptor antagonists significantly attenuate exercise-induced bronchoconstriction.213,214 MCs release histamine in a hyperosmolar environment,215 and this release is attenuated by cromolyn sodium, providing evidence that could link changes in airway osmolarity to bronchoconstriction.

Aspirin-Triggered Asthma (see Chapter 78).  Aspirin and other nonsteroidal antiinflammatory drugs exacerbate symptoms in approximately 10% of all asthmatics. Asthmatic symptoms usually start 1 to 2 hours after drug ingestion and may be life-threatening. The observation that this occurs with all NSAIDs suggests that manipulation of arachidonic acid metabolism is important. Aspirin-triggered asthma is associated with increased LTC4 in nasal secretions,216 increased LTE4 in the urine,217 and protection afforded by leukotriene synthesis and receptor antagonists.218 This might therefore involve MC leukotriene generation. In support of this, lysine aspirin induced PGD2 release from human cord blood–derived MCs.219 Furthermore there are increased numbers of MC in the airways of these patients, and an increased proportion of these MCs express COX-2.220 In addition, MCs are the predominant cells expressing LTC4 synthase in the airways of aspirin-sensitive asthmatics.221

Asthma Exacerbations.  Asthma exacerbations are a major cause of asthma morbidity and asthma death. Allergen exposure clearly contributes to these in some patients and is particularly evident in pollensensitized individuals during the pollen season and after the dispersal of pollen antigens after thunderstorms.222 Viral infections, particularly those caused by the rhinovirus (RV), induce exacerbations of asthma in many individuals.223,224 Subjects who are both sensitized and exposed to common aeroallergens have more severe exacerbations than nonexposed sensitized subjects, demonstrating an important interaction between virus and allergen.225 Experimental nasal infection with live RV16 increases the frequency of bronchial LARs after inhaled allergen provocation226,227 and enhances both the immediate release of histamine into blood227 and BAL228 (presumably MC-derived) and the later recruitment of eosinophils into the airway after local bronchial allergen challenge.228 In addition, activation of human MCs via TLR3, a receptor

CHAPTER 14  Biology of Mast Cells and Their Mediators for double-stranded viral RNA, induces secretion of both interferon-α and -β, and dual stimulation through TLR3 and FcεRI enhances the release of IL-1β, TNF-α, IL-5, and cysteinyl leukotrienes.119 In keeping with these findings, omalizumab therapy significantly reduces the rate of severe exacerbations in asthmatic subjects.229,230 Taken together this provides strong circumstantial evidence that MCs contribute directly to asthma exacerbations. Further evidence linking direct involvement of MCs to asthma exacerbations arises from pathologic studies of asthma death, which are discussed further in the next section. Virus-specific IgE may also play a direct role in asthma exacerbations. The intensity of the virus-specific IgE antibody response has been correlated with changes in airway function during acute viral infection.231,232 Interestingly, an in vivo animal model of asthma and RSV infection has shown that RSV can induce MC degranulation through the cross-linking of virus-specific IgE on MCs, with associated increases in bronchial hyperresponsiveness (BHR).233

Mast Cell Microlocalization in Asthmatic Airways.  MCs are present in healthy airways adjacent to blood vessels and scattered throughout the lamina propria.49,115,234 The number of MCs in the lamina propria is not increased in asthmatic compared with healthy airways,115,234,235 but in asthma, MCs infiltrate three key structures: the airway epithelium,48,115,234 the airway mucosal glands,236 and the ASM50,58,237–239(Fig. 14.5). This anatomic relocation places activated MCs at the heart of several dysfunctional airway elements, and the local delivery of their mediators is likely to be central to the disordered airway physiology. A summary of key interactions is provided in Fig. 14.6. Mast Cell Infiltration of Airway Smooth Muscle as a Key Determinant of the Asthmatic Phenotype.  The disordered airway physiology and airway wall remodeling characteristic of asthma have long been considered to be consequences of Th2 lymphocyte-driven airway eosinophilic inflammation. However, asthma pathophysiology is undoubtedly more complex than this, and there are many examples where there is little relationship between airflow obstruction and inflammation. This is particularly evident in patients with eosinophilic bronchitis (EB), which is characterized by the presence of a sputum eosinophilia occurring in the absence of variable airflow obstruction or BHR.240 Interestingly, the immunopathology of asthma and EB are virtually identical with

respect to the mucosal inflammatory cell infiltrate, mucosal IL-4 and IL-5 expression, the presence of subbasement membrane collagen deposition, markers of structural airway wall remodeling, and inflammatory mediator concentrations.50,237,241–243 This suggests that many of the Th2-related changes in asthmatic airways that have been considered fundamental in disease pathogenesis may not be so important for the development of airflow obstruction, BHR, and airway remodeling after all. The key difference between the pathology of asthma and EB lies within the ASM. In asthma there is infiltration of the ASM bundles by MCs, but these are almost absent in the ASM of patients with EB and healthy subjects sampled using bronchoscopy.50 In contrast, we have not found T cells or eosinophils in the ASM in any of these subject groups. This suggests that MC infiltration of the ASM in asthma may be critical for the development of BHR and variable airflow obstruction. This hypothesis is supported by the finding of significant correlations between the number of ASM MCs and the severity of BHR.50,237 In fact, stepwise linear regression revealed that a combination of ASM MC density and disease duration best modeled BHR.237 This increase in ASM MCs in asthmatic compared with healthy subjects has been confirmed in several further studies.58,238,239 This feature extends across the spectrum of asthma severity237,238 and is present in both eosinophilic and noneosinophilic patients,239 and thus spans asthma phenotypes. The majority of MCs in the ASM are of the MCTC subset50 and express both IL-4 and IL-13, but not IL-5.43,244 The MCs within the ASM in asthma demonstrate ultrastructural evidence of activation,58 and the number of degranulated MCs within the ASM is increased in fatal compared with nonfatal asthma,245 implicating MCs in the pathogenesis of asthma death. There are also further implications. For example, not only might it clarify why many atopic patients do not have asthma, but it could explain why the presence of asthma is such a strong risk factor for death from anaphylaxis and allergen desensitization. Taken together these observations suggest that an ASM MC “myositis” is a key determinant of the asthmatic phenotype.

Functional Mast Cell–Airway Smooth Muscle Interactions.  The localization of MCs within the ASM in asthma is likely to facilitate specific interactions between these two cell types through the specific targeting of both soluble mediators and signals delivered through direct cell-cell contact. One would predict that the microlocalization of MCs within the ASM will

C

B

A Airway smooth muscle

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Airway epithelium

Mucosal glands

Fig. 14.5  Mast cells (stained red for tryptase) within the airway smooth muscle (A), epithelium (B), and mucosal glands (C) in patients with asthma.

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SECTION A  Basic Sciences Underlying Allergy and Immunology Airway lumen Epithelial denudation activation, permeability

Mucus hypersecretion

Loss of epithelium-derived mast cell inhibitory factor

Tryptase, ?chymase, ?CPA IL-4, IL-13 Epithelium

Subepithelial fibrosis CXCL8 CXCL10 TSLP + CXCL12 IL-33 Histamine, PGD2, LTC4, tryptase, IL-4, IL-6, TNF-α CCL5 IL-13, amphiregulin CCL11 CADM1 adhesion ?juxtacrine signaling CXCR3 Bronchus

Submucosal glands IL-33 CXCR4

Histamine, PGD2, LTC4, tryptase, IL-4, IL-13

TGF-β, tryptase basic FGF

Bronchoconstriction/ hyperresponsiveness

ASM hyperplasia and hypertrophy

CXCR1

ASM mast cell CCR4 recruitment + CXCL10

CCR1

CXCR3 CCR3

Differentiation Smooth muscle

Blood

SCF, IL-6

Circulating progenitors

Mast cell differentiation, survival and activation

Progenitor recruitment (? CXCL12)

Adhesion

Fig. 14.6  An overview of key mast cell interactions with airway smooth muscle and epithelium in the pathogenesis of asthma. ASM, Airway smooth muscle; CADM1, cell adhesion molecule 1; CPA, carboxypeptidase [A]; FGF, fibroblast growth factor; IL, interleukin; LTC4, leukotriene C4; PGD2, prostaglandin D2; SCF, stem cell factor; TGF-β, transforming growth factor β; TSLP, thymic stromal lymphopoietin; TNF-α, tumor necrosis factor α. (Adapted from Bradding P, Walls AF, Holgate ST. The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 2006;117:1277-84.)

contribute to the development of ASM dysfunction expressed as BHR and variable airflow obstruction. In turn, one would predict that the ASM provides a suitable environment for MC growth and survival.

Mechanisms of mast cell recruitment by asthmatic airway smooth muscle.  The mechanism(s) by which MCs are recruited to the ASM

is important, because inhibiting this might offer a novel approach to the treatment of asthma. Whether this involves recruitment of MC progenitors and/or mature tissue-resident cells is not known. The ASM secretes many chemokines and growth factors that exhibit MC chemotactic activity, including CCL11, CXCL8, CXCL10, CXCL12, SCF, and TGF-β. All of these may be involved, but evidence from in vitro and ex vivo studies suggests that the CXCR3/CXCL10 axis is dominant.246 TGF-β is released by ASM after exposure to tryptase, providing a mechanism through which MC might contribute to further MC recruitment via autocrine pathways.247 However, there appears to be an added level of complexity in the regulation of MC migration by ASM in that healthy ASM in vitro also appears to secrete an inhibitor of HLMC migration. Irrespective of the mechanism(s), ASM-dependent HLMC migration is markedly attenuated by blocking the KCa3.1 ion channel, which may prove to be an attractive therapeutic approach.248

Mast cell adhesion, differentiation, survival, and activation in the presence of airway smooth muscle.  Cell-cell adhesion is a fundamental mechanism through which cells communicate, allowing the specific targeting of cell-specific signals.249 It is also important for the retention of cells at particular locations. Resting nonactivated HLMC adhere avidly to resting ASM cells in culture.32 This adhesive process is mediated in part through CADM1 (see previous discussion), and in part via Kit.250 HLMCs in coculture with ASM survive in the absence of exogenous survival factors and proliferate rapidly.33 This is mediated through a cooperative interaction between CADM1, membrane-bound SCF expressed on ASM cells, and soluble IL-6. Another feature of this interaction with ASM is that HLMC exhibit increased constitutive histamine release but still respond to IgE/anti-IgE activation.33 This indicates the presence of an ASM-dependent mechanism for the activation of HLMCs and could explain the presence of chronically activated MCs within the ASM bundles in asthma.58 Because SCF uncouples the β2-adrenoceptor on HL MCs, we investigated whether this occurs when HLMCs and ASM cells are in coculture.251 Again, constitutive HLMC histamine release was increased in

CHAPTER 14  Biology of Mast Cells and Their Mediators HLMC-HASMC coculture, but this was enhanced rather than inhibited by β2-AR agonists. Inhibition of FcεRI-dependent HLMC mediator release by β2-agonists was greatly attenuated in HLMC-HASMC coculture. These effects were reversed by neutralization of SCF but also by blocking adhesion through CADM1. β2-AR agonists did not prevent HASMC contraction in collagen gels when HLMCs were present, but this was reversed by fluticasone. These effects were associated with β2-AR phosphorylation at Tyr350 within a few minutes in both HLMCs and HASMCs when the cells were cocultured, and this was inhibited by neutralizing SCF or CADM1. These data suggest that HLMC interactions with HASMCs via CADM1 and Kit inhibit the potentially beneficial effects of β2-AR agonists on these cells via phosphorylation of the β2-AR. In turn, this might explain the potentially adverse effects of β2-ARs agonists evident in some patients with asthma. The biologic effects of mast cells on airway smooth muscle.  The interaction of MCs with ASM cells is clearly bidirectional. Immunohistochemical and electron microscopic analyses suggest that the MCs within the asthmatic ASM form intimate contact with ASM cells and are present in an activated state.58 Therefore the effects of MC mediators on ASM are likely to be profound. The classical MC autacoid mediators histamine, PGD2, and LTC4 are all potent agonists for ASM contraction. MCs may also impair protective airway mechanical responses. Normally, when a subject takes a deep inspiration, this induces bronchodilation and therefore provides protection against airway narrowing, but in asthma this protective mechanism is impaired or lost altogether.252 This loss of protection from deep inhalation is correlated with MC density within the ASM bundles.253 Tryptase induces bronchoconstriction and the development of BHR in animals, and in vitro potentiates the contractile response of sensitized bronchi to histamine.112 It stimulates cytokine release from ASM, and is also a potent ASM mitogen in vitro.254 In coculture, MC-derived tryptase promotes ASM secretion of TGF-β1, which in turn upregulates α smooth muscle actin (α-SMA) expression in an autocrine manner, leading to enhanced contractility.255 MC density in the asthmatic ASM correlates with the intensity of α-SMA staining in the same or adjacent ASM bundle, providing some evidence that MCs might drive ASM toward a more contractile phenotype in vivo.255 Chymase has been less extensively studied than tryptase but is expressed by those MCs infiltrating the ASM in asthma (MCTC).50 Unlike tryptase, chymase inhibits human ASM proliferation. The effects of the joint expression of these proteases and other MC mediators in vivo therefore remains unclear, but it is noteworthy that ASM proliferation in asthma in situ has rarely been observed.58,256,257 IL-4 and IL-13 may contribute to the severity of BHR.258 IL-4 and IL-13 also enhance the magnitude of agonist-induced intracellular Ca2+ responses in cultured human ASM. Because MCs within the asthmatic ASM express both IL-4 and IL-13, this may represent a further important pathway through which MCs contribute to the severity of BHR. The mechanism by which HLMC-HASMC coculture uncouple the β2-AR in HASMCs is unclear, but it is plausible that this involves the release of growth factors stimulating RTKs in HASMCs. Mast cell infiltration of the airway epithelium in asthma.  MCs infiltrate the airway epithelium in asthma,48,115,234 placing them at the portal of entry of noxious stimuli such as aeroallergens. This might facilitate an effector role in the ongoing immunologic response (antigen presentation, Th2 cell differentiation, IgE synthesis), and result in important bidirectional MC-epithelial interactions. The majority of MCs in the airway epithelium in mild steroid-naïve asthma express tryptase but rarely chymase43,48 and were traditionally thought to represent the classic MCT phenotype. However, it is now apparent that intraepithelial MCs in asthma also expresses carboxypeptidase A3 (CPA3) that had previously been identified only in

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association with chymase.48 This might indicate the presence of a new MC phenotype, the tryptase/carboxypeptidase cell, which has been named MCT/CPA, but perhaps all MCs express CPA3. IL-13-stimulated airway epithelial cells produce increased quantities of SCF and support the survival of the MCT/CPA phenotype but suppress chymase expression, thus providing a possible mechanism for these observations.48 The number of epithelial MCs in steroid-naïve asthmatic subjects varies widely, providing further evidence of pathologic disease heterogeneity, and represents a continuum rather than a dichotomy.48,115,234 Epithelial MC density correlates strongly with the expression of IL-4 protein in the airway epithelium,115 and the number of epithelial MCs correlates with an epithelial Th2 gene signature.48 The degree of epithelial mRNA expression for both tryptase and CPA3 correlates strongly with the improvement in lung function in response to corticosteroid treatment.48 This is in keeping with previous work suggesting that the response to corticosteroids in asthma is greatest in those patients with eosinophilic “Th2-high” disease.239,259 The presence of intraepithelial MCs therefore predicts a steroid-responsive pathology in steroid-naïve subjects. Both oral and inhaled corticosteroids reduce airway epithelial and submucosal MC numbers in steroid-naïve asthma,260,261 but only the MCT (probably MCT/CPA) phenotype.262 However, one study looking at MC phenotype in severe refractory asthma has shown the persistence of MCs in the airway epithelium in refractory disease with a high proportion of MCTC cells.263 However, this conflicts with another recent study demonstrating that intraepithelial MC density declines with increasing disease severity,238 which could be explained readily by the effect of increasing doses of corticosteroids.

Functional Mast Cell–Epithelial Interactions Mechanisms of mast cell recruitment by asthmatic airway epithelium.  The mechanisms by which MCs are recruited to the airway epithelium in human asthma are not known. However, like the ASM, the airway epithelium secretes many growth factors (SCF, TGF-β) and chemokines, which have chemotactic activity for human MCs. Potential airway epithelial-derived chemoattractants are summarized in Fig. 14.6.

Mast cell adhesion, differentiation, survival, and activation in the presence of airway epithelium.  Human lung MCs adhere to

airway epithelial cells in part via Kit.250 The adhesion of HLMCs to both HASMCs and airway epithelial cells via Kit appears to involve a domain on Kit that is outside the SCF-binding domain.250 Interestingly, when HLMCs are cocultured with BEAS-2B human airway epithelial cells, both constitutive and IgE-dependent histamine release are markedly attenuated.264 This is in stark contrast to the activation of HLMC induced by ASM cells.33 In asthmatic airways, though, intraepithelial MCs are clearly degranulated. It has been proposed that the role of the healthy intact bronchial epithelium is to maintain MCs in a relatively quiescent state, but during tissue insults such as those caused by infective agents, or that present in asthma, epithelial damage and denudation removes this suppressive activity, leading to MC priming and activation as part of the innate immune response.265 If this hypothesis is substantiated, the unleashing of MC activity through the loss of an epithelial brake may be central to the pathogenesis of asthma. In addition to loss of MC suppression, the dysfunctional proinflammatory asthmatic airway epithelium may also activate MCs. TSLP is highly expressed in asthmatic airway epithelium across the spectrum of disease severity compared with healthy controls,238 and TSLP derived from human airway epithelial cells induces the release of numerous cytokines and chemokines from human MCs, including IL-13.105 There is, therefore, a potential positive feedback loop between MC-derived cytokines, including IL-13, which then stimulates epithelial TSLP production,105,266 and the epithelial production of TSLP, which in turn

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drives MC activation. In support of this, MC activation was required for the increased TSLP production by airway epithelium in a mouse model of rhinitis.267 Human MCs are also able to produce TSLP and express TSLP in asthmatic airways,238,268 but HLMC proteases rapidly cleave TSLP,269 suggesting that they may serve to tightly regulate TSLP bioavailability. Like ASM, the airway epithelium demonstrates increased IL-33 expression in asthma, and like TSLP, it is expressed by the airway epithelium in increased amounts across the spectrum of asthma severity.101 Coculture of HPBMCs with primary human bronchial epithelial cells induced cytokine release from HPBMCs when exogenous IL-33 was added, with HPBMC-derived IL-13 then initiating a transcriptional programme in epithelium likely to exacerbate airway fibrosis, eosinophilia, and mucous metaplasia.270 It would be interesting to see if the effects of IL-33 on MCs are increased further when the epithelium is wounded. The biologic effects of mast cells on airway epithelium.  Mast cell mediators have several important effects on airway epithelial cells, but coculture experiments examining the epithelial response to MC activation are lacking. However, bearing in mind the points discussed previously, and the further biologic activities listed in Tables 14.3 to 14.5, it seems likely that activated MCs within the airway epithelium in asthma will have a profound effect on its function. For example, tryptase stimulates airway epithelial IL-8 release and can upregulate ICAM-1 expression,26 whereas ozone-induced respiratory epithelial sloughing in mice is entirely MC-dependent.271 The mechanisms behind this and the effects of MC activation on human airway epithelial function is deserving of further investigation.

Mast Cell Microlocalization Within Airway Submucosal Glands.  Severe mucus “plugging” of the airways is a key feature of severe, fatal asthma but to a lesser degree is also present in milder disease. This results from mucus hypersecretion by hyperplastic submucosal glands and epithelial goblet cells. Carroll and co-workers performed a detailed analysis of cartilaginous airways in postmortem lung specimens from patients with fatal asthma, patients with asthma who died from other causes (nonfatal asthma) and subjects without asthma who died of nonpulmonary causes.236 Immunohistochemistry for MC tryptase revealed a significant increase in the number of MCs within the mucosal gland stroma in nonfatal asthma, and a marked increase in the number of degranulated MCs in both fatal asthma and nonfatal asthma compared with normal controls. There were significant correlations between the density of both intact and degranulated MCs within the mucous glands and the degree of mucus obstruction in the airways. This reinforces the concept that the MC as an important contributor to the series of events leading to asthma death.

The biologic effects of mast cells on mucus-secreting cells. 

Numerous MC products have the potential to contribute to both mucous gland hyperplasia and mucus secretion. In terms of the autacoids, their potency for stimulating mucus secretion is in the order LTD4 > LTC4 > prostanoids > histamine. Canine MC chymase is also a potent mucus secretagogue when added to cultures of bovine airway glands.272 IL-6 and TNF-α induce mucous glycoprotein secretion and MUC-2 gene expression by both human bronchial organ explant cultures and airway epithelial cells obtained by bronchial brushing.273 IL-6 also induces expression of MUC5B and MUC5AC.274 This is relevant because MC within the bronchial mucous glands in asthma are known to express IL-6.115 Animal models also indicate important roles for tryptase and IL-13 in mucus hypersecretion. Amphiregulin is another molecule of interest. It is expressed in human progenitor-derived MCs after activation through FcεRI,275 an effect which is not suppressed by dexamethasone. Amphiregulin

expression by MCs in the asthmatic bronchial mucosa is increased compared to healthy controls, and in vitro, MC-derived amphiregulin increases mucin gene expression in the NCI-H292 epithelial cell line.275 Thus MC-derived amphiregulin along with other epidermal growth factor receptor ligands such as TGF-α, and EGF itself contribute to epithelial goblet cell metaplasia and mucus hypersecretion in asthma. In addition, recombinant amphiregulin induces the proliferation of human airway fibroblasts but not ASM cells, suggesting a further mechanism whereby MCs might contribute to subepithelial fibrosis.

Mast Cell Interactions With Airway Fibroblasts.  A characteristic histologic feature of asthma is thickening of the subbasement membrane because of deposition of collagen types I, III, V, and VI in the lamina reticularis. The most likely origin for this collagen is proliferating myofibroblasts whose number correlates with the collagen thickness. MCs in or adjacent to the bronchial epithelium also have the potential to activate subepithelial myofibroblasts; there is long-standing evidence that MCs and fibroblasts interact intimately through several mechanisms (reviewed in reference 276). For example, cultured rat embryonic skin fibroblasts phagocytose rat MC granules, and this is followed by secretion of collagenase and β-hexosaminidase. Histamine, basic FGF, and IL-4 promote fibroblast proliferation in humans. IL-4 is a chemoattractant for human fibroblasts and also induces human fibroblasts to secrete collagen types I and III and fibronectin. IL-13 increases CCL11 release from human airway fibroblasts. Heparin stabilizes basic FGF structurally and preserves its bioactivity by protecting it from degradation and may therefore potentiate fibroblast activation and proliferation indirectly through the regulation of basic FGF bioactivity. In coculture, HLMCs adhere to human fibroblasts in part via CADM1,31 while HCBMCs induced fibroblast collagen secretion and contraction in collagen gels.277 Both HMC-1 cells and human skin MCs augment proliferation of human skin fibroblasts, an effect dependent on this heterotypic cell-cell contact and MC IL-4 expression. IL-4 was secreted by skin MC in low amounts and strictly limited to cell-cell contacts with fibroblasts.

Mast Cells in Animal Models of Asthma.  Several animal models have been developed that aim to induce the airway features of asthma (see Chapter 48). The most widely reported is the mouse model using intraperitoneal antigen sensitization followed by antigen challenge of the airways. This most closely resembles the model of acute allergen challenge in the airways, although the route of sensitization is obviously different. The dependency on MCs in these models with regard to the development of BHR and inflammatory changes in the airways is highly dependent on the model studied and the mode of antigen sensitization. Thus sensitization without adjuvant generates an MC-dependent model, whereas sensitization with adjuvant creates an MC-independent model (reviewed in reference 278). An alternative model uses airway sensitization without adjuvant from the outset and to some extent is more physiologic. In this setting, MCs are again an essential component required for the development of BHR, inflammation, and remodeling (collagen deposition, goblet cell hyperplasia279,280). However, there are inevitably a number of problems in relating these models to the human disease. For example, mouse airways contain very few MCs at baseline, so the changes seen after antigen challenge rely heavily on the recruitment of MC progenitors rather than the activity of resident cells. So it is perhaps not surprising that short-term models using intraperitoneal sensitization do not find a role for MCs in the outcomes commonly measured. In addition, mice have relatively little ASM, and so there is no model described to date in mice that has recapitulated the infiltration of ASM by MCs, a feature that may be key to the development of asthma in humans. So

CHAPTER 14  Biology of Mast Cells and Their Mediators although mouse models are useful for generating hypotheses regarding the pathogenesis of asthma, their findings may also be potentially misleading.281

Pharmacologic Inhibition of Human Mast Cell Activation Several drugs are described as MC “stabilizers” because they inhibit IgE-dependent MC activation in vitro, but in vivo they are of poor efficacy when administered chronically. For example, cromolyn sodium is only a weak inhibitor of IgE-dependent HLMC secretion with maximal inhibition of histamine release in vitro of 10% to 20% when used in the high micromolar range; it also exhibits rapid tachyphylaxis.282 Although for many years its mechanism of action remained a mystery, recently its target has been identified as one of the LPA receptors, GPR 35.283 β2-adrenoceptor agonists such as salbutamol are more potent inhibitors of HLMC mediator release in vitro,282 but again there is rapid tachyphylaxis, and with chronic administration the clinical evidence is that they do not attenuate MC secretion in the asthmatic airway and may even enhance it.93,284,285 Syk inhibitors have shown promise in the laboratory, but clinical studies in rhinitis to date have been disappointing, perhaps because of the pharmacokinetics of the compound used, rather than the target. However, the morphology of MC degranulation in chronic asthma is predominantly piecemeal,58,59 the mechanism of which is unknown, and this may not be susceptible to inhibition by drugs that attenuate classic IgE-dependent anaphylactic degranulation. Identifying effective in vivo inhibitors of MC mediator release therefore remains a major therapeutic goal. Kit and SCF are also obvious targets. This approach has the potential to target pathologic interactions between MCs and airway structural cells mediated by membrane-bound Kit, which is likely to be important in asthma. A study of the Kit inhibitor imatinib in severe asthma suggested that it exerts biologic effects on MCs in the lung, although the clinical effects were limited, and side effects limited treatment.286 Masitinib, another tyrosine kinase inhibitor targeting Kit signaling, may also have some efficacy in severe asthma.287 There is therefore a strong rationale for trialing these further. MCs express several receptors that contain immunoreceptor tyrosinebased inhibition motifs (ITIMs). These regions are phosphorylated upon receptor activation and recruit phosphatases that subsequently dephosphorylate important signaling molecules, thus suppressing cell activation (for a detailed review see reference 228). They are especially active when coligated with FcεRI or Kit, leading to inhibition of FcεRI or Kit-dependent signaling. Examples expressed by human MCs include Siglec-8, CD300a, SIRP1α, CD200R (does not contain classical ITIM), and CD72. There is therefore the potential to inhibit MC activation through the administration of bispecific antibodies targeting both the ITIM receptor and FcεRI or Kit. With respect to FcεRI, whether such an approach would be more effective than the currently available antiIgE monoclonal antibody omalizumab is uncertain. The naturally occurring antiinflammatory lipid molecules lipoxin A4, resolvin D1, and resolvin D2 demonstrated potent inhibition of HLMC FcεRI-dependent degranulation,265 supported by the observation that lipoxin B4 inhibited MC degranulation in a mouse model in vivo.289 It has been proposed that these molecules are deficient in asthma, so restoring the normal antiinflammatory pathways present in the airways may offer a novel approach to restoring MC homeostasis in asthma and other MC-mediated diseases.

CONCLUSION In summary, MCs are multifaceted tissue-resident cells, capable of responding to a variety of noxious stimuli with the secretion of

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numerous multifunctional autacoids, proteases, and cytokines. Current evidence indicates roles in host defense and repair, as well as many diverse diseases. As evident from this chapter, they play a central role in many aspects of allergic disease and asthma, although their activity in these and other disorders involves complex interactions with other immunologic and structural cells. Developing drugs that inhibit pathologic MC secretion when administered regularly should improve the treatment of many patients with asthma and related allergic diseases.

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CHAPTER 14  Biology of Mast Cells and Their Mediators 242. Brightling CE, Symon FA, Birring SS, et al. TH2 cytokine expression in bronchoalveolar lavage fluid T lymphocytes and bronchial submucosa is a feature of asthma and eosinophilic bronchitis. J Allergy Clin Immunol 2002;110:899–905. 243. Brightling CE, Ward R, Woltmann G, et al. Induced sputum inflammatory mediator concentrations in eosinophilic bronchitis and asthma. Am J Respir Crit Care Med 2000;162:878–82. 244. Brightling CE, Symon FA, Holgate ST, et al. IL-4 and IL-13 are co-localised to mast cells within airway smooth muscle in asthma. Clin Exp Allergy 2003;33:1711–16. 245. Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J 2002;19:879–85. 246. Brightling CE, Ammit AJ, Kaur D, et al. The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle. Am J Respir Crit Care Med 2005;171:1103–8. 247. Berger P, Girodet PO, Begueret H, et al. Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis. FASEB J 2003;17:2139–41. 248. Cruse G, Duffy SM, Brightling CE, et al. Functional KCa3.1 K+ channels are required for human lung mast cell migration. Thorax 2006;61: 880–5. 249. Zimmerman GA, Lorant DE, McIntyre TM, et al. Juxtacrine intercellular signaling: another way to do it. Am J Respir Cell Mol Biol 1993;9: 573–7. 250. Gough KC, Maddison BC, Shikotra A, et al. Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells. Respir Res 2015;16:86. 251. Lewis RJ, Chachi L, Newby C, et al. Bidirectional counterregulation of human lung mast cell and airway smooth muscle beta2 adrenoceptors. J Immunol 2016;196:55–63. 252. Kapsali T, Permutt S, Laube B, et al. Potent bronchoprotective effect of deep inspiration and its absence in asthma. J Appl Physiol 2000;89:711–20. 253. Slats AM, Janssen K, van Schadewijk A, et al. Bronchial inflammation and airway responses to deep inspiration in asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;176:121–8. 254. Berger P, Perng DW, Thabrew H, et al. Tryptase and agonists of PAR-2 induce the proliferation of human airway smooth muscle cells. J Appl Physiol 2001;91:1372–9. 255. Woodman L, Siddiqui S, Cruse G, et al. Mast cells promote airway smooth muscle cell differentiation via autocrine up-regulation of TGF-beta 1. J Immunol 2008;181:5001–7. 256. Woodruff PG, Dolganov GM, Ferrando RE, et al. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001–6. 257. Benayoun L, Druilhe A, Dombret MC, et al. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003;167:1360–8. 258. Venkayya R, Lam M, Willkom M, et al. The Th2 lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through direct effects on resident airway cells. Am J Respir Cell Mol Biol 2002;26:202–8. 259. Woodruff PG, Modrek B, Choy DF, et al. T-helper type 2-driven inflammation defines major subphenotypes of asthma. Am J Respir Crit Care Med 2009;180:388–95. 260. Djukanovic R, Wilson JW, Britten KM, et al. Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am Rev Respir Dis 1992;145:669–74. 261. Laitinen LA, Laitinen A, Haahtela T. A comparative study of the effects of an inhaled corticosteroid, budesonide, and a beta 2-agonist, terbutaline, on airway inflammation in newly diagnosed asthma: a randomized, double-blind, parallel-group controlled trial. J Allergy Clin Immunol 1992;90:32–42. 262. 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 1996;153:551–6.

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263. Balzar S, Fajt ML, Comhair SA, et al. Mast cell phenotype, location, and activation in severe asthma. Data from the Severe Asthma Research Program. Am J Respir Crit Care Med 2011;183:299–309. 264. Yang W, Wardlaw AJ, Bradding P. Attenuation of human lung mast cell degranulation by bronchial epithelium. Allergy 2006;61: 569–75. 265. Martin N, Ruddick A, Arthur GK, et al. Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation. PLoS ONE 2012;7:e43545. 266. Kato A, Favoreto S Jr, Avila PC, et al. TLR3- and Th2 cytokine-dependent production of thymic stromal lymphopoietin in human airway epithelial cells. J Immunol 2007;179:1080–7. 267. Miyata M, Hatsushika K, Ando T, et al. Mast cell regulation of epithelial TSLP expression plays an important role in the development of allergic rhinitis. Eur J Immunol 2008;38:1487–92. 268. Ying S, O’Connor B, Ratoff J, et al. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity. J Immunol 2005;174:8183–90. 269. Okayama Y, Okumura S, Sagara H, et al. FcepsilonRI-mediated thymic stromal lymphopoietin production by interleukin-4-primed human mast cells. Eur Respir J 2009;34:425–35. 270. Nagarkar DR, Ramirez-Carrozzi V, Choy DF, et al. IL-13 mediates IL-33-dependent mast cell and type 2 innate lymphoid cell effects on bronchial epithelial cells. J Allergy Clin Immunol 2015;136:202–5. 271. Longphre M, Zhang LY, Harkema JR, et al. Mass: [sic] cells contribute to O3-induced epithelial damage and proliferation in nasal and bronchial airways of mice. J Appl Physiol 1996;80:1322–30. 272. Sommerhoff CP, Caughey GH, Finkbeiner WE, et al. Mast cell chymase. A potent secretagogue for airway gland serous cells. J Immunol 1989;142:2450–6. 273. Levine SJ, Larivee P, Logun C, et al. Tumor necrosis factor-alpha induces mucin hypersecretion and MUC- 2 gene expression by human airway epithelial cells. Am J Respir Cell Mol Biol 1995;12:196–204. 274. Chen Y, Thai P, Zhao YH, et al. Stimulation of airway mucin gene expression by interleukin (IL)-17 through IL-6 paracrine/autocrine loop. J Biol Chem 2003;278:17036–43. 275. Okumura S, Sagara H, Fukuda T, et al. FcepsilonRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells. J Allergy Clin Immunol 2005;115:272–9. 276. Bradding P, Holgate ST. Immunopathology and human mast cell cytokines. Crit Rev Oncol Haematol 1999;31:119–33. 277. Margulis A, Nocka KH, Wood NL, et al. MMP dependence of fibroblast contraction and collagen production induced by human mast cell activation in a three-dimensional collagen lattice. Am J Physiol Lung Cell Mol Physiol 2009;296:L236–47. 278. Williams CM, Galli SJ. Mast cells can amplify airway reactivity and features of chronic inflammation in an asthma model in mice. J Exp Med 2000;192:455–62. 279. Taube C, Wei X, Swasey CH, et al. Mast cells, Fc epsilon RI, and IL-13 are required for development of airway hyperresponsiveness after aerosolized allergen exposure in the absence of adjuvant. J Immunol 2004;172:6398–406. 280. Yu M, Tsai M, Tam SY, et al. Mast cells can promote the development of multiple features of chronic asthma in mice. J Clin Invest 2006;116:1633–41. 281. Holmes AM, Solari R, Holgate ST. Animal models of asthma: value, limitations and opportunities for alternative approaches. Drug Discov Today 2011;16:659–70. 282. Church MK, Hiroi J. Inhibition of IgE-dependent histamine release from human dispersed lung mast cells by anti-allergic drugs and salbutamol. Br J Pharmacol 1987;90:421–9. 283. Yang Y, Lu JY, Wu X, et al. G-protein-coupled receptor 35 is a target of the asthma drugs cromolyn disodium and nedocromil sodium. Pharmacology 2010;86:1–5. 284. Roberts JA, Bradding P, Britten KM, et al. The long-acting beta2-agonist salmeterol xinafoate: effects on airway inflammation in asthma. Eur Respir J 1999;14:275–82.

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285. Giannini D, Carletti A, Dente FL, et al. Tolerance to the protective effect of salmeterol on allergen challenge. Chest 1996;110:1452–7. 286. Cahill KN, Katz HR, Cui J, et al. KIT inhibition by imatinib in patients with severe refractory asthma. N Engl J Med 2017;376:1911–20. 287. Humbert M, de Blay F, Garcia G, et al. Masitinib, a c-kit/PDGF receptor tyrosine kinase inhibitor, improves disease control in severe corticosteroid-dependent asthmatics. Allergy 2009;64:1194–201.

288. Karra L, Levi-Schaffer F. Down-regulation of mast cell responses through ITIM containing inhibitory receptors. Adv Exp Med Biol 2011;716:143–59. 289. Karra L, Haworth O, Priluck R, et al. Lipoxin B(4) promotes the resolution of allergic inflammation in the upper and lower airways of mice. Mucosal Immunol 2015;8:852–62.

CHAPTER 14  Biology of Mast Cells and Their Mediators

242.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following are properties of the adhesion molecule CADM1? a. CADM1 is a Ca2+-dependent integrin. b. CADM1 works independently of Kit to regulate MC survival. c. CADM1 is expressed as a single functional isoform. d. CADM1-dependent adhesion promotes MC survival and proliferation in co-culture with airway smooth muscle cells and lung fibroblasts. e. CADM1 is expressed widely on healthy leukocytes. 2. Which of the following applies to the action of β2-adrenoceptor agonists on mast cells (MCs) and airway smooth muscle cells? a. They are effective at inhibiting MC activation when administered chronically to patients with asthma. b. Their activity is enhanced after MC exposure to stem cell factor (SCF). c. They inhibit IgE-dependent mast cell activation when applied acutely in vitro, but not activation induced by airway smooth muscle contact in vitro. d. They prevent airway smooth muscle contraction in coculture with MCs. e. β2-adrencoeptor agonists do not inhibit human lung MC migration. 3. Which of the following is true regarding mast cell (MC) mediator release? a. MC mediator release is all-or-nothing. b. MC mediator release may be strictly localized to points of cellcell contact.

c. The mechanisms of piecemeal degranulation are well defined. d. After IgE-dependent activation, release of lipid mediators derived from arachidonic acid (e.g., LTC4, PGD2) precedes degranulation. e. Orai3 is the ion channel primarily responsible for mediating IgE-dependent Ca2+ influx. 4. Which of the following is true of mast cells in chronic nonallergic (nonatopic) asthma? a. Mast cells (MCs) are not involved. b. MC FcεRI expression is similar to that in nonatopic healthy controls. c. MC type-2 cytokine expression is similar to that in nonatopic healthy controls. d. MCs do not infiltrate the airway smooth muscle (ASM) bundles. e. In severe nonallergic asthma, MC proteases are elevated in sputum and bronchoalveolar lavage fluid, indicative of ongoing MC activation. 5. Which of the following is true regarding mast cell (MC) interactions with the immune system? a. MCs are capable of presenting antigen to T cells. b. MCs are not capable of stimulating B cells to produce IgE. c. MC exosomes inhibit dendritic cell development. d. MC-derived histamine and PGD2 increase dendritic cell IL-12 production. e. MCs inhibit T cell Th2 differentiation.

15  Biology of Basophils John T. Schroeder

CONTENTS Introduction, 243 Development and Morphology, 243 Functional and Phenotypic Markers, 244 Inflammatory Mediators, 246

SUMMARY OF IMPORTANT CONCEPTS • Basophil granulocytes develop in the bone marrow and are released into the circulation as mature end-stage cells representing less than 1% of blood leukocytes. • Although their exact role in in vivo processes remains an enigma, basophils secrete a variety of mediators and cytokines that are central in allergic disease. • Basophils are particularly capable of generating interleukin 4 (IL-4) and IL-13, cytokines that promote immunoglobulin E (IgE) synthesis. • Although they have a role in IgE-dependent reactions, basophils also express receptors specific for microbial products, thus indicating a role in innate immunity. • Basophils selectively migrate into sites of allergic inflammation (e.g., skin, nose, lung).

Basophil Activation, 247 Pharmacologic Modulation of Secretion, 249 Basophil Involvement in Disease, 250 Summary, 252

Immediate hypersensitivity reactions begin when preformed (e.g., histamine) and newly synthesized (e.g., leukotrienes) mediators are released from basophils and mast cells. Degranulation events resulting in the release of these mediators are preceded by a cascade of intricate intracellular signals resulting from the interaction of allergen with specific IgE molecules bound to the high-affinity IgE receptors (FcεRI) on the surface of these cells (Chapter 24). This IgE-dependent activation also leads to the production of immunomodulatory cytokines. In particular, human basophils have the capacity to produce more IL-4 and IL-13 in response to allergen than any other cell type found in blood— findings that have been substantiated by recent work done in mice. Because IL-4 and IL-13 are critical for the development of atopy and, in fact, are found in allergic lesions, the role of basophils in allergic diseases has taken on a whole new significance. In the overview presented in this chapter, the emphasis is therefore on the more recent developments pertaining to the biology of human basophils, with comments on recent experimental research in mice.

INTRODUCTION

DEVELOPMENT AND MORPHOLOGY

Paul Ehrlich first identified basophil granulocytes in 1879, at which time he noted their distinctive cytoplasmic granules that bore remarkable similarity to those in tissue mast cells, which he had described a year earlier. Nearly 100 years were to pass before it was discovered that basophils, which constitute just around 1% of the circulating blood leukocytes, account for essentially all of the histamine released by blood cells in a reaction requiring immunoglobulin E (IgE). This observation initially prompted the notion that basophils might represent a surrogate with which to study the more elusive tissue mast cell. This belief, however, has since been abandoned, because studies have demonstrated that the two cell types differ more than they are alike. Nonetheless, even though the basophil response has been extensively studied in vitro, the exact role for this cell in biologic processes remains unclear. Recent studies in mice do point to basophils helping to expel helminth parasites and possibly ectoparasites (e.g., ticks).1 However, the focus of this chapter is on both the long-standing and more recent evidence that basophils play a critical role in allergic disease by infiltrating sites of allergic inflammation and releasing mediators and cytokines that perpetuate type 1 (immediate) hypersensitivity reactions.

Like all granulocytes, basophils (Fig. 15.1) are of myeloid origin, developing from pluripotent stem cell precursors found in the bone marrow. Although all the factors important in this differentiation are likely yet to be identified, the cytokine IL-3 plays a critical role. Indeed, in vitro studies show that CD34+ precursor cells, when cultured in the presence of IL-3, differentiate into cells that morphologically and functionally resemble basophils.2-4 As discussed further on, IL-3 also plays a significant role in the survival and activation of mature basophils, which are functional consequences of these cells’ retaining the expression of IL-3 receptors (CD123) at remarkably high levels. On being released from the marrow as mature cells, however, basophils have little capacity (if any) for further development and, as indicated by animal studies, are thought to survive for only days.5 For comparison, mast cell development involves the release of intermediate precursors from the bone marrow that are capable of targeting specific tissue sites for maturation where their survival has been estimated on the order of months. Although recurrent proposals have suggested that human basophils and mast cells are of the same lineage, the general consensus at present is that the two cell types are unrelated developmentally. In fact, recent evidence

243

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SECTION A  Basic Sciences Underlying Allergy and Immunology resulting from the binding of specific antigen or anti-IgE antibody. Piecemeal degranulation initially was used to describe the pattern of degranulation observed in basophils found in certain cell-mediated pathologic conditions, including contact dermatitis, skin graft rejection, Crohn disease, and ulcerative colitis. It is characterized by an induced vesicular transport of granular content that does not involve direct granule extrusion. As discussed later, additional insight regarding the occurrence of piecemeal degranulation has emerged in recent years, with respect to basophil activation markers.

FUNCTIONAL AND PHENOTYPIC MARKERS Adhesion

Fig. 15.1  Human basophil stained with Wright’s. Original magnification ×1000.

TABLE 15.1  General Properties of Human Basophils Property

Basophil-Specific

Origin

Bone marrow

Lineage

Myeloid

Frequency in circulation

1010). Human basophils may express between 5000 and 1 million FcεRI sites per cell, depending on the donor, and it is now well accepted that serum IgE levels regulate this expression.24 For instance, FcεRI levels increase with higher serum IgE concentrations through a mechanism where the receptor half-life on the cell surface is stabilized when occupied by the immunoglobulin. This is an important aspect to consider in light of anti-IgE therapy for the treatment of allergic disease. With just an estimated 200 IgE-receptor cross-links needed to initiate mediator release from basophils (and presumably from mast cells), a minimum reduction in allergen-specific IgE of approximately 96% is required to achieve clinical efficacy. Thus both the amount of drug and the duration for which it is given become very important parameters in achieving this efficacy. Finally, an important point in this context is that all IgE binding to basophils is mediated through FcεRIα, because these cells do not express FcεRII (CD23), which is the low-affinity IgE receptor more commonly expressed on B-lymphocytes. Moreover, basophils are believed not to express the trimeric variant of FcεRI, which lacks the β-subunit (also known as

245

αγ2). This αγ2 variant of FcεRI is expressed on a variety of professional antigen-presenting cells (APCs), including Langerhans cells, monocytes, and dendritic cells. Studies indicate that it mediates a variety of functions on these cells, ranging from antigen-presentation, cytokine secretion, and suppression of innate immune function.25 Human basophils express both the CD32α and CD32β variants of FcRγII, which bind subclasses of IgG antibody that potentially mediate stimulatory and inhibitory signals, respectively. It is clearly shown that CD32 can function on basophils by inducing intracellular signals that oppose those mediated through FcεRI, thereby downregulating IgEmediated responses in basophil (and mast cells). Studies have shown, in fact, that FcγRII/FcεRI costimulation attenuates mediator release and cytokine production by basophils.26 It has been suspected that IgG4 might best mediate this activity, owing to increases in this so-called “blocking antibody” during allergen immunotherapy (IT) and that FcγRII/FcεRI costimulation represents a hypothetical mechanism of IT efficacy. However, a recent study indicates that IgG2 and IgG3 subclasses appear more efficacious than IgG4 in recruiting CD32 during costimulation with FcεRI.27 Basophils are one of just a few cell types that express the cell surface ligand for CD40 (CD40L)28; others include mast cells, activated T cells, platelets, and endothelial cells. Cells expressing CD40L, by interacting with CD40 on the surface of B lymphocytes, can relay signals necessary for the latter cell type to develop into immunoglobulin-producing cells. By expressing CD40L and secreting IL-4 and IL-13, basophils have the potential to provide the two necessary signals for B cells to produce IgE.28 In recent years, the increased surface expression of CD63 and CD203c has been widely used as a means for determining basophil activation. These so-called basophil activation markers are commonly used as surrogates of FcεRI-dependent activation, such as that occurring with allergen. Induced CD63 expression serves as the foundation for the so-called basophil activation test (BAT) assay. This marker is typically expressed within the cytoplasmic granules of nonstimulated basophils. Upon anaphylactic degranulation, whereby cytoplasmic granules fused with one another and then with the outer cell membrane, CD63 gets expressed on the cell surface within minutes and is detectable by flow cytometry. There is, in fact, a general acceptance of this marker (more so than CD203c) being diagnostic for IgE-dependent activation. Furthermore, there is mounting evidence implying that induced CD63 expression is especially good in predicting the severity of adverse reactions in food allergy.29,30 Expression levels of CD203c (also known as ectonucleotide pyrophosphatase/phosphodiesterase) likewise increase following IgE-dependent activation, even though this marker is constitutively expressed on basophils. Recent studies show that baseline expression of CD203c is higher among nut-allergic31 and asthmatic subjects32, implying some level of activation among the circulating basophils in these subjects. Nonetheless, a variety of issues relating to specificity and concentration of stimulus required for inducing the expression of CD63 and CD203c have raised concern regarding their diagnostic application.33 For example, a recent study has suggested that increased CD203c expression tracks with piecemeal degranulation, as opposed to the anaphylactic degranulation common with IgE-dependent activation.34 In addition, CD63 expression is not necessarily specific for IgE-dependent activation, because levels of this marker are also induced on basophils by other substances that cause degranulation (e.g., FMLP and C5a). Although not commonly recognized as an activation marker, CD69 also increases with IgE-dependent activation, but the kinetics are slower (hours), and it is more readily induced after prolonged exposure to certain cytokines, namely IL-3.35,36

Receptors Associated With Innate Immunity.  Basophils express a number of receptors associated with innate immunity. Receptors for

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complement (CR) are found on basophils including CR1 (CD35), CR3 (CD116/CD18), CR4 (CD11c/CD18), and the receptor for the anaphylatoxin C5a (C5aR). At this time, only C5aR seems to be of functional consequence with respect to mediator release; it mediates degranulation induced by C5a (see later). The bacterial peptide formyl-methionylleucyl phenylalanine (fMLP) binds a seven-membrane transverse receptor that structurally resembles those that bind chemokines. It too mediates basophil degranulation (see further on). Basophils reportedly express additional types of innate immunity-associated receptors: (1) toll-like receptors (e.g., TLR1, TLR2, TLR4, TLR6, TLR9),37 (2) nucleotide-binding oligomerization domain–containing protein 2 (NOD2) receptors, both of which bind a variety of microbial products,38 and (3) leukocyte immunoglobulin-like receptors (e.g., LIR3 and LIR7),39 for which the natural ligands have not yet been identified. Receptors in all three families, when ligated using natural ligands or receptor-specific antibodies, have been shown to mediate either inhibitory or stimulatory activity (see later discussion).

may reach those found in mast cells.44 Although the significance of these findings is unclear at present, clearly caution is indicated in evaluating studies that use tryptase as the sole specific marker for identifying the presence of mast cells in tissue using immunohistochemistry.

Other Receptors and Specific Markers

Cytokines

Many other markers and receptors have been characterized or identified on basophils. Receptors for prostacyclin (i.e., prostaglandin I2), plateletactivating factor (PAF), adenosine, and histamine all have been detected on basophils. At least three monoclonal antibodies have been described that immunologically detect proteins unique to basophils or their progenitors. Two such antibodies, 2D7 and BB1, have specificity for proteins found within the cytoplasmic granules. The monoclonal antibody 2D7 recognizes a 72-kD protein that is released upon degranulation,40 whereas BB1 is specific for a large complex (5 × 106 daltons) referred to as basogranulin.41 CD203c antibodies are also used to specifically identify basophils, both mature and immature cells.42 All three antibodies have utility in identifying basophils in allergic lesions. At present, however, no role or function for the proteins they detect has been recognized. Finally, recent evidence suggests that an antibody targeting pro–major basic protein 1 (J175-7D4) specifically recognizes basophils, suggesting that it too may have potential use in identifying basophils in allergic and nonallergic lesion sites.43

Cytokines represent a third class of mediator released by basophils. In particular, basophils are a significant source of IL-4 and IL-13, two Th2 cytokines whose expression is characteristic of allergic lesions and which are now considered critical components in the pathogenesis of allergic disease.36 Of most importance, isotype switching in B cells, from IgM to IgE, is now widely known to be dependent on the biologic activities of IL-4 and to a lesser extent by IL-13. Subsequently, when CD40 on the surface of B cells is engaged with cells expressing CD40L, including basophils themselves, then synthesis of IgE occurs. Both IL-4 and IL-13 upregulate VCAM-1 expression on endothelium, thus playing an important role in the selective recruitment of eosinophils, basophils, and lymphocytes into sites of allergic inflammation. Class II major histocompatibility (MHC) antigens, such as human leukocyte antigen (HLA)DR, also are upregulated on cells exposed to IL-4 or IL-13. Basophil-derived IL-4/IL-13 was recently shown to promote differentiation of alternatively activated macrophages (otherwise known as M2 cells) from monocytes, both in vitro and in vivo.45 With M2 cells implicated in many Th2associated processes ranging from immunity to extracellular parasites to allergic disease and wound healing to cancer, then basophils could conceivably participate in these developments. IL-4 certainly plays an important role in driving naïve T cells toward the Th2 phenotype, and several non–T lymphocyte cell types (including basophils) have been suggested to provide this IL-4. Indeed, the belief that a non–T cell source of IL-4 might amplify Th2 responses and therefore promote allergic disease has sparked considerable debate as to the cellular source of IL-4. At this time, the volume of evidence indicates that basophils are a primary source of IL-4 when conducting in vitro studies using human specimens. Indeed, early studies revealed that both mRNA and protein for this cytokine correlated with the presence of basophils.46 In PBMC cultures, which have been traditionally used to demonstrate T cell cytokine production, the 1% to 2% basophils typically found in these suspensions accounted for essentially all of the IL-4 secreted in response to allergen stimulation.47 This finding has a simple explanation: the frequency of antigen-specific basophils (i.e., those expressing antigen-specific IgE) far outnumbers the antigen-specific T cells, which at best are estimated at 1 in several thousand in PBMC. In contrast, in vivo studies done in mice, while also pointing to the basophil as an important source of IL-4 (see later), further indicate that innate lymphoid type 2 cells (ILC2s) produce this cytokine. Nonetheless, it remains unclear whether human ILC2s possess the same capacity. Certainly, there is greater similarity between the two models with regard to production of IL-5 and IL-13, where both mouse and human ILC2s secrete copious amounts of these cytokines.

INFLAMMATORY MEDIATORS Histamine Basophils constitutively store, on average, approximately 1 pg of histamine per cell, and this amount is remarkably consistent among allergic and nonallergic donor populations. Histamine is synthesized by the actions of histidine decarboxylase, which removes a carboxyl group from l-histidine. Its storage in basophils is mediated through ionic interactions with the highly charged proteoglycan chondroitin sulfate, as opposed to heparin sulfate in the mast cell. These complexes dissociate with changes in pH and ionic strength that occur during the process of degranulation, thus resulting in the release of histamine. The physiologic effects of histamine on smooth muscle, the vasculature, and neural tissues are well documented. As a spasmogen, it is capable of smooth muscle contraction; it causes vascular leakage through its ability to dilate terminal arterioles. The clinical efficacy of histamine H1 receptor antagonists in the treatment of allergic symptoms is partially mediated by their ability to prevent histamine from binding to H1 receptors in the airways and vasculature.

Other Preformed Mediators Although tryptase and major basic protein (MBP) have long been considered mast cell– and eosinophil-specific mediators, respectively, small quantities of these proteins also are stored in basophils. One study has shown that the tryptase levels detected in basophils of some donors

Leukotriene C4 On activation with various stimuli, basophils rapidly metabolize arachidonic acid (AA) through the lipoxygenase pathway to generate leukotriene C4 (LTC4). Thus, unlike histamine, this mediator is not stored in the cytoplasmic granules, but its rate of secretion (i.e., minutes) is only slightly slower than that of histamine, suggesting that it also contributes to the acute symptoms associated with allergic reactions. The amounts of LTC4 generated by basophils and mast cells (i.e., 10−14 to 10−13 g per cell) are far less than the amounts of histamine stored in these cell types (i.e., 10−6 g per basophil). On a molar basis, however, LTC4 is some 6000 times more potent than histamine in contracting airway smooth muscle.

CHAPTER 15  Biology of Basophils Development of the IL-4 reporter mice strains (i.e., G4 and 4get mice) and conditional basophil knockout mice have substantiated the importance of IL-4 production by basophils by providing direct in vivo evidence for these cells driving Th2 polarization.48-50 The 4get mouse strain has eosinophils, mast cells, and basophils that are positive for IL-4 mRNA, but only the basophils in these animals are now thought to secrete significant levels of IL-4 protein—a finding substantiated by the G4 strain. Moreover, the evidence emerging from studies using these mice indicates that the basophil may very well provide the initial source of IL-4 responsible for amplifying Th2 responses (see further on). Animals depleted of mast cells, eosinophils, and T cells still mount a Th2 inflammatory response, which, by all accounts, is dependent on granulocytes resembling basophils. The evidence at this time indicates that human basophils produce only Th2-like cytokines. There are no reports of basophils secreting Th1like cytokines. Basophils rapidly produce IL-3, which is then capable of working in an autocrine fashion to prime these cells for phenotypic and functional responses associated with allergic disease.51 There is mounting evidence that human basophils secrete copious amounts of another Th2-like cytokine, IL-31, which is implicated in the pathogenesis of chronic inflammatory conditions such as atopic dermatitis and chronic urticaria.12 As noted previously, IL-31 modulates basophil function and therefore may also mediate autocrine effects of its own. Other studies have identified IL-17–associated cytokines and growth factors (e.g., VEGF, amphiregulin, GM-CSF, leptin) produced by basophils. In addition, several chemokines (e.g., IL-8/CXCL8, macrophage inflammatory protein [MIP]-1α/CCL3) are secreted by basophils, with implications that they may modulate cell trafficking into lesion sites. The findings are summarized in the previous edition of this chapter and elsewhere.36 Basophils synthesize and release granzyme B, suggesting perhaps yet another class of mediator produced by these cells.52 Although generally considered to be a natural killer (NK) cell product, large quantities of granzyme B were made by basophils in response to IL-3 stimulation, but not in response to IgE receptor activation. The significance of this finding remains undetermined.

BASOPHIL ACTIVATION Immunoglobulin E–Dependent Pathway When allergen interacts with specific IgE occupying FcεRI receptors on the surface of basophils, a succession of intracellular events is initiated that culminates in the secretion of mediators and cytokines. Mediator release induced by antigen has often been depicted as the simple crosslinking of at least two IgE-receptor complexes. In reality, the interaction is far more complex, with the overall response being dependent on the sensitivity of the cell, or the number of receptors needed for aggregation to achieve 50% of maximal release.53 This is further complicated by the fact that basophils can express many different IgE specificities that potentially affect sensitivity by creating changes in spatial availability for antigen binding. As it stands, sensitivities vary significantly among the basophils of different donors, with the number of aggregates required for histamine release ranging from as few as 200 to as many as 30,000. It is not fully understood at this time whether the numbers of aggregates for LTC4 release and cytokine secretion differ significantly from those optimal for histamine release. However, only subtle differences have been found in the sensitivities for the three classes of mediators, despite clear differences in the general parameters important for their release. Under optimal conditions, which include an absolute requirement for calcium, basophils will release histamine, LTC4, and cytokines over a several-log range of antigen concentrations, with typically bell-shaped dose-response curves for all three classes of mediators. Because of the ability of anti-IgE and anti-FcεRIα antibodies to interact indiscriminately

247

with the IgE and FcεRIα, respectively, on basophils in both allergic and nonallergic subjects, investigators often rely on the use of these antiantibodies to activate cells from both donor populations in responses that mimic those occurring with specific antigen. These bivalent stimuli also produce classic bell-shaped dose-response curves with regard to the release of these mediators, although generally over a much narrower range of concentrations. Although all three classes of mediators are released from basophils on IgE-mediated stimulation, considerable differences in the rates at which they are secreted have been recognized. Maximum release of preformed histamine, with either anti-IgE or antigen, is nearly complete within 20 minutes. As noted earlier, LTC4 is not stored as a preformed mediator, but its synthesis and release also are nearly complete within minutes after IgE-mediated stimulation. By contrast, the very different time course for cytokine generation appears to be dependent on signals that initiate transcription and translation for de novo synthesis. For example, increased levels of IL-3 and IL-4 protein are secreted within 1 hour after IgE-mediated activation, are half-maximal at approximately 2 hours, and peak after 4 to 6 hours of incubation.36 Cycloheximide, an inhibitor of protein synthesis, will ablate any further increases in cytokine secretion if added at any time during the culture incubation. Increases in mRNA for IL-4 (and IL-3) are detected within 30 minutes after activation, with levels peaking at about 2 hours. Message levels are back near baseline by 3 to 4 hours, which probably accounts for the waning in the secretion of IL-3 and IL-4 protein at this time. The generation of IL-13 also involves de novo synthesis, but the general consensus at this time is that it begins minutes to hours after IL-4 secretion is initiated and peaks some 18 to 20 hours later. More recent evidence suggests that this longer duration results from the early release of IL-3 that, through autocrine activity and direct stimulation, prolongs IL-13 secretion.51 Several laboratories have shown that mRNA for IL-4 is constitutively expressed (at about 10 copies per cell) in unstimulated or “resting” basophils.46,54 Although this is also true for IL-13 mRNA expression,54,55 this parameter seems more evident with IL-4. In agreement with these findings, small quantities of IL-4 protein are secreted within 5 to 10 minutes after activation, and with time, gradual accumulations are seen when cells are simply placed in medium alone.56 Although the significance of these findings is unclear at this time, the low levels of IL-4 that are constitutively expressed may simply represent a small percentage of basophils that have been activated either while in the donor before blood drawing or during their preparation ex vivo. Although IgE-dependent mediator release and cytokine secretion follow bell-shaped dose-response curves, they do so using very different concentration gradients. Optimal cytokine levels (e.g., IL-3, IL-4, IL-13) are produced when concentrations of the cross-linking stimulus are 10 times less than those required for optimal release of histamine and LTC4. This is particularly true with anti-IgE stimulation, but also with antigen challenge.57 This dissociation in cytokine secretion and mediator release probably means that basophils play a dual role in allergic inflammation, depending on antigen concentrations within the microenvironment. At low concentrations of antigen, basophils may modulate the immune responses of B cells, T cells, and eosinophils through the production of IL-4 and IL-13. As they infiltrate the lesion site, and the concentration of antigen increases, basophils may take on more of an effector role by secreting the two substances (i.e., histamine and LTC4) most responsible for the acute symptoms of allergic reactions. Fig. 15.2 provides a conceptual representation and summary of those parameters recognized as most important in regulating mediator release and cytokine secretion by human basophils. The IgE-dependent release of mediators and cytokines discussed thus far refer to interactions between antigen and antigen-binding regions

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Allergen

FCεRI

IL-3 (other cell sources)

CD123

+

Allergen

+

IL-3 (hours)

ng

+

i Prim

+

↑Expression of various cell surface markers (e.g., CD69, ST2, CD203c), survival, and overall function? IL-13 (hours, prolonged)

Histamine LTC4 IL-4, IL-13

Minutes (high allergen concentration) Hours (low allergen concentration)

Fig. 15.2  A conceptual representation of mediator release and cytokine secretion from basophils after IgE-dependent activation and the importance of IL-3 in augmenting these responses. IgE, Immunoglobulin E; IL, interleukin; LTC4, leukotriene C4.

of specific IgE. Mounting evidence, however, indicates that there are proteins that bind IgE nonspecifically, acting like superantigens or lectins that have the capacity to crosslink IgE/FcεRI complexes to activate basophils. In particular, the gp120 glycoprotein of HIV is reported to bind the variable heavy domain (VHD)-3 of IgE.58 The production of IL-4 and IL-13, which results from this interaction, has been hypothesized to play an important role in regulating the high IgE levels often observed in some HIV-infected patients. Recently, basophils have been shown to release histamine and large quantities of IL-4/IL-13 when co-cultured with the lung epithelial cell line, A549 (an adenocarcinoma).20 The response was dependent on cell-to-cell contact, the expression of IgE by basophils, and of signal transduction associated with FcεRIdependent activation. N-acetyllactosamine (LacNac) inhibited basophil activation in these cocultures, clearly indicating that A549 cells express a lectin(s) capable of binding IgE. At this time, galectin-3 is a likely candidate, given that it is well known to bind IgE and is commonly expressed by epithelial cells, particularly those of cancer origin. Although the clinical significance of this interaction remains to be determined, galectin-3 is implicated in many pathological conditions, including those whereby IL-4–producing basophils have been identified (e.g., asthma, lupus nephritis, and cancer).

Immunoglobulin E–Independent Pathway Studies have long demonstrated that basophils are more reactive than are mast cells to a greater number of substances that induce histamine release independently of IgE/FcεRI cross-linking. This is particularly true of products generated during immune reactions, such as cytokines and complement factors, but more recently has included agonists of specific innate immune responses (see later on). Early studies collectively grouped such substances as histamine-releasing factors (HRFs). However, the development of recombinant DNA technology helped in identifying many HRFs as known proteins (e.g., chemotactic factors). Although their exact significance in the pathogenesis of allergic disease remains unknown, these substances likely play a role in amplifying allergic reactions by inducing mediator release from basophils as they infiltrate allergic lesions during late-phase responses. Specific chemokines are capable of activating basophils, particularly when using cells from allergic subjects. Members of the monocyte

chemotactic protein (MCP) family (e.g., MCP-1/CCL2, MCP-3/CCL7, MCP-4/CCL13) all have been reported to cause degranulation when used at nanomolar concentrations. They mediate this activity by interacting through several CCR receptors. Other CC chemokines, such as RANTES/CCL5, MIP-1α/CCL3, and the eotaxins (types 1/CCL11 and 2/CCL24), have more limited potential for inducing histamine release; mostly from cells obtained from allergic subjects or from cells primed first in IL-3. These chemokines interact with the CCR3 receptor and appear to have a greater role in the selective recruitment of eosinophils, basophils, and lymphocytes into allergic lesions. As noted earlier, SDF-1/ CXCL12 is a potent chemoattractant for basophils and also is capable of inducing histamine release from these cells. To date, there have been no reports of any of these chemokines directly inducing the secretion of IL-4 or IL-13 from basophils. Several cytokines play a role in enhancing basophil responses, in particular, IL-3, IL-33, IL-5, GM-CSF, NGF, and so-called human recombinant histamine-releasing factor (HrHRF). All possess some ability to acutely prime basophils for enhanced histamine release in response to IgE-dependent activation. At relatively high concentrations (approximately 300 nM), IL-3 can act as a complete stimulus for histamine release. Again, IL-3 by itself has essentially no ability to induce LTC4 synthesis and is generally a poor activator of IL-4. However, these products also are released to a greater extent after IgE-mediated activation of cells primed in IL-3. IL-3 and IL-5 also have late priming effects on LTC4 secretion, which are related to intracellular events involving the activation of cytosolic phospholipase A2 (cPLA2).59 Perhaps the most striking activity mediated by IL-3 is its ability to activate basophils for IL-13 secretion directly.54,55 IL-3 is more potent in stimulating IL-13 secretion than is antigen or anti-IgE, which probably reflects difference in the mechanisms used by the two types of stimuli. The secretion of IL-13 in response to IL-3 is relatively slow to start, beginning after 4 hours of incubation; however, it remains ongoing beyond 20 hours in culture (Fig. 15.2). As noted earlier, the evidence that basophils themselves secrete IL-3 could mean that this priming phenomenon is autocrine in nature and occurs as a result of direct contact of circulating basophils with allergen.51 Results from several allergen challenge studies are consistent with this hypothesis but are only correlative at this time.60,61 Overall, it is becoming apparent that IL-3 affects essentially every aspect of basophil biology, including development and maturation, survival, acute or late effects on mediator release, and direct stimulation of IL-13 production. Studies continue to unravel some of the intracellular signaling pathways occurring in basophils after treatment with this cytokine. Table 15.2 summarizes some of the effects IL-3 and other cytokines have on basophil function. As noted previously, a recent study shows evidence that IL-31, in some ways, acts like IL-3.12 For example, it is reported to induce cytokine production (IL-4 and IL-13) without causing degranulation. And, because basophils appear to synthesize IL-31, it too can mediate autocrine effects. To date, only type 1 interferons such as IFN-α and IFN-β have been shown to negatively regulate basophil function.14 These cytokines were shown not to affect FcεRI responses; rather, they mediate only those involving cytokine secretion in response either to IL-3 priming of IgE receptor activation or to the IL-13 secreted in direct response to IL-3. It is not currently reported whether they might also inhibit the effects of IL-31. The anaphylatoxins C5a and C3a and the bacteria-derived peptide fMLP are some of the most potent basophil secretagogues described.62 Unlike the chemokines and cytokines just described, these are not necessarily linked to allergic inflammation, suggesting that basophil involvement in immune responses likely extends beyond that associated with immediate hypersensitivity reactions (see later on). fMLP is a particularly active and rapid inducer of basophil histamine release and mediates

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CHAPTER 15  Biology of Basophils

TABLE 15.2  Selected Cytokines and Chemokines Affecting Human Basophil Development,

Survival, Chemotaxis, and Secretion

SECRETED PRODUCTS

Mediator

Receptor

Development/ Survival

Cytokines IL-3

(CD123)

Potent

—

Alone Priming

± +++

± +++

+ +++

+++ +

IL-33

(ST2)

Weak

—

Alone Priming

+ ++

nr nr

+ ++

+ ++

HRF

(unknown)

nr

—

Aloneb Primingc

++ ++

nr nr

++ ++

− ++

IL-5

(CD125)

Weak

—

Alone Priming

− +

− +

− nr

− nr

IL-31

IL-31RA/OSMR

nr

Potent

Alone Priming

− ±

nr nr

++ ±

++ ±

GM-CSF

(CD-116)

Moderate

—

Alone Priming

− +

− +

− nr

− nr

NGF

(TrkA)

Weak

—

Alone Priming

− +

− +

− ++

+ +++

Chemokines MCP-1, -2, -3, -4 (CCL2, -8, -7, -13)

CCR2, CCR3

—

Potent

Aloneb With IL-3

+ +

± +

− ±

− ±

Eotaxins, RANTES (CCL11, -24, -26, -5)

CCR3

—

Potent

Aloneb With IL-3

+ ++

± ±

− ±

− ±

SDF-1/CXCL12

CXCR4

—

Potent

Alone

+

±

nr

nr

a

Chemotaxis

Activation

HR

LTC4

IL-4

IL-13

GM-CSF, Granulocyte macrophage colony–stimulating factor; HR, histamine release; HRF, histamine-releasing factor; IL, interleukin; LTC4, leukotriene C4; OSMR, oncostatin M receptor; MCP, monocyte chemotactic protein; NGF, nerve growth factor; SDF-1, stromal cell–derived factor 1; Trk A, tyrosine kinase–associated. Activity level: +, relative in vitro response; ±, positive responses rarely observed; −, no activity; nr, no reports. a Alone denotes the capacity to directly activate basophils for the products listed, whereas priming refers to whether it augments FcεRI-mediated secretion of these products. b Cells from selected allergic donors. c Cells from most donors.

degranulation through a G protein–coupled receptor that is structurally related to the seven-membrane transversing chemokine receptors. As a result, the intracellular pathways used by fMLP during degranulation are very different from those culminating after FcεRI-mediated activation using antigen or anti-IgE. This is important to consider, because neither fMLP nor the chemokines that activate basophils for histamine release, when used alone, typically induce IL-4 and IL-13 secretion. These findings support the notion that cytokine generation (particularly IL-4) is primarily a response mediated through FcεRI. As noted earlier, the exception to this is the stimulation of IL-13 in response to IL-3, IL-31, IL-33, or NGF, and in fact, there is evidence that either C5a or fMLP, when combined with IL-3, can stimulate the generation of IL-4 and IL-13 in basophils.63 Like fMLP, C5a does not normally activate basophils for cytokine secretion. Both of these stimuli are, however, capable of promoting LTC4 synthesis; the amount generated in response to C5a is greatly augmented in IL-3–primed basophils. As noted previously, more recent evidence shows that basophils, like many other leukocytes, express various receptors associated with innate immunity and are responsive to specific agonists known to bind these receptors. In particular, TLR2 ligands such as peptidoglycan (PGN)—a major constituent of the cell wall of gram-positive bacteria—have been shown to directly induce IL-4 and IL-13 from human basophils, albeit at levels approximately one-tenth of that typically induced on IgE

receptor stimulation.64 Moreover, PGN and the synthetic TLR2 agonist Pam3Cys markedly augmented mediator release or cytokine secretion, or both, when combined with other stimuli, both IgE-dependent and IgE-independent. By contrast, the same study indicated that basophils were unresponsive to TLR4 agonists such as lipopolysaccharide (LPS), a constituent of gram-negative bacteria, despite expressing mRNA and protein for this receptor. At this time, unresponsiveness to LPS is thought to result from basophils not normally expressing CD14—an important co-receptor in this innate immune response. In related studies, the LIR family of receptors mediated similar differential effects on basophil function. For example, antibodies directed at LIR3 mediated inhibitory activity on basophil secretion, whereas those that cross-linked LIR7 actually caused mediator release and cytokine secretion.39

PHARMACOLOGIC MODULATION OF SECRETION The relative ease of using washed leukocyte suspensions to investigate basophil histamine release has long made it possible for investigators to study the effects of various therapeutic drugs or inhibitors on both IgE-dependent and IgE-independent responses. Likewise, the whole blood BAT assay (both commercial and numerous in-house methods) has become the most widely used approach to monitor basophil degranulation during therapeutic interventions (e.g., oral and sublingual

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SECTION A  Basic Sciences Underlying Allergy and Immunology

immunotherapy for food allergy). And, with the added knowledge that basophils secrete IL-4 and IL-13, and under conditions that differ from those important for histamine release, it is now possible to investigate whether certain drugs differentially affect these responses. Presented in this section is a brief overview of those therapeutic agents currently in use for the treatment of allergic conditions and how they affect mediator release by basophils challenged in vitro. Drugs known to inhibit the release of preformed histamine from basophils are more effective at preventing the generation and secretion of cytokines from these cells. For instance, increases in cytosolic levels of cyclic adenosine monophosphate (cAMP) negatively affect the IgEmediated release of histamine. Thus substances that either stimulate cAMP synthesis through receptor-mediated activation of adenylate cyclase (i.e., β2-agonists, PGE2, histamine) or prevent its metabolism by inhibiting phosphodiesterases (theophylline) have been shown to inhibit, with variable potency, the histamine released in response to anti-IgE stimulation. Gibbs and colleagues have shown that the β2agonist salmeterol is two to nine times more potent at inhibiting IL-4 and IL-13 secretion from basophils than blocking histamine release.65 In the same study, theophylline was found to be nearly 10 times more active at inhibiting IL-4 than either histamine or IL-13. Like the cAMPenhancing agents, various H1 receptor antagonists also are long known to inhibit IgE-mediated histamine release by basophils in vitro. In comparing the inhibitory effects on mediator release versus cytokine secretion, desloratadine was approximately seven times more potent at inhibiting IL-4 than inhibiting either histamine or LTC4.66 Terfenadine also has been shown to inhibit mediator release and cytokine secretion, but with similar potencies.65 Of interest, cetirizine was shown to have a slight enhancing effect on cytokine secretion in this study. Whether the clinical efficacy of these drugs is related in part to their ability to inhibit the basophil response is not currently known. Their relatively greater potency in inhibiting cytokine secretion compared with mediator release has, however, renewed this debate. By contrast, glucocorticosteroids have proven efficacy in the treatment of allergic disease by inhibiting many factors contributing to inflammation. These drugs were shown to inhibit in vitro histamine release from basophils almost four decades ago.67 They did so, however, only after an 8- to 20-hour preincubation period before activation of the basophils with anti-IgE. Studies have since shown that antiinflammatory corticosteroids are far more effective in blocking basophil cytokine secretion; there is an immediate inhibition and at concentrations much lower than those required for the inhibition of mediator release.68 Some of the most potent inhibitors of IgE-mediated cytokine secretion are those agents that block Ca2+-dependent calcineurin activity, such as tacrolimus and cyclosporine. This finding has implied that the nuclear factor of activated T cell (NFAT) family of transcription factors are involved in the transcription of IL-4 and IL-13 initiated with FcεRI activation. Of note, tacrolimus has no effect on the IL-13 mRNA and protein produced by basophils in response to IL-3 stimulation, suggesting that NFAT molecules are not involved in the signaling occurring with this mode of activation.69 Finally, selective inhibitors of upstream signaling events also are reported to inhibit IgE-dependent secretion of mediators and cytokines from basophils—in particular, those targeting syk tyrosine kinase and, more recently, an inhibitor of Bruton’s tyrosine kinase (Btk).34 There are now several biologics capable of targeting basophils, either suppressing their function and/or depleting them from circulation. In particular, there is a plethora of data generated during the past 20 years showing how in vivo administration of omalizumab (e.g., anti-IgE therapy) suppresses basophil function.24 In brief, by binding to the portion of IgE that normally binds to FcεRI, omalizumab effectively neutralizes only unbound (or free) IgE, thus preventing it from sensitizing new basophils entering the circulation. Moreover, unoccupied FcεRIα

receptors are downregulated more rapidly on basophils than are those occupied with IgE. As a consequence, basophils become incapable of activation by allergen if not armed with sufficient levels of specific IgE. Given that the half-life of basophils circulating in blood is estimated at approximately 48 hours, then these effects on the basophil can occur within days after omalizumab administration, depending on baseline IgE serum levels. For comparison, tissue mast cells, which are estimated to live for months and thus turnover IgE/FcεRI more slowly, will downregulate these more slowing following anti-IgE therapy. Omalizumab administration does not alter the numbers of circulating basophils. However, two more recently developed biologics (both humanized monoclonals) have been shown to deplete basophils through enhanced antibody-directed cell cytotoxicity (ADCC). As noted above, benralizumab targets cells bearing the alpha subunit of the IL-5 receptor and was originally designed as a therapeutic to deplete eosinophils. Early studies indicate that it is also quite effective at depleting basophils.13 A new anti–IL-3 receptor antibody (CSL362), which is undergoing clinical trial testing in acute myelogenous leukemia (AML), has been shown to deplete basophils and plasmacytoid dendritic cells in vitro, both of which express IL-3 receptors at high levels.70

BASOPHIL INVOLVEMENT IN DISEASE Correlates of Allergic Disease By the mid-1960s, Lichtenstein and colleagues at Johns Hopkins University had demonstrated that the histamine released by a patient’s basophils challenged in vitro with ragweed allergen could predict the severity of the respiratory symptoms that individual would experience during the ragweed season. Other clinical correlates have since linked the basophil response to disease severity, particularly in conditions such as urticaria, asthma, and food allergy. For instance, most food-allergic children and many asthmatic subjects in general have basophils that spontaneously secrete histamine in vitro. Basophils from allergic asthmatics also possess an overall increased releasability to various stimuli, both physiologic and nonphysiologic. At least one report has demonstrated a twofold to threefold increase in the levels of IL-13 secreted from the basophils of allergic persons compared with nonallergic control subjects after exposure to IL-3 or NGF.71 Likewise, several experimental allergen challenge studies report evidence of phenotypic and functional changes in circulating basophils that are consistent with priming. In particular, increases in IL-13 secretion are noted with use of allergen provocation protocols in both the nose and lung.60,61 The numbers of basophils and their progenitors also are often increased in the blood of asthmatics and in allergic individuals who have been experimentally challenged with allergen.7 From these studies, it seems evident that there is a systemic activation or “priming” of basophils in individuals presenting with clinical inflammatory disease. Although this increased responsiveness could be a manifestation of the overall inflammatory response, new evidence points to self-priming by basophils augmented by autocrine-produced IL-3 on encountering allergen.51 As noted previously, both CD63 and CD203c are phenotypic markers whose expression is increased on basophils after in vitro activation. In addition to being used as surrogates to assess basophil degranulation, more recent studies have linked increases in CD63, CD203c, and CD69 to in vivo activation states. For example, CD203c expression is reportedly higher on basophils from asthmatic subjects who have recently experienced exacerbations as well as from subjects who present with nut allergy.31,32 More recently, it has been shown that basophils from most food-allergic children will upregulate these markers in vitro—a finding that is consistent with earlier findings that these basophils spontaneously release histamine. These responses were further shown to be IgE-dependent and thus transferable (using serum/plasma) to basophils

CHAPTER 15  Biology of Basophils from nonallergic subjects.72 Emerging immunotherapy modalities (e.g., oral and sublingual immunotherapy) show reductions in spontaneous CD63 expression (and histamine release), indicating some value in monitoring these markers during therapeutic intervention.30

In Allergic Disease The development of monoclonal antibodies that specifically detect human basophils in tissue has provided direct evidence that this onceelusive cell is indeed found within sites of allergic inflammation and in naturally occurring diseases. By using a basophil-specific antibody (2D7), Kepley and co-workers confirmed earlier reports that showed large numbers of basophils in the lungs of asthmatics, particularly from those who died from severe asthma, compared with those dying from non–asthma-related deaths.73 The use of basophil-specific antibodies (e.g., 2D7 and BB1) has since confirmed basophil involvement in chronic idiopathic urticaria and atopic dermatitis.

Late-Phase Responses The evidence that best supports the involvement of basophils in allergic inflammatory reactions has come from studies investigating the cellular inflammation and parameters associated with the late-phase response following experimental allergen challenge. These reactions typically occur several hours (6 to 12 hours) after attenuation of the immediate response and are manifested not only by symptoms that resemble those occurring during the early response but also by a selective recruitment of inflammatory cells from the circulation that accumulate at the lesion site. It has long been acknowledged that infiltrating eosinophils and, to a lesser extent, lymphocytes, are a hallmark of these reactions. The identification of basophils infiltrating the late-phase reaction took much longer to evolve, in that early studies never really differentiated between basophils and mast cells; metachromatically stained cells were often simply referred to mast cells. Ultimately, it was discovered from in vitro studies that mast cells and basophils differ with respect to the mediators they release and the way in which they respond to various stimuli. These differences resulted in the development of indirect measures that were often used to differentiate the involvement of the two cells types. For example, mast cells secrete PGD2 on FcεRI-mediated activation, whereas basophils do not. The relative ease of lavaging the site of allergen challenge, at multiple time points, made it possible to profile the mediators released during early and late reactions by analyzing the fluids recovered. The first of such studies performed in the nose showed that PGD2 was released during the immediate reaction, along with other mast cell mediators such as tryptase and histamine. By contrast, the fluids recovered during the late phase response, although containing histamine, did not show detectable levels of PGD2. As a result, these findings played an important role in establishing the concept that the late response is largely mediated by basophils, whereas the immediate response is orchestrated by mast cells. There has since been a renewed appreciation of the participation of basophils in the late-phase response. This interest stems from the knowledge that these cells secrete cytokines in addition to mediators, and that their identification in tissue has been made easier with the development of specific antibodies suitable for immunohistochemistry. Of great interest, these antibodies (both 2D7 and BB1) have detected basophils infiltrating late phase lesions at frequencies significantly higher than those reported in earlier studies (summarized in a review from that period).62 This is particularly true in the skin, where the frequency of basophils has approached 50% of that observed for eosinophils infiltrating these lesions. Although the presence of basophils infiltrating the lung is somewhat less than that described in the skin, they can still account for up to approximately 10% of the eosinophil infiltrate. The sensitivity achieved using these antibodies has further indicated that

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basophil influx into reaction sites of the lung and skin is occurring within 6 to 7 hours and persisting for at least 24 hours. In the nose, basophil numbers are reportedly higher at 1 hour, rather than 24 hours, after allergen challenge. It is thus clear that the involvement of basophils in the late phase response has been underestimated. Several studies indicate that basophils found in the late response in the lung also may be a significant source of IL-4 and IL-13. In recovering BAL cells 18 to 24 hours after segmental allergen challenge (SAC), ongoing or spontaneous secretion of IL-4/IL-13 ex vivo was detected only in the basophil suspensions, suggesting that these cells had been activated in vivo.60,66 In sharp contrast, the lymphocytes and eosinophils that also were recovered from these lesions did not secrete detectable levels of this cytokine. Using dual immunohistochemical staining, NouriAria and colleagues showed that basophils detected by the 2D7 antibody in bronchial biopsy specimens taken 24 hours after SAC accounted for more than 70% of the cells costaining for IL-4 protein and 25% of the cells expressing mRNA for this cytokine.74 It seems probable that these results will prompt further studies evaluating the cells responsible for IL-4/IL-13, because most previous reports have emphasized lymphocytes as the primary source of this cytokine in allergic lesions.

Basophils in Mouse Models Reagents for use in the murine system have greatly substantiated the in vivo biologic significance of basophils as IL-4–producing cells, with several recent studies pointing to a critical role for basophils in the initiation of the Th2 responses that are a hallmark of allergic disease. Foremost, studies in IL-4 reporter mice that make use of enhanced green fluorescence protein (eGFP) in a bicistronic construct (4get mice) or a construct that replaces the IL-4 gene itself (G4 mice) have unequivocally shown a rare fluorescent cell population bearing a basophil-specific phenotype.48,49 The occurrence of these cells even in animals deficient for STAT6 indicates that prior exposure to IL-4 is not necessary for their IL-4–producing ability, such is the case for development of IL-4– producing Th2 lymphocytes. A subsequent study showed evidence that papain, a serine protease similar to those associated with many known allergens and helminthic proteins, activated basophils to produce IL-4 in vitro, implying direct activation. However, this same protease when administered in vivo, may very well induce cytokines (e.g. IL-33, TSLP) from other cell types that subsequently activate basophils for IL-4. Whatever the mode of activation, papain, when administrated in vivo to mice, caused a rapid influx of basophils into the lymph nodes at a time preceding the lymphocytes.75 Moreover, the dependency of basophils in promoting the overall Th2 response was also demonstrated in depletion experiments. With the use of IL-4 reporter mice definitively showing basophils as an important source of IL-4 in vivo, subsequent studies proceeded to report evidence that basophils themselves can act as professional antigen-presenting cells (APCs) critical for the induction of Th2 responses. This concept remains controversial, however, in that the same findings have not been confirmed in other mouse models, nor have they been substantiated in vitro using human specimens. Mouse models that conditionally knock out basophils further point to the importance of basophil-derived IL-4 in vivo that until recently were only predicted by the in vitro properties of IL-4. Most relevant to allergic disease is evidence that basophils facilitate eosinophil transendothelial migration by upregulating VCAM-1 expression and contribute to IgE production by modulating B cell function and by promoting development of ILC2 cells and M2 macrophages.10,50,76

Delayed-Type Hypersensitivity It has been known since the early 1970s that basophils participate in specific delayed-type hypersensitivity reactions that are apparent

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manifestations of cellular immunity. For instance, the Dvoraks and their colleagues described the selective recruitment of basophils into sites of allergic contact dermatitis in humans that resembled the socalled cutaneous basophilic hypersensitivity (CBH) reactions originally reported in the guinea pig model. When given rhus toxoid (the active agent in poison ivy) or dinitrochlorobenzene in a patch test, sensitized subjects developed skin reactions that were characterized by a cellular infiltrate into the dermis consisting of up to approximately 16% basophils by 3 to 6 days after application of the antigen. In fact, it was often noted that basophils were the only granulocytes found in these lesions. However, mononuclear cells accounted for most of the cells infiltrating these lesions, which led to the hypothesis that the selective recruitment of basophils resulted from the secretion of some factor(s) released by T cells. To date, there has been no description of a known chemokine or cytokine that is responsible for the recruitment of basophils in these reactions. The profound changes in the dermal microvasculature that often accompanied these CBH reactions suggested the release of inflammatory mediators. It remains unknown whether the basophils migrating into these reaction sites are secreting cytokines; however, it is intriguing to think that they may be producing IL-13, because the synthesis of this cytokine (unlike IL-4) seems less dependent on signals generated through FcεRI. Ultrastructural analysis of the cells recovered in biopsies taken from lesion sites showed basophils undergoing a degranulation unlike that seen in immediate hypersensitivity reactions, which led the Dvoraks to describe so-called piecemeal degranulation (as discussed earlier).8 Of interest, an increased number of basophils showing an identical morphology also has been described in many other conditions involving a cellular immune component, including skin allograft and tumor rejection, viral hypersensitivity, and Crohn disease. There is a long-held belief that basophils (and eosinophils) are involved in natural immunity to parasitic infections. In particular, studies using the IL-4 reporter mice and other mouse models that selectively deplete basophils have greatly substantiated this concept by showing that IL-4–secreting basophils are critical for the expulsion of these worms.1 Of interest, similar animal models have pointed to the importance of basophils in the expulsion of ectoparasites as well, such as ticks. Although it remains to be determined whether such findings translate to human disease, a recent study has reported for the first time that basophils were seen in biopsies from subjects experiencing a recent tick bite.77 Whereas basophils were not identified in biopsies taken within 12 hours of tick infestation, those obtained after 12 hours showed on average approximately 50 basophils in the vicinity of tick mouthparts. By contrast, a relatively novel belief that also has been proposed is that human basophils may actually play a role in impaired immunity to parasitic infections, which is completely opposite from the work emerging from the animal studies. First, there is a striking relationship between immediate hypersensitivity reactions and parasitic infections in that increased IgE, eosinophils, basophils, and mast cells often are associated with both immune responses. However, parasite antigens seldom if ever induce the clinical manifestations typically seen in immediate hypersensitivity reactions, despite high levels of antigen-specific IgE that are capable of sensitizing basophils and mast cells. It has been shown that basophils from human patients with helminth infections secrete IL-4 and IL-13 in response to antigens derived from specific life stages of parasites. The production of these cytokines by basophils is hypothesized to be “driving” the Th2 response seen in helminthic infections, much like that proposed in immediate hypersensitivity. Interestingly, this apparent favoring of Th2-like responses is associated with impaired immunity to the parasite, suggesting that the production of IL-4 and IL-13 by basophils may create conditions that favor the survival of the organism.78 Consistent with this belief, the translationally

controlled tumor protein (TCTP) of Plasmodium falciparum, the organism responsible for malaria, is homologous to the HRF that causes histamine release and IL-4 secretion described earlier.79 This form of immune mimicry by the organism, which is well known among certain viruses, is thought to benefit the organism by modifying the immune response that normally removes it from the host. Finally, there is new evidence that is challenging the long-held belief that basophils only participate in immediate and delayed hypersensitivities. For example, IL-4-producing basophils have been identified in lupus nephritis, where it is predicted that autoimmune IgE is helping to facilitate this response.80 Just as striking is recent evidence that IL4-producing basophils were found in tumor-draining lymph nodes (TDLN) from subjects with pancreatic cancer.81 Moreover, the number of basophils found in these lymph nodes was predictive of patient survival in that greater numbers correlated with a poor outcome. In the same manuscript, strikingly similar results were shown in a murine model of pancreatic cancer, leading the authors to hypothesize that basophil IL-4 production plays a critical role in promoting a Th2 environment that favors survival of the tumor. This concept certainly raises novel possibilities as to the role of basophils beyond allergic disease, but additional studies are required for confirmation and in identifying the parameters/stimuli inducing basophil activation.

SUMMARY Basophils have long been seen as a surrogate with which to study the more elusive mast cell. This view, however, is no longer valid, because substantial developmental and physiologic evidence now shows that these two cell types have more differences than similarities. Once thought to do little other than to secrete histamine and LTC4, basophils are now well recognized to be the predominant cellular source of IL-4 and IL-13—perhaps the two most important cytokines with a role in the pathogenesis of allergic disease. This information, along with evidence that these cells infiltrate allergic lesions and are capable of responding to a wide variety of stimuli, continues to spark interest in basophils and in their role in allergic inflammation and disease.

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54. Redrup AC, Howard BP, MacGlashan DW Jr, et al. Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures. J Immunol 1998;160(4):1957–64. 55. Ochensberger B, Daepp GC, Rihs S, et al. Human blood basophils produce interleukin-13 in response to IgE-receptor-dependent and -independent activation. Blood 1996;88(8):3028–37. 56. Gibbs BF, Haas H, Falcone FH, et al. Purified human peripheral blood basophils release interleukin-13 and preformed interleukin-4 following immunological activation. Eur J Immunol 1996;26(10):2493–8. 57. Schroeder JT, MacGlashan DW Jr, Kagey-Sobotka A, et al. IgE-dependent IL-4 secretion by human basophils. The relationship between cytokine production and histamine release in mixed leukocyte cultures. J Immunol 1994;153(4):1808–17. 58. Patella V, Giuliano A, Florio G, et al. Endogenous superallergen protein Fv interacts with the VH3 region of IgE to induce cytokine secretion from human basophils. Int Arch Allergy Immunol 1999;118(2–4): 197–9. 59. Miura K, MacGlashan DW Jr. Dual phase priming by IL-3 for leukotriene C4 generation in human basophils: difference in characteristics between acute and late priming effects. J Immunol 2000;164(6):3026–34. 60. Schroeder JT, Bieneman AP, Chichester KL, et al. Pulmonary allergic responses augment interleukin-13 secretion by circulating basophils yet suppress interferon-alpha from plasmacytoid dendritic cells. Clin Exp Allergy 2010;40(5):745–54. 61. Saini S, Bloom DC, Bieneman A, et al. Systemic effects of allergen exposure on blood basophil IL-13 secretion and FcepsilonRIbeta. J Allergy Clin Immunol 2004;114(4):768–74. 62. Schroeder JT, MacGlashan DW Jr, Lichtenstein LM. Human basophils: mediator release and cytokine production. Adv Immunol 2001;77: 93–122. 63. Ochensberger B, Tassera L, Bifrare D, et al. Regulation of cytokine expression and leukotriene formation in human basophils by growth factors, chemokines and chemotactic agonists. Eur J Immunol 1999;29(1):11–22. 64. Bieneman AP, Chichester KL, Chen YH, et al. Toll-like receptor 2 ligands activate human basophils for both IgE-dependent and IgE-independent secretion. J Allergy Clin Immunol 2005;115(2):295–301. 65. Gibbs BF, Vollrath IB, Albrecht C, et al. Inhibition of interleukin-4 and interleukin-13 release from immunologically activated human basophils due to the actions of anti-allergic drugs. Naunyn Schmiedebergs Arch Pharmacol 1998;357(5):573–8. 66. Schroeder JT, Lichtenstein LM, Roche EM, et al. IL-4 production by human basophils found in the lung following segmental allergen challenge. J Allergy Clin Immunol 2001;107(2):265–71.

67. Schleimer RP, Lichtenstein LM, Gillespie E. Inhibition of basophil histamine release by anti-inflammatory steroids. Nature 1981;292(5822):454–5. 68. Schroeder JT, Schleimer RP, Lichtenstein LM, et al. Inhibition of cytokine generation and mediator release by human basophils treated with desloratadine. Clin Exp Allergy 2001;31(9):1369–77. 69. Schroeder JT, Miura K, Kim HH, et al. Selective expression of nuclear factor of activated T cells 2/c1 in human basophils: evidence for involvement in IgE-mediated IL-4 generation. J Allergy Clin Immunol 2002;109(3):507–13. 70. Busfield SJ, Biondo M, Wong M, et al. Targeting of acute myeloid leukemia in vitro and in vivo with an anti-CD123 mAb engineered for optimal ADCC. Leukemia 2014;28(11):2213–21. 71. Sin AZ, Roche EM, Togias A, et al. Nerve growth factor or IL-3 induces more IL-13 production from basophils of allergic subjects than from basophils of nonallergic subjects. J Allergy Clin Immunol 2001;108(3):387–93. 72. Schroeder JT, Bieneman AP, Chichester KL, et al. Spontaneous basophil responses in food-allergic children are transferable by plasma and are IgE-dependent. J Allergy Clin Immunol 2013;132(6):1428–31. 73. Kepley CL, McFeeley PJ, Oliver JM, et al. Immunohistochemical detection of human basophils in postmortem cases of fatal asthma. Am J Respir Crit Care Med 2001;164(6):1053–8. 74. Nouri-Aria KT, Irani AM, Jacobson MR, et al. Basophil recruitment and IL-4 production during human allergen-induced late asthma. J Allergy Clin Immunol 2001;108(2):205–11. 75. Sokol CL, Barton GM, Farr AG, et al. A mechanism for the initiation of allergen-induced T helper type 2 responses. Nat Immunol 2008;9(3):310–18. 76. Yamanishi Y, Miyake K, Iki M, et al. Recent advances in understanding basophil-mediated Th2 immune responses. Immunol Rev 2017;278(1):237–45. 77. Kimura R, Sugita K, Ito A, et al. Basophils are recruited and localized at the site of tick bites in humans. J Cutan Pathol 2017;44(12):1091–3. 78. King CL. Transmission intensity and human immune responses to lymphatic filariasis. Parasite Immunol 2001;23(7):363–71. 79. MacDonald SM, Bhisutthibhan J, Shapiro TA, et al. Immune mimicry in malaria: Plasmodium falciparum secretes a functional histamine-releasing factor homolog in vitro and in vivo. Proc Natl Acad Sci USA 2001;98(19):10829–32. 80. Pellefigues C, Charles N. The deleterious role of basophils in systemic lupus erythematosus. Curr Opin Immunol 2013;25(6):704–11. 81. De Monte L, Wormann S, Brunetto E, et al. Basophil recruitment into tumor-draining lymph nodes correlates with Th2 inflammation and reduced survival in pancreatic cancer patients. Cancer Res 2016;76(7):1792–803.

CHAPTER 15  Biology of Basophils

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SELF-ASSESSMENT QUESTIONS 1. What is the primary degranulation marker evaluated in the basophil activation test (BAT) assay? a. Interleukin 4 (IL-4) b. CD203c c. CD69 d. CD63 e. Leukotriene C4 (LTC4) 2. On average, basophils constitute approximately what percentage of the circulating white blood cells? a. 5% b. 2% c. 10% d. 0.1% e. 1% 3. What Th2 cytokine produced by human basophils is most dependent on activation involving IgE crosslinking? a. Interleukin 2 (IL-2) b. IL-4

c. IL-5 d. Interferon γ (IFN-γ) e. IL-13 4. What cytokine is produced by basophils following IgE-dependent activation and is capable of mediating autocrine activity? a. IL-4 b. IL-8 c. IL-3 d. IL-13 e. Histamine 5. What monoclonal antibody targeting so-called basogranulin is sometimes used for detecting basophils in tissue biopsies? a. 2D7 b. CD123 c. BB1 d. BASO1 e. BA-1

16  Biology of Eosinophils Hirohito Kita, Bruce S. Bochner

CONTENTS Introduction, 255 Eosinophil Morphology, Production, and Tissue Distribution, 255 Eosinophil-Derived Mediators, 257 Eosinophil Phenotype, 259

Eosinophil Recruitment and Accumulation, 261 Eosinophil Activation and Effector Functions, 262 Role of Eosinophils in Host Defense and Disease, 263 Conclusion, 264

INTRODUCTION

vesicles, also referred to as microgranules or tubulovesicular structures, are the most abundant and characterized by their dumbbell-shaped structures. They contain membrane-bound cytochrome b558 (phox-22), CD11b, the α chain of a β2 integrin, and other substances. Eosinophils also contain varying numbers of lipid bodies, a location for leukotriene synthesis, which are nonmembrane-bound, lipid-rich inclusions. The numbers of lipid bodies are increased in activated eosinophils and in eosinophils from patients with eosinophilia.

The eosinophil is a bone marrow–derived, peripheral blood and tissue granulocyte prominent in allergic and inflammatory responses against metazoan helminthic parasites but rarely increased in activity and number in otherwise healthy individuals. It was likely first observed by Wharton Jones in 1846 in unstained preparations of peripheral blood but became so named by Paul Ehrlich in 1879 because of the intense staining of its granules with the acidic dye eosin.1 Since that time the eosinophil has been the subject of extensive investigation. It is strongly associated with disorders involving mucosal surfaces, particularly allergic asthma and rhinitis, which exhibit a significant correlation with the number as well as activation status of infiltrating tissue eosinophils. Also, many disorders of the gastrointestinal system exhibit prominent eosinophilic inflammation in the mucosa. Although these conditions have been characterized clinically as either “allergic” or “nonallergic,” the mechanisms underlying recruitment and activation of eosinophils in both types of disease appear similar. This chapter focuses on the cell biology and biochemical aspects of human eosinophils and their potential roles in human diseases and host defense. Information on eosinophils from other species (e.g., mouse) is included to highlight any important similarities or differences. Other excellent reviews are also available.2-6

EOSINOPHIL MORPHOLOGY, PRODUCTION, AND TISSUE DISTRIBUTION Morphology 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 (Figs. 16.1 and 16.2). These “specific” granules or secondary granules are composed of a crystalline electron-dense core and an electron-lucent matrix. They contain highly charged cationic proteins, including major basic protein (MBP), eosinophil peroxidase (EPX), eosinophil cationic protein (ECP), and eosinophilderived neurotoxin (EDN). Three other types of eosinophil granules have been described. Primary granules are round, uniformly electrondense, and characteristically seen in immature eosinophilic promyelocytes. They are enriched with Charcot-Leyden crystal protein (CLC, galectin-10). Small granules contain acid phosphatase and arylsulfatase. Secretory

Production The life cycle of the eosinophil is divided into bone marrow, blood, and tissue phases. Production of eosinophils involves a cascade of interdependent regulatory events of at least three classes of transcription factors, including GATA-1 (zinc finger family member), PU.1 (ETS family member), and C/EBP members (CCAAT/enhancer-binding protein family). Specifically, expression of GATA-1 distinguishes cells restricted to the mast cell, eosinophil, megakaryocyte, and erythroid lineages from those restricted to the monocyte, neutrophil, and lymphocyte lineages.7 Similarly, PU.1 determines cell lineage fates, with low levels inducing lymphocytic cells and high levels myeloid differentiation. Thus GATA-1 and PU.1 synergistically induce eosinophil lineage differentiation. Of these transcription factors, GATA-1 is likely the most important for eosinophil lineage because mice with a targeted deletion of the highaffinity GATA-binding site show a specific loss of eosinophils.8 A number of unique genes, including the Ikaros-family transcription factor Helios and Aiolos (Fig. 16.3), are expressed by the eosinophil lineage.9 Hematopoietic factors important for eosinophil proliferation and differentiation include interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-5 (IL-5). IL-3 and GM-CSF stimulate proliferation of neutrophils, basophils, and eosinophils. In contrast, IL-5 potently and specifically stimulates eosinophil production in the bone marrow. IL-5 and GM-CSF also delay eosinophil apoptosis and increase their longevity. In the bone marrow, committed eosinophil precursors are recognized by their expression of the IL-5 receptor IL-5Rα (CD125) and the C-C chemokine receptor CCR3. High plasma levels of IL-5 are observed in some patients with blood eosinophilia, including episodic angioedema with eosinophilia, recombinant IL-2 therapy, and parasite infection. The malignant expansion of T cell clones that produce IL-5 in some patients with lymphoma

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induces marked eosinophilia.10 Furthermore, overproduction of IL-5 in transgenic mice results in profound eosinophilia, and deletion of the IL-5 gene causes a reduction, but not elimination, of eosinophils in the blood and tissues. Certain cytokines can inhibit eosinophil progenitor growth and differentiation. For example, interferon-α inhibits

colony formation by human bone marrow multipotential, erythroid, granulocyte-macrophage, and eosinophil progenitor cells in vitro and has been used to treat certain patients with eosinophilia (see Chapter 73). Although cytokines clearly play an important role in eosinophilopoiesis, it is also regulated by an intrinsic pathway involving paired

Fig. 16.1  Photomicrograph of purified peripheral blood eosinophils using Romanowsky (Giemsa-type) stain (Diff-Quik).

Fig. 16.2  Electron photomicrograph of a peripheral blood eosinophil. Major basic protein is located in the electron-dense crystalline cores of the secretory granules. (×7000.)

Fig. 16.3  Eosinophil lineage development within mouse bone marrow. Eosinophils develop from GATA-1+ pre-GMPs (granulocyte-macrophage progenitors) in the bone marrow. These pre-GMPs give rise to GMPs that (at least in mice) respond to IL-33 through the ST2 receptor, which promotes eosinophil development and IL-5 receptor α (IL-5Rα) expression. GMPs express higher levels of processed XBP1 mRNA, which is essential later in development. These GMPs give rise to Siglec-F+IL-5Rα+ mouse eosinophil precursors (EoPres). Additionally, IL-33 promotes eosinophil development by inducing IL-5 expression from other bone marrow cells, acting on EoPres and EoPs, which then follow EoPres in lineage development. EoPs express higher levels of Helios and Aiolos, members of the Ikaros family of transcription factors, which can play a role in regulating gene expression during eosinophil development and remain highly expressed in mature mouse eosinophils. Proper granule maturation requires expression of the transcription factor XBP1, inhibition of cysteine protease activity by cystatin F, and crystallization of the granule protein MBP-1 in a nontoxic form. Improper granule maturation can lead to loss of cell viability and blockade of eosinophil development. The long noncoding RNA Morrbid is highly expressed in eosinophils and other short-lived myeloid cells and has been found to prevent cell death by inhibiting transcription of the proapoptotic Bcl2 family member Bim. (Illustration by Jacqueline Schaffer. Modified from O’Sullivan JA, Bochner BS. Eosinophils and eosinophilassociated diseases: an update. J Allergy Clin Immunol 2018;141:505–17.).

CHAPTER 16  Biology of Eosinophils immunoglobulin-like receptor A (PIR-A) and PIR-B.11 By recognizing major histocompatibility complex class I, PIR-B blocks the proapoptotic activity of RIP-A and allows IL-5-induced expansion of eosinophils. Eosinophil development is also linked to successful biogenesis of their granules. The transcription factor XBP1 regulates the unfolded protein response by promoting the transcription of genes encoding stressresponse factors. Deletion of Xbp1 in the hematopoietic lineage disrupts granule formation and specifically reduces both eosinophil precursors and mature eosinophils.12 Similarly, loss of the endogenous cysteine protease inhibitor cystatin F impairs integrity of granules and reduces survival of eosinophils (Fig. 16.3).13

Tissue Distribution Although a formed element of the peripheral circulation, the eosinophil is primarily a tissue-dwelling cell; in humans the tissue eosinophil-toblood ratio is about 100 : 1. Normally, eosinophils are present in the gastrointestinal tract (but not the esophagus), regulated by the constitutive tissue expression of the chemoattractant eotaxin-1 (CCL11). Eosinophils also home into the thymus, mammary gland, and uterus. The mean bone marrow maturation and storage time is about 4.3 days. Once the eosinophil has entered the blood, it has a short half-life of 8 to 18 hours. The normal range of blood eosinophils is 0 to 0.5 × 103/ mm3 and exhibits diurnal variation in humans. The lowest and highest levels are seen in the morning and evening, respectively. The tissue life span of eosinophils ranges from 2 to 5 days, depending partly on the tissue studied. However, cytokines, such as IL-5, increase eosinophil survival up to 14 days. A long noncoding RNA Morrbid prolongs the life-span of myeloid cells, including eosinophils (Fig. 16.3), by inhibiting the transcription of the proapoptotic gene Bcl2l11 (Bim).14 Mechanisms for clearing the tissues of eosinophils include shedding of cells across mucosal surfaces into the lumen of the gut or the respiratory tract, engulfment of apoptotic eosinophils by macrophages, and lysis or degranulation with accompanying cell degeneration.

EOSINOPHIL-DERIVED MEDIATORS Eosinophils are unique among circulating leukocytes in their prodigious capacity to produce a variety of mediators, including granule proteins, cytokines, lipids, oxidative products, and enzymes (Table 16.1). These molecules are closely involved in the biologic and effector functions of eosinophils.

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TABLE 16.1  Eosinophil-Derived Mediators Granule Proteins Major basic protein (MBP) MBP homolog (MBP2) Eosinophil cationic protein (ECP) Eosinophil-derived neurotoxin (EDN) Eosinophil peroxidase (EPX) Charcot-Leyden crystal (CLC) protein Secretory phospholipase A2 (sPLA2) Bactericidal/permeability-inducing protein (BPI) Acid phosphatase Arylsulfatase β-Glucuronidase Lipid Mediators Leukotriene B4 (negligible) Leukotriene C4 5-HETE 5,15- and 8,15-diHETE 5-oxo-15-hydroxy-6,8,11,13-ETE Platelet-activating factor (PAF) Prostaglandin E1 and E2 Thromboxane B2 Oxidative Products Superoxide radical anion (OH−) Hydrogen peroxide (H2O2) Hypohalous acids Enzymes Collagenase Metalloproteinase-9 Indoleamine 2,3-dioxygenase (IDO)

Cytokines* IL-1α IL-2 IL-3 IL-4 IL-5 IL-6 IL-9 IL-10 IL-11 IL-12 IL-13 IL-16 Leukemia inhibitory factor (LIF) Interferon-γ (IFN-γ) Tumor necrosis factor–α (TNF-α) GM-CSF APRIL Chemokines CXCL8 (IL-8) CCL2 (MCP-1) CCL3 (MIP-1α) CCL5 (RANTES) CCL7 (MCP-3) CCL11 (eotaxin-1) CCL13 (MCP-4) Growth Factors Nerve growth factor (NGF) Platelet-derived growth factor (PDGF) Stem cell factor (SCF) Transforming growth factor (TGF-α, TGF-β)

APRIL, A proliferation-inducing ligand; ETE, eicosatetraenoic acid; GM-CSF, granulocyte-macrophage colony-stimulating factor; HETE, hydroxyeicosatetraenoic acid; IL, interleukin. *Physiologic significance of these cytokines needs to be confirmed.

Granule Proteins The specific granules contain MBP, which is localized in the crystalloid core, as well as ECP, EPX, EDN, β-glucuronidase, and secretory phospholipase A2 (sPLA2), all localized in the matrix (Fig. 16.4). Certain cytokines are preformed and stored in the core and matrix of the specific granules. Several molecules involved in the secretory process, including CD63 and vesicle-associated membrane proteins (VAMPs), are localized on the granule membrane. Human major basic protein is a 13.8-kD single polypeptide rich in arginine and five unpaired cysteines, with a calculated isoelectric point (pI) of 11.4. MBP is translated as a 23- to 25.2-kD proMBP with a calculated pI of 6 to 6.2. The 9.9-kD propiece of proMBP might protect the cell during transport of cytotoxic MBP from the Golgi apparatus to the eosinophil granule. Mature MBP is stored in the granules as a nondeleterious nanocrystal, which is acidified and becomes cytotoxic during the degranulation process.15 Because human MBP binds to and disrupts the cellular membrane, it is capable of directly damaging helminths, such as Schistosoma mansoni and Trichinella spiralis, and certain bacteria, such as Staphylococcus aureus and Escherichia coli. Human MBP is also toxic to tumor cells and other mammalian cells by

Lipid bilayer membrane CD63 VAMP-7 VAMP-8

Crystalline core Major basic protein (MBP) Cytokines

Matrix (internum) Eosinophil peroxidase (EPX) Eosinophil cationic protein (ECP) Eosinophil-derived neurotoxin (EDN) Cytokines/chemokines/growth factors

Fig. 16.4  Structure of the eosinophil crystalloid granule. This membranebound organelle is a major site of storage of eosinophil cationic granule proteins as well as a number of cytokines, chemokines, and growth factors.

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disrupting the integrity of lipid bilayers. MBP released into the skin tissues persists for up to 6 weeks. MBP2, the MBP homolog, has a calculated pI of 8.7 and is about 100 times less basic than MBP. MBP2 has effects similar to MBP but likely with reduced potency. Human eosinophil cationic protein is a basic protein with pI of 10.8 and consists of a single polypeptide chain of 15.5 kD. The ECP amino acid sequence has 32% identity to human pancreatic ribonuclease (RNase) A. ECP possesses RNase activity and is also known as RNase-3. The RNase activity of ECP is required for its neurotoxic and antiviral properties of the protein, but not for its antibacterial and antihelminthic activities. Human eosinophil-derived neurotoxin is a powerful neurotoxin that can severely damage myelinated neurons when injected intrathecally into experimental animals. Along with ECP, EDN belongs to the pancreatic RNase A superfamily and is also known as RNase-2 or RNase Us. EDN shows marked amino acid sequence homology (67%) with ECP but has about 100 times more RNase activity than ECP. Similarly to ECP, EDN has antiviral activities and decreases infectivity of respiratory syncytial virus. However, RNase by itself does not produce neurotoxicity or antiviral activities, suggesting unique functional properties of these proteins. In addition, EDN is a chemoattractant and activator of dendritic cells (DCs). As a consequence, EDN enhances helper T cell type 2 (Th2) responses through a Toll-like receptor 2 (TLR2)–dependent mechanism. The RNase gene family has among the highest rate of mutation in the primate genome, ranking with gene families of immunoglobulins and T cell receptors,16 suggesting that evolutionary constraints, perhaps microbial exposure, are acting on the ECP/EDN superfamily. Human eosinophil peroxidase (EPX) is a member of a mammalian peroxidase family and is the only granule protein found exclusively in eosinophils and no other cells. EPX 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 a pI greater than 11. Myeloperoxidase (from neutrophils and monocytes) in the presence of hydrogen peroxide (H2O2) and halide chloride kills bacteria, viruses, Mycoplasma, and fungi. EPX shows similar antimicrobial activity but prefers bromide over chloride as the source of halide. EPX is a central participant in generating reactive oxidants and radical species by activated eosinophils. Indeed, eosinophil activation in vivo shows oxidative damage of proteins through bromination of tyrosine residues in patients with asthma.17 Considerable evidence links these eosinophil granule proteins to human diseases. For example, the concentrations of MBP in the bronchoalveolar lavage (BAL) fluid from patients with asthma and from monkeys are correlated with the severity of bronchial hyperreactivity. MBP has been localized to sites of damaged bronchial epithelium in patients with asthma and chronic rhinosinusitis. Instillation of human MBP and human EPX provokes bronchoconstriction, and MBP increases airway responsiveness to inhaled methacholine.18 Interestingly, polyglutamic acid antagonizes MBP’s ability to increase respiratory resistance and bronchial hyperreactivity in cynomolgus monkeys, suggesting that the cationic nature of MBP contributes to damage and physiologic changes in the airway. In vitro, MBP acts as an antagonist for M2 muscarinic receptors. Many eosinophils are localized close to nerves, with extracellular MBP adhering to the nerves. Furthermore, neutralization of endogenously secreted MBP, either with a polyanionic peptide or with antibodies to MBP, can prevent antigen-induced bronchial hyperreactivity in guinea pigs.

Cytokines Eosinophils are a source of a number of regulatory or proinflammatory cytokines and chemokines. For example, eosinophils produce cytokines that act on eosinophils themselves, the so-called autocrine cytokines, including GM-CSF.19 Human eosinophils infiltrating tissues express transforming growth factor-α (TGF-α) and TGF-β1, suggesting that

eosinophils may contribute to the remodeling of tissues. Eosinophils can also produce osteopontin, vascular endothelial cell growth factor (VEGF), and nerve growth factor (NGF). IL-4 protein has been localized to eosinophils in airway and skin tissue specimens from patients with allergic diseases. Eosinophils also produce TNF-α and chemokines such as MIP-1α (CCL3) and RANTES (CCL5). Murine eosinophils were shown to produce APRIL, IL-6, and TGF-β.20 Thus eosinophils can produce various cytokines and chemokines, suggesting that eosinophils may be involved in diverse biologic responses. However, given that eosinophils make relatively small quantities of many of these proteins, the contribution of eosinophil-derived cytokines, chemokines, and growth factors to the development and maintenance of inflammatory reactions associated with allergic reactions remains difficult to determine, particularly in humans.

Lipid Mediators Eosinophils produce a wide variety of lipid-derived mediators. The main substrate for these mediators is arachidonic acid (AA), which is specifically liberated from membrane phospholipids. In general, PLA2 hydrolyzes membrane phospholipids, phosphatidylcholine (PC), and/ or phosphatidylethanolamine (PE) to produce AA and lyso-PC and/or lyso-PE. AA may be metabolized by cyclooxygenase or 5-lipoxygenase/5LO–activating protein (FLAP) into prostaglandins or leukotrienes, as well as lipoxins and 5-hydroxyeicosatetraenoic acid (5-HETE). Lyso-PC is acetylated to form platelet-activating factor (PAF). In eosinophils the predominant metabolite via the 5-lipoxygenase (5-LO) pathway is leukotriene C4 (LTC4), which in turn is metabolized extracellularly to LTD4 and LTE4. Human eosinophils are particularly rich in LTC4 synthetase and account for 70% of all LTC4 synthetase–positive cells in the airway mucosa of normal and asthmatic individuals.19 This finding is in contrast to 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. In eosinophils, 15-lipoxygenase metabolites are also detectable, including 5,15- and 8,15-diHETEs and a novel chemotactic lipid, 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid. Eosinophils contain high levels of ether phospholipids (the stored precursor to PAF), suggesting that the eosinophil is a good PAF producer. PAF has a number of important pharmacologic activities, including the activation of platelets and neutrophils and induction of bronchoconstriction. The cyclooxygenase pathway is also prominent in eosinophils, and eosinophils are capable of producing prostaglandin E1 (PGE1), PGE2, and thromboxane B2.

Oxidative Products Respiratory burst is defined as the increase in cell metabolism and oxygen consumption, coupled with the release of reactive oxygen species (ROS). The principal product of the respiratory burst is superoxide (O2−), which dismutates into more reactive ROS, including H2O2, the hydroxyl radical (OH−), and hypohalous acids. The O2− production is mediated through the activation of the NADPH oxidase complex. Overproduction of the NADPH oxidases is cytotoxic to the tissues as well as to microorganisms and has been implicated in the pathogenesis of many eosinophil-related disorders, including asthma. The ability of eosinophils to release a large amount of O2− extracellularly is thought to be the result of high levels of expression of the NADPH oxidase complex and preferred assembly of NADPH oxidase at the cell membrane. This is in contrast to neutrophils, which show an intracellular NADPH oxidase assembly.

Other Eosinophil Mediators A variety of other enzymes of potential biologic importance have been associated with the eosinophil. The Charcot-Leyden crystal (CLC) protein

CHAPTER 16  Biology of Eosinophils Eosinophil-derived substances

Proposed effects in asthma

Granule proteins (MBP, ECP, EDN, EPX) Galectin-10 (CLC protein) MMPs (e.g., MMP-9) LTC4 Cytokines and chemokines (e.g., GM-CSF, RANTES) Growth factors (e.g., TGF-β)

Effects of anti-IL-5 treatment on asthma*

Epithelial injury

Improved

Atelectasis

Unknown

Tissue damage and remodeling

Improved

Smooth muscle contraction

Eosinophil recruitment and survival; Immune modulation

Tissue remodeling and fibrosis Oxidative products (e.g., H2O2, O2–) Cellular injury

259

Exacerbations reduced; Lung function unchanged or slightly improved; Bronchial hyperreactivity unchanged Reduced Unknown Decreased matrix protein deposition Unknown

Fig. 16.5  Summary of the eosinophil’s biologic effects in asthma. MMPs, Matrix metalloproteinases. *Based primarily on studies of asthmatic patients with persistent disease activity and sputum eosinophilia despite ongoing use of inhaled corticosteroids.

forms hexagonal bipyramidal crystals. The appearance of CLC crystals in the sputa has been regarded as a hallmark of the eosinophil as early as 1872. CLC belongs to a family of galactose-binding lectins (so-called galectins) and is also named galectin-10.21 CLC constitutes 7% to 10% of total eosinophil protein content. Messenger RNA (mRNA) for CLC as well as that for EDN and ECP are among the most abundantly expressed by mature peripheral blood eosinophils,22 suggesting potential de novo synthesis of these proteins. Nonetheless, the biologic functions of CLC remain poorly understood. The CLC protein shows no affinity for β-galactosides but binds mannose avidly, suggesting it may bind carbohydrates expressed on microorganisms or other biologic molecules. The phospholipase A2 superfamily of structurally discrete enzymes consists broadly of three subfamilies: (1) 85-kD cytosolic PLA2 (cPLA2, group IV); (2) 14-kD secretory PLA2 (sPLA2, groups I to III, V, and IX); and (3) 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 AA at the sn-2 position of membrane phospholipids and generation of various lipid mediators in human eosinophils. Specific blockade of cPLA2 inhibited secretion of EPX, 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 leukocytes. sPLA2 may degrade phospholipids of gram-negative bacteria and cause airway inflammation and airway smooth muscle contraction. Eosinophils are a source of a 92-kD matrix metalloproteinase (MMP-9), gelatinase B. Gelatinase B can cleave type XVII collagen, a transmembrane molecule of the epidermal hemidesmosome, suggesting that production and release of gelatinase by eosinophils contributes to tissue damage. Furthermore, in vitro 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. Human eosinophils from the blood of allergic donors constitutively express indoleamine

2,3-dioxygenase (IDO), an enzyme that catalyzes the oxidative catabolism of tryptophan to kynurenines. Kynurenines inhibit proliferation and promote preferential apoptosis of Th1 cells. Together, eosinophils can provide a strikingly wide variety of biologic molecules, suggesting that eosinophils are potentially involved in diverse biologic responses, from host defense and tissue remodeling to activation of resident and infiltrating immune cells in many diseases, including asthma (Fig. 16.5).

EOSINOPHIL PHENOTYPE Eosinophils express a wide range of cell surface receptors through which they communicate with their surrounding environment and other cells. These include Fc receptors for immunoglobulins, complement receptors, receptors for soluble mediators (e.g., lipids, cytokines, chemokines), cell adhesion molecules, pattern recognition receptors, inhibitory receptors, and others. Although no one surface structure uniquely identifies human eosinophils, several are often used to distinguish and isolate eosinophils from other granulocytes. (For an extensive list of CD markers on eosinophils and other cells, see Appendix A.) Several non-CD cell surface molecules are expressed on eosinophils and are also discussed here (Table 16.2).

Cytokine Receptors As previously mentioned, eosinophil growth and differentiation are highly dependent on IL-5, and eosinophils selectively express the IL-5 receptor (CD125/CD131), along with related receptors for GM-CSF (CD116/CD131) and IL-3 (CD123/CD131). Indeed, both IL-5 and its receptor have been targeted using approved biologics for eosinophilic asthma.23 IL-5R levels are reduced in cells recruited to the lung and in blood of patients with marked eosinophilia, the latter in association with reciprocally increased serum soluble IL-5R levels. Although less cell-type selective, eosinophils can directly respond to many other cytokines, including IL-1, IL-4, TNF-α, and IFN-γ, presumably all through traditional cytokine receptors. More recently, it was determined that

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TABLE 16.2  Non-CD Surface Molecules

Expressed by Human Eosinophils and Their Ligands* Surface Molecule

Ligands

C3a receptor

C3a

CysLT1 receptor

Cysteinyl leukotrienes

CysLT2 receptor

Cysteinyl leukotrienes

EMR1

Unknown

†

FcεRI

Immunoglobulin E (IgE)

Histamine H4 receptor

Histamine

IL-33 receptor (ST2)

Interleukin-33

KIR2DL3

Human leukocyte antigen A (HLA-A)

LTB4 receptor

Leukotriene B4

LIR3

Unknown

P2X purinergic receptor

Various nucleotides

P2Y purinergic receptor

Various nucleotides

PAR-2

Serine proteases

PAF receptor

Platelet-activating factor

Siglec-8

6′-sulfo-sialyl Lewis X and 6’-sulfo-sialyl-Nacetyl-D-lactosamine

Siglec-10

α2,3-linked and α2,6-linked sialylated glycans Vascular adhesion protein 1

εBP (Mac-2)

IgE

*See Appendix A for a complete listing of CD molecules expressed by human eosinophils and other cells. † Controversial findings; see text.

eosinophils also possess receptors for IL-33 (ST2 and IL-1 receptor accessory protein), and IL-33 enhances eosinophil hematopoiesis and survival, as well as other responses (Fig. 16.3).24,25

Immunoglobulin Receptors Eosinophils express receptors recognizing the Fc portion of various immunoglobulins (FcR). Cross-linking of immunoglobulin (Ig) receptors on eosinophils has been shown to be highly effective at inducing a respiratory burst and degranulation in eosinophils. Flow cytometric analyses and degranulation studies demonstrated that eosinophils have functional IgA receptors (FcαR or CD89). Beads coated with IgA or secretory IgA (sIgA) induce degranulation of eosinophils, and eosinophils from allergic individuals display enhanced FcαR expression. The hierarchy of effectiveness in degranulation is in the order of sIgA ≥ IgA > IgG ≫ IgE.26 IL-3, IL-5, and GM-CSF enhance Ig-mediated degranulation. Collectively, these findings, along with the localization of eosinophils at epithelial surfaces, suggest an important role for sIgA in mediating eosinophil effector function in vivo. Receptors recognizing the Fc portion of IgG (FcγR) on human eosinophils have been identified. They possess FcγRII (CD32), especially the FcγRIIA (CD32A) activating isoform, although mouse eosinophils instead appear to express the inhibitory isoform FcγRIIB (CD32B)27 but eosinophil activation in vivo or by cytokines such as IFN-γ induces the expression of FcγRIII (CD16), a phosphatidylinositol-linked form, as well as FcγRI (CD64). On eosinophils, FcγRs have been studied for their ability to mediate phagocytosis, antigen-dependent cytotoxicity, oxygen metabolism, LTC4 production, and degranulation. Many of these studies have been performed using IgG-coated targets, but FcγR can also directly mediate eosinophil cytotoxicity of IgG-coated target cells

through FcγRII. The interaction between IgG and FcγRII may also be important in eosinophil degranulation responses in allergic diseases. For example, the sera from patients with allergic rhinitis contain 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 a monoclonal antibody. Also, the lack of FcγRIII (CD16) expression is useful experimentally. Human eosinophils are frequently isolated from peripheral blood by density-gradient centrifugation to create a mixture of eosinophils and neutrophils. Immunomagnetically based negative selection targeting CD16 is then used to remove neutrophils that, unlike normal eosinophils, strongly express CD16. Controversy still surrounds the presence of high-affinity receptors for IgE (FcεRI) and other lower-affinity IgE receptors on human eosinophils, but it is widely accepted that mouse eosinophils do not express FcεRI. Some reports describe IgE-dependent human eosinophil cytotoxic function and degranulation, such as killing of Schistosoma mansoni. However, most reports suggest that the number of high-affinity IgE receptors expressed on the surfaces of human eosinophils from allergic subjects or airway eosinophilia is minimal or undetectable, and ligation of FcεRI does not result in measurable eosinophil degranulation. Eosinophils do synthesize detectable levels of α-chain protein of FcεRI, but this protein is released extracellularly and is usually not detectable on the cell surface. Unlike other FcεRI-bearing cells (mast cells, DCs, basophils), eosinophil surface expression of FcεRI is not enhanced in the presence of extracellular IgE. Eosinophils from patients with eosinophilia express a low-affinity IgE-binding molecule, Mac-2/εBP, and the cytotoxic function of eosinophils against parasites was abolished by an antibody against this molecule. In some cases, low levels of FcεRII (CD23) can also be detected, but its exact function on eosinophils is uncertain. Thus the nature of human eosinophil IgE binding and its functional significance to human disease remain to be clarified.

Complement and Platelet-Activating Factor Receptors Eosinophils express the CR1 (CD35) and CR3 (Mac-1, CD11b/CD18) complement receptors, and levels of expression can be rapidly increased via mobilization to the cell surface from preformed intracellular stores by many eosinophil-activating stimuli. The C3a receptor is constitutively expressed on human eosinophils but not on neutrophils. The C5a receptor (CD88) is expressed on both eosinophils and neutrophils. The complement-derived anaphylatoxins, C3a and C5a, induce elevations in intracellular Ca2+, degranulation and production of oxygen radicals from eosinophils, and induce their shape change and chemotaxis. Eosinophils respond to PAF (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine), one of their most potent and effective chemoattractants. It preferentially induces the migration of eosinophils over neutrophils. PAF activation evokes the release of granule proteins, ROS, and LTC4 from eosinophils. Despite these in vitro activities, and with a growing interest in the role of PAF in anaphylaxis, the specific role of PAF compared with other mediators in eosinophilic inflammation still needs to be elucidated.

Receptors for Arachidonic Acid Metabolites Eosinophils, along with basophils and Th2 lymphocytes, express a receptor for prostaglandin D2 (PGD2) known as CRTH2 or DP2. This sevenspanner receptor mediates eosinophil migration and activation.28 Ongoing work with oral CRTH2 antagonists and a biologic targeting this receptor will be enlightening for fully defining its role in mediating eosinophilic inflammation. Cysteinyl leukotrienes (cysLTs; LTC4, LTD4, and LTE4) interact with specific receptors, CysLT1 and CysLT2, to cause their cellular effects.29 Both these receptors are expressed by eosinophils, and their migration

CHAPTER 16  Biology of Eosinophils

261

in response to cysLTs can be blocked by CysLT1 antagonists, which explains at least some antieosinophil properties seen clinically as a result of CysLT1 antagonist therapy. Eosinophils also respond to 5-lipoxygenase products from arachidonic acid, such as 5-Oxo-ETE (5-oxo-6,8,11,14eicosatetraenoic acid), via the OXE receptor. There are at least two mammalian receptors for LTB4, the high-affinity BLT1 and low-affinity BLT2, both of which are G protein–coupled receptors (GPCRs). LTB4 induces several functional responses in eosinophils, including chemotaxis.

Siglec-8, and Siglec-10.35 The most extensively studied is Siglec-8, in which cross-linking with antibodies or a synthetic glycan ligand induces cell death through a β2 integrin and ROS-dependent death pathway.36 More modest proapoptotic effects are seen when Siglec-8’s closest mouse paralog, Siglec-F, is targeted on murine eosinophils by antibodies or sialoglycans, such as those on specific airway mucins.37 Other ways to induce receptor-dependent eosinophil apoptosis include exposure to glucocorticosteroids and activation of CD30 or FAS (CD95).

Chemokine Receptors

Other Receptors

Chemokine receptors are members of the GPCR superfamily (see Chapter 7). Among the CC subfamily, eosinophils most prominently and selectively express CCR3, along with lower levels of CCR1 and CCR2. CCR3 binds the eotaxins (CCL11, CCL24, and CCL26); RANTES (regulated on activation, normal T cells expressed and secreted, CCL5) binds CCR1, CCR3, and CCR5; MCP-3 (CCL7) and MCP-4 (CCL13) bind both CCR1 and CCR3; and macrophage inflammatory protein-1α (MIP-1α, CCL3) binds CCR1. Importantly, CCR3 is expressed by eosinophils, basophils, and mast cells but not by other leukocytes, which explains the interest in the development of CCR3 antagonists for clinical use. Human eosinophils also express CXCR3 and CXCR4 that bind to IFN-γ/ inducible protein-10 (IP-10, CXCL10) and stromal cell–derived factor1α (SDF-1α, CXCL12), respectively, but these latter cytokines appear to play more of a role in noneosinophilic inflammation.

A few other cell surface molecules are mentioned here because of their potential diagnostic and clinical relevance. Human eosinophils, among other cells, express CD52, and an antibody to this molecule (alemtuzumab) has been used as a treatment for refractory hypereosinophilic syndrome, with most patients achieving hematologic remission, but most still relapse.38 The epidermal growth factor (EGF) seventransmembrane-spanning cell surface molecule F4/80 is a marker of murine macrophages, but the human ortholog of F4/80, an EGF-like module containing mucin-like hormone receptor 1 (EMR1), was unexpectedly found to be uniquely expressed on eosinophils.39 Eosinophils express additional cell surface structures that alter their migration, including the type 4 histamine receptor (histamine H4 receptor) and various purinergic receptors, including those belonging to the P2X and P2Y receptor families (e.g., P2X7, P2Y2). Finally, more so in mice than in humans, there is growing evidence suggesting that there may be distinct eosinophil subsets in tissues.40,41

Pattern Recognition Receptors This family of pattern recognition receptors (PRRs; see Chapter 1) includes Toll-like receptors (TLRs), C-type lectin receptors, nucleotidebinding oligomerization domain (NOD)–like receptors, and the receptor for advanced glycation end products (RAGE). The PRRs function to recognize and trigger innate immune responses to pathogen-associated molecular patterns (PAMPs) associated with microbe-derived material or cellular stress, as well as damage-associated molecular patterns (DAMPs) associated with cell-derived material released during their damage. Some PRRs are cell surface receptors, whereas others are intracellular. Ligands include lipids (e.g., lipoproteins, lipopolysaccharides), proteins (e.g., flagellin), intracellular nucleotide, PAMPs derived from invading viruses (e.g., single- and double-stranded RNA), and humanderived danger signals. Human eosinophils express several PRRs, including TLRs 1 to 5, 7, and 9, NOD1, NOD2, and RAGE.30 Receptor engagement induces a range of eosinophil responses, including enhanced cell survival, production of ROS, activation of the inflammasome, augmented cell adhesion, and release of cytokines, chemokines, and eosinophil granule proteins. Their expression of a variety of PRRs suggests that eosinophils can participate in the immune response to some bacteria, viruses, and fungi. Indeed, lipopolysaccharide activates cytokine-primed eosinophils to release mitochondrial DNA, which in turn kills bacteria.31 Other studies implicate antiviral and antifungal activities of eosinophils as well, although protease-activated receptors also contribute to the latter responses.32

Inhibitory and Proapoptotic Receptors The most common types of inhibitory receptors contain so-called immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. Engagement of these receptors typically transduces a negative cell signal by recruiting SRC homology 2 domain–containing phosphatases. Eosinophils express several ITIM-containing surface molecules, including leukocyte inhibitory (Ig-like) receptor type 3 (LIR3, CD85a), killer cell Ig-like receptor, two domains, long cytoplasmic tail 3 (KIR2DL3), CD300a and CD300f.33,34 Eosinophils also express four members of the sialic acid–binding immunoglobulin-like lectin (Siglec) subfamily of the Ig superfamily: Siglec-3 (CD33), Siglec-7 (CD328),

EOSINOPHIL RECRUITMENT AND ACCUMULATION Eosinophils are predominantly tissue cells, and their major target organ for homing in the healthy individual is the gastrointestinal tract, likely because of their role in innate defense against parasites. In disease states, eosinophils also home to other tissues, including the lungs, skin, heart, and other organs. Once they enter target tissues, eosinophils do not return to the blood circulation, although studies in mice suggest that endobronchial eosinophils can travel to regional lymph nodes and act as antigen-presenting cells. Eosinophil numbers can remain high in tissues even when peripheral numbers are low, suggesting that their survival is enhanced after extravasation. Curiously, pathogen-free laboratory animals have no eosinophils in their blood, and tissue eosinophils are difficult to find, suggesting that their production may be related to microbial colonization. The selective tissue recruitment of eosinophils occurs across the vascular endothelium and into tissues (Fig. 16.6).42 Most migration through endothelium occurs at postcapillary venules. Each of these steps is controlled by a complex network of chemotactic factors and adhesion molecules that collectively and selectively direct the movement of the eosinophil into the tissues (see Chapters 6 and 7). Selectins and their sialylated glycan ligands, along with α4 integrins, are important in eosinophil tethering and rolling, whereas α4 and β2 (CD18) integrins mediate firm adhesion. The transmigration step is primarily regulated by β2 integrins, as well as CC chemokines such as the eotaxins (CCL11, CCL24, and CCL26). Cytokines and chemokines are elaborated by surrounding tissues to modulate the transmigration of eosinophils into tissues. For example, IL-4 and IL-13 appear to be involved in upregulating eosinophil adhesion, primarily through selective induction of vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells.43 IL-5 also upregulates eosinophil, but not neutrophil, adhesion to unstimulated endothelium. IL-5 and GM-CSF activate transendothelial migration of eosinophils through intercellular adhesion molecule 1 (ICAM-1) through decreased β1 and increased β2 integrin function and expression. Similarly, stimulation of eosinophil CCR3 with a chemokine such as CCL11,

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Blood vessel (capillary)

Tethering

Endothelium

Rolling

Flattening, integrin activation and adhesion

E- and P-selectins VCAM-1

CCR3-active chemokines ICAM-1 VCAM-1

Mediator release Asthma Allergic bronchopulmonary aspergillosis Churg-Strauss syndrome Helminthiasis/Helminthiases

Activation in situ

Transmigration (diapedesis)

Chemotaxis CCR3-active chemokines ICAM-1 VCAM-1 PECAM-1 OR

Accumulation of quiescent cells Eosinophilic bronchitis (?) Idiopathic pulmonary eosinophilia (?)

CCR3-active chemokines Fibronectin Laminin

Fig. 16.6  Eosinophil tethering, rolling, adhesion, transmigration, and chemotaxis in response to inflammatory signals in tissues. During chemotaxis, eosinophils may become activated in response to local inflammation and release mediators, as in asthma and other related conditions, or they accumulate in tissues in the apparent absence of mediator release.

which can be presented on endothelial cells, also increases β2 integrin activity, resulting in preferential binding to ICAM-1. Upon adherence to vascular endothelium, eosinophils commence diapedesis and accumulate in adjacent connective tissue. Changes in the binding affinity for adhesion molecules and extracellular matrix proteins are thought to contribute to cell movement on a substratum. Cytokines and chemokines also influence the binding of eosinophils to tissue surfaces. Other chemotactic factors, such as CCL5, CCL7, and C5a, can alter eosinophil β1 integrin affinity. The balance of these factors determines the rate and direction of eosinophil migration.44 The most potent and effective eosinophil chemoattractants include PAF, LTD4, C5a, and CC chemokines such as CCL11/eotaxin-1 and CCL26/eotaxin-3. CCR3-active chemokines are increased at sites of allergic inflammation and are produced by many cells, including endothelial cells, epithelial cells, parasympathetic nerves, T cells, and fibroblasts. Indeed, CCL26 is the gene most highly upregulated in mucosal biopsies of patients with eosinophilic esophagitis.45 Yet to date, clinical testing of oral antagonists to CCR3 has been disappointing, raising doubts about the importance of this pathway in asthma.46

EOSINOPHIL ACTIVATION AND EFFECTOR FUNCTIONS Eosinophils in human blood are not a homogenous population but represent various levels of activation. Eosinophils isolated from blood of patients with asthma demonstrate many enhanced proinflammatory properties compared with those from normal individuals, including enhanced adhesion, chemotaxis, transendothelial migration, and LTC4 production.47 These phenotypic and functional changes in human eosinophils are likely the result of enhanced cellular responsiveness termed priming, which can also occur when eosinophils are recruited into the tissues. The treatment of asthma patients with a combination of inhaled glucocorticoids and β-adrenergic agonists reduces markers of eosinophil priming. Anti-IL-5 antibody reduced the numbers of airway eosinophils, but it did not affect their activated phenotype.48

Exposure of eosinophils ex vivo to various cytokines mimics in vivo primed eosinophils. For example, IL-5 activates LTC4 and O2− generation, phagocytosis, and helminthotoxic activity, as well as Ig-induced degranulation. GM-CSF; IL-3; Th2 cytokines, such as IL-4 and IL-13; and a Th1 cytokine IFN-γ also activate eosinophils.19 IL-33 and TSLP are cytokines produced by tissue cells, and they are implicated in Th2type immune responses.49 Both TSLP and IL-33 activate eosinophil effector functions such as adhesion to matrix proteins, cytokine production, and degranulation. Interestingly, IL-33 does not affect neutrophil functions, suggesting that IL-33 may regulate functions of eosinophils specifically in airway mucosa. Several molecules inhibit eosinophil functions. For example, TGF-β, a prototypic antiinflammatory cytokine, decreases the number of eosinophils in human bone marrow suspension cultures. TGF-β also inhibits eosinophil survival induced by the eosinophil hemopoietins in vitro.50 Eosinophils change their function and phenotype as they become activated. For example, activated eosinophils express HLA-DR and increased amounts of ICAM-1. In vitro stimulation of blood eosinophils with IL-3, IL-5, or GM-CSF induces the expression of activation antigen CD69 and CD25 and upregulates the expression of the β2 integrin, CD11b. Peripheral blood eosinophils from patients with helminth infection and BAL eosinophils from patients with asthma show increased expression of ICAM-1, HLA-DR, CD11b, CD11c, CD44, CD66, CD66b, CD69, and CD81.47 Importantly, expression of αMβ2 integrin (CD11b/ CD18) and an activation epitope of αMβ2 are increased in airway eosinophils from patients with asthma who are exposed to airway allergens. Thus the activation status of eosinophils differs among individuals and among diseases and disease activity. Extracellular release of granule contents is considered to be one of the major effector functions of eosinophils and is implicated in host defense as well as disease processes. The eosinophil exhibits three forms of granule release. The first is classical exocytosis of crystalloid granules, in which their contents are released by the granules fused with plasma membrane. This type of release is typically seen in vitro and can be demonstrated electrophysiologically using a patch-clamp technique that measures change in membrane capacitance. The second mode of

CHAPTER 16  Biology of Eosinophils eosinophil degranulation is by piecemeal degranulation (PMD). PMD was first reported as the appearance of numerous small vesicles in the cytoplasm coupled with the apparent loss of crystalloid granule core and matrix components, creating a “mottled” appearance on electron microscopy. PMD was thought to be caused by small vesicles budding off from the larger secretory granules and moving to the plasma membrane for fusion, thereby causing gradual emptying of the granule contents. Finally, eosinophils in airway and skin tissue often appear necrotic, as if they were undergoing cytolytic degranulation. This process is likely regulated by the receptor-interacting protein kinase 3–mixed lineage kinase-like signaling pathway and may represent a pathologic event to release cytotoxic granule proteins into surrounding tissues.51 Exosomes that contain a variety of eosinophil proteins, including MBP and EPX, are also released by activated eosinophils.52 Several in vitro models have been used to investigate the immunologic mechanisms of eosinophil degranulation. Secretory IgA is the most potent among all immunoglobulins in inducing degranulation. Interestingly, eosinophil granule proteins themselves, including MBP and EPX, stimulate eosinophils and cause degranulation, suggesting an autocrine mechanism of eosinophil degranulation. The other effective stimuli for eosinophil degranulation include cytokines, serum-opsonized zymosan, fMLP (N-formyl-methionyl-leucyl-phenylalanine), PAF, the complement fragments C5a and C3a, and naturally occurring peptides, such as substance P and melittin. Further studies are necessary to elucidate the roles of these molecules in eosinophil degranulation in the disease. Notably, an integrin, Mac-1 (CD11b/CD18, αMβ2), likely plays pivotal roles in regulating eosinophil degranulation; thus this molecule is not only important for eosinophil recruitment but also crucial for eosinophil effector functions. Historically, receptor ligands immobilized to relatively large surfaces, such as IgG-coated Sepharose beads and parasites, but not to particulate ligands, such as aggregated IgG and bacteria, are effective stimuli for eosinophil degranulation. Later it was found that β2 integrins, in particular αMβ2, play a crucial role in the activation of eosinophils stimulated by IgG, when IgG is immobilized onto tissue culture plates or Sepharose beads.26 Similarly, the eosinophil functional response to PAF or cytokines is greatly influenced by the availability of cellular adhesion mediated by αMβ2.19 The ability of human eosinophils to effectively engage their integrins and to respond to microbes/molecules with a large surface, such as helminths and fungi, likely distinguishes this leukocyte from other granulocytes.

ROLE OF EOSINOPHILS IN HOST DEFENSE AND DISEASE In understanding disease pathophysiology, at least four approaches are used to explore the role of a particular cell or molecule in a particular disorder. For evaluating the role of eosinophils and eosinophil-derived mediators in this way, approaches include (1) correlating eosinophil numbers with disease activity, (2) determining whether therapies that selectively reduce eosinophils are beneficial, (3) exploring whether there are genetic conditions affecting eosinophils that result in disease, and (4) using mouse models, especially those in which eosinophils or eosinophil-derived mediators have been targeted or eliminated. This section summarizes how these approaches have, or have not, implicated eosinophils and their mediators in various immune responses and disease states.

Immune Regulation and Homeostasis Previously, eosinophils have been considered an end-stage effector cell. However, accumulating evidence suggest that eosinophils can perform various immune regulatory functions, likely through production of a range of cytokines and other immunomodulatory molecules. Earlier,

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the immunomodulatory functions of eosinophils in vivo were demonstrated in murine models of allergen sensitization and challenge with ovalbumin (OVA) and helminth infection. For example, in IL-5/eotaxin double-knockout mice, in which eosinophil numbers in both blood and tissues are severely decreased, IL-13 production of Th2 cells in response to OVA challenge is attenuated. This defect in Th2 cells was restored by eosinophil reconstitution,53 suggesting regulation of adaptive Th2-type immune response by eosinophils. The roles of eosinophils in Th2-type airway inflammation were addressed subsequently by using eosinophil-deficient animals. Eosinophils were depleted by using an eosinophil-specific promoter to drive expression of a cytocidal protein, diphtheria toxin A chain (so-called PHIL mice),54 or by developing mice harboring a deletion of a highaffinity GATA-binding site in the GATA-1 promoter (Δdbl-GATA), which led to the ablation of the eosinophil lineage.55 When sensitized and challenged with OVA, Th2-type airway inflammation and asthma-like pathology (e.g., mucus production, airway remodeling) were attenuated in both PHIL mice and Δdbl-GATA mice, and these responses were restored by reconstitution of eosinophils. Although the mechanisms to explain how eosinophils modulate Th2 responses are not fully understood, the deficiency in eosinophils resulted in the defect of airway chemokine responses and decreased expression of genes regulating the coagulation cascade, suggesting that eosinophils may be involved in priming of tissue environment for effective mobilization of Th2 cells. Although the connection to humans remains uncertain, mouse studies also showed that eotaxin-1 (CCL11)–deficient mice, which lack eosinophils in some tissues at baseline, demonstrate a 2-week delay in the onset of estrus, along with a delay in the first age of parturition, suggesting a role for eosinophils in preparing the mature uterus for pregnancy and in blastocyst implantation. Deletion of the eotaxin-1 gene also resulted in a reduction in terminal-end bud formation and reduced branching complexity of the ductal tree, suggesting a role for eosinophils in postnatal mammary gland development. Eosinophils also may play roles in long-term maintenance of plasma cells in bone marrow in mice by providing their survival factors, APRIL and IL-6.20 In the peripheral tissues, the absence of eosinophils is associated with decreased production of secretory IgA in the intestinal mucosa, and thereby alters intestinal microbiome and integrity of mucosal barrier.56 These data suggest that the steady-state presence of eosinophils in certain tissues may play important roles in morphogenesis and maintenance of mucosal organs, as well as immune homeostasis in bone marrow and other tissues.

Helminth Infections Infection with helminths is a common cause of eosinophilia. Studies in the late 1970s demonstrated that eosinophils have the capacity to kill parasitic targets. Eosinophils are capable of phagocytosing and killing bacteria and other small microbes in vitro but cannot effectively defend the host against bacterial infections when neutrophil function is deficient. Rather, eosinophils appear to defend against large, nonphagocytosable organisms, most notably multicellular helminthic parasites. Indeed, several in vivo mouse models suggested protective roles for eosinophils in parasitic infection.57 However, the precise role of eosinophils in helminth infections remains controversial.19 For example, although treatment with anti–IL-5 reduced the number of circulating and tissue eosinophils, there was no evidence for change in the nature or extent of helminth infection. Similar conclusions were reached in several studies using genetically altered mice, including eosinophil-less mice. It is difficult to discern a role for eosinophils in mouse models for several reasons, most importantly because these experiments are performed with human pathogens that do not naturally infect rodents. Therefore the mouse immune system may not have been exposed to evolutionary pressure to develop optimal responses to these pathogens.

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Questions also remain as to whether eosinophils participate in immunity against the helminths, in the pathology of the disease, or both.

Innate Immunity Several lines of evidence indicate that eosinophils may be involved in immunity against bacterial and viral infections. As noted, eosinophils constitutively express many Toll-like receptors. Eosinophils do not express common fungus receptors, such as dectin-1, but use their versatile β2 integrin molecule, αMβ2, to recognize and to adhere to a major cell wall component, β-glucan. A unique antibacterial activity of human eosinophil is also recognized. Human eosinophils rapidly release mitochondrial DNA in response to exposure to bacteria.31 The traps contain the granule proteins ECP and MBP and display antimicrobial activity. These mitochondrial DNA and the granule protein bind and kill bacteria in the extracellular space in vitro and in vivo. Indeed, after cecal ligation and puncture, eosinophil-rich IL-5 transgenic, but not wild-type, mice are protected from microbial sepsis. 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 concentrations of eosinophil granule proteins are increased in the respiratory tracts of patients with respiratory syncytial virus (RSV), an RNA virus. As previously described, EDN and ECP are RNases, and purified eosinophils, EDN, and ECP added to RSV suspensions reduce the viral titer, an effect dependent on their RNase activities. Furthermore, in guinea pigs infected with parainfluenza, pretreatment with anti–IL-5 with a reduction in eosinophils strikingly 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.

Rhinosinusitis and Allergic Inflammation Evidence for the involvement of eosinophils in rhinosinusitis is mainly circumstantial.58,59 Eosinophils, eosinophil-active chemokines, and eosinophil granule proteins increase in number in nasal secretions during the allergy season, after allergen challenge, and in chronic rhinosinusitis, and use of effective medications such as corticosteroids reduces eosinophil numbers. Two studies found benefit of anti–IL-5 treatments in some patients with nasal polyposis. These biologics, when given for the treatment of asthma, appear to work particularly well in those who also have concomitant nasal polyposis; most effective among antibody therapies tested to date, however, was the use of an anti-IL-4/IL-13 biologic.60,61

Asthma, Airway Remodeling, and Airway Hyperreactivity Ever since the study showing that adjustment of the use of inhaled corticosteroids based on sputum eosinophil counts was a particularly effective strategy for improving asthma control, efforts to try antieosin­ ophil therapies in asthma have been eagerly awaited.62 Although first disappointing, recent studies enrolling patients with persistent airways eosinophilia despite ongoing use of corticosteroids have yielded favorable outcomes. This has resulted in three approved biologics for the treatment of moderate to severe eosinophilic asthma, namely mepolizumab, reslizumab, and benralizumab, by showing reduced exacerbations, corticosteroid-sparing activity, and in some studies improved lung function (but without changes in airways hyperreactivity).63 Also quite exciting are favorable results of a controlled trial of anti-IL-5 in eosinophilic granulomatosis with polyangiitis, the most definitive data so far to implicate eosinophils in the pathophysiology of this rare disease.64

Other Disorders Although covered in greater detail elsewhere (see Chapters 65 and 73), studies of hypereosinophilic syndrome38 and eosinophilic esophagitis

are worth mentioning in the context of the specific role of eosinophils in each disorder. On the one hand, anti–IL-5 antibody treatment was shown to be effective by having corticosteroid-sparing properties in certain hypereosinophilic syndromes, but their benefits in eosinophilic esophagitis have so far been inconsistent and thus mostly disappointing.65 In the context of potential effects of targeting eosinophils, recent developments in mouse models of obesity have suggested a beneficial role for IL-5–activated eosinophils in promoting energy expenditure in beige adipocytes.66 Eosinophil-deficient mice fed with a high-fat diet increased their body fat and acquired glucose intolerance and insulin resistance.67 Similarly, eosinophil-derived IL-4 is implicated in regeneration of injured skeletal muscles and liver in mice. Whether similar beneficial processes occur in humans, and whether targeting eosinophils adversely alters body weight or tissue repair, is unclear.

Conundrum of Comparing Mouse and Human Eosinophils Mice have been used extensively and often successfully to study mechanisms of Th2-type inflammation in asthma, parasitic infection, and other conditions, as well as the roles of eosinophils in these disease models. However, significant differences exist between mice and humans in immune system development, activation, and response to antigen challenge, in both the innate and the adaptive arm.68 Such differences may not be surprising given the two species diverged 65 to 75 million years ago, differ hugely in both size and life span, and have evolved in different ecologic niches where widely different pathologic challenges need to be met. Therefore overlooking aspects of human immunology that do not occur or cannot be modeled in mice is always a risk. Unfortunately, eosinophils are not exempt from these challenges. For example, mouse eosinophil–associated RNases are remarkably divergent (>50%) orthologs of human EDN and ECP.2 Among various molecules involved in activation and effector functions of human eosinophils previously discussed, FcαRI (IgA receptor) and FcγRIIA and C (activating IgG receptors) cannot be identified in the mouse genome, nor can eotaxin-3 or Siglec-8. Lack of eosinophil degranulation in mouse models of asthma and Th2-type airway inflammation is consistently seen.69 Furthermore, mice do not develop the reversible airway obstruction central to asthma pathogenesis in humans. It is difficult to determine whether similarities and differences seen in mouse asthma models are caused by inherent properties and contributions of mouse eosinophils or by a limitation of models used to reproduce eosinophilic inflammation, or both. However, it is crucial to consider the species differences when extrapolating results of experiments using mouse models to human disease. Different immune responses to parasites and other antigens observed among different mouse strains add to this complexity.70

CONCLUSION The eosinophil remains an enigmatic cell that continues to intrigue biomedical scientists after more than a century. Its precise function in allergic inflammation and asthma is controversial and requires further study. However, recent advances in molecular biology, immunology and pharmacology are uncovering the mechanisms of eosinophil proliferation, recruitment, activation, homeostasis, and effector function. With the availability of eosinophil-deficient laboratory mice and ongoing clinical studies of therapies that specifically target eosinophils, their beneficial and pathologic roles in health and disease are being elucidated. The mechanisms of blood and tissue eosinophilia associated with disease, although not yet fully understood, are likely controlled at the level of the interactions between innate and adaptive immune responses and the subsequent elaboration of mediators that exert both direct and indirect effects on these inflammatory cells. It seems inevitable that a

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complex biologic system that includes eosinophils and their products may participate in a cascade of events leading to inflammation and disease, including asthma.

22. Nakajima T, Matsumoto K, Suto H, et al. Gene expression screening of human mast cells and eosinophils using high-density oligonucleotide probe arrays: abundant expression of major basic protein in mast cells. Blood 2001;98:1127–34.

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46. Neighbour H, Boulet LP, Lemiere C, et al. Safety and efficacy of an oral CCR3 antagonist in patients with asthma and eosinophilic bronchitis: a randomized, placebo-controlled clinical trial. Clin Exp Allergy 2014;44:508–16.

Eosinophil Activation and Effector Functions 47. Na HJ, Hamilton RG, Klion AD, et al. Biomarkers of eosinophil involvement in allergic and eosinophilic diseases: review of phenotypic and serum markers including a novel assay to quantify levels of soluble Siglec-8. J Immunol Methods 2012;383:39–46. 48. Kelly EA, Esnault S, Liu LY, et al. Mepolizumab attenuates airway eosinophil numbers, but not their functional phenotype, in asthma. Am J Respir Crit Care Med 2017;196:1385–95. 49. Hammad H, Lambrecht BN. Barrier epithelial cells and the control of type 2 immunity. Immunity 2015;43:29–40. 50. Alam R, Forsythe P, Stafford S, et al. Transforming growth factor beta abrogates the effects of hematopoietins on eosinophils and induces their apoptosis. J Exp Med 1994;179:1041–5. 51. Radonjic-Hoesli S, Wang X, de Graauw E, et al. Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3 (RIPK3)-mixed lineage kinase-like (MLKL) signaling pathway, which is counterregulated by autophagy. J Allergy Clin Immunol 2017;140:1632–42. 52. Mazzeo C, Canas JA, Zafra MP, et al. Exosome secretion by eosinophils: a possible role in asthma pathogenesis. J Allergy Clin Immunol 2015;135:1603–13.

Role of Eosinophils in Host Defense and Disease 53. Mattes J, Yang M, Mahalingam S, et al. Intrinsic defect in T cell production of interleukin (IL)-13 in the absence of both IL-5 and eotaxin precludes the development of eosinophilia and airways hyperreactivity in experimental asthma. J Exp Med 2002;195:1433–44. 54. Lee JJ, Dimina D, Macias MP, et al. Defining a link with asthma in mice congenitally deficient in eosinophils. Science 2004;305:1773–6. 55. Yu C, Cantor AB, Yang H, et al. Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo. J Exp Med 2002;195:1387–95. 56. Chu VT, Beller A, Rausch S, et al. Eosinophils promote generation and maintenance of immunoglobulin-A-expressing plasma cells and contribute to gut immune homeostasis. Immunity 2014;40:582–93.

57. Specht S, Saeftel M, Arndt M, et al. Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection. Infect Immun 2006;74:5236–43. 58. Stevens WW, Ocampo CJ, Berdnikovs S, et al. Cytokines in chronic rhinosinusitis. Role in eosinophilia and aspirin-exacerbated respiratory disease. Am J Respir Crit Care Med 2015;192:682–94. 59. Schleimer RP. Immunopathogenesis of chronic rhinosinusitis and nasal polyposis. Annu Rev Pathol 2017;12:331–57. 60. Bachert C, Mannent L, Naclerio RM, et al. Effect of subcutaneous dupilumab on nasal polyp burden in patients with chronic sinusitis and nasal polyposis: a randomized clinical trial. JAMA 2016;315:469–79. 61. Chiarella SE, Sy H, Peters AT. Monoclonal antibody therapy in sinonasal disease. Am J Rhinol Allergy 2017;31:93–5. 62. Wechsler ME, Fulkerson PC, Bochner BS, et al. Novel targeted therapies for eosinophilic disorders. J Allergy Clin Immunol 2012;130:563–71. 63. O’Sullivan JA, Bochner BS. Eosinophils and eosinophil-associated diseases: an update. J Allergy Clin Immunol 2018;141:505–17. 64. Wechsler ME, Akuthota P, Jayne D, et al. Mepolizumab or placebo for eosinophilic granulomatosis with polyangiitis. N Engl J Med 2017;376:1921–32. 65. Schoepfer AM, Straumann A, Safroneeva E. Pharmacologic treatment of eosinophilic esophagitis: an update. Gastrointest Endosc Clin N Am 2018;28:77–88. 66. Brestoff JR, Kim BS, Saenz SA, et al. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity. Nature 2015;519:242–6. 67. Wu D, Molofsky AB, Liang HE, et al. Eosinophils sustain adipose alternatively activated macrophages associated with glucose homeostasis. Science 2011;332:243–7. 68. Mestas J, Hughes CC. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–8. 69. Denzler KL, Borchers MT, Crosby JR, et al. Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation. J Immunol 2001;167:1672–82. 70. Walsh ER, Sahu N, Kearley J, et al. Strain-specific requirement for eosinophils in the recruitment of T cells to the lung during the development of allergic asthma. J Exp Med 2008;205: 1285–92.

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SELF-ASSESSMENT QUESTIONS 1. Which proteins are most unique to eosinophils? a. Siglec-8 and eosinophil cationic protein b. Major basic protein and CCR3 c. Eosinophil peroxidase and EMR1 d. Eosinophil-derived neurotoxin and C5aR e. CLC protein and IL-5R 2. All of the following cytokines have been associated with eosinophil hematopoiesis, activation, or blood or tissue eosinophilia, except: a. IL-5 b. IL-12

c. IL-13 d. IL-33 e. GM-CSF 3. Selective accumulation of eosinophils into tissue sites of inflammation is mediated by: a. CCR3 b. VCAM-1 c. IL-13 d. ICAM-1 e. All of the above

17  Biology of Neutrophils Katherine J. Baines, Jodie L. Simpson, Michael Fricker, Peter G. Gibson

CONTENTS Introduction, 267 Neutrophil Migration, 267 Mediators Released by Activated Neutrophils, 270

Neutrophil Clearance and Death, 271 Neutrophils in Asthma, 272 Summary, 276

SUMMARY OF IMPORTANT CONCEPTS

NEUTROPHIL MIGRATION

• Neutrophils play a key role in innate immune defenses. • Respiratory triggers and insults result in neutrophil accumulation in the airways. • Airway neutrophils can release newly synthesized and preformed inflammatory mediators, which are cytotoxic and can support angiogenesis and remodeling processes. • Inappropriate or uncontrolled neutrophil responses contribute to airway disease pathogenesis.

Neutrophils migrate from the blood to the airways, where they play an important role during infection and inflammation. This complex process requires the maturation of neutrophils in the bone marrow and their release into the bloodstream, from which they migrate to the airways under the influence of chemotactic factors and adhesion molecules. Mature neutrophils do not undergo cell division. They are generated continuously from the bone marrow (at a rate of 1 to 2 × 1011 cells/ day in steady state), and their numbers, although tightly regulated, can be greatly amplified in times of stress, such as during infection. The maturation of neutrophils in the bone marrow involves the highly controlled process of myelopoiesis. During maturation, neutrophil granules are formed, which contribute to the inflammatory response in the fight against microorganisms. A variety of newly synthesized and preformed compounds exist in neutrophil granules, including serine and metalloproteinases, reactive oxygen species, lipid mediators, defensins, and cytokines (Fig. 17.1). Approximately 300 different proteins reside within neutrophil granules. These toxic molecules are released from activated neutrophils and have the ability to cause significant tissue damage to the lung and airways in asthma. Such damage occurs when neutrophils accumulate in large numbers and their activation is inappropriate or uncontrolled.

INTRODUCTION Neutrophils play a crucial role in innate inflammatory responses that are critical for host defense against infection; however, uncontrolled activation of these cells can cause tissue damage and contribute to the pathogenesis of chronic inflammatory conditions that involve the sinuses and respiratory tract. Neutrophils are produced in the bone marrow through proliferation and differentiation of stem cells and constitute the most abundant cell type in the peripheral blood, comprising 50% to 75% of circulating leukocytes in humans. They are the first circulating cells to migrate to the site of infection. Through phagocytosis, the production of reactive oxygen intermediates (ROIs), and the release of cytotoxic granule contents and extracellular traps, these cells function to contain and eliminate invading microorganisms. Representing a major mechanism of innate immunity, neutrophils also release cytokines and chemokines that initiate and amplify inflammation, as well as contributing to the development of the acquired immune response. In keeping with the toxic nature of an activated neutrophil, these cells are associated with the pathogenesis of inflammatory airway diseases, including chronic obstructive pulmonary disease (COPD) and asthma, as well as acute parenchymal lung conditions such as pneumonia and acute lung injury. This chapter examines the biology of the neutrophil, with a focus on its contribution to airway inflammation in asthma.

Myeloid Development Neutrophils are generated continuously from hematopoietic stem cells of the bone marrow; this process is referred to as myelopoiesis, and more specifically granulopoiesis. This process begins with the differentiation of pluripotent stem cells into myeloid progenitors, which then develop into myeloid precursors that under certain conditions continue development to become mature neutrophils. Developing neutrophils can be divided into six subtypes: the myeloblast; the promyelocyte, in which primary (azurophilic) granules appear; the myelocyte, in which cell division ceases and secondary (specific) granules appear; the metamyelocyte, in which tertiary (gelatinase) granules appear; band cells; and finally, mature neutrophils, characterized by their multilobed nucleus and cytoplasm containing granules.

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Newly Synthesized

Preformed

Cytokines e.g., IL-1β, TNF-α

Azurophilic granules e.g., elastase, defensins, myeloperoxidase, cathepsin G

Chemokines e.g., IL-8 Growth factors e.g., G-CSF, GM-CSF Reactive oxygen species e.g., superoxide

Gelatinase granules e.g., gelatinases (including MMP-9)

Airway Lumen

Epithelium

Adhesion CD11b/CD18

Specific granules e.g., flavocytochrome b558

Fig. 17.1  Neutrophil mediators. The neutrophil is a source of a range of preformed and newly synthesized mediators. G-CSF, Granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colonystimulating factor; IL, interleukin; MMP-9, matrix metalloprotease 9; TNF-α, tumor necrosis factor-α.

Many factors influence the development of neutrophils in the bone marrow, including interactions with other cells, components of the extracellular matrix, adhesion molecules, and growth factors. Granulocyte colony–stimulating factor (G-CSF) is a particularly important growth factor for neutrophil proliferation. Its production is regulated by interleukin (IL)-23 and IL-17A (i.e., cytotoxic T lymphocyte–associated antigen [CTLA]-8). Also important in this process are specific changes in gene expression patterns controlled by transcription factors such as C/EBPs (CCAAT/enhancer-binding proteins) and GFI-1 (growth factor independent-1 protein), as well as microRNAs (miRs) such as miR-125a. During maturation, neutrophils increase their mobility, deformability, and responsiveness to chemokines. Neutrophil maturation in the bone marrow takes approximately 10 to 15 days and is followed by the detachment of the cells from the marrow microenvironment and the mechanical “pumping” of the cells into the bone marrow sinuses. The chemokine receptor CXCR4 and its ligand CXCL12 (stromal cell–derived factor 1) are essential for the retention of neutrophils in the bone marrow. Immature neutrophils can be released prematurely into the circulation in times of infection or inflammation during exposure to inhalants such as cigarette smoke, and these cells preferentially sequester within the lung microvessels. This process is influenced by stimulation of the receptors CXCR2, G-CSF receptor (G-CSFR), and toll-like receptors (TLRs) and contact with cytokines (e.g., G-CSF, GM-CSF, IL-1) and chemokines (e.g., CXCL8 [IL-8]). Once released into the bloodstream, neutrophils have a half-life of 4 to 10 hours and can migrate into the tissues.

Neutrophil Trafficking and Margination Peripheral blood neutrophils are divided between a circulating pool, present in large and small blood vessels, and a marginating pool that is arrested in capillaries. Margination in the systemic circulation is regulated by selectin-mediated capture from the bloodstream. The pulmonary capillary bed is the main site containing marginating neutrophils, the concentration of which is 20 to 60 times that in large systemic blood vessels. Neutrophils may traverse this vast capillary network without signs of rolling or tethering.1 However, most neutrophils have to deform and elongate to travel through the pulmonary capillaries, which increases their transit time, resulting in a higher concentration of neutrophils in this space. Normal margination should not be confused with neutrophil sequestration, which is defined as amplified intravascular neutrophil numbers induced by inflammatory mediators and complement factors. Initial stages of sequestration are thought to involve cytoskeletal rearrangements, and prolonged sequestration of neutrophils requires CD11b/

Postmigration ICAM-1

Migration CD47 SIRPα JAMs

Chemotactic gradient, e.g., IL-8 Extravascular Tissue

Endothelium

IL-8 Firm adhesion Rolling B2 integrins Selectins ICAM-1 and -2

Migration Cell polarity PECAM-1 JAMs

Blood Fig. 17.2  Neutrophil migration to the airways. The migration of neutrophils from the bloodstream requires neutrophil rolling, activation, and firm adhesion to endothelial cells; this is followed by migration through the endothelial layer, the basement membrane, and the epithelial interface, where the cells accumulate in the airway lumen. ICAM, Intercellular adhesion molecule; IL, interleukin; JAMs, junction adhesion molecules; PECAM-1, platelet–endothelial cell adhesion molecule-1; SIRPα, signal regulatory protein-α.

CD18. The migration of neutrophils into tissues involves neutrophil capture, rolling, slow rolling, arrest or firm adhesion, postadhesion strengthening, intravascular crawling, and paracellular or transcellular migration through the endothelium, the basement membrane, and the epithelial interface, with accumulation in the airway lumen (Fig. 17.2).2 These events involve complex interactions between neutrophils and the endothelium, extracellular matrix, and epithelium and are mediated largely by cellular adhesion molecules (CAMs) such as the β2 integrins. This process gradually changes the functional state of the neutrophil from a passive circulating cell into a highly activated effector cell of innate immunity.

Cellular Adhesion Molecules Neutrophil adherence to the endothelium involves cellular adhesion molecules on the neutrophil and endothelial cell, whose expression is tightly regulated. Interaction among P-selectin ligand 1 (PGSL-1), L-selectin, and CD44 on the neutrophil and between P- and E-selectin on the endothelial cells mediates the capture and rolling of the neutrophils and, together with chemokines through G protein–coupled receptors, regulates integrin adhesiveness. Activation of neutrophil β2 integrins through binding to the endothelial intercellular adhesion molecules (ICAM)-1 and ICAM-2 is required for firm adhesion to the endothelium.

Integrins.  The integrins are a family of heterodimeric transmembrane glycoproteins that mediate direct cell-cell, cell–extracellular matrix, and cell-pathogen interactions. They contain two functional units: α and β chains. The β2 integrins are expressed on neutrophils and consist of

CHAPTER 17  Biology of Neutrophils four different heterodimers: CD11a/CD18, or leukocyte function– associated antigen-1 (LFA-1); CD11b/CD18, or macrophage 1 antigen (Mac-1); CD11c/CD18, or p150,95; and CD11d/CD18. The functional state and presence of integrins on neutrophils are regulated by lipid, cytokine, and chemokine signaling molecules as well as “cross-talk” from other adhesion molecules. Multiple mechanisms, including conformational change (affinity regulation) and clustering associated with the cytoskeleton (avidity regulation), are responsible for integrin activation, arising from or caused by ligand binding. Of interest, the ability of the extracellular domains of integrins to bind ligands can be activated in less than 1 second by means of signals from within the cell (inside-out signaling). Integrin activation on the neutrophil initiates several signaling pathways that induce adhesion strengthening, transmigration, and respiratory burst.

Endothelial Cell Interactions.  Transendothelial migration of neutrophils is either paracellular (between endothelial junctions) or transcellular (directly through endothelial cells). The paracellular route requires the tight junctions between endothelial cells to loosen, and this is mediated by platelet–endothelial cell adhesion molecule (PECAM)-1 and junction adhesion molecules (JAMs) expressed at intercellular tight junctions of endothelial and epithelial cells, along with LFA-1 and Mac-1 on the neutrophil. The transcellular route is mediated by transmigratory cups high in ICAM-1 and vascular cell adhesion molecule 1 (VCAM-1), which bind crawling neutrophils and allow them to make their way through the cell. Epithelial Cell Interactions.  Neutrophil migration across the epithelium involves three stages: epithelial adhesion, migration, and postmigration. Transepithelial migration of neutrophils involves both cell-cell interactions that include adhesion molecules and signaling events to open the epithelial tight junctions, allowing the passage of cells without disturbance of the epithelial barrier. Initially, neutrophil firm adherence to the basolateral epithelial membrane is mediated exclusively by CD11b/ CD18 on the neutrophil’s surface to several molecules on the epithelial surface, such as fucosylated glycoproteins, and JAMs, particularly JAM-C. Neutrophils then crawl along the epithelium; CD47 and signal regulatory protein-α (SIRPα) are important in this process. Within the tight junctions, neutrophil JAM-L binds epithelial CAR (coxsackie and adenovirus receptor)—probably important in the migratory process and in the formation of a seal around migrating cells to preserve barrier function. Interactions between neutrophils and the epithelium postmigration include neutrophil CD11b/CD18 and epithelial ICAM-1, Fc receptors to apical antigens, and probably binding of decay accelerating factor (DAF) to CD97.

Chemotactic Mediators Once through the endothelial basement membrane, neutrophils migrate along a chemotactic gradient. Neutrophil chemotactic proteins include chemokines (e.g., CXCL8), bacterial products (e.g., N-formyl methionyl peptides), lipid mediators (e.g., leukotriene B4), and complement split products (e.g., C5a). There is a functional hierarchy that exists to prioritize inflammatory responses; for example, neutrophils adhere to the endothelium in response to chemokines including CXCL8; however, while adhered they must respond to other bacterial chemoattractants such as fMLP. This occurs through activation of alternate signaling cascades, with CXCL8 stimulating PI3K-phosphatase and tensin homolog (PTEN) versus fMLP activating the p38 MAPK pathway, which dominates over the former.1

Chemokines.  The chemokine family comprises approximately 50 low-molecular-weight proteins that exert their effects through activation

269

of one of the 19 G protein–coupled chemokine receptors. The two main subfamilies of chemokines, CXC and CC, are classified according to the position of the first two cysteines in their amino acid sequence (separated by one amino acid—CXC, or adjacent CC). Chemokines are produced by inflamed tissues and activate signal cascades in the neutrophil that lead to an increase in cell motility, adhesion, and survival. CXCL8 is a potent chemotactic factor for neutrophils, acting via its receptors CXCR1 and CXCR2. Other members of this family include epithelial cell–derived neutrophil activator-78 (ENA-78), growth regulatory genes Gro-α and Gro-β, neutrophil-activating peptide-2 (NAP-2), and granulocyte chemotactic protein-2 (GCP-2). Proteolytic enzymes also influence the chemotaxis and activation of neutrophils indirectly; for example, neutrophil elastase (NE) can induce CXCL8 production, and MMP-9 can enhance CXCL8 potency by augmenting amino-terminal processing of CXCL8.

Lipid Mediators.  Lipid mediators derived from fatty acids such as arachidonic acid play important roles in physiologic and pathologic processes involving neutrophils. These substances include the eicosanoids, prostaglandins, thromboxane, and leukotrienes. Leukotriene B4 is a potent chemoattractant and activator of neutrophils and has been linked to airway diseases including asthma. Resolvins are a family of lipid mediators that are derived from omega-3 essential polyunsaturated fatty acids, which are important in the resolution of inflammation mediated through antiinflammatory effects on neutrophils.3

Neutrophil Activation Neutrophil activation usually occurs in two stages, whereby the neutrophil first becomes primed and then is activated upon a second stimulation. Activation of neutrophils, a major mechanism of innate immunity, involves the detection of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs)—alarmins or danger signals generated after tissue injury4—by pattern recognition receptors (PRRs). PRRs of importance for neutrophils include the toll-like receptor (TLR) family, formyl peptide receptors (FPRs), C-type lectins (CLECs), cytoplasmic ribonucleic sensors RIG-I and MDA-5, and nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs), which are components of the inflammasome (Fig. 17.3). All but 1 (TLR3) of the 10 TLRs are expressed in neutrophils. Activation of both TLR2 and TLR4 regulates several important proinflammatory neutrophil functions through the nuclear factor (NF)-κB pathway; these include neutrophil activation, migration, and survival. Stimulation of these receptors on neutrophils leads to activation of effector functions, including mediator production, degranulation, and oxidative burst. Macrophage IL-1 production is particularly important in sensing sterile inflammation and necrotic cell death, leading to acute neutrophil infiltration.5 Neutrophils also release active IL-1β, as a result of either inflammasome activation or protease cleavage. Neutrophils contain both NLR pyrin containing 3 (NLRP3) and absent in melanoma 2 (AIM2) inflammasomes, whose activation leads to production of IL-1β and IL-18.6 MicroRNAs also regulate neutrophil activity. MiR-223–knockout mice demonstrate enhanced proliferation and differentiation of neutrophils that are hyperresponsive and infiltrate the lung and cause tissue damage. MiR-9 is induced by TLR4, TLR2, and TLR7/8 agonists, as well as by proinflammatory cytokines IL-1β and tumor necrosis factor α (TNF-α) through NF-κB and acts as a controller of NF-κB responses. Recent studies have identified heterogeneity within circulating neutrophils. Three subsets of neutrophils have been identified in the circulation after an inflammatory challenge, including banded neutrophils, normal neutrophils, and hypersegmented neutrophils.7 Each subtype had a distinct nuclear morphology and pattern of surface adhesion molecule expression, with hypersegmented neutrophils showing increased

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Reactive Oxygen Species

TLR2/4 Signal transduction Inflammasome formation MyD88 NLRP3 ASC Procaspase-1

Transcription

Caspase-1 IL-8 pro-IL-1β/ IL-18

IL-1β and IL-18

IL-8

Neutrophils

MMP-9

NE

LTB4

Fig. 17.3  Innate immune activation pathway leading to accumulation of neutrophils and neutrophil activation. ASC, Apoptosis-associated speck-like protein containing a CARD; ATP, adenosine triphosphate; IL, interleukin; LTB4, leukotriene B4; MMP-9, matrix metalloprotease 9; NE, neutrophil elastase; NLRP3, nucleotide-binding oligomerization domainlike receptor 3; TLR, toll-like receptor.

The respiratory burst of neutrophils involves the activation of (reduced) nicotine adenine dinucleotide phosphate (NADPH) oxidase, which is an enzymatic complex composed of cytosolic proteins p40phox, p47phox, and p67phox and flavocytochrome b558, which is composed of membrane proteins p22phox and gp91phox. Flavocytochrome b558 is located between the plasma membrane and the membrane of the specific granules and is incorporated into the phagocytic vacuole, where it pumps electrons from NADPH in the cytosol to oxygen in the vacuole. When neutrophils are activated, p47phox is phosphorylated to cause cytosolic components to migrate to the plasma membrane, where they are able to associate with flavocytochrome b558, assembling the active oxidase. Reactive oxygen species are generated as a result of NADPH oxidase activity to produce superoxide (O2−). Superoxide can be rapidly converted into hydrogen peroxide (H2O2) by the enzyme superoxide dismutase. Superoxide and hydrogen peroxide also can form to create the highly reactive hydroxyl radical (HO−). In addition, myeloperoxidase (MPO) generates hyperchlorous acid (HOCl) from hydrogen peroxide. Exposure to reactive oxygen species can result in pulmonary injury. They can activate granule proteins, interact with various signaling cascades, and modulate neutrophil functions. Superoxide can activate granule proteins through the recruitment of K+ to the phagosome, thus allowing cationic proteases of the azurophilic granules to go from a highly organized intragranule structures into solution where they can kill ingested microbes. Reactive oxygen species can inhibit a variety of protein tyrosine phosphatases through oxidation of key residues and can disrupt intercellular tight junctions, increasing the permeability of the endothelial barrier.

Defensins capacity for oxidative burst along with a unique ability to suppress T lymphocytes.8

MEDIATORS RELEASED BY ACTIVATED NEUTROPHILS Proteases The serine neutrophil proteases are important components of the azurophilic granules of neutrophils that have important functions in the innate immune response. Serine proteases include neutrophil elastase, cathepsin G, and proteinase 3. The primary function of serine proteases is to degrade and kill engulfed microorganisms in the phagolysosome; these molecules, however, also participate in extracellular matrix degradation, tissue remodeling proteolysis of surfactant proteins, activation of matrix metalloproteinases (MMPs), proteolytic cleavage of cytokines and receptors, and cell signaling. Production of protease inhibitors also is important in limiting their actions; for example, α1-antitrypsin (AAT) is the major endogenous serine protease inhibitor that counterbalances NE activity. MMPs are a family of zinc-dependent endopeptidases that are characterized by their ability to degrade components of the extracellular matrix, such as collagen. MMP-9 is a major component of neutrophil gelatinase granules. MMP-9 is released as a proenzyme that can be activated by a number of mechanisms, including those mediated by other MMPs, bacterial proteases, reactive oxygen species, and other neutrophil proteins including lipocalin. An imbalance of MMP-9 activity and the inhibitory action of tissue inhibitor of metalloproteinase (TIMP)-1 is implicated in multiple diseases, including asthma and COPD.

Defensins are small arginine-rich cationic peptides that have antimicrobial activity against a broad range of pathogens including bacteria, fungi, and some enveloped viruses and exert this antimicrobial effect through membrane permeabilization of the microbe. Six α-defensins have been identified, including four neutrophil defensins, also known as human neutrophil peptides, designated HNP1 to HNP4. These HNPs are produced predominantly by neutrophils, stored in neutrophil azurophilic granules, and released in large quantities upon neutrophil activation. α-Defensins can stimulate neutrophil influx into the airways, most likely through CXCL8 and IL-1β production, and NF-κB binding activity. α-Defensin-1 can increase mucin gene and protein expression, which may contribute to excess mucus production. There is higher expression of neutrophil defensins in the blood and sputum of subjects with neutrophilic asthma.9,10

Cytokine Synthesis The neutrophil is both a target and a source of a vast and diverse repertoire of cytokines. These include proinflammatory cytokines (e.g., IL-1), antiinflammatory cytokines (e.g., IL-1 receptor antagonist [IL-1RA]), immunoregulatory cytokines (e.g., IL-12), TNF superfamily members (e.g., TNF-α), and growth factors (e.g., G-CSF). Neutrophils were formerly thought to be devoid of transcriptional activity or protein synthesis. It is now clear, however, that neutrophils are an important source of newly synthesized cytokines and growth factors. Cytokine production by neutrophils is increased by inflammatory stimuli, bacterial endotoxin (lipopolysaccharide [LPS]) being the most potent. For production of some cytokines, stimulation with more than one agonist is required, such as with interferon (IFN) and LPS needed for IL-12 production.

CHAPTER 17  Biology of Neutrophils

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Fig. 17.4  Sputum NET fluorescence microscopy. This figure is a representative image displaying profuse NETs detected in the sputum of a patient with severe asthma (20× objective). NETs are the weblike structures stained with SYTOX green, DAPI in blue, and NE stained in red. (Reproduced from Wright TK, Gibson PG, Simpson JL, McDonald VM, Wood LG, Baines KJ. Neutrophil extracellular traps are associated with inflammation in chronic airway disease. Respirology 2016;21:467-75.)

Neutrophil Intracellular Killing Once neutrophils have recognized a pathogen, phagocytosis can take place, and subsequent bacterial killing occurs in the phagolysosome, through oxygen-dependent or independent mechanisms. The oxygendependent mechanisms are mediated by ROS downstream of superoxide formed by the NADPH complex, which metabolizes the phagosome into highly bactericidal end products. The oxygen-independent mechanisms are regulated by azurophilic granule products that are delivered to the phagolysosome upon fusion with the bacteria-containing phagosome.

of NETs (through natural DNases) must be regulated, because they are toxic to the endothelium and epithelium, can expose autoantigens and immunostimulatory proteins, activate plasmacytoid dendritic cells to release interferon, and participate in organ damage through undefined mechanisms.15 Neutrophils generally undergo cell death during the formation of NETs, although some studies suggest anuclear neutrophils are capable of phagocytosis.12 NETs have recently been identified in the sputum of subjects with neutrophilic asthma and are associated with heightened innate immune responses (Fig. 17.4).9

Neutrophil Extracellular Traps (NETs)

NEUTROPHIL CLEARANCE AND DEATH

In addition to the production of classical mediators, impressive images of NETs were first reported in 2004.11 NETs are composed of decondensed DNA in complex with modified extracellular histones and neutrophil granule proteins including the antimicrobial α-defensins and LL-37. NET formation (NETosis) is triggered by both infectious and noninfectious stimuli,12 including airway pathogens such as Haemophilus influenzae,13 and bacterial components (e.g., LPS), which are important in neutrophilic asthma. NETosis occurs via disintegration of the neutrophil nuclear envelope and mixing of chromatin with granule contents containing neutrophil antimicrobial proteins and proteases. Under some conditions NETs can also be formed from mitochondrial DNA,14 which is a powerful stimulator of innate immunity. Although some pathogens are susceptible to damage and containment within NETs, others counteract this or even incorporate NETs into their support structures, where they escape phagocyte uptake and clearance within NET biofilms. Removal

After killing and digesting invading microbes, neutrophils at the inflammatory site will die via one of four mechanisms: (1) NETosis, as described previously; (2) apoptosis (programmed cell death), comprising chromatin condensation, nuclear collapse, cytosolic vacuolation, and cell shrinkage; (3) necrosis, in which the cell bursts and releases its toxic contents, or (4) phagoptosis, in which activation-induced surface exposure of the “eat me” signals direct uptake of viable neutrophils by tissue-resident professional phagocytes including macrophages (Fig. 17.5).16 The regulation of neutrophil apoptosis and/or phagocytosis is crucial to maintain neutrophil numbers, as well as for the effective removal of invading pathogens and the resolution of inflammation, limiting neutrophils’ destructive capability. Apoptotic neutrophils become instantly recognizable to alveolar macrophages, resulting in their removal through efferocytosis (i.e., apoptosis and subsequent clearance of inflammatory cells), whereby the neutrophil retains its granule contents. This process

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Apoptosis, activation “Don’t eat me” signals CD31

CD31

CD47

SIRP1α

PAI1

“Eat me” signals CRT

LRP

PS

PS

PS

PS

MFG-E8

Gas6, Protein S

Annexin A1

VR

MerTK

FPR2

PS exposure

PS

Tim4, BAI1, Stabilin 1&2

Neutrophil target

Macrophage phagocyte

Actin cytoskeleton rearrangement, phagocytosis, anti-inflammatory signaling Fig. 17.5  Regulation of phagocytic clearance of neutrophils by “don’t eat me” and “eat me” signaling. Neutrophils differentially expose a number of “don’t eat me” and “eat me” signals on their cell surface, dependent on their activation status, age, and viability. “Don’t eat me” signals inhibit, whereas “eat me” signals promote phagocytic signaling in macrophages, allowing tight control of neutrophil turnover. Activation of neutrophils and induction of apoptosis can both result in increased “eat me” signal exposure and subsequent induction of phagocytosis. BAI1, brain-specific angiogenesis inhibitor 1; CRT, calreticulin; FPR2, formyl peptide receptor 2; LRP, low density lipoprotein receptor-related protein; MerTK, Mer tyrosine kinase; MFG-E8, milk fat globulin E8; PAI1, plasminogen activator inhibitor 1; PS, phosphatidylserine; SIRP1α, signal-regulatory protein alpha; VR, vitronectin receptor.

also can induce changes in the activation phenotype of lung macrophages, suppressing the release of proinflammatory mediators. Neutrophil apoptosis is an active process that can be modulated by various signals and mediators. Inflammatory stimuli, such as growth factors (e.g., GM-CSF, G-CSF), cytokines (e.g., IL-1, IL-6), chemokines (e.g., CXCL8), and bacterial products (e.g., LPS), and processes such as adhesion and transmigration can delay neutrophil apoptosis. A prolonged lifespan of the neutrophil is necessary for the effective removal of pathogens and also for the regulation of immune responses. Macrophages can recruit neutrophils and control the lifespan and activity of the recruited cells.4 Corticosteroids delay neutrophil apoptosis, influencing the persistence of neutrophilic inflammation. This effect is further potentiated by the addition of β2-agonists, suggesting that current mainstay asthma therapies may in fact be promoting airway neutrophilia.17 By contrast, theophylline increases apoptosis of both neutrophils and eosinophils. NE can inhibit apoptotic cell clearance in bronchiectasis through cleavage of macrophage phosphatidylserine receptors. Conversely, TNF-α and Fas ligand (Fas-L) can increase the rate of neutrophil apoptosis. Neutrophil apoptosis is induced by activation of cellular caspases and can occur through two main pathways. The first is the death receptor (DR) pathway, by which the clustering of TNF and Fas receptors activates the caspase cascade beginning with cleavage of procaspase 8. The second is an intrinsic pathway, consisting of mitochondrial cytochrome c and members of the Bcl-2 family, which results in formation of an apoptosome which activates caspase 9. Cell stress related to exposure to reactive oxygen species, DNA damage, or lack of growth factors can result in apoptosis, induced by the release of cytochrome c. Activated caspases 8 and 9 can then activate caspase 3 to cleave proteins essential for cell survival. Neutrophil clearance by host cells including macrophages is regulated by extensive cell-to-cell communication, involving opposing “don’t eat me” and “eat me” signaling. The best understood “eat me” signals are surface exposure of phosphatidylserine (PS) and calreticulin (CRT), which are bound by bridging opsonin molecules

or directly by phagocyte surface receptors (Fig. 17.5). Recognition of “eat me” signals by phagocytes triggers intracellular signaling cascades culminating in the directed cytoskeletal rearrangements required to orchestrate the engulfment and internalization of the target neutrophil. “Don’t eat me” signals on the neutrophil surface, including CD47 and PAI1, impose an inhibitory brake on the phagocytic process to ensure uptake and clearance occurs only when necessary. Neutrophils constitutively expose the “eat me” signal CRT, and the “don’t eat me” signals CD47 and PAI1. PS exposure, mediated by caspase activation during apoptosis, but also by caspase and cell death–independent mechanisms triggered during neutrophil activation, can tip the balance of signaling in favor of clearance of neutrophils. The process whereby apoptotic neutrophils are phagocytosed and cleared is termed efferocytosis, and the process whereby viable neutrophils are phagocytosed and cleared is termed phagoptosis.16 Efficient efferocytosis of apoptotic neutrophils by tissue macrophages is thought to exert important antiinflammatory and resolving functions by: (1) preventing release into the extracellular milieu of proinflammatory factors as neutrophils undergo delayed secondary necrosis; and (2) stimulating the adoption of inflammationresolving phenotypes in the phagocytosing macrophage. The impact of phagoptosis of viable neutrophils on macrophage-related inflammatory signaling is not understood.

NEUTROPHILS IN ASTHMA Understanding of the pathophysiology of asthma has increased in the past decade, and the disorder is now recognized to be a heterogeneous, multidimensional disease, with several identifiable phenotypes.18 Of importance, asthma phenotypes are related to clinical outcomes and treatment responsiveness. Inflammatory phenotypes of stable asthma are now well known and include a persistent neutrophilic bronchitis, termed neutrophilic asthma, in a subgroup of patients. Neutrophilic asthma is defined as symptomatic asthma and airway hyperresponsiveness in the presence of a neutrophilic bronchitis (Fig. 17.6), with sputum

CHAPTER 17  Biology of Neutrophils

A

273

B

C Fig. 17.6  Microscopic appearance of sputum cytospin samples in different asthma inflammatory phenotypes. (A) Neutrophilic asthma. (B) Eosinophilic asthma. (C) Paucigranulocytic asthma. (Reproduced from Simpson JL, Scott R, Boyle M, Gibson PG. Inflammatory subtypes in asthma: assessment and identification using induced sputum. Respirology 2006;11:54-61, with permission.)

neutrophil counts greater than 61%. Neutrophilic asthma represents between 10% and 30% of cases of stable asthma in adults. Patients with neutrophilic asthma are older than those with normal levels of neutrophils and tend to have less severe airway hyperresponsiveness. They are similar, however, in terms of gender, atopy, smoking history, and lung function.19 In neutrophilic asthma, a number of unique inflammatory abnormalities distinguish the disorder from typical eosinophilic asthma (in which sputum eosinophils are outside the normal range) and paucigranulocytic asthma (where sputum granulocytes are within normal ranges). Additionally, a small subgroup of patients with increased sputum neutrophils in conjunction with eosinophils has been recognized; this phenotype is termed mixed granulocytic asthma. Neutrophilic asthma is an important inflammatory phenotype of the disease. Neutrophils infiltrate or predominate in the airway in more severe forms of asthma,20 including life-threatening asthma, asthma exacerbations, acute severe asthma, stable persistent asthma, and severe

refractory asthma. Neutrophils are increased in a number of airway compartments, including the lumen and submucosa. Neutrophil numbers and products are increased during exacerbations in both adults and children with asthma, and such changes have been associated with damaged epithelium. Inhalation of a variety of triggers, such as ozone, endotoxin, cigarette smoke, and dust, is associated with neutrophil accumulation and airways hyperresponsiveness. The precise cause of neutrophil accumulation and dysfunction in asthma remains unclear; in people with neutrophilic asthma, however, the persistence of these cells in the airway is a critical component of the disease. In addition, several factors are known to worsen neutrophilic inflammation, including environmental exposures such as endotoxin, infection, smoking, air pollution, and diets heavy in fats and lacking antioxidants; and other comorbid conditions such as obesity and systemic inflammation also are important. Controversially, even asthma medications, particularly corticosteroids, can potentiate the presence and activity of neutrophils in the airway.

274

SECTION A  Basic Sciences Underlying Allergy and Immunology Neutrophilic Asthma

BOX 17.1  Etiology of Neutrophilic

Bronchitis in Asthma

Persistent Neutrophilic asthma Allergic bronchopulmonary aspergillosis Smoking

Bacterial products, inflammatory cytokines, corticosteroids

TLR2

Transient Viral exacerbations Bacterial bronchitis Pollutants Occupational settings

TLR4

NLRP3, ASC Procaspase-1

Increased IL-8, IL-1β

Pathophysiology and Mechanisms of Neutrophilic Asthma Persistent neutrophilic bronchitis in asthma (neutrophilic asthma) may represent a chronic condition that is driven by distinct immune and inflammatory mechanisms involving innate immune dysfunction. In general, on exposure to pathogens, the respiratory epithelium controls microbial invasion by innate defenses including mucociliary clearance. In some situations, a controlled inflammatory response may be required, which involves movement of leukocytes into the bronchial lumen. If this response is insufficient to eliminate the invading pathogen, the resulting inflammation may become chronic, leading to tissue damage (Box 17.1). Evidence of dysregulation of innate immunity in neutrophilic asthma has emerged. Key parts of the innate immune response are altered in neutrophilic asthma (Fig. 17.7). For example, several PRRs are changed, such as upregulation of TLR219 and deficiency of RAGE (receptor for advanced glycation end-products),21 consistent upregulation of proinflammatory cytokines CXCL8 and IL-1β, members of the TNF-α/NF-κB and IL-1 pathways,22 neutrophil proteases, and NETs.10 Other findings include high levels of airway endotoxin and colonization with bacteria in neutrophilic asthma, particularly Haemophilus influenzae,23 which may represent a key activator of airway neutrophilia in asthma mediated by IL-17.24 Higher airway endotoxin levels are associated with worsened airway obstruction and elevated sputum neutrophils, indicating a role for endotoxin in stimulating a neutrophilic inflammation and worsening airway disease (Fig. 17.7). Systemic inflammation also is an important feature of neutrophilic asthma. In other airway diseases characterized by airway neutrophilia, particularly COPD, low-grade systemic inflammation is recognized as a key pathogenic feature of the disease. The presence of inflammatory phenotypes of asthma is based on the number of inflammatory cells present in peripheral blood,25 suggesting there may also be systemic manifestations of the airway inflammatory response in asthma. Circulating C-reactive protein (CRP) and IL-6 also are increased in neutrophilic asthma.26 Isolated peripheral blood neutrophils display a distinct gene expression profile and release more CXCL8 in neutrophilic asthma than in eosinophilic asthma.27 Gene expression of peripheral blood showed upregulation of neutrophil proteases, including NE and cathepsin G, and α-defensins in neutrophilic asthma.9 This microarray study provided the first demonstration of a difference between mixed granulocytic asthma and neutrophilic asthma. This observation suggests that the neutrophilia observed in mixed granulocytic asthma is a local airway response, as opposed to the neutrophilia in neutrophilic asthma, which has a systemic component involving circulating neutrophil dysfunction.

CXCL8

MMP-9

NE

Mucus hypersecretion? Impaired efferocytosis? Bacterial persistence?

Asthma Fig. 17.7  Current known pathomechanisms in neutrophilic asthma. IL, Interleukin; MMP-9, matrix metalloprotease 9; NE, neutrophil elastase; TLR, toll-like receptor.

Obesity, along with dietary intake, also can modify neutrophilic airway inflammation in asthma. An increase in neutrophilic inflammation is associated with obesity in asthma, particularly in female patients, and serum fatty acids in male patients are important predictors of neutrophilic asthma.28 Direct investigation into the effects of a highfat diet showed that consumption of a single high-fat meal can increase sputum neutrophils and TLR4 gene expression.29 Depletion of dietary antioxidants for 14 days in people with asthma induces upregulation of genes involved in the innate immune response, particularly the IL-1 pathway in airway samples. These findings indicate that a diet low in antioxidants and high in saturated fat may worsen neutrophilic inflammation in asthma through activation of the innate immune response. In addition to increased recruitment of neutrophils, decreased neutrophil cell clearance could play a role in driving neutrophilic airway inflammation in asthma. The efferocytosis of neutrophils by macrophages collected from the airways of asthma patients is impaired, in particular in macrophages derived from patients with more severe asthma, obese asthma patients, and patients with noneosinophilic asthma, all of which are linked to the neutrophilic asthma phenotype.30 The mechanisms underlying this impairment of neutrophil clearance are poorly understood, but therapeutic approaches to restore normal neutrophil clearance capacity hold some clinical promise.

CHAPTER 17  Biology of Neutrophils Decreased mucociliary clearance and mucus hypersecretion are features of COPD, bronchiectasis, and asthma, which could potentiate neutrophil retention in the airways. In patients with asthma and mucus hypersecretion, asthma therapies have little effect on mucociliary clearance, and neutrophil proteases such as MMP-9 are increased in bronchoalveolar lavage (BAL) fluid. In COPD, mucus hypersecretion is significantly associated with FEV1 decline, increased risk of need for hospitalization, and death related to pulmonary infection. Respiratory insults such as infection and cigarette smoke can increase mucus production in the airways, most likely through the production of proinflammatory cytokines, proteases, and defensins. Excess release of NETs may also contribute to the increased gel-like nature of mucus, because DNA is an extremely viscous polyanion.

Triggers of Neutrophilic Asthma Several triggers and other factors that influence neutrophilic inflammation in the airways have been recognized, including endotoxin, particulate air pollution, respiratory viruses, and smoking (Box 17.2). Endotoxin (i.e., LPS) is a component of household dusts and grain dusts and has been implicated in occupational respiratory disease. Endotoxins are found in the outer membrane of gram-negative bacteria, and the molecule consists of a hydrophilic polysaccharide region and a hydrophobic lipid region (lipid A). Exposure to LPS, signaling through TLR4, can cause clinical signs and symptoms such as fever, coughing, dyspnea, and a reduced FEV1. In very high concentrations, endotoxin is lethal, resulting in septic shock; however, only small quantities are required to stimulate an immune response. Inhalation of particulate matter from air pollution can cause exacerbations of asthma in both adults and children. Inhalation of larger-size particles (PM2.5-10 [i.e., “coarse” particulate matter, ranging from 2.5 to 10 µm in diameter]) can cause airway inflammation. This particulate matter contains both bacteria and fungi, in addition to their degradation products such as LPS. It is thought that the bacterial component of pollutants is what causes the inflammatory response. Cytokine production in response to exposure to bacteria found in particulate matter was inhibited by blocking CD14, an essential protein required for responses to LPS. In addition, both TLR2 and TLR4 were activated in response to exposure to particulate matter that was contaminated by bacteria. Respiratory viruses constitute an important trigger of asthma exacerbations. The viruses that have been implicated in such events include influenza virus, respiratory syncytial virus (RSV), and rhinovirus. In children, exacerbations are caused by viruses in 85% of cases, the most common viruses isolated being rhinovirus and RSV. RSV infection is an important respiratory tract infection in infants, accounting for more than 70% of all cases of infantile bronchiolitis, and may be linked to the development of asthma. More than one-third of asthmatic patients are smokers, and more have a past history of smoking. Active smoking in asthma results in an

BOX 17.2  Factors Influencing Airway

275

increase in symptoms, decreasing lung function, and an impaired response to corticosteroids. The mechanisms underlying this appear to be linked with the neutrophilic airway inflammation. Smokers with asthma have increased airway neutrophils, which are associated with lower lung function. With cessation of smoking, an improvement in lung function and a reduction of neutrophilia are seen within 6 weeks, suggesting that alterations in airway obstruction can be reversed by mitigating neutrophilia.

Neutrophils and Corticosteroids Research recognizing phenotypes of asthma clearly shows that the best responses to corticosteroids are largely limited to patients with a Th2driven eosinophilic inflammation.18 Not only do corticosteroids show little impact on noneosinophilic inflammation, but they potentially promote neutrophilic inflammation,31 one mechanism being increased neutrophil survival. The presence of airway neutrophils is also related to corticosteroid responsiveness. Patients with more severe asthma and those who smoke are less responsive to corticosteroids, and these patients are also more likely to have neutrophilic inflammation. Triggers of neutrophilic airway inflammation, such as endotoxin exposure and cigarette smoking, also can affect the responsiveness of subjects to inhaled corticosteroid therapy.32 The mechanisms of corticosteroid unresponsiveness are not fully understood but may involve reduced corticosteroid binding to the glucocorticoid receptor, reduced glucocorticoid receptor expression, enhanced activation of inflammatory pathways, or lack of corepressor activity.33 This line of evidence demonstrates the importance of the identification of asthma phenotypes in guiding treatment strategies for successful outcomes and highlights the unmet need for more effective treatments for neutrophilic asthma.

Options for Therapy in Neutrophilic Asthma Unlike targeting Th2-dominant eosinophilic asthma, few neutrophildirected approaches have been developed in recent years. Several options have been investigated and reviewed34 as add-on therapies to the standard inhaled or oral corticosteroids, for example macrolides, theophylline, long-acting beta agonists (LABA), and antagonists to CXCR2 and IL-1β. Potential therapeutic options for neutrophilic asthma are summarized in Box 17.3. The antiinflammatory effects of macrolide antibiotics are well established. Macrolide antibiotics such as azithromycin have separate and distinct antibiotic and antiinflammatory actions. As antibacterial agents, macrolides have been widely used in the treatment of infections caused by atypical bacteria, which are associated with obstructive airway diseases such as asthma and COPD. Macrolide antibiotics have become first-line drugs for the treatment of diffuse panbronchiolitis, with potential benefits in a range of conditions including cystic fibrosis, bronchiolitis obliterans, and COPD. A recent clinical trial concluded that

Neutrophilia in Asthma

BOX 17.3  Options for Therapy in

• Endotoxin • Particulate matter • Respiratory viruses • Bacterial infection • Smoking • Corticosteroids • Diet—high in fats, low in antioxidants • Occupational exposures

• Reduce inhaled corticosteroid dosage • Bacterial eradication • Macrolide antibiotics • Anti-CXCL8 antibodies • Theophylline • Recombinant IL-1RA (anakinra) • Recombinant DNase (dornase alpha)

Neutrophilic Asthma

276

SECTION A  Basic Sciences Underlying Allergy and Immunology

azithromycin add-on treatment of patients with uncontrolled, moderate to severe asthma resulted in a significant reduction in annualized exacerbation rate, as well as improvement in asthma symptom score.35 Importantly, this therapeutic effect was also observed in patients with neutrophilic asthma. The mechanisms by which macrolides reduce airway inflammation are not well understood. These agents can reduce the expression of proinflammatory cytokines, improve lung macrophage function, and promote mucus secretion and mucociliary transport. Clarithromycin treatment of patients with asthma for 8 weeks resulted in reduced sputum CXCL8, neutrophils, and NE, as well as significant improvements in some quality of life parameters.36 Although modulation of neutrophilic airway inflammation was not observed in a 48-week trial of azithromycin in asthma,35 there was a reduction in the level of IL-1β protein.37 In COPD, azithromycin was shown to reduce inflammatory mediators and to improve macrophage efferocytosis. The improvement in efferocytosis was observed in conjunction with increased expression of the mannose receptor, which provides further evidence supporting a defect in the innate immune response in patients with obstructive airway disease.38 Several other treatment strategies of potential benefit for patients with neutrophilic asthma have emerged, though they need further investigation. In COPD, improvements in dyspnea followed treatment with a human monoclonal CXCL8 antibody.39 Low-dose theophylline inhibits the influx of neutrophils and can reduce CXCL8 levels in COPD.40 LABA can attenuate neutrophilic airway inflammation through reducing CXCL8 production.34 A recent clinical trial of a CXCR2 antagonist in patients with persistent, uncontrolled asthma did not achieve a reduction in annualized exacerbation rate, raising doubts as to the potential of this approach.41 The association of proinflammatory cytokine IL-1β with neutrophilic asthma has led to suggestions it could be targeted for therapeutic gain in this phenotype. Novel therapeutic approaches to inhibit IL-1β–mediated inflammation include the IL-1R antagonist, anakinra, the IL-1β monoclonal antibody, canakinumab, and newly designed inflammasome inhibitors, which inhibit airway neutrophilic inflammation and restore corticosteroid responsiveness in preclinical mouse models of neutrophilic airways disease.42 The identification of NETs in neutrophilic asthma also suggests that targeting this pathway may be of benefit. Nebulized recombinant human DNase (dornase alpha) is widely used to treat CF and is thought to work by breaking down the DNA backbone in NETs and reducing sputum viscosity.

SUMMARY It is clear that neutrophils are an important part of innate immune defense against infections; however, uncontrolled or inappropriate neutrophil responses contribute extensively to the pathogenesis of a number of inflammatory lung diseases, including neutrophilic asthma. Neutrophils are a source of both preformed and newly synthesized inflammatory mediators and are recruited to the lungs after exposure to respiratory triggers such as viruses, endotoxin, particulate air pollution, and cigarette smoke. Current asthma treatments, in particular, corticosteroids, are not effective at combating neutrophilic inflammation in asthma and may potentially promote this type of inflammation by delaying neutrophil apoptosis. A better understanding of the mechanisms modulating the recruitment and activation of neutrophils in neutrophilic asthma will have an impact on future therapeutic strategies, which may include use of macrolide antibiotics.

REFERENCES 1. Aulakh GK. Neutrophils in the lung: “the first responders”. Cell Tissue Res 2018;371(3):577–88.

2. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 2013;13:159–75. 3. Uddin M, Levy BD. Resolvins: natural agonists for resolution of pulmonary inflammation. Prog Lipid Res 2011;50:75–88. 4. Soehnlein O, Lindbom L. Phagocyte partnership during the onset and resolution of inflammation. Nat Rev Immunol 2010;10:427–39. 5. McDonald B, Kubes P. Cellular and molecular choreography of neutrophil recruitment to sites of sterile inflammation. J Mol Med 2011;89:1079–88. 6. Bakele M, Joos M, Burdi S, et al. Localization and Functionality of the Inflammasome in Neutrophils. J Biol Chem 2014;289:5320–9. 7. Pillay J, Kamp VM, van Hoffen E, et al. A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1. J Clin Invest 2012;122:327–36. 8. Leliefeld PH, Koenderman L, Pillay J. How neutrophils shape adaptive immune responses. Front Immunol 2015;6:471. 9. Baines KJ, Simpson JL, Wood LG, et al. Systemic upregulation of neutrophil α-defensins and serine proteases in neutrophilic asthma. Thorax 2011;66:942–7. 10. Wright TK, Gibson PG, Simpson JL, et al. Neutrophil extracellular traps are associated with inflammation in chronic airway disease. Respirology 2016;21:467–75. 11. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532–5. 12. Peschel A, Hartl D. Anuclear neutrophils keep hunting. Nat Med 2012;18:1336–8. 13. Juneau RA, Pang B, Weimer KE, et al. Nontypeable Haemophilus influenzae initiates formation of neutrophil extracellular traps. Infect Immun 2011;79:431–8. 14. McIlroy DJ, Jarnicki AG, Au GG, et al. Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery. J Crit Care 2014;29:1133. 15. Cheng O, Palaniyar N. NET balancing: a problem in inflammatory lung diseases. Front Immunol 2013;4:1–13. 16. Brown G, Vilalta A, Fricker M. Phagoptosis - Cell death by phagocytosis - Plays central roles in physiology, host defense and pathology. Curr Mol Med 2015;15:842–51. 17. Perttunen H, Moilanen E, Zhang X, et al. Beta2-agonists potentiate corticosteroid-induced neutrophil survival. COPD 2008;5:143–5. 18. Svenningsen S, Nair P. Asthma Endotypes and an Overview of Targeted Therapy for Asthma. Front Med 2017;4:158. 19. Simpson JL, Grissell TV, Douwes J, et al. Innate immune activation in neutrophilic asthma and bronchiectasis. Thorax 2007;62:211–18. 20. Ray A, Kolls JK. Neutrophilic Inflammation in Asthma and Association with Disease Severity. Trends Immunol 2017;38:942–54. 21. Sukkar MB, Wood LG, Tooze M, et al. Soluble RAGE is deficient in neutrophilic asthma and COPD. Eur Respir J 2012;39:721–9. 22. Baines KJ, Simpson JL, Wood LG, et al. Transcriptional phenotypes of asthma defined by gene expression profiling of induced sputum samples. J Allergy Clin Immunol 2011;127:153–60. 23. Simpson JL, Daly J, Baines KJ, et al. Airway dysbiosis: Haemophilus influenzae and Tropheryma in poorly controlled asthma. Eur Respir J 2016;47:792–800. 24. Essilfie AT, Simpson JL, Horvat JC, et al. Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease. PLoS Pathog 2011;7:e1002244. 25. Nadif R, Siroux V, Oryszczyn MP, et al. Heterogeneity of asthma according to blood inflammatory patterns. Thorax 2009;64:374–80. 26. Wood LG, Baines KJ, Fu J, et al. The neutrophilic inflammatory phenotype is associated with systemic inflammation in asthma. Chest 2012;142:86–93. 27. Baines KJ, Simpson JL, Bowden NA, et al. Differential gene expression and cytokine production from neutrophils in asthma phenotypes. Eur Respir J 2010;35:522–31. 28. Scott HA, Gibson PG, Garg ML, et al. Airway inflammation is augmented by obesity and fatty acids in asthma. Eur Respir J 2011;38:594–602. 29. Wood LG, Garg ML, Gibson PG. A high-fat challenge increases airway inflammation and impairs bronchodilator recovery in asthma. J Allergy Clin Immunol 2011;127:1133–40.

CHAPTER 17  Biology of Neutrophils 30. Simpson JL, Gibson PG, Yang IA, et al. Impaired macrophage phagocytosis in non-eosinophilic asthma. Clin Exp Allergy 2013;43:29–35. 31. Cowan DC, Cowan JO, Palmay R, et al. Effects of steroid therapy on inflammatory cell subtypes in asthma. Thorax 2010;65:384–90. 32. Barnes PJ. Glucocorticosteroids. Handb Exp Pharmacol 2017;237: 93–115. 33. Adcock IM, Barnes PJ. Molecular mechanisms of corticosteroid resistance. Chest 2008;134:394–401. 34. Chang HS, Lee TH, Jun JA, et al. Neutrophilic inflammation in asthma: mechanisms and therapeutic considerations. Expert Rev Respir Med 2017;11:29–40. 35. Gibson PG, Yang IA, Upham JW, et al. Effect of azithromycin on asthma exacerbations and quality of life in adults with persistent uncontrolled asthma (AMAZES): a randomised, double-blind, placebo-controlled trial. Lancet 2017;390:659–68. 36. Simpson JL, Powell H, Boyle MJ, et al. Clarithromycin targets neutrophilic inflammation in refractory asthma. Am J Respir Crit Care Med 2008;177:148–55.

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37. Simpson JL, Powell H, Pabreja K, et al. Sputum IL-1β is reduced with Azithromycin add-on therapy in patients with poorly controlled asthma. Eur Respir J 2017;50(Suppl. 61):PA4029. 38. Hodge S, Hodge G, Jersmann H, et al. Azithromycin improves macrophage phagocytic function and expression of mannose receptor in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2008;178:139–48. 39. Mahler D, Huang S, Tabrizi M, et al. Efficacy and safety of a monoclonal antibody recognizing interleukin-8 in COPD. Chest 2004;126:926–34. 40. Ford PA, Durham AL, Russell RE, et al. Treatment effects of low-dose theophylline combined with an inhaled corticosteroid in COPD. Chest 2010;137:1338–44. 41. O’Byrne PM, Metev H, Puu M, et al. Efficacy and safety of a CXCR2 antagonist, AZD5069, in patients with uncontrolled persistent asthma: a randomised, double-blind, placebo-controlled trial. Lancet Respir Med 2016;4:797–806. 42. Kim RY, Pinkerton JW, Essilfie AT, et al. Role for NLRP3 inflammasome– mediated, IL-1β–dependent responses in severe, steroid-resistant asthma. Am J Respir Crit Care Med 2017;196:283–97.

CHAPTER 17  Biology of Neutrophils

277.e1

SELF-ASSESSMENT QUESTIONS 1. The process of neutrophil margination refers to: a. The production by neutrophils of small arginine-rich peptides termed defensins. b. The exit of neutrophils from mainstream blood circulation through contact with endothelial cells in the capillary bed. c. The production by neutrophils of a fatty mucous layer spread upon target cells. d. The amplification of intravascular neutrophil numbers induced by inflammatory mediators and complement factors. e. The formation of the multilobe nucleus during neutrophil maturation. 2. Which of the following are not produced by neutrophils to mediate bacterial target killing? a. Extracellular traps composed of DNA b. Reactive oxygen species c. Proteases d. Cytokines e. Antibodies

3. CD47/SIRPα signaling plays important roles in which two processes? a. Targeting of neutrophils to specific organs during inflammation b. Production of NETs c. Adhesion and rolling on epithelial cell surfaces d. Release of neutrophil granules e. Prevention of phagocytic clearance of neutrophils 4. Neutrophilic asthma may be triggered by or commonly associated with: a. Exposure to tobacco smoke and/or air pollution b. Bacterial or viral lung infection c. Obesity d. Fatty diets lacking antioxidants e. All of the above

18  Biology of Monocytes and Macrophages Marc Peters-Golden, William J. Janssen

CONTENTS Introduction, 278 Monocytes and Macrophage Subsets in the Lung, 278 Origins of Monocytes and Macrophages, 279 Phagocytosis, 280 Elaboration of Mediators, 281

SUMMARY OF IMPORTANT CONCEPTS • In the airways and alveoli, macrophages serve as sentinel immune cells that orchestrate both inflammatory responses to diverse challenges and their subsequent resolution necessary to restore homeostasis. • Resident alveolar macrophages are embryonically derived and self-renew by replication; however, during inflammatory responses circulating monocytes are recruited to the lungs and differentiate to macrophages. • Macrophages use phagocytic receptors to recognize and ingest microbes and particulates which promote inflammation, and dying cells, which promotes an antiinflammatory program. • Macrophages secrete a vast array of molecules, including cytokines, chemokines, enzymes, growth factors, lipids, reactive species, and nucleic acids to carry out their pleiotropic effector responses. • Macrophages are highly plastic cells that can polarize along a phenotypic continuum from M1 cells, specialized for antimicrobial defense, to M2 cells, equipped to mediate allergic inflammation, fibrosis, and resolution. • Macrophages are increasingly appreciated to play key roles in dictating inflammatory responses in asthma, with resident alveolar macrophages mainly promoting homeostasis and recruited monocytes mainly promoting type 2 inflammation.

INTRODUCTION Macrophages are found in every organ and tissue of the human body. Their phagocytic function has been recognized since Élie Metchnikoff ’s seminal discoveries of the late 1800s, and they have long been recognized as essential participants in innate as well as adaptive immune responses. It is now known that they also play central roles in organ development, homeostasis, and tissue repair. Thus they play beneficial and sometimes harmful roles in myriad acute and chronic diseases.1 Historically, macrophages have been considered a part of the larger mononuclear phagocyte system (MPS), composed of monocytes, macrophages, and dendritic cells (DCs). For decades, dogma held that cells of the MPS derived from circulating monocytes, which continuously replaced effete tissue macrophages and DCs. However, more recent studies2,3 have led to a new paradigm in which tissue-resident

278

Macrophage Activation and Polarization, 282 Inflammation and Resolution, 283 Macrophages in Asthma, 285 Summary, 285

macrophages arise from embryonic precursors and self-replicate in situ without large-scale replacement from monocytes. In this context, it is important to note that although macrophages from different organs share many common features and functions, they also adopt tissuespecific characteristics that are influenced by local environmental factors. In this chapter, we will primarily focus on macrophages in the lungs, but many of the concepts discussed apply to macrophages in other sites. We describe the origins of various subsets of lung macrophages, factors that influence their activation, their phenotypic plasticity, the surface and secreted molecules that mediate their interactions with foreign substances and other cells, and the roles that they play in innate immune and inflammatory responses, including those pertinent to asthma.

MONOCYTES AND MACROPHAGE SUBSETS IN THE LUNG Lung macrophages and monocytes are best conceptualized using a framework that incorporates anatomic location and cell ontogeny. Anatomically, the lung can be divided into three compartments: the airways and airspaces, the vasculature, and the interstitium. The lumen of the airways and airspaces provides a direct conduit to the outside world. Macrophages that occupy this compartment are collectively termed alveolar macrophages (AMs). A large fraction of AMs reside in the alveolar spaces, where they are bathed in lung surfactant and are in intimate contact with the alveolar epithelium. Accordingly, they help protect the epithelial surface by engulfing inhaled pathogens and particulates and by regulating inflammatory processes. Smaller numbers of macrophages are also found in the lumen of the small and large airways. Although these macrophages are also referred to as AMs, it is unknown whether they have different functions and a different origin than their counterparts in the alveoli. AMs are characterized by cell surface markers that are common to macrophages in other organs, as well as several markers that distinguish them from interstitial macrophages (IMs) (Table 18.1). The vascular compartment of the lungs contains a large number of leukocytes, including monocytes. Many of these leukocytes are marginated rather than free flowing, and it has been estimated that the

CHAPTER 18  Biology of Monocytes and Macrophages

279

TABLE 18.1  Lung Macrophage Markers Designation

Alternative Names and Functions

Cell Specificity

Markers on All Lung Macrophages CD45 Protein tyrosine phosphatase receptor type C (PTPRC)

All leukocytes

HLA-DR

Human leukocyte antigen DR Major histocompatibility complex class II molecule used for antigen presentation

Universal for macrophages Dendritic cells, B lymphocytes

CD11b

Integrin alpha M (ITGAM) Subunit of Mac-1 or complement receptor 3

All myeloid cells, natural killer cells

MerTK

MER proto-oncogene, tyrosine kinase Multiple functions including phagocytosis

Universal for macrophages

CD16

Fc-γ receptor III Binds immunoglobulin G (IgG) with low affinity

Universal for macrophages Neutrophils, some monocytes, NK cells

CD32

Fc-γ receptor II Binds IgG with low affinity

Universal for macrophages B lymphocytes, some dendritic cells

CD64

Fc-γ receptor 1 (FcɣRI) Binds IgG with high affinity

Universal for macrophages On some monocytes

CD68

Macrosialin Associates with lysosomes and endosomes

Universal for macrophages

CD169

Sialoadhesin Siglec-1. Binds sialic acid residues on glycoproteins

High expression on AMs Low expression on IMs

CD206

Mannose receptor C-type lectin and pattern recognition receptor involved in phagocytosis

All lung macrophages On some dendritic cells

Markers for Alveolar Macrophages CD200R Receptor for OX-2 membrane glycoprotein (CD200) Inhibits inflammatory signaling

Alveolar macrophages

CD123

IL-3 receptor α subunit

Alveolar macrophages Basophils, some dendritic cells

CD43

Leukosialin, sialophorin Transmembrane sialoglycoprotein

Alveolar macrophages Lymphocytes, nonclassical monocytes

Markers for Interstitial Macrophages C1q Complement component High intracellular protein expression in interstitial macrophages CX3CR1

CX3C chemokine receptor 1, fractalkine receptor Involved in cell adhesion, migration

Interstitial macrophages Interstitial macrophages Nonclassical monocytes

AM, Alveolar macrophages; IM, interstitial macrophages; NK, natural killer.

pulmonary capillaries contain two to three times more monocytes than the total circulating blood pool.4 During homeostasis, a small fraction of monocytes constitutively traffics into the tissues and ultimately into the draining lymph nodes where they can present antigen.5 Perhaps more importantly, the marginated pool of monocytes provides a reservoir of cells that can be rapidly recruited to the lungs in response to injury or infection. The interstitium is the space that lies between the blood vessels and airspace lumen. Macrophages that reside in this compartment are termed IMs. The alveolar interstitium is remarkably thin, and in the healthy lung it is unlikely that a substantial number of IMs reside there. In comparison, the interstitium at the level of the conducting airways comprises a larger area. IMs can easily be identified around the airways and vessels on histologic sections. Based on transcriptional and functional profiling, at least three subpopulations of IMs exist in the mouse,6 and similar subpopulations are likely to exist in the human lung. Although the precise roles played by IM subsets remain unclear, as a group they appear to be important for regulating epithelial immune responses, vascular homeostasis and, in conjunction with fibroblasts, matrix composition. They have also been shown to dampen airway DC function and to suppress asthmatic responses.7

ORIGINS OF MONOCYTES AND MACROPHAGES Monocytes originate from hematopoietic stem cells in the bone marrow that give rise to a series of progressively more differentiated progenitor cells. The final uncommitted progenitor in the series is the monocyte-DC precursor, which gives rise to either the common DC precursor in the presence of Flt3 ligand, or the monocyte precursor in the presence of M-CSF. Three subsets of monocytes exist in the human circulation, as distinguished by flow cytometry. These include classical monocytes (CD14high CD16−), nonclassical or patrolling monocytes (CD14dim CD16high), and an intermediate population (CD14high CD16low).8 Similar populations exist in the mouse. Classical monocytes are released directly from the bone marrow and constitute 80% to 95% of the monocytes in the circulation. They express high levels of CC-chemokine receptor 2 (CCR2) and low levels of CX3C-chemokine receptor 1 (CX3CR1). A small percentage of classical monocytes differentiate into intermediate monocytes in the circulation. The intermediate monocytes account for 2% to 11% of circulating monocytes and express modest levels of CCR2 and high levels of CX3CR1. Intermediate monocytes can further differentiate into nonclassical monocytes. These comprise 2% to 8% of monocytes

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in the circulation and express low levels of CCR2 and high levels of CX3CR1. Classical and intermediate monocytes are recruited to areas of inflammation by CCL2 and CCL5. Although their range of functions are diverse and dependent on local environmental stimuli, it is generally thought that classical monocytes participate in phagocytosis and promote angiogenesis. In comparison, intermediate monocytes produce proinflammatory cytokines (e.g. IL-1β and TNF-α) and may play roles in antigen presentation. Nonclassical monocytes patrol vessel walls during homeostasis and invade them during tissue injury in response to CX3CL1 and CCL3. Depending on the setting, each subset has the potential to promote inflammation or foster tissue repair. Moreover, each subset can mature into functional macrophages. AMs are embryonic in origin and arise from fetal liver monocytes. They self-replicate and maintain a continuous pool that under normal circumstances is not replenished by circulating monocytes. During homeostasis, proliferation of AMs occurs at a low rate and is promoted by locally produced growth factors, namely GM-CSF and M-CSF. Upregulation of the transcription factor PU.1 and inactivation of transcription factors MafB and cMaf are also required.9 Notably, if the AM pool is depleted (e.g., from irradiation used for bone marrow transplantation, or experimentally by administration of clodronate-containing liposomes), monocytes will migrate to the lung and give rise to new macrophages that are virtually indistinguishable from embryonically derived ones. Changes in morphology and ultrastructure happen within hours to days, whereas transcriptional changes take several weeks. The origins of IMs are less well understood. Although it is clear that the IM pool arises during embryogenesis, emerging evidence suggests that at least some IMs are replaced by circulating monocytes during homeostasis. Furthermore, under some circumstances, IMs can give rise to AMs.10 However, the specific relationships among monocytes, IMs, and AMs under various conditions remain to be fully elucidated. During inflammation, macrophage numbers in the lung expand markedly, fueled both by replication of resident tissue macrophages and recruitment of monocytes from the circulation (Fig. 18.1).11 The cues that stimulate proliferation of resident AMs and IMs during inflammation are incompletely understood and are likely to be context-dependent. In the pleural space and peritoneum, high levels of IL-4 can drive macrophage proliferation independently of M-CSF and GM-CSF.12 Whether IL-4 can also stimulate AM and IM proliferation remains unknown. The migration of monocytes to inflammatory sites is driven by chemokine gradients. CCL2 appears to be a major player in the lungs, and in murine models of inflammation, genetic deletion of CCL2 or its receptor, CCR2, significantly reduces monocyte recruitment. Although it is clear that classical monocytes migrate to infected or injured lung tissue, it remains unknown whether nonclassical monocytes do as well. In the heart, recruitment of monocytes to infarcted tissue occurs in sequential waves, with classical monocytes arriving first, followed by nonclassical monocytes;13 the former play critical roles in phagocytosis of dying cells, whereas the latter promote tissue repair and beneficial scar formation. As mentioned previously, both classical and nonclassical monocytes have the capacity to mature and differentiate to macrophages. The factors that regulate the monocyte-to-macrophage transition remain incompletely defined, but both M-CSF and IL-4 have been implicated.9 As inflammation resolves, macrophage numbers return to baseline levels. Programmed cell death followed by local clearance of dying cells appears to be the main driver, but expectoration of dead cells may also contribute.

PHAGOCYTOSIS The principal function of the lungs is gas exchange. Accompanying the approximately 10,000 liters of air exchanged in the lungs each day are approximately 100 billion inhaled particles. Most particles are large

Homeostasis

Inflammation Recruited macrophage

Monocyte Airway

Resident interstitial macrophage

Proliferation Recruited macrophage

Monocyte

Alveolus Self-renewal

Resident alveolar macrophage

Proliferation Recruited interstitial macrophage

Fig. 18.1  Macrophage origins. During homeostasis, the lungs contain resident alveolar and interstitial macrophages (shown in blue). Alveolar macrophages exist primarily in the alveoli but may also be found in the airways. They arise during embryogenesis and maintain their numbers by self-renewal without replacement from circulating monocytes. Interstitial macrophages are found at the level of the airways and are unlikely to exist in the alveolar interstitium in substantial numbers. They arise during embryogenesis, but some may also derive from circulating monocytes. During inflammation, macrophage numbers expand. This is driven both by proliferation of resident macrophages and migration of monocytes from the circulation that mature into recruited macrophages (shown in green). Monocytes may immigrate directly into the alveolus and mature there, or they may mature in the interstitium before migrating to the airspaces.

enough to get trapped in the nasopharynx and mucociliary apparatus of the trachea and large airways. However, particles less than approximately 3 µm deposit in the lung periphery, where they are ingested by AMs. For many years AMs were thought to patrol the alveolar surfaces in search of inhaled particles, but recently they have instead been found to be largely sessile or stationary.14 This new paradigm raises the possibility that AMs depend on bulk movement of alveolar fluid to deliver microbes and particulates to them. Under most circumstances, such particles are cleared efficiently and silently—without inducing inflammatory responses that can injure delicate gas exchange structures. However, when the burden of microbes or particulates is high, AMs can initiate robust inflammatory responses. As discussed in more detail later, the unique environment of the lung facilitates these dual roles. Macrophages engulf particles and pathogens using a number of endocytic mechanisms (reviewed in reference 15). Of these, phagocytosis is the most common and is the dominant mechanism by which bacteria,

CHAPTER 18  Biology of Monocytes and Macrophages

TABLE 18.2  Phagocytic Receptors on

Lung Macrophages Designation

Ligands and Functions

Pathogen Receptors Complement Receptors   CR1 (CD35)   CR3 (αMβ2)   CR4 (αXβ2)

Binds C1q-, C4b-, C3b-opsonized particles iC3b-opsonized particles, β glucans iC3b-opsonized particles

Fc-Receptors  FcαRIII (CD89)  FcγRI (CD64)  FcγRII (CD32)  FcγRIII (CD16)  FcεR (CD23)

IgA opsonized particles IgG opsonized particles IgG opsonized particles IgG opsonized particles IgE opsonized particles

C-type Lectins   CD206 (Mannose receptor)   Dectin-1 (CLEC7a)  Dectin-2  Mincle

Mannan β Glucans (fungi) α-Mannans (fungi) α-Mannans (fungi)

Scavenger Receptors Class A Scavenger Receptors Lipopolysaccharide, lipoteichoic acid e.g. Macrophage Receptor (bacteria) with Collagenous Structure Bacteria, environmental particulates Macrophage Receptor with Collagenous Structure (MARCO) CD14

LPS, peptidoglycans

Efferocytosis Receptors MerTK Axl Stabilin-1 αvβ3 integrin αvβ5 integrin TIM-4 CD36 CD91 (Low-density lipoprotein receptor)

Phosphatidylserine (PS) via Gas-6, protein S PS via Gas-6 PS PS via MFG-E8 PS via MFG-E8 PS Thrombospondin-1 Cells opsonized with surfactant proteins A, D, or c1q with calreticulin as a promoter

fungi, and environmental particulates are cleared. When a macrophage encounters a phagocytic target, specialized receptors on its surface are engaged (Table 18.2). These trigger actin-dependent protrusion of cell membrane pseudopods that “zipper” tightly around the target until it is fully enclosed. The target is then internalized into a membrane-bound phagosome and degraded. Dying cells, which accumulate in the airspaces during inflammation and injury, are cleared by a specialized form of endocytosis, termed efferocytosis. As with phagocytosis, specialized receptors are required.16 Endocytic receptors recognize specific molecules on targets (e.g., phosphatidylserine on apoptotic cells or fungal glucans), or bind to opsonins or “bridge” molecules that have coated the target. Examples of opsonins include immunoglobulins, complement, and surfactant proteins. Notable bridge molecules include protein C and S, milk fat globule E8, and Gas6. Internalization of bound targets requires signal transduction events that promote cytoskeletal rearrangement. Although individual receptors exhibit variable capacity to initiate signaling, engulfment is often facilitated by clustering of multiple different receptors in a “phagocytic synapse.” Resident (embryonically derived) and recruited (monocyte derived) macrophages demonstrate differential expression of some endocytic receptors. Although differential receptor expression

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may correlate with functional differences in the capacity of macrophages of different origins to ingest various targets, the exact nature of any such relationships is complex and likely to vary between models and disease states.

ELABORATION OF MEDIATORS Macrophages and monocytes have the capacity to secrete a vast repertoire of bioactive molecules. These secreted molecules can modulate the functions of either the source cells themselves (autocrine effect) or of neighboring cells (paracrine effect) such as epithelial cells, fibroblasts, or other leukocytes. Considering that the frequency of AMs in the mammalian lung is less than one per alveolus14,17 and that they are largely stationary14 underscores the importance of such paracrine mechanisms in the ability of AMs to communicate with other cells in the distal lung. As the resident immune cells in the lower respiratory tract, AMs must carefully calibrate their own activation threshold—maintaining quiescence to facilitate gas exchange unless antimicrobial defense demands full-scale activation.18 This need for context-dependent versatility is facilitated by the macrophage’s ability to secrete both activating and suppressive mediators, whose balance can be shifted on demand. Moreover, in keeping with the paradigm of tissue specificity as a dominant determinant of macrophage function, it is not surprising that the secreted mediator profile of resident AMs favors quiescence and resolution of inflammation, whereas those produced by IMs and blood monocytes favor inflammation. Major molecular classes of macrophage-derived mediators (Table 18.3) will be discussed, including proteins, lipid mediators, reactive species, and extracellular vesicles. The best studied of these secreted substances are proteins, including cytokines and chemokines, growth factors, and enzymes. Cytokines, chemokines, and growth factors typically act by binding to receptors on the surface of target cells, with the subsequent activation of signaling pathways and of downstream transcriptional responses. In comparison, enzymes act directly on their substrate targets. Macrophage-derived cytokines TNF-α, IL-1β, IL-6, and IFN-γ are well-recognized contributors to inflammatory responses in the lung, and these together with IL-12 are also important in antimicrobial defense. In contrast, their elaboration of IL-10 and TGF-β restrains inflammatory responses. A variety of chemokines produced by macrophages contribute to recruitment and activation of leukocytes, including neutrophils (e.g., CXCL8), monocytes (e.g., CCL2), eosinophils (e.g., CCL11), T lymphocytes (e.g., CCL18), and DCs (e.g., CCL19). Macrophages secrete a number of growth factors that activate fibroblasts and contribute to wound healing as well as pathologic tissue fibrosis (e.g., TGF-β, fibroblast growth factor, platelet-derived growth factor), whereas secreted GM-CSF has autocrine- and paracrine-activating effects on leukocytes and protects epithelium from oxidant injury. Enzymes secreted by macrophages are exemplified by proteases that degrade matrix proteins such as elastin (e.g., MMP-12), hydrolyze bacterial peptidoglycans (e.g., lysozyme) or fungal cell wall chitin (e.g., chitinase), activate proenzymes (e.g., angiotensin-converting enzyme and plasminogen activator), and elicit shedding or cleavage of membrane proteins (e.g., ADAMs). Macrophages also secrete a large number of lipid mediators that exert autocrine and paracrine actions. These are generally the products of sequential enzyme-mediated transformation reactions from a parent lipid molecule. Lipid mediators act by binding to G protein– coupled receptors on the surfaces of target cells and activating downstream signaling events. Those that have been the best studied are eicosanoids—oxygenated metabolites of arachidonic acid—which include prostaglandins and leukotrienes. Leukotrienes promote inflammation, immune responses, and tissue remodeling, and are implicated in asthma as well as antimicrobial defense. Prostaglandin E2 (PGE2),

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SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 18.3  Bioactive Molecules Secreted by Alveolar Macrophages Cytokines and Chemokines  IL-1β, IL-6, IL-10, IL-12, IL-17, IL-18, IL-23

  Platelet-activating factor

  CXCL1, CXCL2, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11

  Lysophosphatidic acid

  CCL2, CCL3, CCL4, CCL5, CCL7, CCL11, CCL18, CCL19

 Sphingosine-1-phosphate

 IFN-α, IFN-β, IFN-γ  TNF-α  RELM-α (FIZZ1)  GM-CSF Enzymes   ADAM proteins   Angiotensin-converting enzyme   Acid hydrolases   Aryl sulfatase   Chitinases (e.g., acid mammalian chitinase)   Chitinase-like molecules (e.g., CHl3L1, CHl3L2)   Matrix metalloproteinases (e.g., MMP-12)   Cysteine proteases (e.g., cathepsin C)  Lysozyme   Plasminogen activator Growth Factors  TGF-α, TGF-β   Fibroblast growth factor   Platelet-derived growth factor   Insulin-like growth factor Lipid Mediators   Leukotrienes (LTB4 and the cysteinyl leukotrienes LTC4, LTD4, and LTE4)   Prostaglandins (PGE2, PGF2α, PGI2, PGD2) and thromboxane A2

the major prostaglandin in most tissues, is conventionally regarded as a proinflammatory molecule, but its actions are highly context-dependent and in the lung are largely salutary as it inhibits leukocyte activation, immune responses, and fibroblast activation. A family of oxygenated lipid mediators derived from arachidonate as well as fatty acids enriched in fish oils has also been implicated in inhibition and resolution of inflammation and includes lipoxins, resolvins, protectins, and maresins.19 Additional lipid mediators with predominantly mitogenic and chemotactic activities include sphingosine 1-phosphate and lysophosphatidic acid. Another category of extracellular mediators elaborated by macrophages includes reactive species and gases. Reactive oxygen species contribute to microbial killing and injurious inflammatory tissue responses. Nitric oxide likewise promotes bacterial killing but also acts as a vasodilator and has the capacity to either increase or dampen inflammatory responses. Carbon monoxide similarly attenuates vascular tone and inflammatory responses. Macrophages can also release products within extracellular vesicles (EVs), small lipid membrane-delimited packets that can be internalized by neighboring cells. Two major types of EVs include exosomes and microvesicles (also termed microparticles). Exosomes are approximately 30- to 150-µm diameter vesicles originating from endosomal membranes. In comparison, microvesicles are approximately 100- to 1000-µm diameter vesicles that bud from the plasma membrane. Both types of EVs

  Proresolving lipids (e.g., lipoxins, resolvins, protectins, maresins)

Reactive Species and Gases   Nitric oxide   Carbon monoxide   Hydrogen sulfide   Reactive oxygen species   Reactive nitrogen species Other Bioactive Molecules   Coagulation factors   Tissue inhibitors of metalloproteinases   Complement factors   Dihydroxyvitamin D3  Endothelin  Fibronectin  Glutathione  Transferrin   α2-Macroglobulin   α1-Protease inhibitor Extracellular Vesicle (Exosomes and Microvesicles) Cargo Molecules   Proteins (e.g., IL-1β, SOCS proteins, lipoxygenase enzymes)   Nucleic acids (e.g., mRNAs, microRNAs, long noncoding RNAs)   Lipids (e.g., fatty acids, ceramide, sphingomyelin, leukotrienes, prostaglandins, proresolving lipids)

can harbor protein molecules, lipid mediators, and nucleic acids such as microRNAs—either decorating their surface or within them. Because many of these cargo molecules are typically considered to be intracellular and not subject to conventional secretion, EVs markedly expand the spectrum of molecules that can be elaborated by a source cell. Once bound or internalized by the recipient cell, the cargo is able to exert biologic actions. EVs, then, represent the most recently recognized vector of cell-cell communication. In the lung, bidirectional EV transfer between macrophages and epithelial cells has been identified and implicated in both homeostasis and tissue inflammation and injury.20 Packaging of cargo within EVs, release of EVs by source cells, and the uptake of EVs by recipient cells are all subject to modulation, providing dynamic control of this means of communication.

MACROPHAGE ACTIVATION AND POLARIZATION In contrast to T lymphocytes, macrophages demonstrate remarkable plasticity and can quickly change their programming in response to environmental cues. This is most readily and classically evidenced by in vitro studies demonstrating that activation with IFN-γ and/or lipopolysaccharide (LPS) versus IL-4 results in markedly different programs of cell surface markers; transcriptional profiles; and secreted cytokines, chemokines, and growth factors. To parallel historic T cell polarization paradigms, macrophages stimulated with IFN-γ and/or LPS were termed

CHAPTER 18  Biology of Monocytes and Macrophages Resting macrophage

Classical activation (M1)

283

Alternative activation (M2) Allergens

Bacterial infection

LPS IFNγ

IL-4 IL-13 M2

M1

Markers IL-1R TLR4 CD86 CD80

Helminth infection

STAT-6

STAT-1

Pro-inflammatory cytokines IL-1β IL-6 TNF-α IL-12

Th1 responses

Markers CD163 Transglutaminase 2 IL-4Rα

Reactive oxygen species

IL-10

Growth factors TGF-β VEGF PDGF

Anti-inflammatory Tissue repair

Fibrosis

Fig. 18.2  Polarization of macrophages. Macrophages can become activated quickly after exposure to stimuli in the local environment. Although a spectrum of activation states exist, the most commonly described are classical (M1) and alternative (M2) activation. Classical activation occurs in states characterized by high levels of LPS and/or IFN-γ, such as bacterial infection. Classical activation requires phosphorylation of the transcription factor STAT-1 and leads to production of proinflammatory cytokines, release of reactive oxygen species, and promotion of Th1 responses. Alternative activation occurs in states characterized by high levels of IL-4 and/or IL-13, such as allergen exposure or helminthic infection. Alternative activation requires activation of STAT-6 and leads to production of IL-10 and growth factors such as transforming growth factor β (TGF-β), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF). Depending on the context, the profile of mediators released from alternatively activated macrophages can suppress inflammation, promote tissue repair, or accentuate fibrotic responses. Although classically and alternatively activated macrophages may differentially express cell markers, these are not absolute and should not be relied on as the sole means to determine macrophage activation status or phenotype.

M1 or classically activated macrophages, whereas those stimulated with IL-4 were termed M2 or alternatively activated. It is now recognized that these dichotomous states do not adequately capture the complexity of macrophage activation and that macrophage activation states exist on a continuum.21 This holds especially true for the in vivo setting in which the macrophage is exposed to a panoply of signals. Nonetheless, the M1 versus M2 concept will be discussed here because it provides a useful starting point for understanding macrophage plasticity (Fig. 18.2). M1 macrophages are most often associated with bacterial infection and are characterized by production of high levels of proinflammatory mediators (e.g., IL-1β, IL-18, TNF-α, IL-6, and IL-12), antimicrobial defense, release of reactive oxygen and nitrogen species, and promotion of Th1 responses. M1 polarization requires phosphorylation and activation of signal transducer and activator of transcription 1 (STAT-1). Cellular metabolism is considered to be an essential regulator of macrophage function and for M1 polarization is characterized by glycolytic metabolism and high enzymatic activity of inducible nitric oxide synthase, which converts arginine to nitric oxide.22 Typical M1 markers include CD86, CD80, IL-1R, and TLR4. M2 macrophages are commonly associated with helminthic infections and allergic diseases characterized by high levels of IL-4 and IL-13. They produce IL-10, TGF-β, vascular endothelial growth factor, plateletderived growth factor, and insulin-like growth factor 1.23 Depending on context, this mediator profile may be considered antiinflammatory

or tissue reparative, but also profibrotic. M2 activation requires STAT-6 and is promoted by interferon regulatory factor 4, peroxisome proliferator-activated receptor γ (PPARγ), and Kruppel-like factor 4. Metabolic features include downregulation of glycolysis, an active tricarboxylic acid cycle fueled by fatty acids, and upregulation of the enzyme arginase-1, which shunts arginine metabolism away from nitric oxide production and toward synthesis of ornithine and proline (an essential component of collagen).23 Common M2 markers include CD163, transglutaminase 2, IL-4Rα, resistin-like molecule α, and in the mouse Fizz1 and chitinase-like 3 (YM1). The mannose receptor (CD206) and CD200R are classically recognized as M2 macrophage markers, but are expressed on lung macrophages, even at baseline. Some investigators have attempted to divide M2 macrophages into subsets, but these designations are unavoidably artificial and of limited utility, especially in the complex environment of the lung. It is more useful to instead describe macrophages by their pertinent functions or programs. This approach is illustrated later in which contributions of M1 and M2 macrophages to the pathogenesis of asthma are considered.

INFLAMMATION AND RESOLUTION Inflammation denotes the complex biologic response to harmful stimuli such as infections and wounds. This surely evolved as a protective response to defend the host, but when inflammation is dysregulated or

SECTION A  Basic Sciences Underlying Allergy and Immunology TREM2 Resident AM PPARγ Expression of inflammatory mediators

IL-10 TGF-β

SIRP- SOCS CD200R CD200

inappropriately persistent, chronic disease or fibrosis often result. Again, the magnitude of this challenge and its physiologic stakes are especially high in the lung. As sentinel immune cells, macrophages play critical and dynamic roles in the initiation and resolution of inflammatory responses. Beyond their classical role in phagocytosis, macrophages are now recognized to participate in far more complex ways that permit coordination with other immune system components. Both the plasticity of macrophages and the functional differences between resident and recruited macrophages facilitate their pleiotropic and dynamic participation in all phases of inflammation. Evidence for an essential role of AMs in innate inflammation derives from studies showing that depletion of these cells by intrapulmonary administration of clodronate-containing liposomes—which induce apoptosis in macrophages that ingest them—reduces the acute inflammatory response in mice subjected to lung infection with various viruses or bacteria. Besides the phagocytic receptors discussed previously, a family of pattern recognition receptors (PRRs) on macrophages recognize specific microbial lipid, protein, carbohydrate, and nucleotide moieties, termed pathogen-associated molecular patterns (PAMPs). Often acting in a combinatorial manner, PRRs drive broad activation of inflammatory responses via both autocrine and paracrine means.24 PRRs include toll-like receptors (TLRs), C-type lectin receptors (CLRs), nucleotide-binding oligomerization domain (NOD)–like receptors (NLRs), and retinoic acid inducible gene-I (RIG-I)–like receptors (RLRs). TLRs are the best studied of the PRRs, and this 10-member mammalian family is exemplified by TLR4, which recognizes gram-negative LPS or endotoxin. The CLRs are exemplified by dectin-1 (CLEC7a), which is primarily involved in antifungal defense. NLRs comprise a large family of cytosolic receptors that recognize a variety of PAMPs. Once engaged, most TLRs, CLRs, and NLRs initiate signaling cascades that result in activation of two key transcription factors, nuclear factorκB (NF-κB) and AP1. Acting individually as well as cooperatively, NF-κB and AP-1 result in transcriptional activation of a program of proinflammatory genes encoding cytokines such as TNF-α, IL-12, IFN-γ, and IL-6. These promote differentiation toward classically activated M1 macrophages specialized for antimicrobial effector functions and also activate those functions in other cells such as Th1 cells and neutrophils. Innate defense functions are further amplified by the parallel biosynthesis of numerous lipid mediators in response to PRR binding. NLRs have an additional and unique function—promoting assembly of a multimeric protein complex called the inflammasome; this activates caspase-1, which results in the processing and maturation of proinflammatory cytokines IL-1β and IL-18. Finally, RLRs are cytosolic receptors that recognize viral DNA and elicit signaling responses culminating in the generation of antiviral type I IFNs. In addition to their essential role in microbial recognition and defense, macrophage PRRs also become engaged during sterile insults. Such insults are characterized by exposure to an array of molecular motifs termed damage-associated molecular patterns (DAMPs). These include both endogenous products of tissue injury (e.g., nucleic acids, ATP, hyaluronan fragments, cholesterol or monosodium urate crystals, and the chromatin-associated protein high mobility group box 1) as well as exogenous substances (e.g., asbestos, silica, alum). TLRs and NLRs are most important in DAMP recognition, and the activation of these PRRs by DAMPs stimulates diverse acute inflammatory processes as it does during infections. In these circumstances, innate responses that evolved to be protective instead initiate or amplify injury. Lung macrophages are far less effective at antigen presentation than are macrophages in most other organs, ceding this initiating role in adaptive immunity instead to DCs. For this reason, antigen presentation is not considered here, but is comprehensively addressed in Chapter 13. Furthermore, numerous studies using clodronate depletion of resident

Surfac ta protein nt

284

EVs

• Lipoxin and resolvin • PGE2

Alveolar space or airway lumen

Expression of inflammatory mediators

Alveolar or airway epithelial cells Fig. 18.3  Antiinflammatory mechanisms of action for resident alveolar macrophages. Resident alveolar macrophages (AMs) at baseline tend to restrain inflammatory signaling at the alveolar surface both by dampening their own activation and that of epithelial cells or other leukocytes such as T cells. These antiinflammatory mechanisms can be further upregulated when AMs ingest apoptotic cells (efferocytosis). Cell surface receptors on AMs that inhibit inflammatory signaling and induction of inflammatory mediators include CD200R, which recognizes CD200 on the surface of epithelium, signal-regulatory protein-α (SIRP-α), which recognizes surfactant proteins, and triggering receptor expressed on myeloid cells 2 (TREM2), whose ligand is unknown. AMs have high level expression of the transcription factor peroxisome proliferatoractivated receptor γ (PPARγ) and the suppressor of cytokine signaling (SOCS) protein family members, both of which oppose inflammatory gene transcription. AMs secrete antiinflammatory or immunoregulatory molecules TGF-β, IL-10, and the lipid mediators PGE2 as well as lipoxins and resolvins. Acting on cell surface receptors in the AM, these exert autocrine restraint on AM activation. The lipid mediators also act in a receptor-dependent manner on neighboring epithelial cells or leukocytes to inhibit activation of inflammatory pathways. Finally, AMs can secrete extracellular vesicles (EVs) containing a variety of cargo molecules that can be internalized by epithelial cells. Such transcellular delivery of AMderived cargo, such as SOCS proteins, can likewise inhibit inflammatory signaling in recipient epithelial cells.

AMs—some combined with adoptive transfer of naïve macrophages— suggest a predominantly suppressive role for these resident cells in adaptive immune responses to intrapulmonary antigen challenge. Indeed, a similar role for resident AM-mediated suppression or even resolution has been observed in a variety of other mouse models of immune and nonimmune lung inflammation or injury. This antiinflammatory or proresolving potential of resident AMs reflects the contributions of a diverse set of negative regulatory molecules expressed by these cells (Fig. 18.3).18 First, AMs have high expression of certain molecules that serve as brakes on their own proinflammatory activation; these include cell surface receptors such as CD200 receptor, triggering receptor expressed on myeloid cells 2 (TREM2), and signalregulatory protein α (SIRPα); the transcription factor PPARγ; and members of the suppressors of cytokine signaling (SOCS) protein family. Moreover, they can secrete a number of molecules that act in both autocrine fashion as well as on neighboring epithelial cells and other leukocytes to exert antiinflammatory actions; these include IL-10, TGF-β, PGE2, lipoxins and resolvins, and EV-encapsulated SOCS

CHAPTER 18  Biology of Monocytes and Macrophages proteins.25 Certain of these suppressive signals have been shown to be upregulated above basal levels after inflammation. In addition, the antiinflammatory and proresolving programs associated with efferocytosis are in large part related to elaboration of many of these same negative regulatory factors.

MACROPHAGES IN ASTHMA The recognized importance of type 2 inflammation in allergic asthma prompted investigators to focus on cells such as DCs, lymphocytes, eosinophils and mast cells, while macrophages, historically linked to innate type 1 inflammation, were relatively ignored. Recently, however, complex and important roles for macrophages in asthma have emerged.26 The initial insight was that type 2 cytokines prevalent during allergic inflammation (such as IL-4) polarized macrophages to an alternatively activated or M2 phenotype; as noted previously, these cells can exhibit a variety of properties that, depending on context, include inhibiting classical activation, promoting eosinophilic inflammation, and augmenting tissue remodeling. M2 macrophage–derived TGF-β has been implicated in epithelial damage and subepithelial fibrosis seen in chronic airway remodeling. Although the presence of M2 macrophages correlates with the severity of airway inflammation in models of asthma, whether these cells are the cause or the consequence of allergic inflammation remains to be clarified. It is also increasingly appreciated that in both mouse models of asthma and humans with asthma, airway biopsies actually reveal a mix of M2 and M1 macrophages.27 Although M1 cells might be expected to oppose allergic sensitization, some data suggest that they can interact with M2 cells in complex ways and can favor the development of more severe asthma. Certainly, classically activated M1 macrophages are logical participants in Th2-low or neutrophilpredominant endotypes of asthma, which may be associated with a corticosteroid-resistant phenotype. M1 cells may also be expected to mediate responses initiated or exacerbated by respiratory viruses or other microbes, or by LPS and other PAMPs contaminating organic dusts (e.g., farm dust) or cigarette smoke. As with other acute inflammatory insults, aeroallergen challenge is associated with CCR2-dependent recruitment to the airways of inflammatory monocytes, which subsequently differentiate to macrophages. In parallel, increased proliferation of macrophages occurs. In a model of LPS-induced inflammation, proliferation is limited to the resident macrophage pool, but it is not known if this also applies to allergen challenge. Importantly, the roles of resident vs. recruited monocytederived macrophages in the immunologic response to allergen challenge are contradictory, because depletion of airway and alveolar macrophages by intrapulmonary administration of clodronate worsens type 2 inflammatory responses, whereas depletion of monocytes by intravenous clodronate ameliorates them.28 The latter finding is consistent with a growing recognition for a pathogenic role for inflammatory monocytes in a variety of disease states and models. The suppressive actions of resident AMs suggested by the former finding can be explained by the actions of a number of inhibitory mediators discussed previously, whose expression is, moreover, diminished in asthma. For example, fewer IL-10+ macrophages were found in mouse models of asthma and in airway biopsies of asthmatics than in healthy mice and humans.27 Reduced AM generation of lipoxin A4 has also been observed in asthma. Findings such as these may be related to the observation that macrophage efferocytosis is also reported to be diminished in asthma.

SUMMARY As the resident immune cells of the challenging and ever-changing milieu of the respiratory mucosa, macrophages are charged with

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dictating the appropriate balance between quiescence and activation. Over the last 10 years, new insights into the origins, plasticity, determinants, functions, and fate of lung macrophages have emerged, permitting a more thorough and nuanced view of their roles in the initiation, perpetuation, and resolution of immune-related respiratory diseases. Contrary to longstanding assumptions, we now recognize that resident AMs arise not from bone marrow–derived monocytes but from embryonic precursors and proliferate in situ. These cells promote homeostasis by contributing to innate immune responses to microbes, clearing debris and apoptotic cells, and—in certain settings—dampening and promoting resolution of inflammatory responses. Although monocytes can replenish the AM pool when it is depleted, they are increasingly appreciated to mediate inflammatory and injurious responses to more harmful perturbations. The diverse and dynamic abilities of monocytes and macrophages to interact with offending stimuli and with neighboring structural cells or recruited leukocytes reflect their considerable repertoire of cell surface receptors as well as secreted bioactive molecules. The functional divergence between monocytes and resident AMs noted previously is manifest in allergic asthma, where the former tend to promote and the latter to suppress allergic inflammatory responses. Despite these advances, a number of important gaps in knowledge remain. We still have much to learn about the biology of IMs, and their ontogenic and functional relationships with AMs. We know little about airway macrophages; in particular, do they represent effete AMs that are in the process of being cleared via the mucociliary escalator, or do they have a distinct origin? Are they functionally distinct from AMs? Certainly, we also know little about the macrophages that reside in the sinus and nose. Finally, knowledge of lung macrophages may provide targets or strategies for therapeutic intervention; examples include inhibiting monocyte recruitment, modulating macrophage polarization, stimulating macrophage proliferation, or therapeutic administration of macrophages themselves or of mediators or EVs derived from macrophages.

REFERENCES 1. Vannella KM, Wynn TA. Mechanisms of organ injury and repair by macrophages. Annu Rev Physiol 2017;79:593–617. 2. Hashimoto D, Chow A, Noizat C, et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 2013;38:792–804. 3. Guilliams M, De Kleer I, Henri S, et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. J Exp Med 2013;210: 1977–92. 4. Doerschuk CM, Downey GP, Doherty DE, et al. Leukocyte and platelet margination within microvasculature of rabbit lungs. J Appl Physiol 1990;68:1956–61. 5. Jakubzick C, Gautier EL, Gibbings SL, et al. Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity 2013;39:599–610. 6. Gibbings SL, Thomas SM, Atif SM, et al. Three unique interstitial macrophages in the murine lung at steady state. Am J Respir Cell Mol Biol 2017;57:66–76. 7. Bedoret D, Wallemacq H, Marichal T, et al. Lung interstitial macrophages alter dendritic cell functions to prevent airway allergy in mice. J Clin Invest 2009;119:3723–38. 8. Grage-Griebenow E, Flad HD, Ernst M. Heterogeneity of human peripheral blood monocyte subsets. J Leukoc Biol 2001;69:11–20. 9. Sieweke MH, Allen JE. Beyond stem cells: self-renewal of differentiated macrophages. Science 2013;342:1242974-1-7. 10. Landsman L, Jung S. Lung macrophages serve as obligatory intermediate between blood monocytes and alveolar macrophages. J Immunol 2007;179:3488–94.

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11. Mould KJ, Barthel L, Mohning MP, et al. Cell origin dictates programming of resident versus recruited macrophages during acute lung injury. Am J Respir Cell Mol Biol 2017;57:294–306. 12. Jenkins SJ, Ruckerl D, Cook PC, et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science 2011;332:1284–8. 13. Swirski FK, Nahrendorf M, Etzrodt M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 2009;325:612–16. 14. Westphalen K, Gusarova GA, Islam MN, et al. Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity. Nature 2014;506:503–6. 15. Conner SD, Schmid SL. Regulated portals of entry into the cell. Nature 2003;422:37–44. 16. Penberthy KK, Ravichandran KS. Apoptotic cell recognition receptors and scavenger receptors. Immunol Rev 2016;269:44–59. 17. Hyde DM, Tyler NK, Putney LF, et al. Total number and mean size of alveoli in mammalian lung estimated using fractionator sampling and unbiased estimates of the Euler characteristic of alveolar openings. Anat Rec A Discov Mol Cell Evol Biol 2004;277:216–26. 18. Hussell T, Bell TJ. Alveolar macrophages: plasticity in a tissue-specific context. Nat Rev Immunol 2014;14:81–93. 19. Buckley CD, Gilroy DW, Serhan CN. Proresolving lipid mediators and mechanisms in the resolution of acute inflammation. Immunity 2014;40:315–27.

20. Nana-Sinkam SP, Acunzo M, Croce CM, et al. Extracellular vesicle biology in the pathogenesis of lung disease. Am J Respir Crit Care Med 2017;196:1510–18. 21. Xue J, Schmidt SV, Sander J, et al. Transcriptome-based network analysis reveals a spectrum model of human macrophage activation. Immunity 2014;40:274–88. 22. Ghesquiere B, Wong BW, Kuchnio A, et al. Metabolism of stromal and immune cells in health and disease. Nature 2014;511:167–76. 23. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 2016;44:450–62. 24. Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell 2010;140:805–20. 25. Bourdonnay E, Zaslona Z, Penke LR, et al. Transcellular delivery of vesicular SOCS proteins from macrophages to epithelial cells blunts inflammatory signaling. J Exp Med 2015;212:729–42. 26. Draijer C, Peters-Golden M. Alveolar macrophages in allergic asthma: the forgotten cell awakes. Curr Allergy Asthma Rep 2017;17:12-1-8. 27. Draijer C, Boorsma CE, Robbe P, et al. Human asthma is characterized by more IRF5+ M1 and CD206+ M2 macrophages and less IL-10+ M2-like macrophages around airways compared with healthy airways. J Allergy Clin Immunol 2017;140:280–3.e3. 28. Zaslona Z, Przybranowski S, Wilke C, et al. Resident alveolar macrophages suppress, whereas recruited monocytes promote, allergic lung inflammation in murine models of asthma. J Immunol 2014;193:4245–53.

CHAPTER 18  Biology of Monocytes and Macrophages

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SELF-ASSESSMENT QUESTIONS 1. During homeostasis, resident alveolar macrophages derive from: a. Circulating monocytes b. Interstitial lung macrophages c. Embryonic precursors d. Dendritic cells e. The bone marrow monocyte-dendritic cell precursor 2. M1 or classically activated macrophages produce high levels of all of the following, EXCEPT: a. IL-10 b. IL-6 c. TNF-α d. IL-1β e. IL-12

3. Efferocytosis generally promotes an antiinflammatory or pro-resolution molecular program. Efferocytosis describes the process by which macrophages ingest: a. Fungi b. Helminths c. Toll-like receptors d. Apoptotic cells e. Necrotic neutrophils

19  Airway Epithelial Cells Darryl Knight, Jeremy Hirota

CONTENTS Introduction, 287 Anatomy of the Airway Epithelium, 287 Barrier Function of the Airway Epithelium, 289 Airway Epithelium Repair Processes, 290 Airway Epithelium Immune Responses, 290

The Airway Epithelium in Asthma, 295 Influence of Asthma Medications on the Asthmatic Epithelium, 297 Summary, 298

SUMMARY OF IMPORTANT CONCEPTS

ANATOMY OF THE AIRWAY EPITHELIUM

• The epithelium plays an important role as a physical and innate immune barrier. • The epithelium plays an active role in regulating airway homeostasis through the production of a multitude of damage-associated molecular patterns (DAMPs), cytokines, chemokines, lipid mediators, and growth factors. • Epithelial cells bridge the innate and adaptive immune response by translating environmental exposures into disease phenotypes. • Epithelial cell structure and function are abnormal in asthma. • Changes to epithelial cell structure and function occur early in disease pathogenesis.

Development of the Lung, Airways and Epithelium

INTRODUCTION The respiratory epithelium is the interface between the respirable environment and the submucosa and acts as a physical and immune barrier against inhaled noxious agents, aeroallergens, and viruses. The epithelium of the conducting airways is pseudostratified, consisting of ciliated columnar epithelial cells, goblet cells, intermediate cells, side population cells, serous cells, and basal cells. The epithelium of asthmatics is characterized by several structural abnormalities suggestive of dysregulated differentiation, including a greater proportion of resident progenitor cells, fewer ciliated cells, and abnormal junctional protein expression compared with the epithelium of healthy individuals. Functionally, this is associated with increased permeability and susceptibility to oxidant-induced stress, abnormal cytokine and extracellular matrix (ECM) release,1 and a deficient innate immune response.2-4 Both genetic susceptibility and environmental risk factors influence asthma,5 and the majority of genome-wide association studies have shown a central role for epithelial genes in disease pathogenesis and severity. Similarly, environmental challenges can affect epithelial gene expression through epigenetic regulation including DNA methylation, histone modifications, and regulation by noncoding RNAs. Cumulatively, these pathways play important roles in airway epithelial homeostasis. Conversely, abnormalities will adversely impact epithelial repair and regeneration, leading to defective maintenance of the epithelial barrier and its normal function.

Lung development follows a branching morphogenesis pattern specified by interactions between the embryonic endoderm and the surrounding mesoderm, in which the mesoderm directs epithelial proliferation, differentiation, and development. This bidirectional interaction between the airway wall components is referred to as the epithelial-mesenchymal trophic unit (EMTU)6 and requires coordinated and exquisitely regulated interaction between a number of factors, including fibroblast growth factor (FGF), epidermal growth factor (EGF), bone morphogenic protein (BMP) families, and wnt/β-catenin and Sonic Hedgehog pathways.7 Through the use of specific therapeutic antibodies to block the Notch pathway, an inherent requirement for Notch activity in club cells has been shown to be necessary to maintain their cell fate.8 Signaling by many of these pathways, together with extracellular matrix components and integrin signaling pathways, also directs lung epithelial cell differentiation. This process initiates in the trachea and progresses distally toward the terminal airways in a unidirectional manner. Until this process is complete, the epithelium contains a mixture of both mature and immature cell types. In humans, ciliated cells differentiate first in late gestation and first appear overlying the smooth muscle side of the trachea. Subsequently, nonciliated cells containing secretory granules and submucosal glands initially develop on the cartilaginous portions of the trachea. This indicates that the proximity to and robustness of mesenchymal signals directs the pattern of epithelial differentiation. Recent evidence also suggests a key role for the Notch family of signaling molecules in silencing ciliated and promoting secretory cell lineage decisions. Basal and small mucous granule cell populations appear last, suggesting that there are several different progenitor cells that give rise to the multiple cell types. By adulthood, at least eight morphologically distinct epithelial cell types are present in human respiratory epithelium.9,10

Major Cell Types of the Airway Epithelium The tissue lining the conducting airways is considered to be a pseudostratified epithelium, indicating that all epithelial cells make contact with the basal lamina (Fig. 19.1). From the trachea to the fifth generation of bronchi, the most abundant cell types are ciliated and secretory

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Ciliated columnar epithelial cell Goblet cell Side population cell Intermediate columnar Pulmonary neuroendocrine cell

Serous cell Basal cell Macrophage Dendritic cell Neuron

Fig. 19.1  Anatomy of and cell types associated with pseudostratified airway epithelium in proximal airways. The airway epithelium consists of multiple cell types (see text) including ciliated columnar epithelial cells, goblet cells, intermediate columnar epithelial cells, side population cells, serous cells, and basal cells (see legend). The junctions between the epithelial cell types consists of the apical junctional complex (green connectors), adherens junctions (gray connectors), and desmosomes (red connectors). The pseudostratified airway epithelium is arranged so that each cell type has connections to the underlying basal membrane (curved red lines beneath basal cells). Pulmonary neuroendocrine cells and neurons also are interspersed within the airway epithelium. Lastly, immune cells involved in antigen presentation including but not limited to dendritic cells or macrophages can be located within intimate reach of the airway lumen.

(goblet) columnar cells; undifferentiated columnar cells, which are neither secretory nor ciliated; and basal cells. In the smaller airways, down to the 23rd generation, the epithelium is composed of a higher proportion of ciliated cells, and secretory club cells replace goblet cells. Beyond this, the airways enter the terminal bronchiolar region, where alveolar cell types (type I and type II) predominate.9 The luminal surface of the epithelium is primarily composed of columnar epithelial cells, which include both ciliated and goblet cells. These cells lie perpendicular to the basal lamina. Ciliated cells, which contain an average of 300 cilia per cell, sweep mucus consisting of a low viscosity sol phase covered by a high viscosity gel phase. Mucus is produced from submucosal glands and goblet cells to create a liquid trap for inhaled foreign substances. Collectively the cilia and mucus create the mucociliary ladder, which escalates trapped foreign substances toward the pharynx and ultimately the esophagus where they are swallowed. In the large conducting airways, 80% of the luminal surface area is covered by cilia. Goblet cells are unicellular paracrine glands, which arise from epithelial progenitor cells. Goblet cells are specialized for mucus production and have a high content of cytoplasmic granules. They make up less than 3% of the total number of cells in the trachea and are generally not found in distal airways. However, the number of these cells increases substantially and generally at the expense of ciliated cells in several airway diseases. The mucus produced by these cells, as well as that from submucosal glands, works in conjunction with the adjacent ciliated cells to enable mucociliary clearance of inhaled particles. Basal cells are defined by the orientation of their nuclei, which lie parallel to the basal lamina; expression of the intermediate filament

proteins, cytokeratins 5 and 14; and expression of the transcription factor p63. This transcription factor is a homolog of p53 and its germline inactivation results in neonatal death related to agenesis of organs such as skin, breast, salivary glands, and prostate.11 More recently, p63 has been shown to play a critical role in the development of tracheobronchial epithelium and in particular appears to control the commitment of stem cells into basal cells.12 Basal cells are anchored to the basal lamina via hemidesmosomes. These attachment points have been shown to be made up from 10 or more molecular components, including α6β4 integrins, CD151, and plectin. Thus the hemidesmosomal complex provides robust structural support to the more luminal cells, which are attached to basal cells via desmosomes. The basal cell compartment, although making up less than 1% of the total cell number, constitutes more than 50% of the proliferative potential of the epithelium and as a consequence is thought to harbor a progenitor cell population.9,10 The airway epithelium also hosts a number of nonepithelial, functionally relevant cell types, including pulmonary neuroendocrine cells (PNECs), nerves, and inflammatory/immune cells. Pulmonary neuroendocrine cells (PNECs) exist as either solitary cells or clustered into neuroepithelial bodies (NEBs). These cells are so named due to their dual expression of both neural and endocrine markers such as serotonin and neural peptides, and their expression also appears to be controlled by Notch signaling.13 Pulmonary neuroendocrine cells play an important role in lung development and also function as hypoxia-/ hypercapnia-sensitive chemoreceptors.14 Although PNECs are found in relative abundance in human airways, NEBs are rare.15

CHAPTER 19  Airway Epithelial Cells Many nerves lie adjacent to the airway epithelium, including both sensory afferent and autonomic efferent neurons. These are responsible for transmission of irritant signals as well as release of stimulatory neuropeptides such as calcitonin-gene–related peptide and tachykinins such as substance P and neurokinin A. Inflammatory and immune cells including mast cells, T and B lymphocytes, dendritic cells, macrophages, and innate lymphoid cells often reside within the epithelium. Dendritic cells are crucial in determining the outcome of encounters with inhaled antigens: They integrate signals derived from the antigen with its level of danger and the host environment into a signal that can be translated into an effective immune response.9 Dendritic cells are discussed in more detail later (and in Chapter 14). Innate lymphoid cells are a recently discovered subset of lymphoid cells that secrete cytokines similar to T cells formed during adaptive immune responses.

Epithelial Stem Cells Because of the relative functional specialization of each region, the proximal airways, distal airways, and alveolar regions have distinct resident stem (progenitor) cell populations.16 In rodents, several cell populations within the trachea and bronchi have been reported to be enriched for stem (progenitor) cell activity, including basal cells, Clara cells, cells lining submucosal glands, and NEBs. In the more distal airways, cells residing within niches such as the bronchoalveolar duct junction and more recently parabronchial smooth muscle have been shown to be essential for epithelial repair.10 However, different populations of progenitor cells replenish the airway epithelium under differing conditions. In uninjured and modestly injured airways, single randomly distributed progenitor cells maintain epithelial homeostasis, whereas severe injury results in expansion of large clonal cell patches associated with stem cell niches residing in NEBs and bronchoalveolar duct junctions. Importantly, airway epithelial repair mediated through activation of local stem cells led to loss of progenitor cell diversity.17 In contrast to the degree of understanding in rodents, little is known about resident epithelial stem (progenitor) cells in human airways.18 Cells within the basal cell compartment harbor a progenitor cell population with the capacity to generate a multilayered differentiated epithelium in vitro and in xenograft models in vivo. Using flow cytometry, a “side population” (SP) of airway epithelial progenitor cells has been identified in human airway epithelium.19 As expected for a progenitor, these cells are uncommon, constituting less than 0.1% of the total epithelial cell population, and most do not express hematopoietic lineage markers. However, they do exhibit sustained colony-forming capacity, stable telomere length, and, importantly, the ability to form a multilayered differentiated epithelium in vitro.11 SP cells can be identified in normal airway epithelium but exist in greater numbers in the airways of patients with asthma.

BARRIER FUNCTION OF THE AIRWAY EPITHELIUM Cell-Cell Communication The epithelium constitutes the interface between the external environment and the internal milieu of the lung and as such is the site of first contact with respirable particles, pollutants, respiratory viruses, and airborne allergens. Under normal circumstances, the airway epithelium forms a highly regulated, semipermeable barrier, through the formation of a series of protein complexes: the apical-junctional complex (AJC) composed of tight junctions (TJ), adherens junctions (AJ), and desmosomes.20 TJs are the most apical AJC structure, constitute a semipermeable barrier that regulates paracellular movement of ions and solutes between cells, and physically separate the apical and basolateral membrane domains. Depending on species, more than 40 different proteins have been identified in TJs, although the main components are the

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tetraspannin proteins; occludin; and members of the claudin family, together with the cytoplasmic scaffolding proteins zonula occludens (ZO)-1, -2, and -3; junctional adhesion molecule (JAM)-1; and cingulin.21 In mammals, the claudin family consists of 24 members, which exhibit variable patterns of expression. The extracellular domains of the claudins homotypically interact with those on adjacent cells to form the paracellular seal. The intracellular C-terminus of the claudins also contains binding sites for ZO-1, -2 and -3.22 AJs initiate cell-cell contacts and mediate the maturation and maintenance of the contact and consist of the transmembrane protein E-cadherin and intracellular components p120-catenin, α, β and γ-catenin and α-catenin actinin. Through these interactions, E-cadherin is dynamically coupled to the actin cytoskeleton. The assembly of AJs appears to be required for the formation TJs, because the antibodies that disrupt E-cadherin also block the formation of TJs.22 Desmosomes provide the epithelium with mechanical strength and resistance to shearing forces. Desmosomes are most commonly viewed as dense cytoplasmic plaques composed of a complex of intracellular anchor proteins such as plakoglobin and desmoplakin that are responsible for connecting intermediate filaments such as cytokeratins to transmembrane adhesion proteins.23 Recent studies of AJCs have provided new insights into the molecular mechanisms involved in the generation, function, and maintenance of the epithelium. For example, the formation of TJs and AJs also serves to reinforce polarity of cells by forming an intramembranous diffusion fence preventing the admixing of apical and basolateral membrane proteins. More recently it has been demonstrated that TJs and AJs can regulate epithelial cell proliferation and differentiation through various transcription factors including ZO-1–associated nucleic acid binding protein (ZONAB) and cyclin dependent kinase (CDK)-4.22 After wounding, epithelial cells must reestablish cell-cell contact through proliferation, migration, and differentiation. It is the loss of the AJC along with inflammatory stimuli released from damaged neighboring cells that signals to the structural cytoskeleton of a cell at the leading edge of a wound to protrude lamellipodia and filopodia to migrate and initiate cell-cell contacts. These initial cell-cell contacts are termed spot-like junctions, which are specifically enriched for the AJC proteins, E-cadherin, ZO-1, and JAM-1. These proteins need to be fully associated within the AJC complex to switch off proliferation and migration to allow the cell to polarize and differentiate and reform a patent epithelial barrier. In asthma, the Th2 cytokine profile environment that is commonly observed negatively influences epithelial barrier function, because a significant decrease in expression of ZO-1 and occludin have been shown in epithelial cell lines after exposure to IL-4/IL-13 accompanied by increased permeability.24 House dust mite allergens with proteolytic activity and proteases from pollen have been shown to disrupt epithelial integrity through direct extracellular cleavage of TJ proteins. These proteolytic allergens appear only to affect the paracellular pathways, because they do not cause cell damage or death.25 Similar effects have also been seen after exposure to rhinovirus.26,27 The evidence with other viruses is less conclusive: Influenza infection results in significant barrier damage, particularly in lower airway and alveolar epithelium.28 RSV has been shown paradoxically to increase barrier function in air-liquid interface (ALI) cultures from healthy volunteers and have no effect on ALI cultures from asthmatics.29

Cell–Extracellular Matrix Communication In addition to intercellular adhesion molecules, adhesion to the ECM plays a key role in maintaining a patent mucosal barrier, as well participating in regulating epithelial cell phenotype and reparative potential. The effects of ECM proteins are mediated through specific cell surface

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receptors, the integrins consisting of heterodimeric transmembrane glycoprotein α and β chains, with ligand specificity conveyed through the α chain and signal transduction through the β chain. At the time of writing, 18 α and 8 β subunits with more than 24 integrin combinations have been identified. Epithelial cells express nine separate integrins; two of these, α3β1 and α6β4, recognize the components of the basement membrane and function as specific adhesion molecules. The remaining six recognize ECM proteins that are not normally present in close proximity with these cells. Data from preclinical models of airway disease suggest that integrins expressed on epithelial cells may regulate cellular responses to injury and inflammation. For example, the αvβ6 integrin activates latent TGF-β1 by interacting with the latency-associated peptide which normally sequesters and keeps TGF-β1 inactive.30 Airway epithelial hemidesmosomes morphologically resemble desmosomes and, like desmosomes, connect to intermediate filaments and act as focal points to distribute tensile strength through the epithelium. However, instead of joining adjacent epithelial cells, hemidesmosomes, which are primarily composed of the integrin α6β4, link the basal surface of cells to the laminin-rich ECM.

AIRWAY EPITHELIUM REPAIR PROCESSES In a normal setting, the half-life of an airway epithelial cell in the trachea and lower airways has been estimated to be 6 and 17 months, respectively. Damage to the epithelium caused by noxious agents or aero­ allergens triggers a cascade of inflammatory and cell signaling events that can lead to regeneration or repair of the epithelium. Regeneration is the outcome of processes that returns the tissue to its normal structure and function. By contrast, repair regulates the stability of a tissue, but fails to restore full structural or functional capacity, and in most cases, results in excessive wound healing that ultimately leads to pathologic remodeling and fibrosis. Thus ordered regeneration is critical in maintaining barrier integrity and normal function of the epithelium.31 Irrespective of the anatomic location along the tracheobronchial tree, analogous repair processes are performed after injury. Immediately after insult, an acute inflammatory event is initiated with IL-1β, IL-6, and TNFα production (see Airway Epithelium Immune Responses). Simultaneously, nearby progenitor cells spread to cover the wound within 24 hours, proliferate, and begin to differentiate and effectively cover the wound by 5 days. The temporality of wound repair depends on the degree of insult, with greater insults taking longer to repair. Additionally, damage to pseudostratified airway epithelium may take longer to repair than single cell layers that line terminal bronchi. Early cell spreading and migration to cover the wound is regulated by proteins that modulate the cytoskeleton and actin-myosin complexes, including RhoA and Rac1. The cytoskeleton of the cells binds to focal adhesions that are bound to the extracellular matrix, creating an anchor point for the cells to attach to a pivot from.32 In contrast to isolated wounds, repeated wound/repair cycles facilitate structural changes to the airway epithelium, including goblet cell metaplasia and different secretion of basement membrane proteins by adjacent fibroblasts and myofibroblasts.31 The cell signaling cascades that control epithelial repair processes are not clearly defined, although E-cadherin has been implicated in playing a vital role by regulating β-catenin stabilization, function, and nuclear translocation. In turn, β-catenin is responsible for regulating the differentiation state and proliferation capacity of basal cells involved in the repair process. β-catenin performs this function by acting as a transcription factor that can bind to either CBP or p300, the former skewing basal cell phenotype toward proliferation, whereas the latter skews toward differentiation.33 TGF-β1 is released by airway epithelial cells and modulates not only antiviral innate immune responses, but also cell phenotype in a paracrine/

autocrine manner to facilitate epithelial mesenchymal transition (see later), which can also contribute to wound repair in scenarios of chronic insult. EGF and related ligands are also released by airway epithelium and can mediate migration, proliferation, and spreading of these cells after wounding.32

AIRWAY EPITHELIUM IMMUNE RESPONSES Persistent exposure of the epithelium to the external environment necessitates the airway epithelium to function as a physical barrier but also to react to inhaled agents, communicating and coordinating recruitment and activation of other cells. In this way, the epithelium acts as a bridge between the innate and adaptive immune systems.

Innate Immunity The airway epithelium is clearly well equipped to perform mechanical barrier functions through the mucociliary ladder and TJs to prevent unwanted access to the internal lung. An epithelium with only mechanical barrier function is insufficient to counter the myriad of stimuli inhaled on a daily basis, including particulate matter, allergens, virus, and bacteria. To complement mechanical impedances, the airway epithelium has become equipped with an innate immune component that exploits various pattern recognition receptors (PRRs) that recognize evolutionary conserved pathogen-associated molecular patterns (PAMPs) on inhaled foreign substances and damage-associated molecular patterns (DAMPs) from self. On airway epithelium, like other cells of the body, PRRs are germline encoded, constitutively expressed, and do not rely on immunologic memory.34 The production of antimicrobial peptides by the airway epithelium is in addition to the gel/sol layer that makes up the mucus protecting the airways (Table 19.1).35 Secreted antimicrobials mix with the mucus to create an unfavorable environmental for foreign antigens. Antimicrobials including β-defensins, lactoferrin, lysozyme, and secretory leukocyte protease inhibitor (SLPI) are constitutively expressed and are upregulated in disease states.36 The broad-spectrum nature of these varied compounds allows for chelating key elements required for pathogen survival (lactoferrin), insertion into pathogen membrane for disruption of ionic gradients (β-defensins), enzymatic destruction of pathogen membranes (lysozyme), or protection from pathogenic proteases (SLPI). The airway epithelium may be a source of many more antimicrobial peptides that have yet to be explored37,38 (Fig. 19.2). The PRRs that make up the innate immune system include toll-like receptors (TLRs), nucleotide-binding oligomerization domain containing proteins (NODs), nucleotide-binding domain and leucine-rich repeat containing proteins (NLRs), RNA helicases, and C-type lectin receptors. Each of these categories are expressed in airway epithelium and can be further subdivided into more specific innate immune molecules.34,39 The TLR family was the first group of innate immune receptors to be discovered and have been described in detail elsewhere. The structure of a TLR consists of an extracellular C terminal leucine-rich repeat (LRR) motif attached to an intracellular toll/IL-1 receptor homology domain (TIR). The family of TLRs now includes 11 distinct receptors, although the expression levels on airway epithelium are not fully characterized. TLRs 1, 2, 4, 5, and 6 are expressed with their LRR on the extracellular surface, whereas TLRs 3, 7, 8, and 9 are localized to intracellular vesicles with their LRR on the intravesicle side. Consistent with the IL-1 receptor, most TLRs can activate NF-κB pathways through accessory molecules including MyD88, TIR-associated protein (TIRAP), TIR-domain-containing adaptor protein-inducing IFN-β (TRIF), or TRIF-related adaptor molecule (TRAM), although TLR3 may also signal through interferon regulatory factor 3 (IRF3). The role of these innate PRRs is indispensable

CHAPTER 19  Airway Epithelial Cells

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TABLE 19.1  Antimicrobial Molecules Secreted by Human Airway Epithelial Cells Molecule

Function

Permeabilizing Peptides Defensins (α and β), cathelicidin, bacterial permeability–increasing protein, palate, lung and nasal epithelium–associated proteins (PLUNC)

Peptides with high antimicrobial activity against a broad spectrum of pathogenic agents, including bacteria, fungi, and viruses

Enzymes Complement protein C3a, phospholipases, lysozyme

Antibacterial and antifungal activity

Collectins SP-A, SP-D

C-type lectins are pattern recognition molecules that interact with macrophages, antigen-presenting cells, and T cells to link innate and adaptive immunity

Pentraxins PTX-3, CRP

Recognition, elimination, and/or neutralization of pathogenic agents

Protease Inhibitors SLPI, Elafin, WAPs

Bind and inhibit specific proteases released during inflammation, bind to bacterial lipopolysaccharide, and inhibit viral replication

Small Molecules ROS, NO

Important for airway antibacterial activity and bacterial clearance; prevent growth of bacteria, fungi, and viruses; preserve sterility of secretions

Binding Proteins Mucin, lactoferrin

Scavenge, immobilize and/or kill pathogens

for host protection and development of initial immune responses to viruses (TLRs 2, 3, 4, 7, 8, and 9), bacteria (TLRs 2, 4, 5, and 6), fungi (TLRs 2, 4, and 6), and parasites (TLRs 2, 4, and 9). Clearly, an airway epithelium that is consistently exposed to inhaled pathogens requires the wide array of these receptors, although the precise characterization remains to be determined spatially throughout the lung.34,38 More recently, intracellular pattern recognition receptors have been identified as an additional layer of innate immunity. Nucleotide-binding oligomerization domain–containing proteins 1 and 2 (NOD1 and NOD2) were originally described to respond to distinct molecular patterns within peptidoglycan molecules, a key component in bacterial cell walls. NOD1 and NOD2 are similar in structure to the TLRs, having a LRR, which is attached to a nucleotide-binding domain, and an effector domain, which handles additional protein-protein interactions. NOD1 and NOD2 signal through the adaptor protein, receptor-interacting protein-2 (RIP2), followed by NF-κB activation to induce proinflammatory signaling. NOD1 and NOD2 have kept their original names as literature has evolved, although subsequent discovery of new members has resulted in a nomenclature being defined. The nucleotide-binding domain and leucine-rich repeat containing (NLR) family of proteins covers these intracellular PRRs, including NOD1 and NOD2.40 Unlike NOD1 and NOD2, which have been characterized in airway epithelium, little is known about the expression of the other 18 members of this family. NOD1 and NOD2 are also distinct in that they signal through NF-kB, whereas the remaining NLRs converge on caspase-1, through apoptosis-associated speck-like protein containing a caspase-activation and recruitment domain (ASC), to generate cleaved mature IL-1β or IL-18. The multiprotein intracellular complexes of NLRP3 and caspase-1 are called inflammasomes and facilitate innate immune responses to a variety of stimuli.41 To date, the NLRP3 inflammasome has been extensively characterized in hematopoietic cells and shown to be the intracellular sensor of the adjuvant Alum, uric acid crystals, reactive oxygen species, changes in potassium concentration, nanoparticles, and asbestos fibers. The presence of an NLRP3 in human airway epithelium has been very recently reported.42 Exposure of primary cultures of human airway epithelium to urban particulate matter activated the NLRP3

inflammasome, leading to caspase-1 dependent IL-1β production. Exposure of wild type mice to this same particulate matter induced IL-1β release, neutrophil recruitment, and dendritic cell maturation. In contrast, exposure of NLRP3-deficient mice had no such response, supporting a central role of the epithelial inflammasome in coordinating the innate responses, which may affect downstream adaptive immunity (Fig. 19.3). It remains to be determined whether other members of the NLR family of PRRs are present in the airway epithelium and how they contribute to normal and undesired immune responses such as virus-induced airway disease exacerbations. RNA helicases represent another arm of the innate immune system important for airway epithelium responses to external insults.43 The RNA helicase, retinoic acid–inducible protein I (RIG-I), has been shown to recognize intracellular dsRNA that, due to its location, cannot be processed by endosomal TLRs. RIG-I integrates the signal from cytoplasmic viral dsRNA through adaptor proteins resulting in NF-κB activation. Infections of the airway epithelium by clinically relevant RNA viruses including influenza demonstrate the need to better understand the role of RIG-I and other RNA helicases, because these molecules may prove to be an important step in immune recognition and downstream inflammatory signaling.44 The C-type lectin receptors have also been poorly explored in the airway epithelium. Few reports exist about the presence of dectin-1 and dectin-2 functioning in response to fungal and bacterial infections.45 More work remains to be performed on this area, in light of airway diseases that include colonization of airways by fungus and bacteria (e.g., cystic fibrosis [Aspergillus fumigatus], chronic obstructive pulmonary disease [nontypeable Haemophilus influenzae]). In addition to responding to external environmental signals via innate receptors, airway epithelial cells may also be a source of DAMP signals that can affect innate and adaptive immune responses. Specifically, human airway epithelial cells have been reported to release a number of alarmins including IL-25, TSLP, IL-33, ATP, HMGB1,46 dsDNA and mitochondrial DNA,47 galectin-3,48 and uric acid.49 The release of these diverse molecular signals can occur actively via including channels and transporters or, alternatively, via cell death pathways. However, the

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SECTION A  Basic Sciences Underlying Allergy and Immunology Innate Immune Receptors

Ctype lectin

Innate Immune System Downstream NF-κB Signaling Cascade LPS

TNFR

TLRs

TLR4

IRAK-4 TLRs

IRAK-1

MYD88 TLR9

IRAK-1

dsRNA Downstream Endosome/ signaling lysosome (see right panel zoom)

TRAF6

TRAF6

RIP2

Kinase signaling

NODs

PG NOD1

Phosphorylation P

Nucleus NLRP3

RIG-I

IκB

Ubiquination

NF-κB

IκB Degredation

Nuclear translocation

NF-κB

Transcription

Fig. 19.2  Innate immune system responses of the airway epithelium include localization of innate immune receptors (left) and the downstream nuclear factor-κB (NF-κB) signaling cascade (right). dsRNA, Double-stranded RNA; IκB, inhibitor of kappa B; IRAK-1/4, interleukin-1 receptor–associated kinase 1/4; MYD88, myeloid differentiation primary response gene 88; NLRP3, nucleotide-binding leucine-rich repeat-containing protein 3; NOD, nucleotide-binding oligomerization domain proteins; PG, peptidoglycan; RIG-I, retinoic acid–inducible genelike protein encoded by the DDX58 gene; RIP2, receptor-interacting protein 2; TLR, toll-like receptor; TNFR, tumor necrosis factor-α receptor; TRAF6, TNF receptor–associated factor 6.

end result of the immune response induced by DAMPs is dependent on the effector immune cells present and the Th1, Th2, and Th17 cytokine environment at the site of inflammation (see Influence on Adaptive Immunity).

Influence on Adaptive Immunity Despite the strengths of a tight mechanical barrier and an evolutionary conserved innate immune system, the airway epithelium has additional weaponry to orchestrate immune responses. Recognition of ligands at innate immune receptors results in pathogen-specific signaling pathways, de novo gene expression, and protein production. The result is secretion of cytokines, chemokines, and additional growth factors to recruit and activate cells of the adaptive immune system. Specifically, the airway epithelium can influence T cells, B cells, dendritic cells, innate lymphoid cells, eosinophils, mast cells, and neutrophils; essentially all types of cells from the immune system that can lead to adaptive immune responses. The airway epithelium therefore lies at the nexus between linking innate and adaptive immune responses37 (Fig. 19.4).

Airway Epithelial Cell–Lymphocyte Crosstalk.  The airway epithelium is a source of chemokines and cytokines for Th1, Th2, and Th17 cell recruitment and activation.50,51 Th1 responses are observed

in response to intracellular pathogen exposure, including bacteria and viruses, and are dominated by interferon signaling. The airway epithelium can recruit Th1 cells through CXCL9 (monokine-induced by gamma interferon [MIG]), CXCL10 (interferon gamma–induced protein [IP-10]), and CXCL11 (interferon-inducible T-cell alpha attractant [ITAC]) chemokines, which bind to CXC class chemokine receptors. Airway epithelial production of interferon-β subsequently helps polarize newly recruited Th1 cells. Recently airway epithelium production of CXCL16 has been identified after IFN-β exposure, contributing to maintenance of a Th1 inflammatory profile. Recruitment of lymphocytes into the airway epithelium occurs via mucosal microvascular beds. The airway epithelium can express four members of the B7 family of T cell costimulatory molecules that facilitate ligand-specific interactions on T cells to induce activation or inhibition. The B7-H1 and B7-DC members are ligands for PD-1, which produces an inhibitory signal in T cells. In contrast, B7-H2 and B7-H3 activate memory T cells and CD4+/CD8+ cells, respectively. Upregulation of the B7 family members and intercellular adhesion molecule-1 (ICAM-1/ CD54) can occur after virus infection, suggesting that the airway epithelium contributes to appropriate virus responses by helping orchestrate T cell activation and recruitment. Furthermore, lymphocyte expression of CD103 can interact with epithelial e-cadherin, leading to recruitment

CHAPTER 19  Airway Epithelial Cells

Particulate matter

Dendritic cell maturation

Virus

Crystals

ASC

NLRP3

ProPro-

Inflammatory signaling

Caspase 1

IL-1β IL-18

IL-1β IL-18

IL-1β IL-18 MYD88

GM-CSF CCL-20 TSLP??? IL-33???

293

NF-κB

IL-1RI IL-1RAcP

Translation

Transcription

Fig. 19.3  Proposed model for airway epithelial cell NLRP3 inflammasome activation and downstream activation of adaptive immune responses. ASC, Caspase activation and recruitment domain; GM-CSF, granulocytemacrophage colony-stimulating factor; IL, interleukin; MYD88, myeloid differentiation primary response gene 88; NF-κB, nuclear factor-κB; NLRP3, nucleotide-binding leucine-rich repeat-containing protein 3; TSLP, thymic stromal lymphopoietin.

IL-6 CCL3 CXCL8

TSLP GM-CSF IL-15 IL-33 CCL20

INF-γ CXCL9 CXCL10 CXCL11

CCL1 CCL17 CCL22 IL-33 IL-β IL-4 IL-11

Neutrophil Dendritic cell

IL-β IL-6?? TGF-β??

Th17 cell

Th1 cell

BAFF APRIL

B cell

Th2 cell Fig. 19.4  Interaction between airway epithelial cell–derived cytokines and inflammatory cells. APRIL, A proliferation-inducing ligand; BAFF, B cell–activating factor of the TNF family; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN-γ, interferon-γ; IL, interleukin; TGF-β, transforming growth factor-β; Th, T helper cell subset; TSLP, thymic stromal lymphopoietin.

and retention of the latter cells within the mucosal barrier. Interactions between airway epithelial cells and T cells through E-selectin are less clear, and vascular adhesion molecule-1 (VCAM-1/CD106) is not expressed on airway epithelial cells.37 Th2 responses are observed in allergic diseases and may be perpetuated by recruitment of this class

of cells through CCL1, CCL17 (thymus and activation-regulated chemokine [TARC]), and CCL22 (macrophage derived chemokine [MDC]) chemokine secretion by the airway epithelium and binding to CC class chemokine receptors on T cells. Secretion of IL-4, IL-13, IL-33, and TSLP cytokines by the airway epithelium into the extracellular milieu creates a Th2 environment for newly recruited T cells and resident innate lymphoid type 2 cells.52 Recruited T cells and resident innate lymphoid type 2 cells are capable of secreting Th2 cytokines (IL-5/ IL-13) that promote allergic airway inflammation in asthma.53,54 Th17 lymphocytes represent a category of effector T cell that varies in cytokine production relative to the traditional Th2 lymphocytes found in allergic asthma. Appearance of these cells requires TGF-β1 and IL-6 and are differentiated from other T lymphocytes by expression of the Th17-specific transcription factor, RORγT, and release of IL-17, which upregulates epithelial expression of CXCR2 chemokines from the epithelium,55 thereby amplifying and perpetuating leukocyte recruitment to the airways. In addition to influencing T cell responses, the airway epithelium can directly influence B cell responses, including antibody production. Although the main inducer of Ig-class switching in B lymphocytes is interaction with T cells, recent evidence suggests that TLR3 ligands, interferons, and TNF-α can induce airway epithelial cells to produce the B cell–inducing molecule, B cell–activating factor of the TNF family (BAFF), and a proliferation-inducing ligand (APRIL).80 BAFF and APRIL can affect B cell maturation, survival, and proliferation and ultimately lead to the production of IgG and IgA. Epithelial cells also facilitate the transport of B cell–produced immunoglobulins IgM and IgA to the mucosal surface via the polymeric Ig receptor.56

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Airway Epithelial Cell–Dendritic Cell Crosstalk.  Studies have emerged linking innate pattern recognition by the airway epithelium with adaptive immune responses through recruitment of dendritic cells. The flattened cell bodies of dendritic cells are located on or underneath the basal lamina and extend long processes apically through the extracellular matrix, between epithelial cells, and into the airway lumen. Upon encountering antigen, dendritic cells process antigen and display peptide fragments on MHC molecules on the cell surface and upon trafficking to lymph nodes will display this antigen to other immune cells such as T and B lymphocytes. In doing so, the airway epithelium facilitates the ability of the immune system to remain cognizant of the external environment. Epithelial-derived GM-CSF, CCL-20, IL-33, and TSLP coupled with antigenic stimulation are capable of initiating both innate and adaptive immune responses resulting in airway inflammation and an allergic reaction.57 Airway epithelium production of GM-CSF, CCL-20, IL-33, and TSLP seems to be tightly linked to IL-1β signaling through NF-κB pathways. In vitro studies exposing airway epithelium to recombinant IL-1β or stimulants that induce endogenous IL-1β production (urban particulate matter, house dust mite) both result in production of GM-CSF, CCL-20, IL-33, and TSLP.58 These cytokines and chemokines can bind to their receptors on immature dendritic cells59 and, in the presence of antigen, lead to activation and effective antigen presentation to T cells. Of these molecules, TSLP has received considerable interest as it interacts with its receptor on DCs by upregulating CD40, OX40, and CD80, thereby enhancing Th2 polarization in addition to its ability to directly activate mast cells for cytokine secretion (see Epithelium–Mast Cell Interactions).60 Tying structure to function further, disruption of E-cadherin leads to release of TSLP and CCL17 in an epidermal growth factor receptor (EGFR)–dependent manner. Furthermore, loss of E-cadherin may affect a subset of epithelial cells that express CD103, which bind to E-cadherin to localize to the epithelium.61 Concomitant with antigen acquisition in the airway mucosa, DCs are particularly receptive to local signals derived from epithelial cells, given the close proximity of these two cell types. Epithelial cells express a variety of adhesion molecules and are a rich source of mediators (including TGF-β1, IL-10, PGE2, chemokines, NO) through which they are likely to modulate DC function within the airway. Consequently, examination of these regulatory pathways in the normal epithelium, and how regulation is disturbed in asthma, will be highly relevant to understanding immune responses to inhaled antigens or allergens and the pathogenesis of asthma. The extent of crosstalk between epithelial and dendritic cells has been most closely examined in the skin and gastrointestinal tract. There is strong evidence from the dermatology literature that epithelial-derived factors modulate the differentiation and function of dermal DC and the specialized epidermal DC, the Langerhans cell. Differentiation of Langerhans cells appears to be TGF-β1 dependent, because they are absent from the epidermis in TGF-β knockout mice. Moreover, blood-derived DC precursors give rise to typical epidermal Langerhans cells when cocultured with normal human keratinocytes, whereas ligation of E-cadherin actively suppresses their maturation. In the gut, dendritic cells are able to open the tight junction proteins between enterocytes, extending their processes between adjacent epithelial cells to sample bacteria from the gut lumen without compromising the integrity of the epithelial barrier. Formation of these transepithelial dendrites is dependent on the chemokine receptor CX3CR1 (fractalkine receptor) expressed on DCs.62,63 Environmental particulates containing fungal and microorganism fragments may also influence the epithelial-dendritic cell interaction by acting through PRRs, including TLRs, on dendritic cells

to enhance and direct their response by also serving as “danger” signals.

Airway Epithelial Cell–Eosinophil and Mast Cell Crosstalk.  In normal individuals without airway disease there are relatively few eosinophils within the lung. Mast cells are likely present in greater number, but both are found in increased numbers in the airways of asthmatics and are often intimately associated with the airway epithelium. As effector cells, eosinophils and mast cells are capable of producing Th2 and other cytokines, chemokines, lipid mediators, and growth factors and are also capable of causing an increase in mucus production from the airway epithelium, which is facilitated by their close proximity to each other and expression of required receptors. It is now appreciated that eosinophils and mast cells work in tandem with resident innate lymphoid type 2 cells to promote Th2 inflammation.54 Eosinophilderived granule proteins such as major basic protein and eosinophil cationic protein have been the focus of attention since they have been shown to directly induce induced epithelial damage, which may precede the development of subepithelial fibrosis. In addition, eosinophils contain a substantial amount of oxidases (threefold to fourfold more than neutrophils) as part of the respiratory burst response and all of which have direct cytotoxic potential, which could damage the airway epithelium.64 The recruitment of eosinophils to the airways is regulated by the chemokines eotaxin 1 (CCL11), 2 (CCL24), 3 (CCL26), and RANTES (CCL5) that are released primarily from the epithelium. CCL11 is able to activate eosinophils directly by inducing respiratory burst and actin polymerization. Recent data suggest that epithelial differentiation is an important determinant in Th2 cytokine-induced CCL24 and CCL26 release by airway epithelial cells. The precise contribution of eosinophils to the pathogenesis of asthma is still being revealed, but clearly the communication between the epithelium and this cell exists and remains a promising therapeutic target at least for specific endotypes of asthma.65 Biopsy studies have also shown increased numbers of mast cells residing in close proximity to the epithelium in asthma. Under normal conditions the epithelium inhibits mast cell degranulation and activation, through the release of PGE2, plasminogen activator inhibitor 1, and lipoxins.66 However, this inhibitory effect is compromised in asthma and the release of granule proteins; in particular, chymase and tryptase may then directly disrupt epithelial cell junctions. Additionally, TSLP production from the airway epithelium may mediate direct mast cell activation resulting in the release of proinflammatory cytokines and lipid mediators. How mast cells traffic to the lungs and airway epithelium is still not well understood. Mast cells express the chemokine receptors CCR3 and CXCR3, suggesting they can respond to both Th1 and Th2 chemokines produced by airway epithelium. Further investigation of chemokine receptor expression on both mast cells and basophils in asthma will determine which chemokine ligand/receptor axis might provide a suitable therapeutic target.67

Airway Epithelial Cell–Neutrophil Crosstalk.  Although not classically considered part of the adaptive immune system, the airway neutrophil mediates early acute responses to inhaled antigens in the airway, being the first cell recruited after insult. The airway epithelium is the major source for the release of interleukin-8 (CXCL-8), a potent neutrophil chemokine and activator, although other cells including endothelial cells, fibroblasts, macrophages, and neutrophils themselves can produce this mediator. Damage to the airway epithelium, which may occur in asthma in response to allergen inhalation, mast cell degranulation, and eosinophil degranulation, results in release of CXCL-8 that then binds to CXCR1 and CXCR2 receptors on neutrophils. Consistent with this mechanism, neutrophils have been demonstrated in

CHAPTER 19  Airway Epithelial Cells the airways of asthmatic subjects, especially in the presence of severe disease with severe airflow obstruction,68 although recent evidence suggests that intraepithelial neutrophils may be protective in pediatric severe asthma.69

THE AIRWAY EPITHELIUM IN ASTHMA Genetic and Epigenetic Factors Although alterations in the adaptive immune response are undoubtedly contributing to asthma, increasing evidence suggests that immune dysfunction may be downstream of, or at least parallel to, the initiating events that occur at the epithelial-environmental interface (Fig. 19.1); indeed, the importance of tissue-specific mechanisms that regulate the expression of allergic inflammation has been highlighted by data showing that although atopy affects up to half of the adult population, the vast majority of atopic subjects do not develop asthma. Large genome-wide association studies (GWAS) have reproducibly identified a number of new candidate loci, including DENND1B, CHI3L1, IL1RL1/IL18R1, PDE4D, TSLP, RAD50, IL33, SMAD3 and ORMDL3/GSDM A/B, DPP10, and CDHR3. Interestingly, little overlap was found among the top loci associated with asthma and those that regulated total serum IgE levels, indicating that the two traits are inherited independently.70,71 That many of these susceptibility genes are expressed predominantly in the epithelium suggests that asthma is dependent on epithelial dysfunction in susceptible individuals. Although GWAS and related studies provide key data on diseaseassociated genes, epigenetic regulation is becoming rapidly recognized as an important mechanism through which the environment influences gene expression. Epigenetics refers to the regulation of gene expression that are not dependent on alterations in gene sequence and include direct DNA methylation, histone modifications, and regulation by noncoding RNAs such as long noncoding RNA and microRNA.72 Studies examining epigenetic control of epithelial function in asthma are increasing in number, and gene-specific epigenetic patterns are emerging, although the questions of how, when, and why remain. For example, Nicomedius-Johnson and colleagues showed that a single 48-hour exposure to IL-13 induced substantial changes to the DNA methylation of more than 6500 CpG sites in cultured AECs.73 Intriguingly, hypermethylation was primarily seen adjacent to purported asthma susceptibility genes and importantly more than 75% of these changes were seen in AECs obtained from asthmatics. Clifford and colleagues74 showed that although a single exposure to allergen or diesel or a combination had a modest impact on epithelial DNA methylation, sequential exposures, even when separated by 4 weeks, had a substantial impact on global methylation. Although these examples suggest that DNA methylation is altered in asthma, its role in asthma pathogenesis is undoubtedly fluid and complex. Even less is known about histone modifications and their downstream impact. Expression of HDAC 1 and 9 is higher in epithelium of asthmatics, whereas HDAC 1 and 2 expression is lower.75 Stefanowicz and colleagues showed that several histone-modifying enzymes are differentially expressed in the epithelium of asthmatics compared with healthy controls,76 including an increase in the levels of histone H3 lysine 18 (H3K18) acetylation.77 This epigenetic mark enhances the expression of the transcription factors ΔNp63 and STAT6, as well as EGFR, all of which are dysregulated in the epithelium of asthmatics.77 Another form of epigenetic regulation of gene expression is mediated through noncoding RNAs (lnc-RNA, miRNA, piRNA). To date, most studies of the role of noncoding RNAs have focused on micro­ RNAs,78 and the few array-based studies to examine miRNA on a global level have shown that dramatic differences of airway epithelial cell miRNA levels are a common feature of asthma and that this signature

295

is only modestly corrected by inhaled corticosteroids.79 At a cellular level, dysregulated epithelial proliferation and wound repair are hallmarks of severe asthma, and multiple miRNAs have been associated with these processes. For example, miR-19a, which directly targets TGFβR2, is upregulated in the bronchial epithelium of severe asthmatics and also enhances proliferation.44 A number of miRNAs such as miR-44980 have been shown to play crucial roles in normal cell fate decisions and, although likely to be important in asthma, have not been examined in this context. Examination of lncRNA expression and its impact in the epithelium of asthmatics has been slow, although the cystic fibrosis (CF) airway epithelium shows clear lncRNA expression abnormalities.81 These epigenetic pathways are clearly involved in regulating responses to therapeutic treatment. As an example, the glucocorticoid receptor cannot function at full capacity in an environment where HDAC2 expression is limited.82 The acetylation status of histone 4 (H4) is also important in inflammation and glucocorticoid action. NF-κB induces H4 acetylation at K8 and K12 at the granulocyte macrophage colonystimulating factor (GM-CSF) promoter resulting in gene expression, whereas glucocorticoids not only reduce the expression of NF-κB–induced GM-CSF and inhibit histone acetylation (including K8 and K12) at the GM-CSF promoter but also recruit HDAC2 to the site, resulting in deacetylation of lysine residues.83

Morphologic and Structural Changes of the Epithelium Transmission electron microscope studies conducted in the early 1990s demonstrated that the airway epithelium in patients with asthma have fewer intracellular adhesion complexes compared with nonasthmatics. However, the molecular mechanisms were not described. It has taken nearly 25 years to demonstrate that E-cadherin and ZO-1, components of the APC, are altered in asthmatic airways. However, at a gross level, the composition of the asthmatic airway epithelium is different from that of nonasthmatics (Fig. 19.5). For example, goblet cell hyperplasia and excessive mucus production are common features of asthma and contribute significantly to morbidity and mortality.84 Recent studies have shown that IL-13 and EGFR-dependent expression of Forkhead transcription factors Fox-A2 and Fox-A3 is a central step in epithelial cell mucin production in asthma. Another potential mediator of this process is the SAM-pointed domain-containing ETS transcription factor (SPDEF), which in mice induces the differentiation of club cells into goblet cells in response to house dust mite allergen or IL-13.85 One of the targets for this transcription factor is Foxj1, a master regulator of ciliated cell differentiation. Recent evidence has also shown that the epithelium-specific thyroid transcription factor 1 (alternatively known as NK2 homeobox 1; NKX2.1) is a key negative regulator of SPDEF and mucus production. Interestingly, expression of NKX2.1 is reduced in the airway epithelium of asthmatics.84 It is now well accepted that the asthmatic epithelium is fragile and displays a loss of columnar, ciliated cells, and this is observed both in adults and children. This is coincident with reduced expression of AJC proteins such as E-cadherin and ZO-1 and, importantly, this is observed both in adults and children. Epithelial damage is not only confined to the lower airways, because disrupted desmosome formation has also been shown in nasal polyps from asthmatic children. The asthmatic epithelium also displays dyssynchronous proliferation after injury. However, somewhat surprisingly, asthmatic epithelial cells display attenuated wound repair in vitro, indicating an intrinsic inability to initiate repair processes in a constantly damaged epithelium. Similarly, expression of the adhesion molecule CD44 is also upregulated on basal cell membranes in asthmatic subjects and in areas of localized cell damage. In the asthmatic epithelium, expression of the EGFR is markedly increased, especially in areas where columnar cells have been shed.86

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SECTION A  Basic Sciences Underlying Allergy and Immunology

EGFR

IFN-γ IFN-β

Nucleolin

IFN-γ IFN-β

EGFR Nucleolin Nucleolin EGFR Nucleolin Nucleolin EGFR

pediatric and adult asthmatic patients.88 Thus the available evidence suggests that the asthmatic epithelium is fragile and has an altered phenotype of epithelial cells present, which could occur because of excessive damage or a compromised ability to differentiate. IL-6 PGE2

p38 ERK1/2

Normal airway

Asthmatic airway

Fig. 19.5  Intrinsic differences between nonasthmatic and asthmatic airway epithelium. The asthmatic epithelium is susceptible to virus infection, which may be associated with a deficient interferon γ and β response. Additionally, the receptor for respiratory syncytial virus, nucleolin, may be upregulated on airway epithelium from asthmatics, facilitating greater infection. The airway epithelium from asthmatics is also more fragile because of the reduced expression of junctional proteins. Lastly, an altered inflammatory profile is observed in asthmatics with increased IL-6 and PGE2 observed, which is accompanied by goblet cell metaplasia and increased basement membrane thickening. EGFR, Epidermal growth factor receptor; ERK, extracellular signal–regulated kinase.

This is seen in both adults and children with moderate to severe asthma, although its ligands were not found to be upregulated under nonstimulated conditions. Furthermore, increased expression of EGFR in asthmatics occurs even in areas of intact epithelium and does not correlate with the proliferative response of repairing cells. The expression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 is also increased in the asthmatic epithelium, which may explain the apparent discrepancy between EGFR levels and proliferative response. Exposure of juvenile nonhuman primates to house dust mite allergen with or without ozone induced an asthma-like remodeling of the EMTU.6 These remodeling features include reduced airway number, epithelial cell hyperplasia, an increase in mucus-secreting cells, airway hyperresponsiveness, interrupted postnatal basement membrane zone differentiation, reorganization of the airway vasculature, a modified epithelial distribution of nerves and Th2 skewing of the immune system, providing further evidence that the EMTU either remains active after birth or can become reactivated in a disease such as asthma.6 These morphologic changes are also mirrored at the molecular level in cultured asthmatic epithelial cells, which are more susceptible to oxidant-induced “stress” and demonstrates abnormal expression of proinflammatory transcription factors as well as heat shock proteins. Normal differentiation also appears to be compromised. In freshly isolated bronchial brushings obtained from children with asthma, decreased protein expression of the differentiated epithelial cell marker ck-19 has been observed, together with an increase in the number of cells expressing basal cell markers ck-5 and p63.87 Importantly, this phenotype is maintained over repeated passage, suggesting it is an intrinsic feature. Similar observations have been made using airway sections from both

Functional Changes Although defective tight junction expression leads undoubtedly to compromised barrier function (Fig. 19.6),89 the epithelial cell defects in asthma extend far beyond their physical barrier function. For example, epithelial cells isolated from specific asthmatic phenotypes have a deficient innate antiviral immune response after rhinovirus (RV) infection.3,90 Epithelial cells from asthmatics also undergo necrosis rather than apoptosis in response to IFN-β, which results in greater viral loads. Supplemental IFN-β rescues the apoptosis response to RV and viral loads decrease to normal levels upon exogenous IFN-β treatment.91 The induction of IFN-λ by RV infection is also deficient in the airway epithelial cells of asthmatics, and the extent of the deficiency is highly correlated with the severity of RV-induced asthma exacerbations. However, the mechanism underlying this remains unknown. A comprehensive and complete survey of IFNs produced by virus-infected normal and asthmatic bronchial epithelial cells (BECs) has not been performed to date, but given that it appears that BECs from asthmatic children are characterized by greater expression of types I and III interferons and interferon-stimulated genes than BECs from healthy children in response to RSV,92 it is urgently required. Epithelial cells obtained from asthmatic children also have aberrant cytokine profiles relative to cells from healthy subjects93 and lipid mediators compared with nonasthmatic patients. These mediators are also increased by mechanical stress, injury, virus infection, or interaction with inflammatory cells such as neutrophils and eosinophils, all of which can be abundant in asthma.94

Changes in Epithelial Cell Crosstalk Restitution of damaged epithelium is associated with alterations in cell phenotype as well as disruption of the normal patterns of proliferation and apoptosis, reflecting inflammation and repair processes. An emerging and exciting concept of cell jamming has been recently put forward.95 In this context, cell “jamming” is associated with a mature and quiescent cell layer, with intact barrier function. In contrast, the unjammed phase is associated with an immature and more plastic cell layer. In in vitro experiments at least, it appears that the transition to jamming is delayed in cells derived from asthmatic donors. These processes are spatially and temporally controlled by local signals generated by a plethora of growth factors and integrins. The description of pores in the basement membrane of the airway point to new insights regarding the possibilities of epithelial cell–mesenchymal cell interactions.96,97 It is also likely that altered growth factor production by repairing epithelial cells induces proliferation and activation of subepithelial fibroblasts and their differentiation into myofibroblasts.98 Myofibroblasts, which are characterized by expression of contractile proteins such as α-smooth muscle actin (α-SMA), are centrally involved in wound healing and remodeling, through the release of growth factors that drive autocrine proliferation and deposition of extracellular matrix proteins, as well as influencing epithelial function. Several studies have reported a correlation between myofibroblast numbers and the thickened lamina reticularis characteristic of asthma, although it is unclear whether the myofibroblasts themselves are hyperproliferative.99 The communication in the EMTU is aberrant in asthma, showing reactivation associated with airway remodeling. Although the EMTU interacts with cells and mediators associated with inflammation, many of which are corticosteroidsensitive, processes involved in driving the EMTU assume a more active role in driving remodeling particularly in severe and chronic disease.100 The significance of this is highlighted, showing that these processes are

CHAPTER 19  Airway Epithelial Cells Nonatopic normal

297

Atopic asthma

XY

ZO-1

37.5 µm

37.5 µm

XZ ZO-1 Nuclei 25 µm

25 µm

Fig. 19.6  Confocal immunofluorescent microscopic view of a bronchial biopsy from a normal airway (left) and one from active asthma (right) to show the breakdown in tight junctions (TJ) in asthma as shown by immunostaining with a monoclonal antibody recognizing ZO-1, an integral component of TJs. Top, planar view; bottom, cross-section (XZ) view. (Figure courtesy, Professor Stephen Holgate.)

not influenced by corticosteroids and are likely to be central to the long-term structural changes within the airways and as a consequence, accelerated decline in airflow obstruction.

extent to which differentiation programs are inhibited or dysregulated and its impact on epithelial function is the subject of growing investigation.104

Epithelial–Mesenchymal Transition (EMT)

INFLUENCE OF ASTHMA MEDICATIONS ON THE ASTHMATIC EPITHELIUM

In asthma, increased deposition of collagen, fibronectin, and other ECM proteins in the basement membrane contribute to subepithelial fibrosis and airway hyperresponsiveness. The most likely cells responsible for the increased deposition of ECM are fibroblasts and myofibroblasts; the number of these cells correlates with the magnitude of subepithelial thickening. However, the exact mechanisms for the expansion of the mesenchymal cell population are unknown, although expansion of the resident fibroblast population, recruitment of fibrocytes from the circulation and more recently the smooth muscle bundle101 have both been implicated. Epithelial cells undergoing molecular reprogramming associated with EMT have been shown to play a prominent role in fibrogenesis in several tissues such as the lung, liver, and kidney, contributing more than one-third of the interstitial fibroblast population.102 With respect to the airway epithelium, EMT-like responses have been shown in transformed airway epithelial cells in response to BMP-4 or TGF-β. Interestingly, in this latter study, the effect of TGF-β was enhanced by the addition of house dust mite allergen. This is likely related to the TGF-β1-facilitated uncoupling of EGFR from E-cadherin, leading to prolonged EGFR signaling. EMT has been reported in the small airways of clinically stable lung transplant recipients and those with posttransplant bronchiolitis obliterans syndrome as well as in mice in response to bleomycin, TGF-β, or house dust mite antigen. EMT has also been shown to occur in epithelial cells obtained from asthmatics.103 This process was shown to be Smad 3–dependent and reversible. In differentiated, multilayered air-liquid interface cultures generated from both asthmatic and nonasthmatic donors, EMT was only observed in basal cells expressing ck-5 and p63. Intriguingly, there were significantly greater numbers of ck-5+/p63+ basal cells in asthmatic epithelium. Although it is becoming increasingly clear that the asthmatic airway epithelium displays an altered phenotype, the

Although it seems logical that the epithelium would yield appropriate molecules for therapeutic targeting, only a limited number of studies have evaluated the effects of current asthma therapies on epithelial function.105 For example, although β2-adrenoceptor agonists and phosphodiesterase inhibitors impair acute healing, they have little effect on repair of chronically damaged epithelium in vitro.106 The effects of glucocorticoids (GCS), however, are controversial. Chronic treatment with dexamethasone impairs migration and repair in wounded differentiated bronchial epithelial cell cultures but is able to extend the lifespan of these cells, ultimately enhancing reparative potential. In contrast, other studies have shown that exposure to three different steroids (dexamethasone, beclomethasone or budesonide) produced a time- and concentration-dependent cell death in primary cultures and transformed epithelial cell lines.107 The impact of combination β2-agonist/ GCS treatment on epithelial cells suggests that these two asthma medications synergize to induce antiinflammatory responses that can be further modulated by mechanisms that increase intracellular cAMP level.8,108 Interestingly, treatment with the short-acting β2-agonist albuterol inhibited dexamethasone-induced apoptosis completely but did not inhibit apoptosis induced by Fas receptor activation. Although the effect of corticosteroids on the bronchial epithelial cell barrier has not been studied, in cells of the intestine, GCS treatment has been shown to induce retightening of tight junction defects in patients with Crohn disease. However, there was no effect on basal barrier function, suggesting that the usefulness of GCS in the restoration of epithelial barrier function in patients with asthma may be limited. However, the concept that GCSs spare or enhance innate immunity while suppressing adaptive immunity is gaining momentum.109

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SUMMARY Apart from serving as a physical barrier, the airway epithelium is now known as a dynamic structure playing a pivotal role in controlling many airway functions and intricately involved in airway homeostasis and disease. There is convincing and consistent genetic and genomic data from children and adults that the airway epithelium of asthmatics is intrinsically abnormal and this imparts dysregulated structure and function phenotype. As a consequence, a deranged epithelium will have substantial effects on airway inflammation, immunity, and remodeling in response to common inhaled stimuli. This places the epithelium at the forefront of asthma pathogenesis, and thus understanding the mechanisms that underlie these abnormalities will no doubt have both short- and long-term clinical significance for the treatment of this disease.

Acknowledgments This work is funded by Canadian Institute of Health Research (CIHR) and The National Health and Medical Research Council (NHMRC).

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CHAPTER 19  Airway Epithelial Cells 41. Franchi L, Eigenbrod T, Munoz-Planillo R, et al. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis. Nat Immunol 2009;10(3):241–7. 42. Hirota JA, Hirota SA, Warner SM, et al. The airway epithelium NLRP3 inflammasome is activated by urban particulate matter. J Allergy Clin Immunol 2012;129:1116–25. 43. Slater L, Bartlett NW, Haas JJ, et al. Co-ordinated role of TLR3, RIG-I and MDA5 in the innate response to rhinovirus in bronchial epithelium. PLoS Pathog 2010;6(11):e1001178. 44. Yu M, Levine SJ. Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses. Cytokine Growth Factor Rev 2011;22(2):63–72. 45. Heyl KA, Klassert TE, Heinrich A, et al. Dectin-1 is expressed in human lung and mediates the proinflammatory immune response to nontypeable Haemophilus influenzae. MBio 2014;5(5):e01492-14. 46. Mitchell PD, O’Byrne PM. Biologics and the lung: TSLP and other epithelial cell-derived cytokines in asthma. Pharmacol Ther 2017;169:104–12. 47. Pouwels SD, Zijlstra GJ, van der Toorn M, et al. Cigarette smoke-induced necroptosis and DAMP release trigger neutrophilic airway inflammation in mice. Am J Physiol Lung Cell Mol Physiol 2016;310(4):L377–86. 48. Pouwels SD, Hesse L, Faiz A, et al. Susceptibility for cigarette smoke-induced DAMP release and DAMP-induced inflammation in COPD. Am J Physiol Lung Cell Mol Physiol 2016;311(5):L881–92. 49. Gold MJ, Hiebert PR, Park HY, et al. Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization. Mucosal Immunol 2016;9(3):809–20. 50. Robinson DS. The role of the T cell in asthma. J Allergy Clin Immunol 2010;126(6):1081–91, quiz 92-3. 51. Witherden DA, Havran WL. Molecular aspects of epithelial gammadelta T cell regulation. Trends Immunol 2011;32(6):265–71. 52. Gordon ED, Simpson LJ, Rios CL, et al. Alternative splicing of interleukin-33 and type 2 inflammation in asthma. Proc Natl Acad Sci USA 2016;113(31):8765–70. 53. Ealey KN, Moro K, Koyasu S. Are ILC2s Jekyll and Hyde in airway inflammation? Immunol Rev 2017;278(1):207–18. 54. Dunican EM, Fahy JV. The role of type 2 inflammation in the pathogenesis of asthma exacerbations. Ann Am Thorac Soc. 2015;12(Suppl. 2):S144–9. 55. Traves SL, Donnelly LE. Th17 cells in airway diseases. Curr Mol Med 2008;8(5):416–26. 56. Xu W, He B, Chiu A, et al. Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI. Nat Immunol 2007;8(3):294–303. 57. Hammad H, Lambrecht BN. Dendritic cells and airway epithelial cells at the interface between innate and adaptive immune responses. Allergy 2011;66(5):579–87. 58. Lee HC, Ziegler SF. Inducible expression of the proallergic cytokine thymic stromal lymphopoietin in airway epithelial cells is controlled by NFkappaB. Proc Natl Acad Sci USA 2007;104(3):914–19. 59. Kallal LE, Schaller MA, Lindell DM, et al. CCL20/CCR6 blockade enhances immunity to RSV by impairing recruitment of DC. Eur J Immunol 2010;40(4):1042–52. 60. Allakhverdi Z, Comeau MR, Jessup HK, et al. Thymic stromal lymphopoietin is released by human epithelial cells in response to microbes, trauma, or inflammation and potently activates mast cells. J Exp Med 2007;204(2):253–8. 61. Sung SS, Fu SM, Rose CE Jr, et al. A major lung CD103 (alphaE)-beta7 integrin-positive epithelial dendritic cell population expressing Langerin and tight junction proteins. J Immunol 2006;176(4):2161–72. 62. Wells JM, Rossi O, Meijerink M, et al. Epithelial crosstalk at the microbiota-mucosal interface. Proc Natl Acad Sci USA 2011;108(Suppl. 1):4607–14. 63. Niess JH, Brand S, Gu X, et al. CX3CR1-mediated dendritic cell access to the intestinal lumen and bacterial clearance. Science 2005;307(5707):254–8. 64. Venge P. The eosinophil and airway remodelling in asthma. Clin Respir J. 2010;4(Suppl. 1):15–19.

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65. Flood-Page PT, Menzies-Gow AN, Kay AB, et al. Eosinophil’s role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167(2):199–204. 66. Yang W, Wardlaw AJ, Bradding P. Attenuation of human lung mast cell degranulation by bronchial epithelium. Allergy 2006;61(5):569–75. 67. Balzar S, Fajt ML, Comhair SA, et al. Mast cell phenotype, location, and activation in severe asthma. Data from the Severe Asthma Research Program. Am J Respir Crit Care Med 2011;183(3):299–309. 68. Persson C, Uller L. Transepithelial exit of leukcocytes: inflicting, reflecting or resolving airway inflammation? Thorax 2010;65(12):1111–15. 69. Andersson CK, Adams A, Nagakumar P, et al. Intraepithelial neutrophils in pediatric severe asthma are associated with better lung function. J Allergy Clin Immunol 2017;139(6):1819–29.e11. 70. Ober C, Yao TC. The genetics of asthma and allergic disease: a 21st century perspective. Immunol Rev 2011;242(1):10–30. 71. Loxham M, Davies DE. Phenotypic and genetic aspects of epithelial barrier function in asthmatic patients. J Allergy Clin Immunol 2017;139(6):1736–51. 72. Yang IV, Lozupone CA, Schwartz DA. The environment, epigenome, and asthma. J Allergy Clin Immunol 2017;140(1):14–23. 73. Nicodemus-Johnson J, Naughton KA, Sudi J, et al. Genome-wide methylation study identifies an IL-13-induced epigenetic signature in asthmatic airways. Am J Respir Crit Care Med 2016;193(4):376–85. 74. Clifford RL, Jones MJ, MacIsaac JL, et al. Inhalation of diesel exhaust and allergen alters human bronchial epithelium DNA methylation. J Allergy Clin Immunol 2017;139(1):112–21. 75. Wawrzyniak P, Wawrzyniak M, Wanke K, et al. Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients. J Allergy Clin Immunol 2017;139(1):93–103. 76. Stefanowicz D, Ullah J, Lee K, et al. Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts. BMC Pulm Med 2017;17(1):24. 77. Stefanowicz D, Lee JY, Lee K, et al. Elevated H3K18 acetylation in airway epithelial cells of asthmatic subjects. Respir Res 2015;16:95. 78. Pua HH, Ansel KM. MicroRNA regulation of allergic inflammation and asthma. Curr Opin Immunol 2015;36:101–8. 79. Solberg OD, Ostrin EJ, Love MI, et al. Airway epithelial miRNA expression is altered in asthma. Am J Respir Crit Care Med 2012;186(10):965–74. 80. Marcet B, Chevalier B, Luxardi G, et al. Control of vertebrate multiciliogenesis by miR-449 through direct repression of the Delta/ Notch pathway. Nat Cell Biol 2011;13(6):693–9. 81. McKiernan PJ, Molloy K, Cryan SA, et al. Long noncoding RNA are aberrantly expressed in vivo in the cystic fibrosis bronchial epithelium. Int J Biochem Cell Biol 2014;52:184–91. 82. Bhavsar P, Ahmad T, Adcock IM. The role of histone deacetylases in asthma and allergic diseases. J Allergy Clin Immunol 2008;121(3): 580–4. 83. Barnes PJ. Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 2009;6(8):693–6. 84. Maeda Y, Chen G, Xu Y, et al. Airway epithelial transcription factor NK2 homeobox 1 inhibits mucous cell metaplasia and Th2 inflammation. Am J Respir Crit Care Med 2011;184(4):421–9. 85. Yu H, Li Q, Kolosov VP, et al. Interleukin-13 induces mucin 5AC production involving STAT6/SPDEF in human airway epithelial cells. Cell Commun Adhes 2010;17(4–6):83–92. 86. Holgate ST. The sentinel role of the airway epithelium in asthma pathogenesis. Immunol Rev 2011;242(1):205–19. 87. Kicic A, Sutanto EN, Stevens PT, et al. Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma. Am J Respir Crit Care Med 2006;174(10):1110–18. 88. Hackett T-L, Singhera GK, Shaheen F, et al. Intrinsic phenotypic differences of asthmatic epithelium and its inflammatory responses to RSV and air pollution. Am J Respir Cell Mol Biol 2011;45:1090–100. 89. Xiao C, Puddicombe SM, Field S, et al. Defective epithelial barrier function in asthma. J Allergy Clin Immunol 2011;128(3):549–56.e1–12.

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90. Ritchie AI, Jackson DJ, Edwards MR, et al. Airway epithelial orchestration of innate immune function in response to virus infection. A focus on asthma. Ann Am Thorac Soc 2016;13(Suppl. 1):S55–63. 91. Cakebread JA, Xu Y, Grainge C, et al. Exogenous IFN-beta has antiviral and anti-inflammatory properties in primary bronchial epithelial cells from asthmatic subjects exposed to rhinovirus. J Allergy Clin Immunol 2011;127(5):1148–54.e9. 92. Altman MC, Reeves SR, Parker AR, et al. Interferon response to respiratory syncytial virus by bronchial epithelium from children with asthma is inversely correlated with pulmonary function. J Allergy Clin Immunol 2018;142(2):451–9. 93. Mitchell PD, O’Byrne PM. Epithelial-derived cytokines in asthma. Chest 2017;151(6):1338–44. 94. Hallstrand TS, Henderson WR Jr. An update on the role of leukotrienes in asthma. Curr Opin Allergy Clin Immunol 2010;10(1):60–6. 95. Park JA, Kim JH, Bi D, et al. Unjamming and cell shape in the asthmatic airway epithelium. Nat Mater 2015;14(10):1040–8. 96. Howat WJ, Barabas T, Holmes JA, et al. Distribution of basement membrane pores in bronchus revealed by microscopy following epithelial removal. J Struct Biol 2002;139(3):137–45. 97. Shariff S, Shelfoon C, Holden NS, et al. Human rhinovirus infection of epithelial cells modulates airway smooth muscle migration. Am J Respir Cell Mol Biol 2017;56(6):796–803. 98. Thompson HG, Mih JD, Krasieva TB, et al. Epithelial-derived TGF-beta2 modulates basal and wound-healing subepithelial matrix homeostasis. Am J Physiol Lung Cell Mol Physiol 2006;291(6):L1277–85. 99. Ward JE, Harris T, Bamford T, et al. Proliferation is not increased in airway myofibroblasts isolated from asthmatics. Eur Respir J 2008;32(2):362–71. 100. Davies DE, Holgate ST. Asthma: the importance of epithelial mesenchymal communication in pathogenesis. Inflammation and the

airway epithelium in asthma. Int J Biochem Cell Biol 2002;34(12):1520–6. 101. Kelly MM, O’Connor TM, Leigh R, et al. Effects of budesonide and formoterol on allergen-induced airway responses, inflammation, and airway remodeling in asthma. J Allergy Clin Immunol 2010;125(2):349–56.e13. 102. Chapman HA. Epithelial-mesenchymal interactions in pulmonary fibrosis. Annu Rev Physiol 2011;73:413–35. 103. Hackett TL, Warner SM, Stefanowicz D, et al. Induction of epithelial-mesenchymal transition in primary airway epithelial cells from patients with asthma by transforming growth factor-beta1. Am J Respir Crit Care Med 2009;180(2):122–33. 104. Loffredo LF, Abdala-Valencia H, Anekalla KR, et al. Beyond epithelial-tomesenchymal transition: common suppression of differentiation programs underlies epithelial barrier dysfunction in mild, moderate, and severe asthma. Allergy 2017;72(12):1988–2004. 105. Gras D, Chanez P, Vachier I, et al. Bronchial epithelium as a target for innovative treatments in asthma. Pharmacol Ther 2013;140(3):290–305. 106. Wadsworth SJ, Nijmeh HS, Hall IP. Glucocorticoids increase repair potential in a novel in vitro human airway epithelial wounding model. J Clin Immunol 2006;26(4):376–87. 107. Dorscheid DR, Wojcik KR, Sun S, et al. Apoptosis of airway epithelial cells induced by corticosteroids. Am J Respir Crit Care Med 2001;164(10 Pt 1):1939–47. 108. Huff RD, Rider CF, Yan D, et al. Inhibition of ABCC4 potentiates combination beta agonist and glucocorticoid responses in human airway epithelial cells. J Allergy Clin Immunol 2018;141(3):1127–30. 109. Zhang N, Truong-Tran QA, Tancowny B, et al. Glucocorticoids enhance or spare innate immunity: effects in airway epithelium are mediated by CCAAT/enhancer binding proteins. J Immunol 2007;179(1):578–89.

CHAPTER 19  Airway Epithelial Cells

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SELF-ASSESSMENT QUESTIONS 1. Immune responses can be triggered in human airway epithelial cells in response to inhaled viruses, bacteria, fungi, and environmental pollutants. Which class of receptors orchestrate innate immune responses to these stimuli? a. Interleukin-1 receptor family b. Adrenergic receptors c. Pattern recognition receptors d. Glucocorticoid receptors 2. The airway epithelium forms a pseudostratified barrier tissue that consists of multiple cell types. Which of the following cell types are not part of the pseudostratified epithelium? a. Innate lymphoid cells b. Basal cells

c. Columnar cells d. Club cells 3. Intrinsic abnormalities exist in the airway epithelium of subjects with chronic respiratory disease, including asthma. Which of the following abnormalities are not observed in airway epithelial cells of asthmatics? a. Decreased expression of cytokeratin-19 b. Increased expression of the transcription factor p63 c. Impaired type I interferon responses d. Decreased epidermal growth factor receptor expression

20  Airway Smooth Muscle in Asthma Cynthia J. Koziol-White, Reynold A. Panettieri, Jr.

CONTENTS Introduction, 301 Cellular and Molecular Mechanisms Regulating Smooth Muscle Cell Growth, 301 Factors Regulating Airway Smooth Muscle Growth in Asthma, 301

SUMMARY OF IMPORTANT CONCEPTS • Airway smooth muscle (ASM), the pivotal tissue regulating bronchomotor tone, serves an immunomodulatory and remodeling role in allergic airway disease. • Evidence suggests both a hyperproliferative and hypercontractile phenotype in the airway smooth muscle of subjects with asthma, which may be driven by intrinsic abnormalities in the smooth muscle. • Airway smooth muscle responds to a variety of inflammatory stimuli, including Th1 and Th2 cytokines and extracellular matrix, by altering its contractile state, growth, and ability to release inflammatory mediators and extracellular matrix. This contributes to facets of remodeling, airway hyperresponsiveness, and airflow obstruction characteristically associated with asthma. • The combination of a β2AR agonist with a corticosteroid is a mainstay therapeutic that differentially modulates ASM inflammatory responses. • Modulation of contractile responses of ASM, either by blocking bronchoconstriction or stimulating bronchodilation, alleviates asthma symptoms. • Bronchial thermoplasty improves exacerbation rates in severe asthma and may decrease ASM in asthma.

Signaling Pathways Affecting Airway Smooth Muscle Growth and Proliferation, 302 Airway Smooth Muscle Contraction, Airway Hyperresponsiveness, and Relaxation, 303 Summary, 309

regulating tone of the airways, a greater understanding of mechanisms underlying development of a hypercontractile and hyperproliferative phenotype, as well as increased immunomodulatory capacity of these cells, are important in the treatment of asthma.

CELLULAR AND MOLECULAR MECHANISMS REGULATING SMOOTH MUSCLE CELL GROWTH Investigators have reported an increased amount of ASM in the airways in asthma occurs through hypertrophy and/or hyperplasia of the cells. An increase in ASM mass because of hyperplasia and/or hypertrophy likely physically contributes to increased occlusion and decreased elasticity of the airways associated with severe asthma and correlates with increased airway hyperresponsiveness and airway resistance. Even in mild asthma, ASM hyperplasia has been shown to occur. One of the first studies to examine ASM growth rates reported increased proliferation of cells derived from asthmatic biopsies.8 Alterations in mechanisms of ASM proliferation have mainly been studied in vitro comparing ASM derived from subjects with asthma with ASM from nonasthma subjects.

INTRODUCTION

FACTORS REGULATING AIRWAY SMOOTH MUSCLE GROWTH IN ASTHMA

Increasing evidence suggests that factors present early in airway development, including growth factors and mechanical stimuli affecting airway smooth muscle (ASM), influence lung growth but also increase the incidence of asthma. ASM is likely to play a role in the development of structure and function of the airways. Studies show that peristalsis occurs in vitro and that there are waves of calcium that occur in fetal ASM, which promote branching and elongation of the airways. Factors present in the cells and tissue that allow ASM to sense changes in its environment can be examined to understand how the milieu of the developing airway influences the growth and responses of ASM. Interestingly, on top of the normal environment present during airways development, effects of toxicant exposure, respiratory pathogen exposure, and various other insults most certainly influence not only the development of the airways to contribute to the development of asthma, but may also alter the phenotype of ASM. With ASM being the pivotal cell

Conventional thought suggests that remodeling of the airways was a response to chronic inflammation, but more recent evidence suggests that events including stretch of the airway parenchyma and bronchoconstriction of the airways may evoke airway remodeling. Studies have shown that both cyclic stretch and bronchoconstriction induce increased ASM growth.5,9,10 In line with the idea that contraction of ASM modulates growth, there is sufficient evidence that contractile agonists including histamine, endothelin-1, serotonin, α-thrombin, thromboxane A2, and leukotriene D4 all stimulate ASM growth.11,12 Growth of ASM can also be modulated by a lack of factors that control proliferation, including prostaglandin E2 deficiency, vitamin D deficiency, and low levels of inhibitory transcription factors like CCAAT/enhancer binding protein alpha (C/EBP-α) which normally slow proliferation. Growth factors like epidermal growth factor (EGF), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and insulin-like growth

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SECTION A  Basic Sciences Underlying Allergy and Immunology

factor (IGF), which are elevated in bronchoalveolar lavage (BAL) from asthma subjects, modulate ASM growth via similar mechanisms stimulating growth of other cell types like fibroblasts, such as activation of receptor tyrosine kinases associated with growth factor receptors. Even interactions between ASM and other cell types, including T cells, can stimulate ASM proliferation.13,14 The surrounding matrix also has effects on ASM growth and proliferation. Fibronectin and collagen enhance growth of ASM, whereas laminin diminishes it.15,16 Additionally, ASM itself is a rich source of autocrine-secreted extracellular matrix (ECM) that acts to augment the proliferative capacity of the cells. When ASM from asthma subjects is grown on ECM from asthma subjects, the growth rate is significantly higher than ASM from nonasthma subjects grown on nonasthma ECM. Growth on asthma ECM can induce a higher growth rate in nonasthma ASM compared with growth on nonasthma ECM.17 Additionally, enzymes associated with turnover of ECM, including ADAM33, have been shown to modulate ASM growth.18 Inflammation associated with asthma induces release of mediators that stimulate ASM growth. Cytokines, chemokines, and lipid mediators all modulate ASM growth. Cyclooxygenase (COX) products like prostaglandin E2 inhibit DNA synthesis.19 Inflammatory cytokines such as IL-4 and interferons (type I and II) also inhibit ASM proliferation by various downstream signaling mechanisms.20 IL-13 and IL-4 induce MMP-1 production from ASM.21 Insulin-like growth factor binding protein (IGF-BP) is degraded by active MMP-1, releasing IGF, consequently stimulating ASM proliferation and migration.

SIGNALING PATHWAYS AFFECTING AIRWAY SMOOTH MUSCLE GROWTH AND PROLIFERATION Many extracellular triggers modulating ASM growth act through different receptor-dependent mechanisms. Growth factors work through activation of receptors with intrinsic receptor tyrosine kinase (RTK) activity, contractile agonists mediate their effects through G protein– coupled receptors (GPCRs), and cytokines signal through receptor chains that oligomerize and are coupled to nonreceptor tyrosine kinases like SRC, LYN, and JAK. Despite the array of different receptor types that modulate ASM growth, evidence suggests that certain signaling pathways act as points of convergence to promote proliferation. One of these points is at RASA1, a repressor of the oncogenic protein Ras, which can work downstream through activation of PI3Kα/β and MAPK/ERK signaling cascades to regulate mitogenic signaling. Signaling pathways implicated in the hyperproliferative nature of asthma-derived ASM are mitogen-activated protein kinases (MAPKs) (ERK, MAPK1, MAPK3, MAPK6, MAPK11, MAPK12, MAPK13, MAPK14) and phosphatidylinositol 3-kinase (PI3K). These pathways serve as the predominant pathways mediating ASM growth whether it occurs through activation of RTKs, GPCRs, or cytokine receptors (Fig. 20.1). To affect ASM growth, these pathways impinge upon components of the cell cycle either to move the cell through the cycle or to arrest it. Stages of the cell cycle are composed of phases named G1, S (where DNA synthesis occurs), G2, and M (mitosis) that are common among all cell types, including ASM. Studies primarily examining the effects of external stimuli on ASM growth and proliferation have been performed in vitro utilizing serum deprivation of the cells to synchronize them at the G0 or early G1 phase of the cell cycle. In response to stimulation, the cells progress through the G1 phase of the cell cycle through expression of D-type cyclins (D1, D2, and D3) in association with cyclin-dependent kinases (CDKs 4 and 6). Activation of the cyclin 3/CDK2 complex, which phosphorylates retinoblastoma protein 1 (RB1), occurs as the cells approach the G1/S transition. Upon activation RB1 releases the elongation factor E2F, which activates DNA polymerase, thereby committing the cell to undergo

Extracellular

RTK

Cytokine receptor

GPCR

Src

Gi

Shc GRB SOS Intracellular

RASA

PI3K

Raf-1

MEK1

S6 kinase

Rac1

Cdc42

MAPK/ERK

ADM cell proliferation Nucleus Fig. 20.1  Schematic representation of signal transduction mechanisms that regulate airway smooth muscle (ASM) cell proliferation. ASM mitogens act through receptor tyrosine kinases (RTKs), cytokine receptors, or G protein–coupled receptors (GPCRs) to activate the small guanidine triphosphate hydrolyzing enzyme (GTPase) RAS p21 protein activator 1 (RASA1). RASA1 proteins then interact with the downstream effectors RAF1 and PI3K. RAF1 activates MEK1, which phosphorylates MAPK/ ERK. PI3K activates downstream effectors, such as p70 ribosomal S6 kinase (S6 kinase) or members of the RHO family GTPases, RAC1, and CDC42 (although whether CDC42 acts upstream of RAC1 or crosstalk exists is unknown [dashed lines]). MAPK/ERK, PI3K, and the downstream effectors of PI3K regulate cell cycle proteins, and the MAPK/ERK and PI3K pathways are considered to be two major independent signaling pathways regulating ASM growth. Src, Family of nonreceptor tyrosine kinases. (Reproduced with permission from Ammit AJ, Panettieri RA Jr. Signal transduction in smooth muscle. Invited review: the circle of life—cell cycle regulation in airway smooth muscle. J App Physiol 2001;91:1431.)

DNA synthesis and mitosis. At each phase of the cell cycle, CDK activity can be constrained by CDK inhibitors: Inhibitors of CDK4 and CDK6 include CDKN2A, CDKN2B, CDKN2C, and CDKN2D; cyclin D, E, and A-dependent kinase inhibitors include CDKN1A (p21), CDKN1B (p27), and CDKN1C (p57). Thymidine incorporation is commonly used to measure growth and proliferation of ASM, as it detects the S phase of the cell cycle because of its incorporation into DNA that is undergoing the synthesis phase.12,22 Additionally, interactions between ASM and activated T cells can induce ASM proliferation.14

Phosphatidylinositol 3-Kinase Pathway The PI3K family of protein kinases are divided into four classes based upon structure and substrate specificity: class I (1A isoforms are 110 kD α, β, or δ and 1B consists of a γ isoform; all are catalytic subunits that complex with an 85 kD adaptor protein and can be activated by receptor tyrosine kinases and nonreceptor associated kinases), class II (associated with phospholipid membranes including the cell membrane and trans-Golgi network), and class III (involved in autophagy and vesicular sorting). Human ASM expresses all types of PI3K proteins with the exception of PI3K p110γ.23 The use of PI3K inhibitors has shown that PI3Kα/β activation is critical for cell cycle progression in human ASM.24 Additionally, constitutively active class IA PI3K injected

CHAPTER 20  Airway Smooth Muscle in Asthma or transfected into human ASM markedly increased DNA synthesis, which was substantially less than that induced by receptor-mediated pathways.25 These studies suggest that there are other pathways modulating ASM growth responses in addition to PI3K pathway-dependent modulation of DNA synthesis. PI3K inhibition does not alter MAPK/ ERK activation,24 suggesting that the two pathways work in parallel to modulate DNA synthesis in ASM. Overexpression studies using the catalytic subunit of PI3K (p110 CAAX box) demonstrated that this motif activated the cyclin D1 promoter, which could be attenuated by inhibitors of RAC1 signaling.26 Additionally, overexpression of RAC1 induced transcription of cyclin D1, and a dominant negative allele of RAC1 inhibited PDGF-induced cyclin D1 expression.27 CDC42, another GTPase similar to RAC1, induced transcription at the cyclin D1 promoter.28 Activation of the cyclin D1 promoter by PI3K p110 CAAX,26 RAC1,26 and CDC4228 all occur through a CREB/ATF2 binding site in the promoter. In human ASM inhibition of S6 kinase, a downstream target of PI3K, with rapamycin attenuates growth factor–induced DNA synthesis.24 Regulation of cell cycle progression in the G1 phase occurs through S6 kinase-dependent phosphorylation of the 40S ribosomal protein, which upregulates translation of mRNAs that contain an oligopyrimidine tract at their start site. These moieties encode proteins necessary for cell cycle progression, including E2F.29

Extracellular Signal–Regulated Kinase Pathway Activation of MAPKs occurs through RAF1 kinase activation, which phosphorylates MEK1. Activated MEK1 directly phosphorylates and activates ERK1 and ERK2. Growth and proliferation of human ASM after EGF, PDGF-BB, or thrombin exposure is characterized by significant sustained activation of ERK1/2 and is attenuated by MEK1 inhibition.30 In ASM, there is evidence that suggests that MAPK/ERK activation induces cyclin D1. The promoter region of cyclin D1 contains binding sites for transcription factors that include simian virus 40 protein 1 (SP1), activator protein 1 (AP-1), signal transducers and activators of transcription (STATs), nuclear factor-κB (NF-κB), and cyclic adenosine monophosphate (cAMP) response-element binding protein/activating transcription factor 2 (CREB/ATF2).31,32 In human ASM, Orsini and colleagues showed that mitogen-activated MAPK/ERK activation, thymidine incorporation, and ELK1 and AP-1 reporter activity were all abrogated by inhibition of MEK1.30 Furthermore, studies showed that using an MEK1 inhibitor or a dominant negative mutant of MEK1 or MAPK/ERK abolished PDGF-induced cyclin D1 promoter activity and expression.33 RASA1 also induces MAPK/ERK activation and transcriptional activation of the cyclin D1 promoter when overexpressed in bovine ASM,34 suggesting a role for this protein in regulating MAPK/ERK signaling. MAPK/ERK pathways have also been shown to regulate cyclin-dependent kinase inhibitors, which remain expressed at high levels in nondividing cells. In human ASM, the GPCR agonist SPP increased cyclin D1 levels and decreased the cyclin-dependent kinase inhibitor CDKN1B levels through a MAPK/ERK-dependent pathway.35 SPP was found to augment EGF- and thrombin-induced DNA proliferation through increases in cyclin D1 and decreases in CDKN1C expression.36

AIRWAY SMOOTH MUSCLE CONTRACTION, AIRWAY HYPERRESPONSIVENESS, AND RELAXATION Inflammatory disorders of the airways, such as asthma, and exacerbations are driven in part by secretion of mediators that not only modulate the responses of the immune system, but also significantly affect airway structural cells. ASM is the pivotal cell modulating bronchomotor tone,

303

and its contractile status is modulated by the Th2 inflammatory milieu present in asthma. Shortening of ASM is primarily induced by GPCR agonists and is modulated by downstream pathways that are both calcium (Ca2+)-dependent and independent (Fig. 20.2). In the canonical Ca2+ mobilization pathway, phospholipase β (PLCβ) activation generates inositol triphosphate (IP3) that then binds to the IP3 receptor on the sarcoplasmic reticulum (SR) to elicit [Ca2+]i release. The increased [Ca2+]i activates calmodulin (CaM) and myosin light chain kinase (MLCK) to phosphorylate myosin light chain (MLC) and induce actin-myosin crossbridge cycling and HASM shortening. In parallel, increased expression of CD38 generates cyclic ADP-ribose (cADPR) that then binds to the ryanodine receptor (RyR) to promote SR [Ca2+]i release. The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) refills the SR with the cytosolic [Ca2+]i inhibiting HASM shortening (as reviewed in reference 37). Contractile agonists like methacholine, thrombin, histamine, and leukotriene D4 modulate [Ca2+]i release in ASM. Changes in Ca2+ handling in ASM have been impugned as potential mechanisms by which airway hyperreactivity is increased in asthma. Inflammatory cytokines release in asthma and exacerbations of asthma increase the sensitivity of ASM to contractile agonists. TNF-α, a prominent cytokine present in the airways during allergic airway inflammation,38 evokes AHR.39 Recently, evidence suggests that HASM derived from subjects with asthma expresses higher levels of TNF-α-induced CD38 expression compared with HASM derived from nonasthma subjects.40 TNF-α has been demonstrated to increase CD38 expression/ cADPR activity and augment contractile agonist-induced [Ca2+]i release in ASM.23,40 IL-13 produced by Th2 cells, mast cells, basophils, and eosinophils induces hyper-responsiveness of rabbit tracheal smooth muscle and human small airways to methacholine.41,42 Additionally, TNF-α augments methacholine-induced airway narrowing in a model of human small airways.41 IL-13 and IL-4 induce MMP-1 production from ASM that can alter collagen type I matrix deposition and airway contractility.21 Additionally, increased levels of the metalloproteinase ADAM33 have been shown to increase cell stiffness43 and increase contraction of ASM.44,45 IL-13 augmented [Ca2+]i release in ASM induced by bradykinin, histamine, and methacholine.46 Underlying changes in sensitivity to Ca2+ may be how airway hyperreactivity is precipitated in asthma. Accordingly, Mahn and colleagues showed that ASM derived from asthma subjects exhibited a sustained [Ca2+]i release as well as attenuated expression of SERCA2 compared with ASM derived from nonasthma subjects,6 suggesting the inflammatory environment of asthma may potentially alter the contractile phenotype of ASM. Calcium sensitization pathways that are relatively Ca2+-independent are mediated by activation of RhoA, stimulating Rho kinase, which phosphorylates myosin light chain phosphatase target subunit (MYPT1) to inactivate it. Under resting conditions, MYPT1 is active and limits HASM shortening; upon its phosphorylation and hence its inhibition, HASM shortening is enhanced. In ASM, it was demonstrated that RhoA and ROCK-associated airway hyperresponsiveness is associated with exposure to inflammatory cytokines,47–51 sphingolipids,52,53 and mechanical stress.54 IL-13 has been shown to augment both canonical calcium mobilization pathways46 and enhance calcium sensitization pathways.55 Human ASM derived from subjects with severe asthma exhibited higher ROCK activity compared with ASM derived from nonasthma subjects, suggesting that there may be an intrinsic hypercontractile phenotype associated with asthma.4 Additionally, inhibition of RhoA induced relaxation of methacholine-constricted human small airways.56 Cytokines and chemokines directly modulate ASM function, but other inflammatory mediators including lipid mediators influence the inflammatory environment created and maintained by ASM, as well as alter the contractile status of the ASM. Prostaglandins (PG), products of metabolism of arachidonic acid (AA) by cyclo-oxygenase (COX)

304

SECTION A  Basic Sciences Underlying Allergy and Immunology Soluble mediators (cytokines, lipid mediators) Contractile agonist

CD38 PLC-β α β

γ

RhoA-GDP

cADPR PIP2 IP3

GEF

RhoA-GTP

DAG

IP3R

PI3Kδ

RyR

SR Ca2+-

Rho kinase

CaM

SERCA

MLCK

Ca2+ Ca2+

MYPT1 MLC MLC

P

CaM

MLCK Ca2+-CaM

Actin-myosin crossbridging HASM

Force generation

Fig. 20.2  Model illustrating calcium-dependent and calcium sensitization pathways modulating airway smooth muscle (ASM) contraction. Contractile agonists bind G protein–coupled receptors (GPCRs) to activate phospholipase C (PLC) β, which converts phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3). IP3 binds the IP3 receptor (IP3R) on the sarcoplasmic reticulum (SR) to elicit calcium release. Calcium binds calmodulin (CaM), which activates myosin light chain kinase (MLCK) to phosphorylate myosin light chain (MLC). Once phosphorylated, MLC promotes actin-myosin crossbridging to generate contraction of the cell. Inflammatory mediators also upregulate CD38, producing cyclic adenosine phosphate ribose (cADPR), which binds to the ryanodine receptor (RyR) to elicit calcium release from the SR. The effects of calcium are potentiated through calcium-sensitization pathways (blue box). RhoA-GDP is converted to RhoA-GTP through a guanine nucleotide exchange factor (GEF) to activate it. RhoA-GTP activates phosphoinositide 3-kinase (PI3K) δ, which activates Rho kinase. Rho kinase inactivates myosin phosphatase target subunit (MYPT1), which normally dephosphorylates MLC to inactivate it. Therefore MLC remains phosphorylated and promotes ASM contraction. The sarcoplasmic/endoplasmic reticulum calcium-ATPase (SERCA) acts to reload the SR with cytosolic calcium, thereby diminishing the contractility of ASM.

enzymes, are mediators associated with different aspects of allergic airway inflammation. PGE2 abrogates early- and late-phase responses to antigen challenge; in murine models of allergen-induced AHR, the lack of EP3 receptors for PGE2 increases airway inflammation and AHR to ovalbumin (OVA).57 Other prostaglandins like PGD2 and thromboxane (TXB), however, act as bronchoconstrictors that may enhance allergenmediated airway eosinophilia.58 TXB has been found to be a more potent bronchoconstrictor than methacholine and can enhance methacholine responsiveness in asthma subjects.59 Receptors for leukotrienes LTC4 and LTD4, including CysLT1, are expressed on ASM.12 IFN-γ, a mediator increased in viral exacerbations of asthma, increased cell surface expression of CysLT1 receptors and markedly increased contraction of ASM in culture.60 Another membrane-derived lipid that modulates airway contractility is sphingosine-1-phosphate (S1P). S1P not only promotes contractility of ASM,53,61 but also promotes inflammation and remodeling of the airways via ASM.62–64 Epigenetic modulation of ASM has been impugned to control aspects of ASM function, including airway hyperresponsiveness and inflammation. These types of changes are via either posttranscriptional modification of histone proteins to alter accessibility of DNA for global or local

activation or inactivation of genes or expression of negative regulators or genes via the following mechanisms1: acetylation via histone acetyltransferases (HATs), which is thought to enhance gene expression2; removal of acetylation via histone deacetylases (HDACs), thought to blunt gene expression3; DNA methylation via methyltransferases, which is thought to repress gene expression; and4 micro-RNAs (miRNAs) and noncoding RNAs (ncRNAs), which regulate gene expression to interfere with their function. In inflammatory airway diseases, including asthma, histone deacetylases (HDACs) are downregulated.65–67 The nonselective HDAC inhibitor, Trichostatin A, has been shown to attenuate inflammation68,69 as well as blunt ASM contraction.70 Both upregulation and downregulation of miRNAs have been shown to modulate functions of ASM. Decreased expression of miR-25 after exposure to IL-1β, TNF-α, and IFN-γ led to upregulation of Kruppel-like factor 4 (KLF4), a potent inhibitor of smooth muscle–specific gene expression and mediator of inflammation.71 Additionally, expression of miR-140-3p was attenuated by TNF-α stimulation of both asthma- and nonasthma-derived ASM. This miRNA was found to bind to the 3′-untranslated region (UTR) of the CD38 gene to modulate its expression. CD38 modulates intracellular calcium dynamics and ASM contractility.72

305

CHAPTER 20  Airway Smooth Muscle in Asthma Agonists of the β2 adrenergic receptor (β2AR) induce generation of cAMP and attenuate myosin light chain kinase activity abrogating actinmyosin crossbridging, thereby inducing relaxation of ASM and bronchodilation. This class of therapeutics are mainstays for treatment to reverse bronchoconstriction associated with allergic airways disease. Interestingly, TNF-α synergizes with IL-1β to promote β2AR hyporesponsiveness, an effect ablated by selective COX-2 inhibition, providing evidence to suggest that arachidonic acid derivatives serve to desensitize the airway smooth muscle to bronchodilation. Using cultured ASM, IL-13, TNF-α and IL-1β modulated GPCR-associated signaling pathways by enhancing agonist-induced calcium responses and/or altering cAMP production. Additionally, exposure to IL-13 attenuated β2 agonist-induced dilation of human small airways in an in vitro model,41 which could be reversed by inhibition of PI3Kδ and administration of budesonide.4,73 Recent evidence suggests a potential link between airway remodeling and airway hyperresponsiveness. Studies demonstrate that TGF-β1, a pleiotropic cytokine inducing airway remodeling, also induces human ASM shortening and amplifies contractile agonist-induced force generation.51 These studies show that TGF-β1 alone, and in combination with a contractile agonist, induces activation of calcium sensitization pathways rather than calcium-dependent pathways to elicit ASM contraction.51,74 A guanine nucleotide exchange factor, p115RhoGEF, has been found to be activated by both the M3R and TGF-β1 to mediate contractile responses in ASM.56,74 Therefore this provides a potential link between TGF-β1 and contractile agonist-induced signaling mediating contractility of ASM, as well as a point at which contractile signaling can be amplified. Consequently, targeting TGF-β1 pathways may prevent or reverse sustained airway hyperresponsiveness observed after asthma exacerbations, as well as to abrogate persistent airway obstruction that is a hallmark of remodeling in asthma.

Biased Agonism of the β2AR and Contractile Receptors Engagement of the β2AR activates not only bronchodilatory cAMPdependent pathways, but other signaling pathways that promote downregulation of the receptors, thereby making individuals refractory to these therapeutics (Fig. 20.3). β2 receptors undergo a process of desensitization, which is in part mediated through activation of G protein receptor kinases (GRKs), which phosphorylate the receptor to recruit arrestins in clathrin-coated domains of the membrane. This allows for endocytosis of the receptor and eventual receptor degradation. Chronic β2AR agonist use has been associated with loss of asthma control and, in severe cases, death. Development of novel modulators of signaling pathways downstream of the β2AR, which are based on a greater understanding of receptor conformations, allow for development of drugs to more effectively treat asthma as well as make current therapeutics better. Ligands that selectively engage a receptor to induce conformations that activate one signaling pathway and inhibit another are referred to as “biased agonists” (Fig. 20.4). Activation of the β2AR initiates signaling through G protein (Gαsβγ)- and arrestin-dependent mechanisms (Fig. 20.4A). By elucidating receptor conformations that preferentially signal via one pathway or the other, a given therapy could achieve the effects of traditional β2AR agonist therapy without eliciting the negative regulatory effects that make the therapy ineffective75 (Fig. 20.4B and 20.4C). Examples of this are a 5-HT2cR ligand SB242084, which selectively activates phospholipase C (PLC) but inhibits PLA2 and the release of arachidonic acid.76,77 This ligand, along with biased ligands for the CCK, somatostatin, and µ-opioid receptors, are highly effective but do not induce desensitization of those receptors similarly to how traditional β2AR agonists induce receptor desensitization.78 Studies have shown that inverse agonists like carvedilol, ICI 118551, and propranolol

Uncoupling

AC γ β

α α

p

p

p p p Arres tin

GTP

GDP

γ

ATP

Uncoupling

α

γ β

GTP p

β GRK

p in est Arr

cAMP

PKC

Endocytosis

Degradation

PKA

Decreased contraction/ bronchodilation

p

p p in est Arr

Fig. 20.3  Model illustrating β2 adrenergic receptor (β2AR) signaling pathway activation that modulates bronchodilation or desensitization of the receptor. Ligand binding catalyzes the exchange of GDP for GTP on the α subunit, which activates adenylyl cyclase (AC). AC catalyzes the production of cAMP from ATP, which induces bronchodilation and decreases contraction through activation of protein kinase A (PKA). At the same time, the βγ subunits associated with the β2AR activate G protein receptor kinases (GRKs), which phosphorylate the intracellular loops of the receptor. This allows for recruitment of clathrin and arrestins, which mediate uncoupling of the receptor from its bronchodilatory function (cAMP-dependent signaling) and allows for endocytosis and degradation of the receptor as a form of downregulation.

PKA

306

SECTION A  Basic Sciences Underlying Allergy and Immunology

α GTP

cAMP

Decreased contraction/ bronchodilation

γ β

p p Arresti n

α

γ β

GTP

Endocytosis

cAMP

Decreased contraction/ bronchodilation

Degradation

A β2AR agonist-induced signaling

p p Arresti n

Endocytosis

Degradation

B β2AR agonist-induced signaling–Gαβγ biased

α

γ β

GTP

cAMP

Decreased contraction/ bronchodilation

p p Arresti n

Endocytosis

Degradation

C β2AR agonist-induced signaling–arrestin biased Fig. 20.4  Biased agonism of the β2AR. (A) Signaling pathway activation downstream of the β2AR that mediates bronchodilation and receptor desensitization. (B) Gα-biased signaling that preferentially induces bronchodilation. (C) Arrestin-biased signaling that preferentially induces receptor downregulation and desensitization.

induce activation of β-arrestin-mediated endocytosis of the β2AR, as well as activation of ERK1/2 pathways.79,80 Recent evidence demonstrates that lipidated peptides called pepducins, which are based off the intracellular loops of the β2AR, can serve to selectively activate or inhibit signaling pathways downstream of the β2AR. Pepducins derived from the third intracellular loop (ICL3) of the β2AR activate Gαs-biased signaling, either through stabilizing a specific conformation of the β2AR that interacts with Gαs or through direct interaction with Gαs, and fail to induce β2AR desensitization and endocytosis. Pepducins derived from the intracellular loop 1 (ICL1) were found to mediate signaling through arrestins.81 Modulating bronchoconstriction signaling pathways through targeting of contractile receptors may serve as another strategy to alleviate airway hyperresponsiveness associated with asthma. To date, no strategies to block all contractile receptors have been developed that are selective and easily delivered. Many contractile receptors signal through Gαq to induce contraction of airway smooth muscle. Recent evidence demonstrated that using the pepducins FR900359 and p4PAL, investigators selectively attenuated carbachol- and histamine-induced contraction of human ASM and small airway narrowing in human tissue.82

Airway Smooth Muscle as an Immunomodulatory Cell.  Inflammatory mediators produced by epithelial cells and infiltrating immune cells alter ASM physiology, but several studies have demonstrated that ASM cells also release a number of mediators themselves (Table 20.1). TNF-α stimulates mediator production by ASM in addition to modulating airway responsiveness. IL-33 levels are elevated and coexpressed with TNF-α in tissue derived from endobronchial biopsies of subjects with severe asthma. TNF-α stimulation induced IL-33 in a time- and dose-dependent manner, and these levels synergistically increased with IFN-γ costimulation. Patients with mild asthma express RANTES, a chemokine that recruits leukocytes to sites of inflammation, in ASM derived from biopsies.83 Human ASM also releases RANTES after TNF-α and IFN-γ stimulation in vitro.84,85 After stimulation with TNF-α and IL-1β, ASM secreted eotaxin that activated production of RANTES, as well as IL-8, a known neutrophil chemoattractant.86 TNF-α stimulation increased secretion of IL-6, a cytokine that is thought to induce ASM hyperplasia, and modulates B and T cell proliferation and immunoglobulin secretion. TNF-α exposure of ASM promotes infiltration and adhesion of activated immune cells that, along with IL-1β, modulates inflammatory pathways during exacerbations of asthma.38 IL-1β alone

CHAPTER 20  Airway Smooth Muscle in Asthma

TABLE 20.1 Immunomodulatory

Proteins Expressed by Human Airway Smooth Muscle Cells Cytokines

Chemokines

Cell Adhesion Molecules

Other

IL-1β

IL-8

ICAM-1

CD40

IL-5

RANTES

VCAM-1

FAS

IL-6

MCP1, 2, 3

ALCAM

HLA-DR

IL-11

Eotaxins 1 and 2

CD44

FcγRII

LIF

GM-CSF

LFA-1

FcγRIII

IFN-γ and β

IP-10

α9β1

FcεRI

TSLP

Fractalkine

α5β1

NO

TGF-β

TARC

αV and α6 subunits

PGE2, CysLTR, C3aR, C5aR

IL-33

MMPs, TIMPs VEGF BDNF Collagen, ECM proteins

ALCAM, Activated leukocyte cell adhesion molecule; BDNF, brain-derived neurotrophic factor; CD, cluster of differentiation; CysLTR, cysteinyl leukotriene receptor; ECM, extracellular matrix; GM-CSF, granulocyte macrophage-colony stimulating factor; HLA-DR, major histocompatilibity complex class II-DR; ICAM-1, intercellular adhesion molecule 1; IFN, interferon; IL, interleukin; IP, interferon gamma-induced protein; LFA1, lymphocyte function-associated antigen 1; LIF, leukemia inhibitory factor; MMP, matrix metalloproteinase; PGE2, prostaglandin E2; RANTES, regulated on activation, normal T cell expressed and secreted; TARC, thymus and activation regulated chemokine; TGF-β, transforming growth factor β; TIMP, tissue inhibitor of metalloproteinases; TSLP, thymic stromal lymphopoietin; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor.

induces production of numerous mediators including PGE2; eotaxin; RANTES; GM-CSF; growth-related oncogene (GRO) α; epithelial neutrophil activating protein (ENA)-78; monocyte chemoattractant protein (MCP)-1, -2, and -3; IL-8; and IL-11.87,88 IL-1β alone, as well as in combination with TNF-α, increases production of MMP-12 and MMP-9,89 matrix metalloproteinases known to degrade elastin, type IV collagen, fibronectin, and laminin that promote cell migration, tissue repair, and remodeling responses. A study noted that exposure of HASM derived from subjects with asthma to IL-13, IL-1β, or TNF-α increased eotaxin release 2.5 to 6-fold compared with HASM from healthy donors. The study also showed that blocking of α5β1 integrins abrogated the effect of IL-13-mediated eotaxin release from the asthma tissue.90 Evidence suggests that ASM, both in vivo and in vitro, expresses thymic stromal lymphopoietin (TSLP), a cytokine believed to be necessary and sufficient to induce or maintain allergic airway inflammation. TSLP activates immune cells such as dendritic cells to elicit inflammatory mediator production and promote maturation of T cells into certain phenotypes. Importantly, endobronchial biopsies from subjects with mild asthma express TSLPR on ASM.91 TNF-α stimulation of ASM induces mRNA and protein expression of TSLP, whose expression is dependent on NF-κB and AP-1 activity.92 Interestingly, ASM also expresses functional subunits of the TSLP receptor that promote cytokine-induced release of IL-8, eotaxin, and IL-6. Thymus- and activation-regulated

307

chemokine (TARC), a mediator promoting dendritic cell recruitment to sites of inflammation, evoked airway eosinophilia and AHR in murine models of allergen-induced AHR; TARC levels are also increased in the BAL from allergen-challenged asthma subjects. IL-13 in combination with IL-4 and TNF-α elicits TARC release from ASM, suggesting crosstalk among cytokine pathways in ASM. In response to IL-13, human ASM secretes eotaxin, which requires STAT6 activation.93 Lipid mediators affect ASM physiology but can also be produced by ASM. Activation of 5-lipoxygenase (5-LO) enzyme generates leukotrienes that can be classified into two different classes, the cysteinyl leukotrienes (CysLT) that include LTC2, LTD4, and LTE4, and the dihydroxy leukotriene, LTB4. Studies showed that ozone induces IL-6 production via early enhancement of PGE2.94 Other factors, including bradykinin, IL-1β, TNF-α, IFN-γ, cigarette smoke, LPS, mechanical stretch, and cAMP-elevating agents potentiate PGE2 production by ASM.95 A mixture of TNF-α, IL-1β, and IFN-γ induces cyclooxygenase (COX) activity, which are key enzymes for the production of prostaglandins from arachidonic acid. This cytokine mixture induced PGE2 and 6-keto-PGF1α96 release in human ASM. Interaction of ASM with immune cells may also contribute to airway inflammation. TNF-α increased expression of CAMs on ASM, promoting increased interaction with and adhesion to inflammatory cells.97,98 Adhesion of CD4+ T cells to ASM induced DNA synthesis in ASM.14 CD40, a costimulatory molecule found on antigen-presenting cells, is upregulated on ASM by IFN-γ, and upon ligation increases release of IL-6.99 Activated T cells were incubated with isolated ASM segments, and ASM exhibited enhanced contractile responses to acetylcholine and attenuated the ability of a β2 agonist to relax precontracted ASM segments.100 Given the fact that ASM not only responds to inflammatory mediators but produces them in response to exposure to cytokines/stretch/ etc., this places the muscle at a relatively central position in modulating contractile and noncontractile responses within the inflammatory milieu of asthma.

Cytokine-Mediated Corticosteroid Insensitivity: Effects in Airway Smooth Muscle and Mechanisms Underlying Insensitivity.  Severe asthma and viral infections, either alone or in concert with underlying airway inflammation, exhibit insensitivity to corticosteroid treatment that would normally decrease airway inflammation. TNF-α found in BAL fluid from asthmatics, in combination with IFN-γ released during viral infection, act together to induce corticosteroid insensitivity. TNF-α synergizes with IFN-γ in the production of IP-10 through recruitment of CREB-binding protein and subsequent increased RNA polymerase II activity.101 Such synergism between these normally opposing pathways also increases RANTES, fractalkine, and CD38 expression; however, IFN-γ itself inhibits IL-6, IL-8, and eotaxin production in ASM.20 Inflammatory mediators including IL-33, MIP3β, and TSLP have all been shown to be expressed in ASM in severe asthma subjects even when they were taking oral corticosteroids or high doses of inhaled corticosteroids, suggesting some dysfunction in the way that glucocorticoid receptor (GR) is working. Interactions between inflammatory-induced signaling pathways promote a corticosteroid-insensitive state that is believed to be caused, in part, to alternative splicing of a GR-β isoform. This GR isoform acts as a negative inhibitor of glucocorticoid action that is associated with corticosteroid resistance in subjects with asthma.102 Recent evidence in ASM suggest that corticosteroid insensitivity may also be mediated through accumulation of interferon regulatory factor-1 (IRF-1) in cooperation with glucocorticoid receptor interacting protein-1 (GRIP-1), which occurs in a GR-β-independent manner.103 This protein acts as a coactivator for IRF-1, which binds GRIP-1 in excess to GR to induce a corticosteroid-insensitive state.104 Interestingly, there is a strong

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association between IRF-1 polymorphisms and childhood atopic asthma.105 Studies also suggest that expression of IRF-1 increases after viral infection. This, in combination with the suppressive effect of IRF-1 on GR signaling, would potentially explain the reduced efficacy of corticosteroids in viral-induced exacerbations of asthma.106 Additionally, phosphorylation of GR-α at serine 211 was found to be necessary for optimal transcriptional activity. Costimulation of ASM with TNF-α and IFN-γ impaired corticosteroid-dependent phosphorylation of GR-α. Additionally, in ASM derived from severe asthma donors there was a reduction in GR, as well as a 60% reduction in GR nuclear translocation induced by dexamethasone. Dexamethasone also was less effective at suppressing TNF-α-induced NF-κB (p65 subunit) recruitment to the eotaxin 1 promoter in severe asthma-derived ASM compared with nonasthma-derived ASM.

Therapeutics Affecting Airway Smooth Muscle Function in Asthma.  Targeting of the β2AR remains a mainstay in asthma therapy. Short-acting receptor agonists, such as albuterol for rapid relief of bronchoconstriction, or long-acting receptor agonists, such as formoterol and salmeterol for extended control of airway hyperreactivity, are widely used. These therapeutics work through elevating 3’,5’-cAMP levels in ASM cells. Though mainly used to achieve bronchodilation, β2AR agonists like albuterol107 and fenoterol108 also inhibit mitogen-induced ASM proliferation through their ability to induce cAMP generation.109 Musa and colleagues showed that forskolin, a receptor-independent inducer of cAMP generation, suppressed cyclin D1 expression through phosphorylation of CREB in bovine ASM.110 Mobilizers or stabilizers of cAMP have been shown to inhibit TNF-α-stimulated expression of eotaxin and RANTES in ASM as well.111,112 Additionally, TNF-α-induced ICAM-1 and VCAM-1 expression is partially inhibited by increases in cAMP.113 Though effective at bronchodilation, β2AR agonists induce desensitization of the β2 receptor, which decreases the efficacy of such treatment. Modulation of the pathways downstream of the β2AR has been an attractive approach for developing novel therapeutics. Another mainstay therapeutic for asthma is the use of glucocorticoids, including dexamethasone, fluticasone, and budesonide. Corticosteroids exert their effects through the glucocorticoid receptor α isoform binding to DNA sequences in the promoter regions of genes and suppress inflammatory genes.114 Both fluticasone and dexamethasone arrest human ASM in the G1 phase of the cell cycle after thrombin stimulation.115 Apart from the inhibitory effects on ASM growth, steroids also induce secretion of extracellular matrix components from ASM in the presence of profibrotic factors like TGF-β, as well as induce production of growth factors such as CTGF (as reviewed in reference 116). In ASM derived from subjects with asthma, both budesonide and dexamethasone were unable to inhibit ASM proliferation as they do in normal ASM, which may be attributed to the type of matrix components these cells produce.17 One of the more profound effects of corticosteroids is attenuation of inflammatory mediator production and release. In human ASM, dexamethasone inhibited cytokine-induced release of IL-6, RANTES, and eotaxin, as well as COX2 expression.112,117,118 Modulation of contractile responses is a primary therapeutic target to directly affect airway hyperreactivity. A study of asthma and nonasthma subjects examining inspiratory volume noted that nonasthma subjects dilate their airways to baseline measurements after methacholine provocation, whereas those with asthma were not only unable to dilate their airways well during deep inspiration, but the dilation after provocation was severely diminished. The airways of the nonasthma subjects in the study also recovered faster than the airways of asthma subjects. The contrasting results obtained between asthmatic and nonasthmatic individuals may be attributable to characteristic differences in the ASM phenotypes. A study of isolated human ASM noted increased

velocity of force generation in asthma-derived ASM compared with nonasthma-derived ASM (as reviewed in reference 2). Antimuscarinic agents, including ipratropium, tiotropium and aclidinium bromide, serve to block receptors by binding mainly M3 muscarinic receptors (M3R) on ASM and abrogate the actions of acetylcholine/carbachol/ methacholine when applied topically. Ipratropium is a short-acting anticholinergic, whereas aclidinium and tiotropium bromide are long-acting compounds used in the treatment of asthma and COPD. A recent study showed that administration of aclidinium bromide decreased AHR as well as eosinophilia in a murine model of allergic airway disease.119 It has also been demonstrated that M3R agonists synergize with growth factors to augment the proliferative capacity of the factors, with selective M3R antagonists inhibiting this synergism. Treatment of guinea pig ASM with tiotropium reduced allergen-induced increases in α smooth muscle actin and myosin heavy chain (as reviewed in reference 116), thereby modulating proteins involved in smooth muscle contraction as well as antagonizing the M3R. Antagonism of other receptors that can modulate contractile and noncontractile properties of ASM have been used in the treatment of asthma. Clinically, TNF-α has been proposed as a target for asthma therapy; in patients with severe asthma, elevated levels of TNF-α in bronchoalveolar lavage (BAL) fluid have been reported.120 Etanercept, a TNF-α receptor/IgG fusion protein that is a soluble TNF receptor antagonist, significantly improved methacholine-induced AHR, increased FEV1, and improved quality of life scores.121 CysLT levels are increased in subjects with asthma during exacerbation or after antigen challenge.122 In a murine model of virus-induced AHR, montelukast, a CysLT1 receptor antagonist, blocked action of LTD4 and reduced enhancement of AHR, eosinophilia, and mucus hyperproduction upon reinfection with respiratory syncytial virus (RSV),123 thereby illustrating a role for CysLTs not only in allergic airway inflammation but also during viral exposures. Inhibition of 5-LO or antagonists of the CysLT1 receptor improved FEV1 measurements in asthma. A study comparing montelukast/ fluticasone and salmeterol/fluticasone treatments showed that the rate of exacerbations of asthma were essentially identical when comparing the treatment regimens, whereas montelukast/fluticasone treatment was effective in reducing peripheral eosinophil counts.124 In a study of exercise-induced asthma, a montelukast/fluticasone combination significantly reduced the mean maximum percentage decrease in FEV1 as well as time of response to albuterol rescue, as compared against a salmeterol/fluticasone combination.125 The mitogenic effects of LTD4 on ASM may also support the use of montelukast to attenuate ASM hyperplasia that may contribute to reduction in airflow. Recent studies suggested the use of vitamin D as a potential therapeutic for the treatment of corticosteroid-insensitive asthma. Treatment of ASM with vitamin D3 before costimulation with TNF-α and IFN-γ attenuated production of fractalkine and markedly inhibited RANTES release from ASM. Vitamin D also decreased levels of both RANTES and IP-10 after stimulation with TNF-α alone.126 Studies examining the effect of vitamin D treatment of ASM demonstrated that a number of different genes were altered in expression, including those regulating growth, proliferation, survival, and genes potentially involved in airway remodeling.127–129 IL-10, an antiinflammatory cytokine produced by T regulatory cells in response to corticosteroids, is decreased in patients with corticosteroid-insensitive asthma. Vitamin D3 restored T cell IL-10 responses to corticosteroids in subjects with asthma.130 In human ASM, PDGF-induced proliferation was inhibited by vitamin D, as assessed by decreased DNA synthesis and inhibition of PDGF-induced phosphorylation of Rb.131 Additionally, vitamin D was found to attenuate TNF-α-induced MMP-9 expression and activity in human ASM. Both TNF-α and TGF-β-induced collagen III expression and deposition, as well as proliferation of human ASM, were attenuated by vitamin D

CHAPTER 20  Airway Smooth Muscle in Asthma treatment.132 These studies suggest that vitamin D not only has the ability to suppress growth and proliferation of ASM, but also attenuate immunomodulatory and remodeling functions of ASM. In an effort to alleviate bronchoconstriction associated with increased ASM mass in airway diseases like asthma, bronchial thermoplasty has been reported as a therapeutic approach. The technique uses an instrument that is inserted in a bronchoscope to deliver thermal injury to the airways. Surprisingly, the thermal injury has little long-term effect on other structural cells such as epithelial cells while decreasing methacholine responsiveness.133 In a 5-year study performed in the United Kingdom,134 patients receiving bronchial thermoplasty exhibited no decrease in FEV1 and FVC over the 5-year period. Bronchial hyper­ responsiveness to methacholine improved in those receiving thermoplasty in the second and third years of the study. Rates of exacerbations also decreased in the thermoplasty group compared with control patients treated with inhaled corticosteroids. In a randomized, controlled Research in Severe Asthma (RISA) trial, patients receiving bronchial thermoplasty exhibited decreased use of rescue medication, which manifested as a 15.8% increase in improvement in lung function (FEV1), and reported a greater quality of life (asthma control scores) compared with patients on traditional corticosteroid and β2-agonist therapy.135 Another study noted significant reductions in ASM mass, subepithelial basement membrane thickening, and number of submucosal nerves.136 Apart from the effects on decreasing ASM mass in the airways of asthmatics, a study found that in the BAL fluid both TGF-β1 and RANTES were substantially decreased while TRAIL, a TNF-α-related apoptosis-inducing factor, was increased postthermoplasty.137 Trials of this technique are still underway to determine the safety and efficacy in treating patients whose asthma manifests increased ASM mass. It is important to note that asthma is a disease not only of the larger airways, but also of the small airways. Given this information, the long-term effects of bronchial thermoplasty may not ultimately have a significant impact on airway obstruction because of the limitations of its utility in airways within a given individual but may instead modulate the inflammatory capacity of the cell, thereby decreasing airway hyperreactivity from an inflammatory standpoint rather than via an airway structure perspective.138

SUMMARY A plethora of evidence suggests that ASM plays a pivotal role in both the bronchoconstriction, remodeling, and inflammation associated with asthma. The biology of how ASM is affected by its environment, and in turn influences its environment, is both complex and fascinating. Modulating ASM contractile and immunomodulatory responses can differentially alleviate asthma symptoms. Emerging studies examining the pathobiology of ASM in the context of asthma and airway inflammation provide rich avenues of investigation to better understand the role of ASM in modulating acute inflammation, remodeling, and airway contractility.

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CHAPTER 20  Airway Smooth Muscle in Asthma 72. Jude JA, Dileepan M, Subramanian S, et al. miR-140-3p regulation of TNF-alpha-induced CD38 expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2012;303(5):L460–8. 73. Koziol-White CJ, Cooper P, Zhang J, et al. Budesonide reverses IL-13-induced airway hyper-responsiveness but has little effect on β2 agonist response in human small airways. Eur Respir J 2012;40. 74. Shaifta Y, MacKay CE, Irechukwu N, et al. Transforming growth factor-beta enhances Rho-kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1. J Physiol 2018;596(1):47–66. 75. Carr R 3rd, Benovic JL. From biased signalling to polypharmacology: unlocking unique intracellular signalling using pepducins. Biochem Soc Trans 2016;44(2):555–61. 76. Cussac D, Newman-Tancredi A, Duqueyroix D, et al. Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking. Mol Pharmacol 2002;62(3):578–89. 77. De Deurwaerdere P, Navailles S, Berg KA, et al. Constitutive activity of the serotonin2C receptor inhibits in vivo dopamine release in the rat striatum and nucleus accumbens. J Neurosci 2004;24(13):3235–41. 78. Urban JD, Clarke WP, von Zastrow M, et al. Functional selectivity and classical concepts of quantitative pharmacology. J Pharmacol Exp Ther 2007;320(1):1–13. 79. Azzi M, Charest PG, Angers S, et al. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc Natl Acad Sci USA 2003;100(20):11406–11. 80. Wisler JW, DeWire SM, Whalen EJ, et al. A unique mechanism of beta-blocker action: carvedilol stimulates beta-arrestin signaling. Proc Natl Acad Sci USA 2007;104(42):16657–62. 81. Carr R 3rd, Schilling J, Song J, et al. Beta-arrestin-biased signaling through the beta2-adrenergic receptor promotes cardiomyocyte contraction. Proc Natl Acad Sci USA 2016;113(28):E4107–16. 82. Carr R 3rd, Koziol-White C, Zhang J, et al. Interdicting Gq activation in airway disease by receptor-dependent and receptor-independent mechanisms. Mol Pharmacol 2016;89(1):94–104. 83. Berkman N, Krishnan VL, Gilbey T, et al. Expression of RANTES mRNA and protein in airways of patients with mild asthma. Am J Respir Crit Care Med 1996;154(6 Pt 1):1804–11. 84. Ammit AJ, Hoffman RK, Amrani Y, et al. Tumor necrosis factor-alphainduced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. Am J Respir Cell Mol Biol 2000;23(6):794–802. 85. John M, Hirst SJ, Jose PJ, et al. Human airway smooth muscle cells express and release RANTES in response to T helper 1 cytokines: regulation by T helper 2 cytokines and corticosteroids. J Immunol 1997;158(4):1841–7. 86. Koziol-White CJ, Damera G, Panettieri RA Jr. Targeting airway smooth muscle in airways diseases: an old concept with new twists. Expert Rev Respir Med 2011;5(6):767–77. 87. Clarke D, Damera G, Sukkar MB, et al. Transcriptional regulation of cytokine function in airway smooth muscle cells. Pulm Pharmacol Ther 2009;22(5):436–45. 88. Tliba O, Amrani Y, Panettieri RA Jr. Is airway smooth muscle the “missing link” modulating airway inflammation in asthma? Chest 2008;133(1):236–42. 89. Liang KC, Lee CW, Lin WN, et al. Interleukin-1beta induces MMP-9 expression via p42/p44 MAPK, p38 MAPK, JNK, and nuclear factor-kappaB signaling pathways in human tracheal smooth muscle cells. J Cell Physiol 2007;211(3):759–70. 90. Chan V, Burgess JK, Ratoff JC, et al. Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells. Am J Respir Crit Care Med 2006;174(4):379–85. 91. Shan L, Redhu NS, Saleh A, et al. Thymic stromal lymphopoietin receptor-mediated IL-6 and CC/CXC chemokines expression in human airway smooth muscle cells: role of MAPKs (ERK1/2, p38, and JNK) and STAT3 pathways. J Immunol 2010;184(12):7134–43.

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92. Redhu NS, Saleh A, Halayko AJ, et al. Essential role of NF-κB and AP-1 transcription factors in TNF-α-induced TSLP expression in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2011;300(3):L479–85. 93. Peng Q, Matsuda T, Hirst SJ. Signaling pathways regulating interleukin-13-stimulated chemokine release from airway smooth muscle. Am J Respir Crit Care Med 2004;169(5):596–603. 94. Damera G, Zhao H, Wang M, et al. Ozone modulates IL-6 secretion in human airway epithelial and smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2009;296(4):L674–83. 95. Clarke DL, Dakshinamurti S, Larsson AK, et al. Lipid metabolites as regulators of airway smooth muscle function. Pulm Pharmacol Ther 2009;22(5):426–35. 96. Belvisi MG, Saunders MA, Haddad el B, et al. Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: novel inflammatory role of this cell type. Br J Pharmacol 1997;120(5):910–16. 97. Hughes JM, Arthur CA, Baracho S, et al. Human eosinophil-airway smooth muscle cell interactions. Mediators Inflamm 2000;9(2):93–9. 98. Lee CW, Lin CC, Luo SF, et al. Tumor necrosis factor-alpha enhances neutrophil adhesiveness: induction of vascular cell adhesion molecule-1 via activation of Akt and CaM kinase II and modifications of histone acetyltransferase and histone deacetylase 4 in human tracheal smooth muscle cells. Mol Pharmacol 2008;73(5):1454–64. 99. Lazaar AL, Amrani Y, Hsu J, et al. CD40-mediated signal transduction in human airway smooth muscle. J Immunol 1998;161(6):3120–7. 100. Hakonarson H, Kim C, Whelan R, et al. Bi-directional activation between human airway smooth muscle cells and T lymphocytes: role in induction of altered airway responsiveness. J Immunol 2001;166(1): 293–303. 101. Clarke DL, Clifford RL, Jindarat S, et al. TNFα and IFNγ synergistically enhance transcriptional activation of CXCL10 in human airway smooth muscle cells via STAT-1, NF-κB, and the transcriptional coactivator CREB-binding protein. J Biol Chem 2010;285(38): 29101–10. 102. Christodoulopoulos P, Leung DY, Elliott MW, et al. Increased number of glucocorticoid receptor-beta-expressing cells in the airways in fatal asthma. J Allergy Clin Immunol 2000;106(3):479–84. 103. Tliba O, Damera G, Banerjee A, et al. Cytokines induce an early steroid resistance in airway smooth muscle cells: novel role of interferon regulatory factor-1. Am J Respir Cell Mol Biol 2008;38(4):463–72. 104. Bhandare R, Damera G, Banerjee A, et al. Glucocorticoid receptor interacting protein-1 restores glucocorticoid responsiveness in steroid-resistant airway structural cells. Am J Respir Cell Mol Biol 2010;42(1):9–15. 105. Nakao F, Ihara K, Kusuhara K, et al. Association of IFN-gamma and IFN regulatory factor 1 polymorphisms with childhood atopic asthma. J Allergy Clin Immunol 2001;107(3):499–504. 106. Yamada K, Elliott WM, Hayashi S, et al. Latent adenoviral infection modifies the steroid response in allergic lung inflammation. J Allergy Clin Immunol 2000;106(5):844–51. 107. Tomlinson PR, Wilson JW, Stewart AG. Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture. Br J Pharmacol 1994;111(2):641–7. 108. Stewart AG, Tomlinson PR, Wilson JW. Beta 2-adrenoceptor agonist-mediated inhibition of human airway smooth muscle cell proliferation: importance of the duration of beta 2-adrenoceptor stimulation. Br J Pharmacol 1997;121(3):361–8. 109. Tomlinson PR, Wilson JW, Stewart AG. Salbutamol inhibits the proliferation of human airway smooth muscle cells grown in culture: relationship to elevated cAMP levels. Biochem Pharmacol 1995;49(12):1809–19. 110. Musa NL, Ramakrishnan M, Li J, et al. Forskolin inhibits cyclin D1 expression in cultured airway smooth-muscle cells. Am J Respir Cell Mol Biol 1999;20(2):352–8. 111. Hallsworth MP, Twort CH, Lee TH, et al. Beta(2)-adrenoceptor agonists inhibit release of eosinophil-activating cytokines from human airway smooth muscle cells. Br J Pharmacol 2001;132(3):729–41.

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112. Pang L, Knox AJ. Regulation of TNF-alpha-induced eotaxin release from cultured human airway smooth muscle cells by beta2-agonists and corticosteroids. FASEB J 2001;15(1):261–9. 113. Panettieri RA Jr, Lazaar AL, Pure E, et al. Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF-alphainduced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. J Immunol 1995;154(5):2358–65. 114. Leung DY, Bloom JW. Update on glucocorticoid action and resistance. J Allergy Clin Immunol 2003;111(1):3–22, quiz 3. 115. Fernandes D, Guida E, Koutsoubos V, et al. Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of the extracellular-regulated kinases in human cultured airway smooth muscle. Am J Respir Cell Mol Biol 1999;21(1):77–88. 116. Ammit AJ, Burgess JK, Hirst SJ, et al. The effect of asthma therapeutics on signalling and transcriptional regulation of airway smooth muscle function. Pulm Pharmacol Ther 2009;22(5):446–54. 117. Ammit AJ, Lazaar AL, Irani C, et al. Tumor necrosis factor-alphainduced secretion of RANTES and interleukin-6 from human airway smooth muscle cells: modulation by glucocorticoids and beta-agonists. Am J Respir Cell Mol Biol 2002;26(4):465–74. 118. Vlahos R, Stewart AG. Interleukin-1alpha and tumour necrosis factor-alpha modulate airway smooth muscle DNA synthesis by induction of cyclo-oxygenase-2: inhibition by dexamethasone and fluticasone propionate. Br J Pharmacol 1999;126(6):1315–24. 119. Damera G, Jiang M, Zhao H, et al. Aclidinium bromide abrogates allergen-induced hyperresponsiveness and reduces eosinophilia in murine model of airway inflammation. Eur J Pharmacol 2010;649(1–3):349–53. 120. Gruenberg D, Busse W. Biologic therapies for asthma. Curr Opin Pulm Med 2010;16(1):19–24. 121. Howarth PH, Babu KS, Arshad HS, et al. Tumour necrosis factor (TNFalpha) as a novel therapeutic target in symptomatic corticosteroid dependent asthma. Thorax 2005;60(12):1012–18. 122. Hallstrand TS, Henderson WR Jr. An update on the role of leukotrienes in asthma. Curr Opin Allergy Clin Immunol 2010;10(1):60–6. 123. Han J, Jia Y, Takeda K, et al. Montelukast during primary infection prevents airway hyperresponsiveness and inflammation after reinfection with respiratory syncytial virus. Am J Respir Crit Care Med 2010;182(4):455–63. 124. Bjermer L, Bisgaard H, Bousquet J, et al. Montelukast and fluticasone compared with salmeterol and fluticasone in protecting against asthma exacerbation in adults: one1 year, double blind, randomised, comparative trial. BMJ 2003;327(7420):891. 125. Fogel RB, Rosario N, Aristizabal G, et al. Effect of montelukast or salmeterol added to inhaled fluticasone on exercise-induced

bronchoconstriction in children. Ann Allergy Asthma Immunol 2010;104(6):511–17. 126. Banerjee A, Damera G, Bhandare R, et al. Vitamin D and glucocorticoids differentially modulate chemokine expression in human airway smooth muscle cells. Br J Pharmacol 2008;155(1): 84–92. 127. Bosse Y, Maghni K, Hudson TJ. 1alpha,25-dihydroxy-vitamin D3 stimulation of bronchial smooth muscle cells induces autocrine, contractility, and remodeling processes. Physiol Genomics 2007;29(2):161–8. 128. Song Y, Qi H, Wu C. Effect of 1,25-(OH)2D3 (a vitamin D analogue) on passively sensitized human airway smooth muscle cells. Respirology 2007;12(4):486–94. 129. Himes BE, Koziol-White C, Johnson M, et al. Vitamin D modulates expression of the airway smooth muscle transcriptome in fatal asthma. PLoS ONE 2015;10(7):e0134057. 130. Xystrakis E, Kusumakar S, Boswell S, et al. Reversing the defective induction of IL-10-secreting regulatory T cells in glucocorticoid-resistant asthma patients. J Clin Invest 2006;116(1):146–55. 131. Damera G, Fogle HW, Lim P, et al. Vitamin D inhibits growth of human airway smooth muscle cells through growth factor-induced phosphorylation of retinoblastoma protein and checkpoint kinase 1. Br J Pharmacol 2009;158(6):1429–41. 132. Britt RD Jr, Faksh A, Vogel ER, et al. Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells. J Cell Physiol 2015;230(6):1189–98. 133. Sandstrom T. Effects of pharmacological and non-pharmacological interventions. Clin Respir J 2010;4(Suppl. 1):41–8. 134. Thomson NC, Rubin AS, Niven RM, et al. Long-term (5 year) safety of bronchial thermoplasty: Asthma Intervention Research (AIR) trial. BMC Pulm Med 2011;11:8. 135. Pavord ID, Cox G, Thomson NC, et al. Safety and efficacy of bronchial thermoplasty in symptomatic, severe asthma. Am J Respir Crit Care Med 2007;176(12):1185–91. 136. Pretolani M, Bergqvist A, Thabut G, et al. Effectiveness of bronchial thermoplasty in patients with severe refractory asthma: clinical and histopathologic correlations. J Allergy Clin Immunol 2017;139(4):1176–85. 137. Denner DR, Doeing DC, Hogarth DK, et al. Airway inflammation after bronchial thermoplasty for severe asthma. Ann Am Thorac Soc 2015;12(9):1302–9. 138. d’Hooghe JNS, Ten Hacken NHT, Weersink EJM, et al. Emerging understanding of the mechanism of action of bronchial thermoplasty in asthma. Pharmacol Ther 2018;181:101–7.

CHAPTER 20  Airway Smooth Muscle in Asthma

312.e1

SELF-ASSESSMENT QUESTIONS 1. Airway smooth muscle growth and proliferation is modulated by the following receptors: a. Platelet-derived growth factor receptor b. CysLT receptors c. Histamine receptors d. None of the above e. All of the above 2. Contraction of airway smooth muscle occurs through all of the following except: a. Intracellular calcium increases b. Rho-kinase-dependent mechanisms c. CD38 activation d. Growth factor receptor activation e. G-protein coupled receptor activation 3. Generation of cyclic adenosine monophosphate (cAMP) affects airway smooth muscle (ASM) by altering all of the following except: a. Proliferation b. Contraction

c. Growth factor receptor expression d. Inflammatory mediator release e. Adhesion molecule expression 4. Airway smooth muscle growth is driven primarily by the following pathways: a. PI3K p110 α,β b. PI3K p110 δ c. MAPK/ERK d. Both a and b e. Both a and c 5. Release of inflammatory mediators by airway smooth muscle (ASM) is attenuated by: a. Cyclic adenosine monophosphate generating agents b. Vitamin D c. Glucocorticoids d. Bronchial thermoplasty e. All of the above

21  Pathophysiology of Allergic Inflammation Peter J. Barnes

CONTENTS Introduction, 313 Inflammatory Cells, 314 Nonatopic Allergic Disease, 316 Inflammatory Mediators, 318 Structural Cells, 320

SUMMARY OF IMPORTANT CONCEPTS • Allergic inflammation is due to a complex interplay between several inflammatory cells, including mast cells, basophils, lymphocytes, dendritic cells, eosinophils, and sometimes neutrophils. • Activated inflammatory cells produce multiple inflammatory mediators, including lipids, purines, cytokines, chemokines, and reactive oxygen species. • Allergic inflammation affects several key target cells: epithelial cells, fibroblasts, vascular cells, and airway smooth muscle cells, which become an important source of inflammatory mediators. • Sensory nerves are sensitized and activated during allergic inflammation and produce symptoms. Allergic inflammatory responses are orchestrated by several transcription factors, particularly NF-κB and GATA3. Inflammatory genes are also regulated by epigenetic mechanisms, including DNA methylation and histone modifications. • There are several endogenous antiinflammatory mechanisms, including antiinflammatory lipids and cytokines, that may be defective in allergic disease, thus amplifying and perpetuating the inflammation. • Better understanding of the pathophysiology of allergic inflammation has identified new therapeutic targets, but developing effective novel therapies has been challenging. Antibodies that target specific cytokines involved in allergic inflammation have now been introduced into clinical practice and are effective in select patients.

INTRODUCTION All allergic diseases are characterized by a distinct pattern of inflammation that is largely driven via IgE-dependent mechanisms. Genetic factors have an important influence on whether atopy develops, and several genes have now been identified in asthma and other allergic diseases using genome-wide association studies (GWAS), although few have been reproduced between studies or between different ethnic groups.1 So far the novel genes identified by over 20 GWAS have provided few new insights into the pathophysiology of allergic diseases, although Th2 cytokine genes have been consistently identified. Recent GWAS analysis of patients with atopic diseases (asthma, allergic rhinitis, and atopic dermatitis) have identified at least 130 risk variants, and

Neural Mechanisms, 321 Transcription Factors, 323 Antiinflammatory Mechanisms in Allergy, 325 Therapeutic Implications, 325

these indicate aberrant immunity, explaining why these diseases may often occur together.2 Environmental factors appear to be more important in determining whether an atopic individual develops a particular allergic disease, although genetic factors may exert an influence on how severely the disease is expressed and the extent of the allergic inflammatory response.3 It is likely that epigenetic mechanisms may play an important role in the heritability of allergic diseases. The inflammatory process has several common characteristics shared between different allergic diseases, including asthma, allergic rhinitis/ rhinosinusitis, and atopic dermatitis (eczema).4 Allergic inflammation is characterized by IgE-dependent activation of mucosal mast cells and an infiltration of eosinophils that is orchestrated by increased numbers of activated CD4+ T helper (Th2) lymphocytes as well as group 2 innate lymphoid cells (ILC2).5 However, in patients with more severe disease other cells, such as neutrophils, Th1, Th17, and CD8+ (cytotoxic) lymphocytes may also be involved. The clinical differences between these diseases is largely determined by anatomic differences and the interaction between allergic inflammation and structural cells, such as airway smooth muscle cells in the lower airways resulting in bronchoconstriction, vasodilation in the upper airways leading to nasal blockage and rhinorrhea, and activation of keratinocytes in the skin. The fact that there are common characteristics of allergic diseases suggests that it may be possible to treat these common diseases with single agents, particularly as they often coexist. This chapter focuses on the components of allergic inflammation (inflammatory and immune cells, inflammatory mediators, inflammatory effects), and how allergic inflammation is orchestrated. No single cell or mediator can account for all the features of allergic disease, and different cells and mediators may be more important in one manifestation of allergic disease than another. For example, histamine clearly plays a key role in rhinitis, yet has a relatively minor role in asthma, as judged by the differences in efficacy of antihistamines between these conditions. The allergic inflammatory response has evolved from the inflammatory process mounted in response to parasite and worm infections.6 This inflammatory response not only provides an acute defense against these organisms but is also involved in healing and restoration of normal function after tissue damage as a result of infection or toxins. Helminth

313

314

SECTION A  Basic Sciences Underlying Allergy and Immunology Inhaled allergens

Allergens Sensitizers Viruses Air pollutants??

Epithelial cells SCF

Mast cell Inflammation “Chronic eosinophilic bronchitis”

Airway hyperresponsiveness

Symptoms

Cough Chest tightness

Wheeze Dyspnea

Triggers Allergens Exercise Cold air SO2 Particulates

Histamine cys-LTs PGD2

TSLP IL-33

Bronchoconstriction

IL-9

IgE Fig. 21.1  Inflammation and symptoms of asthma. Inflammation in the airways of asthmatic patients leads to airway hyperresponsiveness and symptoms. SO2, Sulfur dioxide.

CCL11

IL-4, IL-13

Dendritic cell

CCL17 CCL22 CCR4

↓Tregs Th2 cell IL-5

CCR3

B lymphocyte Eosinophils

infections appear to provide protection against the development of allergic diseases, but the nature of this protective immunity is not yet clear. Parasite-derived proteins may have some therapeutic potential in the treatment of allergic diseases.7 In allergic diseases the inflammatory response is activated inappropriately so that it becomes harmful rather than protective. Allergens, such as house dust mite and pollen proteins, activate mast cells and induce eosinophil inflammation, which has evolved to kill the invading parasites. This process would therefore be self-limiting, but in allergic disease the noninfective inciting stimulus persists and the normally acute inflammatory response becomes converted into a persistent inflammation that may have structural consequences in the airways and skin. The relationship between inflammation and clinical symptoms of allergy is not yet well understood. There is evidence that the degree of inflammation is related to airway hyperresponsiveness (AHR), as measured by histamine or methacholine challenge. AHR is an exaggerated airway narrowing in response to many stimuli that is characteristic of asthma, and the degree of AHR relates to asthma symptoms. Inflammation of the airways may increase airway responsiveness, which thereby allows triggers that would not narrow the airways to do so.8 But inflammation may also directly lead to an increase in asthma symptoms, such as cough and chest tightness, by activation of airway sensory nerve endings (Fig. 21.1). In rhinitis nasal blockage is related to vasodilation and nasal discharge is related to plasma exudation and mucus secretion. The characteristic sneezing is a manifestation of hypersensitivity of sensory nerves and is the equivalent of cough in asthma. Skin disruption of the epithelial barrier leads to infection and hypersensitivity of sensory nerves that results in itching.

INFLAMMATORY CELLS Many inflammatory and structural cells are involved in allergic inflammation (Fig. 21.2), but it is not clear how these cells interact or how the inflammatory process is maintained.9

Mast Cells Mast cells appear to play a critical role in initiating allergic inflammation in the airways because they are directly activated by allergens through an IgE-dependent mechanism. This leads to the rapid release of preformed and synthesized mediators that result in bronchoconstriction,

Fig. 21.2  Inflammation in allergy. Inhaled allergens activate sensitized mast cells by cross-linking surface-bound IgE molecules to release several bronchoconstrictor mediators, including cysteinyl-leukotrienes (cys-LT) and prostaglandin D2 (PGD2). Epithelial cells release stem cell factor (SCF), which is important for maintaining mucosal mast cells at the airway or skin surface. Allergens are processed by myeloid dendritic cells, which are conditioned by thymic stromal lymphopoietin (TSLP) secreted by epithelial cells and mast cells to release the chemokines CC-chemokine ligand 17 (CCL17) and CCL22, which act on CC-chemokine receptor 4 (CCR4) to attract T helper 2 (Th2) cells. Th2 cells have a central role in orchestrating the inflammatory response in allergy through the release of interleukin-4 (IL-4) and IL-13 (which stimulate B cells to synthesize IgE), IL-5 (which is necessary for eosinophilic inflammation), and IL-9 (which stimulates mast-cell proliferation). Epithelial cells release CCL11, which recruits eosinophils via CCR3. Patients with allergic disease may have a defect in regulatory T cells (Treg), which may favor further Th2-cell activation.

vasodilation, and plasma exudation, thus leading to symptoms of asthma (wheezing and dyspnea) or allergic rhinitis (nasal blockage and secretions).10 Mast cell activation therefore plays a key role in generating the symptoms of asthma and rhinitis, because mast cells may be activated by many of the triggers causing symptoms, including allergens, cold air, and osmolality changes (which mediate exercise-induced asthma). Mucosal mast cells are maintained at mucosal surfaces in allergic diseases by cytokines such as IL-9 and stem cell factor (SCF).11 SCF interacts with mast cells through its tyrosine kinase receptor c-Kit, and several c-Kit inhibitors, based on imatinib, are in development for asthma and other mast cell–mediated diseases.12 The presence of mast cells in the airway smooth muscle has been linked to AHR.13 Although mast cells account for the early response to allergen in the airways and skin, it is not clear how important they are for the development of late responses and for the chronic inflammatory changes that occur. Allergen-derived T-cell peptides derived from Fel d1 have been shown to induce isolated late responses and AHR without any mast cell activation or eosinophil infiltration in the lower airways through direct activation of CD4+ T cells.9 Mast cells also release several proteases, of which tryptase and chymase are mast cell–specific.14 Tryptase is proinflammatory and may contribute to AHR in asthma, whereas chymase is profibrotic through the activation of transforming growth factor (TGF) β.

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CHAPTER 21  Pathophysiology of Allergic Inflammation Allergen

Macrophages Macrophages, which are derived from blood monocytes, may traffic into the airways in asthma and may be activated by allergen via lowaffinity IgE receptors (FcεRII). The enormous immunologic repertoire of macrophages allows these cells to produce many different products, including a large variety of cytokines that may orchestrate the inflammatory response. Macrophages have the capacity to initiate a particular type of inflammatory response via the release of a certain pattern of cytokines. Heterogeneity of macrophages is now recognized in humans, with some macrophages having a more proinflammatory profile (M1-like), whereas others are antiinflammatory, promote tissue repair, and are more phagocytic (M2-like).15 Alternatively activated macrophages, which are M2-like, driven by IL-4 and IL-13 that secrete IL-13, are described in murine models of asthma, but it is not yet clear that they are important in human asthma, and there are no distinct phenotypic biomarkers.16 Alveolar macrophages normally have a suppressive effect on lymphocyte function, but this may be impaired in asthma after allergen exposure. One antiinflammatory protein secreted by macrophages is IL-10, and its secretion is reduced in alveolar macrophages from patients with asthma.17 Macrophages from normal subjects also inhibit the secretion of IL-5 from T lymphocytes, probably via the release of IL-12, but this is defective in patients with allergic asthma. Macrophages may therefore play an important antiinflammatory role by preventing the development of allergic inflammation. Macrophages may also act as antigen-presenting cells, which process allergen for presentation to T lymphocytes, although alveolar macrophages are far less effective in this respect than dendritic cells.

Dendritic Cells Dendritic cells are specialized macrophage-like cells that have the ability to induce a T cell–mediated immune response and therefore play a critical role in the development of allergic diseases. Dendritic cells in the respiratory tract form a network that is localized to the epithelium and act as very effective antigen-presenting cells.18 Dendritic cells in the skin, known as Langerhans cells, play a critical role in sensitization responses. It is likely that they play a very important role in the initiation and maintenance of allergen-induced responses in allergic diseases. Myeloid but not plasmacytoid dendritic cells have been shown to underlie AHR in experimental models of allergic asthma. Dendritic cells take up allergens, process them to peptides, and migrate to local lymph nodes, where they present the allergenic peptides to uncommitted T lymphocytes, and with the aid of costimulatory molecules, such as B7.1 (CD80), B7.2 (CD86), and CD40, they program the production of allergen-specific T cells. Animal studies have demonstrated that myeloid dendritic cells are critical to the development of Th2 cells and eosinophilia. Immature dendritic cells in the respiratory tract promote Th2 cell differentiation and require cytokines such as IL-12 and interferon-γ to promote the normally preponderant Th1 response. The cytokine thymic stromal lymphopoietin (TSLP) released from epithelial cells in asthmatic patients programs dendritic cells to release chemokines that attract Th2 cells into the airways.19

Lymphocytes Th2 cells play an important role in orchestrating allergic inflammation through the release of cytokines that include IL-4, IL-5, IL-9, and IL-13 (Fig. 21.3).20 Th2 cells are recruited and activated at the sites of allergic inflammation, and a major focus of research has been to understand how topical allergens regulate Th2 cells via antigen-presenting cells. Dendritic cells are the main antigen-presenting cells (APC) that process allergens and present T cell peptides to naïve T cells and play a critical role in the recruitment and activation of Th2 cells through the secretion

MHCII allergen peptide TCR

CD86 (B7-2) CD28

Th9 IL-9

TGF-β

Th0

IL-12 IL-18

Mast cell

IL-4 IgE Th2 IFN-γ

IL-17A IL-17F Th1 IL-22

IL-2 IFN-γ Th17

–

IL-4 IL-13

–

IL-10 TGF-β

Bε

IL-5 –

↓Tregs

Eosinophil

Fig. 21.3  T lymphocytes in allergy. Asthmatic inflammation is characterized by a preponderance of T helper 2 (Th2) lymphocytes over T helper 1 (Th1) cells. Regulatory T cells (Treg) have an inhibitory effect, whereas T helper 17 (Th17) cells have a proinflammatory effect. Th9 cells secrete IL-9 important for mast cell differentiation. Bε, B cell producing epsilon transcripts and IgE; MHCI, Class 1 major histocompatibility complex; IL, interleukin; IFN-γ, interferon gamma; TGF-β, transforming growth factor beta; IgE, immunoglobulin E, Th0, uncommitted T cell; TGF, transforming growth factor.

of chemokines CCL17 and CCL22. IL-4 is necessary for differentiation of Th2 cells but is not secreted by dendritic cells, so accessory cells may be needed as a source of IL-4. There is increasing evidence that basophils, which are c-Kit negative, express FcεR1, MHC Class II, and costimulatory molecules CD80/CD86 and may play a role in antigen recognition and processing, and when recruited to lymph nodes are able to induce Th2 cell differentiation through the release of IL-4. Other cells also have the capacity to process and present allergens, including mast cells, macrophages, and eosinophils. Innate lymphoid cells also play an important role in regulating allergic inflammation and are regulated by epithelial cytokines such as TSLP, IL-25, and IL-33 (alarmins).5,21,22 ILC2 cells may play an important role in nonallergic asthma, because they are not regulated via T cell receptors. Although type-2 immunity (T2) predominates in allergic diseases, and T2 cytokines play a key role in the pathophysiology of these diseases, other types of T cell may also play a role, particularly in more severe disease. Th1 cells and CD8+ cells that secrete T2 cytokines (Tc2 cells) have also been implicated in allergic disease, as have natural killer (NK) cells and invariant natural killer T (NKT) cells.23 Th17 cells are also increased in patients with severe asthma and may orchestrate neutrophilic inflammation by inducing the release of CXCL8 from airway epithelial cells.24,25 Although Th17 cells produce IL-22, a distinct set of CD4+ IL-22–producing cells (Th22) has been described in allergic inflammatory diseases.26 A distinct population of CD4+ cells that produce IL-9 (Th9) are increased in asthma and play a role in maintaining mast cells in the airways.27 B-lymphocytes produce IgE in allergic asthma under the direction of IL-4 and IL-13 but B cells producing IgE locally in the airways have

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also been identified, even in nonatopic asthmatics.28 B cell activating factor of the TNF family (BAFF) is increased after allergen challenge in asthmatic patients and may play a role in increasing IgE production.29 B lymphocytes are also increased in the lungs of patients with chronic obstructive pulmonary disease (COPD), particularly in severe disease. B cells are organized into lymphoid follicles, which are located in peripheral airways and lung parenchyma.30 BAFF, an important regulator of B cell function and hyperplasia, is increased in lymphoid follicles of COPD patients.31 Regulatory T cells (Tregs) may play an important role in suppressing allergic inflammation. Several types of Tregs are now recognized, including CD4+CD25+ innate Tregs that express the transcription factor FoxP3 and inducible Tregs. They may suppress inflammation through secretion of IL-10 or by inducing IL-10 secretion for bystander cells, as well as direct inhibition of cells, such as dendritic cells, by cellcell contact. There is some evidence that Treg function is impaired in patients with allergic diseases and that Treg function is enhanced by specific immunotherapy.32

Eosinophils Eosinophils are common to allergic inflammation at different sites and are orchestrated by T2 cells through the release of IL-5.33 Blocking antibodies to IL-5 and its receptor cause a profound and prolonged reduction in circulating and sputum eosinophils but is not associated with reduced AHR or asthma symptoms.34 Several anti-IL-5 antibodies have now been approved to treat severe eosinophilic asthma and consistently reduce exacerbations but have only modest effects on symptoms and no effect on AHR.35 Eosinophils contribute to the pathophysiology of asthma through the release of cysteinyl-leukotrienes (cys-LT) but are less important as a source of these mediators than mast cells. They may also function as APCs and also produce several Th1 and Th2 cytokines. Eosinophils express high levels of TGF-β, and this has been linked to the characteristic subepithelial fibrosis in asthma, which has also been observed in patients with eosinophilic bronchitis who present with chronic cough and no AHR.36 Cationic proteins released from eosinophils are important in helminth killing but may also be profibrotic and lead to shedding of epithelial cells in asthma, although this does not occur in nasal epithelial cells.

Neutrophils The role of neutrophils in allergic diseases is still uncertain and remains poorly explored. Patients with severe asthma are more likely to have infiltration of neutrophils in bronchial biopsies and sputum, but increased neutrophils in sputum may be seen even in mild asthma. Increased sputum neutrophils are also found in smoking asthmatics and during virally induced exacerbations.37 Patients with neutrophilic asthma appear to be less responsive to corticosteroids, and high doses of corticosteroids may increase neutrophils by reducing apoptosis.38 The mechanism for increased neutrophils may be related to secretion of several neutrophil chemotactic factors, including LTB4, CXCL1, and CXCL8, and may be driven by Th17 cells through the secretion of IL-17A and IL-17F.24 CXCR2 antagonists, which inhibit neutrophilic inflammation in the airways, have no clinical benefit in patients with severe neutrophilic asthma, however, which questions the importance of neutrophils in severe asthma.39,40

Viruses O2, NO2

Allergens

Viruses FcεRII

Macrophage TNF-α, IL-1β, IL-6

Airway epithelial cells GM-CSF CCL11 CCL5 CCL13

Eosinophil survival, chemotaxis

CCL5 IL-16 CCL17, CCL22

Lymphocyte activation

PDGF EGF

Smooth muscle hyperplasia

PDGF FGF IGF-1 IL-11

Fibroblast activation

Fig. 21.4  Airway epithelial cells and inflammation in asthma. Airway epithelial cells may play an active role in asthmatic inflammation through the release of many inflammatory mediators, cytokines, chemokines, and growth factors. CCL, C-C chemokine; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; IL, interleukin; PDGF, platelet-derived growth factor; TNF-α, tumor necrosis factor alpha.

eosinophils (approximately 10 : 1 ratio), and their functional role is unknown.42 Basophils are increased in larger numbers in rhinitis and atopic dermatitis, where their role is more firmly established.43

Platelets There is some evidence for the involvement of platelets in the pathophysiology of allergic diseases, because platelet activation may be observed and there is evidence for activation of platelets in the circulation and in bronchial biopsies of asthmatic patients.44 Platelets from patients with asthma release the chemokine CCL5 (RANTES) and constitutively express the alarmin IL-33. There appears to be an interaction between platelets and eosinophils, with P-selectin from platelets activating β1integrins on eosinophils to adhere to endothelial cells and migrate into tissues.45

Structural Cells Structural cells of the airways, including epithelial cells, endothelial cells, fibroblasts, and airway smooth muscle cells may also be an important source of inflammatory mediators, such as cytokines and lipid mediators in asthma.19,46 Indeed, because structural cells far outnumber inflammatory cells, they may become the major source of mediators driving chronic inflammation in asthma and other allergic diseases. Epithelial cells may have a key role in translating inhaled environmental signals into an airway inflammatory response and are probably a major target cell for inhaled corticosteroids (Fig. 21.4).

Basophils

NONATOPIC ALLERGIC DISEASE

The role of basophils in asthma is uncertain, because these cells have previously been difficult to detect by immunocytochemistry. Using a basophil-specific marker, a small increase in basophils has been documented in the airways of asthmatic patients, with an increased number after allergen challenge.41 However, these cells are far outnumbered by

Some patients with asthma, rhinitis, rhinosinusitis, and eczema have negative skin prick responses to common inhalant allergens and no allergen-specific circulating IgE, yet have clinical features very similar to patients with allergic disease.47 These nonatopic forms of “allergic” disease might give insights into the nature of allergic inflammation.

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(exotoxins), which function as superantigens that have the capacity to stimulate T and B cell proliferation directly (Fig. 21.5). They are also able to induce class-switching to IgE and the production of allergenspecific IgE in mucosal B cells. Specific IgE antibodies against Staphylococcal enterotoxins (termed “superallergens”) have been found in patients with severe asthma, aspirin-sensitive asthma, and nasal polyposis and are able to sensitize mast cells and dendritic cells.52 S. aureus is also capable of invading mast cells and causing the release of cytokines.53 It is therefore possible that patients with nonatopic allergic diseases may in some way be susceptible to colonization of the lower airways with S. aureus, which through the local release of superantigens drives an allergic inflammatory response and local IgE synthesis. Superantigens are associated with more severe disease as the antiinflammatory response to corticosteroids is reduced.47 It is also possible that S. aureus super­ antigens lead to chronic rhinosinusitis, which then affects the lower

Furthermore, the allergic pattern of inflammation with activated mast cells, eosinophils, and Th2 cells is found in the tissues.48 Total circulating IgE is elevated in around 30% of patients with nonatopic (intrinsic) asthma.49 There is evidence for local IgE production in the airways of patients with nonatopic asthma, with local expression of epsilon germline transcripts. Local IgE expression is also seen in chronic rhinosinusitis and nasal polyps.50 This suggests that nonatopic allergic disease may be driven by an unidentified allergen, an endogenous allergen, or an infective agent.

Role of Infective Agents Staphylococcus aureus is thought to be an important amplifying mechanism in allergic and nonallergic atopic dermatitis and chronic rhinosinusitis with nasal polyps but might also be important in asthma.51 S. aureus is able to invade epithelial cells and release its enterotoxins

S. aureus

Epithelial damage exposes cytokeratin-18 leading to IgG autoantibodies

Epithelial cells

Sag IgE switching

Bε cell

T cell

Clonal expansion

Treg

Th17

IL-4 IL-3

IgE

Sag FcεRI Mast cell

IL-4

Th2

CD8 +

IL-5

Bronchoconstriction Eosinophils

Neutrophils

Fig. 21.5  Possible microbial mechanism of intrinsic asthma. Invasion of airway epithelial cells by Staphylococcus aureus (and other microorganisms) causes the release of staphylococcal superantigens (Sag) (and other superantigens), which act on airway B lymphocytes to cause class-switching with the local production of polyclonal IgE, together with IgE directed against SSa (which acts as a “superallergen”). This causes mast cell activation and release of bronchoconstrictor mediators and sensitizes mast cells to activation by triggers, such as exercise and cold air (via changes in surface osmolality). SSa also causes polyclonal expansion of T cells, resulting in increased T helper 2 (Th2) cells and CD8+ cells. Th2 cells release IL-4, which induces the expression of high-affinity IgE receptors (FcεRI) in mast cells and IgE synthesis by B cells (together with IL-13). IL-5 promotes eosinophilic inflammation. SSa may also reduce corticosteroid responsiveness in T cells, resulting in the need for higher doses to control asthma. SSa also inhibits the function of regulatory T cells (Treg), resulting in further enhancement of Th2 and CD8+ cells. SSa may also activate Th17 cells, leading to neutrophilic inflammation. Staphylococcal infection of epithelial cells may also lead to damage and exposure of epitopes on epithelial structural proteins, such as cytokeratin-18, resulting in the formation of cytotoxic IgG antibodies, which further damage epithelial cells, making them more susceptible to further microbial colonization.

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respiratory tract without the need for direct infection of the lower airways.

Superantigens Superantigens are also derived from other bacteria (including Mycoplasma), viruses, parasites and fungi, and all of them are immuno­ stimulatory, acting as Vβ-restricted extremely potent polyclonal T cell mitogens.54 They bind MHC class-II molecules without any prior antigen processing by antigen-presenting cells and stimulate large numbers of T cells on the basis of epitopes specified by this receptor through their unique ability to cross-link MHC class II and the β subunit of the T cell receptor (TCR), forming a tight trimolecular complex. They also appear to cause class-switching in B cells by binding to immunoglobulins, resulting in clonal expansion and expression of polyclonal IgE and specific IgE directed against the superantigen. Some superantigens may even become incorporated into the genome and thus may provide continuous endogenous stimulation. Nasal and inhaled Staphylococcal enterotoxin B (SEB) induces lymphocytic inflammation and eosinophilia in the lungs of mice with increased production of IL-4, together with AHR in both IgE and non-IgE producing strains, demonstrating the capacity of superantigens to induce allergic inflammation independently of any allergen. Inhaled SEB also enhances the eosinophilic inflammatory response to inhaled allergen in sensitized mice. In nasal polyp tissue there are IgE antibodies to SEB, and these are able to activate mast cells.55 Staphylococcal enterotoxin A also results in lung inflammation characterized by increased CD8+ cells and increased secretion of interferon (IFN)-γ.56 These animal studies demonstrate the ability of small concentrations of locally applied super­ antigens to induce complex inflammatory responses in the lungs and suggest a mechanism for intrinsic or nonallergic asthma. Inhaled bacteria may invade airway epithelial cells and result in the release of various superantigens, which may directly activate Th2 cells to release IL-4 and other T2 cytokines, as well as causing class-switching of B cells in the airways to release IgE locally, which then sensitizes mast cells so that they are more easily activated. SEB stimulates the release of several cytokines with a Th2 cell bias, as well as mast cell mediators from nasal polyp tissue in vitro and to a greater extent than from normal nasal tissue, indicating some sort of sensitization to the effects of superantigens.57 There is evidence for local clonal expansion of airway T cells in patients with intrinsic asthma compared with circulating T cells, suggesting that this could be induced by local superantigens.58 Staphylococcal superantigens also inhibit the immunosuppressive activity of Treg cells and may therefore amplify the activity of Th2 and CD8+ cells.59 SEB inhibits CD4+CD25+ Tregs by inducing the protein glucocorticoid-induced TNF receptor–related protein-ligand (GITRL) in monocytes.60 Superantigens induce corticosteroid resistance by activating mitogen-activated protein (MAP) kinase cascades and through the increased expression of an isoform of the glucocorticoid receptor GRβ, which may act as a decoy to block the action of corticosteroids.61 Finally, SEB may stimulate Th17 cells directly.62

Role of Autoantibodies Another possible mechanism of activating IgE signaling without an exogenous allergen may be through the production of autoantibodies that activate this pathway.63 This would also be consistent with the fact that intrinsic asthma often has a late onset and is more commonly seen in women. Antinuclear antibodies are more common in patients with asthma than nonasthmatics, but there is no difference between allergic and nonallergic asthmatics, although patients with aspirin-sensitive asthma have a higher frequency of these antibodies. However, there is no increase in autoantibodies against double-stranded DNA or antineutrophil cytoplasmic antibodies in asthma. Autoantibodies to FcεRI

Inflammatory cells Mast cells Eosinophils Th2 cells Th17 cells Basophils Neutrophils Platelets Structural cells Epithelial cells Smooth muscle cells Endothelial cells Fibroblasts Airway nerves

Mediators Histamine Leukotrienes Prostanoids PAF Kinins Purines Endothelins Nitric oxide Cytokines Chemokines Growth factors

Effects Bronchospasm Plasma exudation Mucus secretion AHR Structural changes

Fig. 21.6  Multiple cells, mediators, and effects. Many cells and mediators are involved in asthma and lead to several effects on the airways. AHR, Airway hyperresponsiveness; PAF, platelet activating factor; Th2, T helper 2 cells.

have been detected in the serum of patients with asthma (approximately 35% compared with 10% in nonasthmatics) but do not appear to be more common in intrinsic compared with extrinsic asthma.64 Circulating antibodies to cytokeratin-18 and α-enolase, which occur in epithelial cells, have been described in patients with intrinsic asthma and more severe asthma.65 IgG autoantibodies from patients with intrinsic asthma have cytotoxic effects on epithelial cells, whereas these antibodies are not found in extrinsic asthma.66 It is possible that these autoantibodies damage airway epithelial cells so that superantigen-producing organisms are able to more easily infect the airway surface.

INFLAMMATORY MEDIATORS Very many different mediators have been implicated in allergic disease, and they may have a variety of effects on the airways or skin, which account for all of the pathologic features of allergic diseases (Fig. 21.6). Mediators such as histamine, prostaglandins, leukotrienes, and kinins contract airway smooth muscle, increase microvascular leakage, increase airway mucus secretion, and attract other inflammatory cells. Because each mediator has many effects, the role of individual mediators in the pathophysiology of allergic diseases is often unclear. The multiplicity and redundancy of effects of mediators makes it unlikely that preventing the synthesis or action of a single mediator will have a major clinical impact in allergic diseases. However, some mediators may play a more important role if they are upstream in the inflammatory cascade and trigger a sequence of downstream events. The effects of single mediators can only be evaluated through the use of potent specific receptor antagonists or mediator synthesis inhibitors. The role of mediators may differ between allergic diseases. Thus antihistamines have a useful clinical effect in allergic rhinitis, whereas they are not useful in the treatment of asthma. On the other hand, antileukotrienes have clinical effects in asthma but appear to be less useful in rhinitis and atopic dermatitis.67

Lipid Mediators Several lipid mediators derived from arachidonic acid (eicosanoids) are involved in the pathophysiology of allergic disease, and this has led to therapeutic strategies to block their production or their receptors on target cells.68,69 Cys-LTs are potent bronchoconstrictors, and antagonism of cys-LT1-receptors provides clinical benefit in asthma.67 Cys-LTs are also involved in eosinophilic inflammation, although antagonists have only weak antiinflammatory effects. LTB4 has also been implicated in

CHAPTER 21  Pathophysiology of Allergic Inflammation asthma, not only as a chemoattractant of neutrophils, which express BLT1-receptors, but also as an important activator of T cells.70 Most of the effects of LTB4 appear to be mediated via the high-affinity BLT1, but a low-affinity receptor BLT2 is also expressed on inflammatory cells, and reducing expression of this receptor by antisense oligonucleotides reduces the inflammatory and AHR response to inhaled allergen in sensitized mice, implicating this receptor in the allergic response to LTB4.71 Prostaglandins have potent and diverse effects on airway function. PGD2 has attracted particular attention because it is released in large amounts from mast cells. It is a potent bronchoconstrictor through the activation of thromboxane receptors (TP) on airway smooth muscle cells but also activates DP1-receptors, which mediate vasodilation and DP2-receptors first identified as chemoattractant homologous receptor expressed on Th2 cells (CRTh2), which is expressed on Th2 cells, ILC2 cells, eosinophils, and basophils and provides a link between mast cell activation and allergic inflammation.72 CRTH2/DP2 antagonists are in development as oral antiallergic therapies.73 One such drug, fevipiprant, reduces sputum eosinophils in patients with allergic asthma,74 although clinical benefits with this drug are only modest.75 Other lipid mediators that are less well investigated in allergic diseases include 5-oxo-eicosateraenic acid (5-oxo-ETE), a product of 5-lipoxygenase that is markedly increased by oxidative stress and which is a potent chemoattractant of eosinophils, which express distinct OXE receptors.76 Oxidation of arachidonic acid by oxidative stress results in the formation of F2-isoprostanes, which are bronchoconstrictors and mediators of plasma exudation.77 Eoxins are generated from arachidonic acid by the 15-liopxygenase pathways and are produced by eosinophils and mast cells.78 Eoxin C4 has proinflammatory effects and causes microvascular leakage.

Cytokine Networks Cytokines play a key role in the orchestration and perpetuation of allergic inflammation and are now targeted in therapy (see Fig. 21.2).79 Allergic inflammation is characterized by the secretion of T2 cytokines, including IL-4, IL-5, IL-9, and IL-13, which are secreted by Th2 and ILC2 cells but now recognized to be secreted by other cells involved in allergic inflammation including mast cells, basophils, eosinophils, and epithelial cells. The use of blocking antibodies has shed new light on the role of individual T2 cytokines.80 IL-4 and IL-13 play a key role in IgE synthesis through isotype switching of B cells and appear to play a critical role in animal models of asthma. So far blocking IL-13 has given disappointing clinical benefit in asthma.81,82 However, blocking their common receptor, IL-4Rα, with dupilumab has given marked clinical improvement in atopic dermatitis, rhinosinusitis, and severe asthma.83-85 IL-5 is of critical importance in the differentiation, survival, and priming of eosinophils. Humanized monoclonal blocking antibodies (mepolizumab and reslizumab) induces a profound decrease in eosinophils in the blood and induced sputum in patients with mild asthma, and in highly selected patients with severe asthma and increased blood and sputum eosinophils, despite high doses of inhaled or oral corticosteroids, these drugs have reduced exacerbations and requirements for oral corticosteroids, but gave little improvement in symptoms or AHR.86,87 Benralizumab is an antibody that blocks the receptor IL-5Rα and is similarly effective to anti-IL-5 blockers.88 Anti-IL-5 therapies are also beneficial in treating eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome)89 but are not very effective in rhinosinusitis or atopic dermatitis. Several proinflammatory cytokines have been implicated in allergic diseases, including IL-1β, IL-6, TNF-α, and GM-CSF, which are released from a variety of cells, including macrophages and epithelial cells, and may be important in amplifying the allergic inflammatory response. Although there is persuasive evidence that TNF-α may be important

319

in patients with severe asthma, and small clinical studies with anti-TNF-α therapies had been promising, a large placebo-controlled trial of an anti-TNF antibody (golimumab) in severe asthma showed no overall benefit.90 However, it is possible that there are some responders and there was a suggestion that patients with greater bronchodilator reversibility showed a reduction in exacerbations. IL-17 is also increased in severe asthma,25 but an anti-IL-17 receptor antibody, brodalumab, was not effective in severe asthma, although patients with neutrophilic inflammation were not selected for this study.91 Interest has now focused on upstream regulatory cytokines in the pathogenesis of asthma, because it is considered that they may have greater therapeutic potential. There has been particular interest in the role of alarmins, which are expressed in airway and nasal epithelial cells and keratinocytes and are released with epithelial injury and infection and include thymic stromal lymphopoietin (TSLP), IL-25, and IL-33.92 TSLP is an upstream IL-7-like cytokine that may initiate and propagate allergic immune responses and plays an important role in immune responses to helminths.93 TSLP is produced predominantly by airways and nasal epithelial cells and by skin keratinocytes and stimulates immature myeloid dendritic cells, which express the heterodimeric TSLP receptor to differentiate into mature dendritic cells (Fig. 21.7). TSLP-activated dendritic cells promote naïve CD4+ T cells to differentiate into a Th2 phenotype and promote the expansion of Th2 memory cells through the release of Th2 chemotactic cytokines CCL17 and CCL22 and expression of the costimulatory molecule OX40 ligand.19 TSLP also promotes allergic inflammation by promoting the differentiation IL-4 transcription in Th2 cells, the production of IL-13 from mast cells, by recruiting eosinophils and by amplifying responses of basophils. TSLP may therefore play a pivotal role in the initiation of allergic asthma, rhinitis, and atopic dermatitis. It is highly expressed in the airways of asthmatic patients, and its expression is correlated with disease severity and the expression of CCL1.94 TSLP is also expressed in epithelial cells of patients with allergic rhinitis and atopic dermatitis. A blocking antibody to TSLP, tezepelumab, reduces the early and late response to allergen95 and is very effective in reducing exacerbations and symptoms in patients with severe asthma, irrespective of circulating eosinophil counts.96

Mast cell

Epithelial cells TSLP Myeloid dendritic cell CCL17 CCR4 Th2 cell IL-5

IL-4, IL-13 IL-4, IL-13 IgE

Eosinophil

B lymphocyte

Fig. 21.7  Thymic stromal lymphopoietin. TSLP is an upstream cytokine produced by epithelial cells/keratinocytes and mast cells that acts on immature dendritic cells to mature and release CCL17, which attracts Th2 cells via CCR4. Th2, T helper 2; CCL, chemokine; CCR, chemokine receptor.

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IL-25 (IL-17E), a member of the IL-17 family of cytokines, induces allergic inflammation through increased production of Th2 cytokines. Although originally shown to be produced by Th2 cells, it is now known to be released from many different cells, including mast cells, basophils, eosinophils, macrophages, and epithelial cells.97 Blocking IL-25 is effective in animal models of allergic disease, and blocking antibodies are now in clinical development. IL-33 is another upstream cytokine and a member of the IL-1 family of cytokines, which is unusual in its localization within the nucleus, where it may regulate chromatin structure and gene expression.98 It appears to be released only on damage to epithelial or endothelial cells and acts as an alarmin and is constitutively expressed at mucosal surfaces, such as the airways. It signals through the ST2 receptor that activates NF-κB and mitogen-activated protein (MAP) kinase pathways. Its relevance to allergic inflammation is that it enhances T2 immunity, leading to eosinophilia, mast cell activation, and mucus hypersecretion and thus may act as a bridge between innate and adaptive immunity in allergic inflammation. It also directly activates eosinophils, mast cells, epithelial cells, and dendritic cells. It appears to switch alveolar macrophages to the alternatively activated form (M2) that has been found in animal models of asthma with increased secretion of CCL17,99 although whether this is relevant to human allergic disease is uncertain. IL-33 shows increased expression in airway epithelium of asthmatic patients, and this is related to disease severity.100 IL-33 is increased in the skin of patients with atopic dermatitis and is released into the circulation during as well as mediating anaphylactic shock. IL-33 is also expressed in mast cells after activation through IgE receptors and also activates mast cells, providing a means of maintaining mast cell activation.101 Antibodies that block IL-33 or ST2 are in clinical development.

Chemokines Many chemokines are involved in the recruitment of inflammatory cells in allergic diseases and work through G-protein–coupled receptors from which small molecule inhibitors have now been developed.79 Chemokines appear to act in sequence in determining the final inflammatory response, so inhibitors may be more or less effective depending on the kinetics of the response. Chemokines, such as CCL11, CCL24, CCL28, CCL5, and CCL13, that are chemotactic for eosinophils activate a single receptor CCR3, so blocking this receptor with a small molecule antagonist was an attractive approach to treating allergic diseases,102 but several selective CCR3 antagonists have failed in toxicology testing. Other chemokine receptors are now being targeted, including CCR4, which is expressed predominantly on Th2 cells.

Oxidative Stress As in all inflammatory diseases, there is increased oxidative stress in allergic inflammation, because activated inflammatory cells, such as macrophages, eosinophils, and neutrophils, produce reactive oxygen species.103 Evidence for increased oxidative stress in asthma is provided by the increased concentrations of 8-isoprostane (a product of oxidized arachidonic acid) in exhaled breath condensates104 and increased ethane (a product of oxidative lipid peroxidation) in exhaled breath of asthmatic patients.105 There is also persuasive epidemiologic evidence that a low dietary intake of antioxidants is linked to an increased prevalence of asthma. Increased oxidative stress is related to disease severity and may amplify the inflammatory response and reduce responsiveness to corticosteroids, particularly in severe disease, in smoking asthma, and during exacerbations.

Nitric Oxide Nitric oxide (NO) is produced by several cells in the airway by NO synthases. NO is a potent vasodilator and may be a key mediator of

vasodilation in allergic inflammation. The level of NO in the exhaled air (fractional exhaled NO–FeNO) of patients with asthma is higher than in normal subjects and is increased further during the late response to inhaled allergen.106 NO production is also increased in allergic rhinitis. Exhaled NO measurements are less useful in rhinitis, because the local production of NO by the nasal mucosa is diluted by the high NO production from paranasal sinuses. However, measurement of FeNO in asthma is increasingly used as a noninvasive way of monitoring the inflammatory process.106 The increased NO in asthma is derived mainly from increased expression of inducible NO synthase (iNOS) in airway epithelial cells and is normalized by selective iNOS inhibitors.107 IL-4 and IL-13 induce iNOS in airway epithelial cells via activation of IL-4Rα,108 and increased FeNO is a good biomarker of response to dupilumab.85

Purines Purines are now recognized to be important mediators of allergic inflammation. Adenosine induces bronchoconstriction by activating A2B receptors on mast cells, and adenosine receptors are a target for new drugs.109 Recently there has been greater interest in adenosine triphosphate (ATP) as a mediator of allergic disease through signaling of P2X receptors (ion channels) and P2Y receptors (metabolic).110 ATP acts as a danger signal that alerts the immune system to tissue damage. ATP is released by danger signals such as oxidative stress. ATP, mainly acting via P2X7 receptors, enhances the chemotaxis and activation of mast cells, eosinophils, and T cells but induces apoptosis of Tregs, thus enhancing allergic inflammation.111 ATP also activates dendritic cells and is increased in bronchoalveolar lavage of asthmatic patients after allergen challenge.96 ATP is also a potent stimulus of mucus secretion and activation of sensory nerves. Inhaled ATP causes bronchoconstriction, cough, and dyspnea in asthmatic patients.112,113 ATP activates P2X7 receptors, which associate with pannexin-1, leading to activation of the NALP3 inflammasome, resulting in cleavage of the precursors of IL-1β, IL-18, and IL-33 by caspase-1 to release these cytokines.114

STRUCTURAL CELLS Although infiltrating and resident inflammatory and immune cells are important sources of inflammatory mediators, it is now apparent that structural cells in the airways and skin play a critical role in the secretion of inflammatory mediators and in maintaining chronic allergic inflammation.

Epithelial Cells Perhaps the most important structural cells are epithelial cells of the airways and skin, which express a wide variety of inflammatory mediators in allergic disease. There are important interactions between epithelial cells and dendritic cells, with the release of key mediators such as granulocyte macrophage–colony stimulating factor (GM-CSF), TSLP, IL-25, and IL-33, which promote a Th2 bias in dendritic cell precursors (see Fig. 21.4).19 In addition, contact between epithelial cells and dendritic cells may involve OX40 ligand and OX40, which also drives a Th2 bias. Surface epithelial cells interact directly with the environment and may be activated by pathogens and endotoxin through various pattern recognition receptors, such as Toll-like receptors (TLR), thus enhancing or triggering an allergic response. Mechanical factors may also be important in releasing inflammatory mediators from epithelial cells.115 For example, compressive forces acting on airway epithelial cells release growth factors, fibrogenic mediators, and inflammatory mediators. Epithelial cells appear to be fragile in asthma and are easily shed, because the intercellular connections are weak. This may be an aspect of failure to repair the structural changes that are a consequence of allergic inflammation in the airway. However, these epithelial changes that are

CHAPTER 21  Pathophysiology of Allergic Inflammation Inflammatory cells

321

Inflammatory mediators

Mediators Airway smooth muscle

Contraction Histamine, cys-LTs, kinins, prostanoids, endothelin

Proliferation PDGF, EGF, endothelin-1

Secretion Cytokines, chemokines, prostanoids

Fig. 21.8  Airway smooth muscle cells. Inflammation has several effects on airway smooth muscle cells, resulting in contraction, proliferation, and secretion of inflammatory mediators. Cys, Cysteinyl; EGF, epidermal growth factor; PDGF, platelet-derived growth factor.

Vasodilatation NO, CGRP histamine, PGE2

Plasma exudation histamine, cys-leukotrienes kinins, PAF

Angiogenesis VEGF, TNF-α

Fig. 21.9  Blood vessels in allergy. Allergic inflammation has several vascular effects, including vasodilation, plasma exudation from postcapillary venules, and new vessel formation (angiogenesis). CGRP, Calcitonin gene-related peptide; NO, nitric oxide; PAF, platelet-activating factor; PGE2, prostaglandin E2; TNF-α, tumor necrosis factor alpha; VEGF, vascularendothelial growth factor.

prominent in asthma are not described in rhinitis or atopic dermatitis, indicating that epithelial cells at different anatomic sites may show different responses.

Airway Smooth Muscle Airway smooth muscle cells play a key role in airway narrowing in asthma, and bronchoconstriction results mainly from the release of bronchoconstrictor mediators from mast cells, including histamine, cys-LTs, and PGD2. But airway smooth muscle mass is increased in asthma through hyperplasia and hypertrophy as a result of various growth factors released from airway epithelial and inflammatory cells. This is particularly pronounced in severe asthma, and there is evidence for a marked increase in proliferation in patients with severe asthma.116 Airway smooth muscle cells also release multiple inflammatory mediators and growth factors and are likely to be an important source of these mediators in the airways (Fig. 21.8). However, it is not certain whether these abnormal smooth muscle cells have altered contractility or response to bronchodilators. The thickened smooth muscle is space occupying and may show a poor relaxation response, thus contributing to the irreversible airway narrowing in severe asthma. Nevertheless selective removal of airway smooth muscle cells by bronchial thermoplasty has rather small effects on airway physiology, perhaps because peripheral airways beyond the reach of the bronchoscope are more important in severe disease.117

Fibroblasts The role of fibroblasts in allergic inflammation appears to depend on the site and therefore the nature of the fibroblast. This has been studied most carefully in asthma, where there is evidence for subepithelial fibrosis in most patients. This may be related to the activity of myofibroblasts, fibroblasts, or infiltrating fibrocytes.118 TGF-β may play an important role in mediating airway fibrosis.26 Myofibroblasts may migrate from airway smooth muscle toward the airway surface, but the origin of these cells is not certain. Eosinophils may be important in activating fibroblasts/myofibroblasts, and subepithelial fibrosis is seen in eosinophil bronchitis as well as in asthma.36 This is underlined by the demonstration that eosinophilic esophagitis is associated with marked fibrosis.

Blood Vessels All allergic diseases are characterized by vasodilation, which is a key component of the inflammatory response (Fig. 21.9). This is often accompanied by microvascular leakage from postcapillary venules, resulting in plasma exudation and local edema. This is prominent in

allergic rhinitis but also seen in asthma and atopic dermatitis. This is most directly observed in the skin, where allergen injection in atopic individuals causes a wheal and flare response. Many mediators directly relax vascular smooth muscle or release NO, a potent vasodilator. Other mediators may also cause plasma leakage and some mediators, such as histamine, PGD2, and cys-LTs cause both.119 Airway blood flow can be measured noninvasively by the inhalation of soluble gases, such as acetylene, and is increased in asthma and correlated with increased breath temperature and FeNO.120 In asthma there is evidence for increased angiogenesis, and this is correlated with increased production of vascular endothelial growth factor (VEGF).121 The role of angiogenesis in other allergic diseases is less certain.

Mucus Hypersecretion Mucus hypersecretion is a prominent feature of allergic inflammation at mucosal surfaces. Mucus secretion is part of the innate response of the respiratory tract, and increased mucus secretion from goblet cells in the epithelium and submucosal glands is a prominent feature of the allergic inflammatory response. Several inflammatory mediators stimulate mucus secretion and cause goblet cell hyperplasia.122 Mucous plugging in asthma is related to an interaction of mucus glycoproteins (MUC5AC, MUC5B) with exuded plasma proteins and is commonly seen in the airways of patients with fatal asthma. T2 cytokines stimulate goblet cell hyperplasia and secretion, linking this response to allergic inflammation. IL-13 is the most potent cytokine that stimulates MUC5AC and MUCB transcription and acts via IL-4Rα, which activates Jak1 and STAT6. STAT6 then switches on MUC genes and causes mucus hyperplasia via a transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF).123 Other cytokines, including IL-4, IL-9, IL-17, and IL-25, also act at least in part via IL-13. One of the most potent stimulants of mucus secretion is ATP, which acts on P2Y2-receptors on goblet cells.124 Epithelial growth factor receptors (EGFR) also regulate mucus hypersecretion and may be activated via its ligand TGF-α and indirectly by oxidative stress and by various proteinases, including neutrophil elastase and MMP9 (Fig. 21.10).125

NEURAL MECHANISMS Neural mechanisms play an important role in the pathophysiology of allergic diseases, and there is a complex interplay between allergic

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Inflammatory mediators Neurotrophins

Oxidative stress

Th2 cell IL-4, IL-13, IL-9

TRPA1 EGF

↑MUC5AC

Mucus hypersecretion

Neuropeptides (SP, CGRP) Neurogenic inflammation Goblet cells

Mucus hyperplasia

Inflammatory cells

Sensory nerves

Fig. 21.11  Neural mechanism in allergy. There is a close interaction between asthmatic inflammation and neural mechanisms. CGRP, Calcitonin gene-related peptide; SP, substance P; TRPA1, transient receptor potential A1 ion channel.

Submucosal gland ACh, SP

Airway nerves

Fig. 21.10  Airway mucus secretion. Increased mucus secretion in allergic disease may be stimulated by T helper 2 (Th2) cytokines and oxidative stress; this may be mediated via epidermal growth factor (EGF), which may result in mucus hyperplasia and increased expression of the mucin gene MUC5AC. Mucus hypersecretion is also enhanced by neural mechanisms through the release of acetylcholine (ACh) and substance P (SP).

inflammation and neural effects. Autonomic nervous control of the airways is complex; in addition to classic cholinergic and adrenergic mechanisms, nonadrenergic noncholinergic (NANC) nerves and several neuropeptides have been identified in the nerves of the upper and lower respiratory tracts.126,127 Several inflammatory mediators may act on prejunctional receptors on airway nerves to modulate the release of neurotransmitters. Thus thromboxane and PGD2 facilitate the release of acetylcholine from cholinergic nerves in canine airways, whereas histamine inhibits cholinergic neurotransmission at both parasympathetic ganglia and postganglionic nerves via histamine H3-receptors.

Sensory Nerves Sensory nerves in the respiratory tract and skin play a key role in producing cough, sneezing, and itch, which are prominent symptoms of allergic disease. Inflammatory mediators may activate sensory nerves to produce these symptoms and may also initiate reflex effects. For example, bradykinin, histamine, and protons (acidity) are potent activators of unmyelinated C-fibers and induce coughing and sneezing when applied locally to the mucosal surface but also activate reflex effects, such as bronchoconstriction and mucus secretion via cholinergic neural pathways. Several inflammatory mediators, including PGE2, bradykinin, and neurotrophins also sensitize sensory nerve endings in the airway epithelium or skin (hyperalgesia), so that the nerves become more easily activated by other stimuli or by stimuli that would not normally elicit any effects. Airway sensory nerves may also be involved in AHR, and one reason why animal models of asthma fail to show the high degree of AHR compared with human asthma is that the animals are usually anesthetized so that the sensory nerve component is absent. Transient potential receptor A1 (TRPA1) ion channels play a key role in the nociception in the airways and skin and may be activated by various irritants to produce coughing, sneezing, and itching.128,129 TRPA1 expression is increased in asthma and with inflammatory cytokines, whereas blocking TRPA1 by gene deletion or a selective inhibitor reduces AHR and airway inflammation in a murine model of asthma.130

Neuropeptides and Neurogenic Inflammation Airway nerves may also release neurotransmitters that have proinflammatory effects. Thus neuropeptides such as substance P (SP), neurokinin A, and calcitonin-gene–related peptide (CGRP) may be released from sensitized inflammatory nerves in the airways or skin to increase and extend the ongoing inflammatory response (Fig. 21.11). There may also be a reduction in the activity of enzymes, such as neprilysin (neutral endopeptidase), that degrade neuropeptides like SP. The role of neurogenic inflammation, either via a classic axon reflex with antidromic release of neuropeptides from sensory nerve endings, or from other nerves via a neural reflex pathway, is uncertain. It has been difficult to demonstrate the presence of neurogenic inflammation in the lower airways, but there is good evidence that it may be important in the nasal mucosa and skin.127,131 Sensory nerves may be very closely associated physically with inflammatory cells, including mast cells and eosinophils. Neuropeptides released from sensory nerves in the skin may cause vasodilation through the release of CGRP and mast cell degranulation via the release of SP. Neuropeptides may also be released from inflammatory and structural cells involved in allergic diseases, and neuropeptide receptors may be expressed on inflammatory cells, so that the boundaries between neural and inflammatory cells have become less distinct. For example, during the late skin response to allergen there is an infiltration of neutrophils expressing the sensory neuropeptide CGRP, which is a potent vasodilator.132

Neurotrophins The role of neurotrophins in allergic diseases is increasingly recognized.133 Nerve growth factor (NGF), brain-derived neurotrophic factor, and neurotrophins 3 and 4/5 are nerve-related cytokines that play an important role in the function, proliferation, and survival of autonomic nerves. In sensory nerves neurotrophins increase responsiveness and may also promote the expression of tachykinins. Neurotrophins may be produced by inflammatory cells, such as mast cells, lymphocytes, macrophages, and eosinophils, as well as structural cells, such as epithelial cells, fibroblasts, keratinocytes, and airway smooth muscle cells. Although neurotrophins have predominant effects on neuronal cells, they may also act as growth factors for inflammatory cells, such as mast cells, as well as increasing chemotaxis and survival of eosinophils.134 NGF induces AHR in various animal models of asthma, including guinea pig, mice, and rats, and this is blocked by antibodies to NGF or its receptor TrkA. In guinea pigs NGF enhances AHR by increasing the release of SP from sensory nerves and increases sensory nerve activation. The role of neurotrophins in human allergic diseases is uncertain. However, NGF levels are increased in bronchoalveolar lavage fluid of asthmatic patients, and there is a further increase after allergen challenge.135

CHAPTER 21  Pathophysiology of Allergic Inflammation

323

Stress and Allergic Inflammation Psychological or social stress is a well-known exacerbating factor in allergic diseases, especially asthma and atopic dermatitis, and may be explained by neuroimmune interactions.136,137 However, the mechanisms are far from clear. Activation of neuroendocrine and the sympathetic nervous system through cortisol and catecholamine secretion may influence the immune system, tipping the balance in favor of T2 responses by reducing IL-12 release.138 The increase in endogenous cortisol might be expected to benefit allergic disease, because corticosteroids are highly effective in therapy, but it is possible that chronic stress induces a state of corticosteroid resistance.139 In a murine model of chronic psychosocial stress there was a delay in resolution of inhaled allergen-induced lung inflammation in sensitized animals with increased T2 cytokines. This was associated with in vitro evidence of a reduced antiinflammatory response to corticosterone, suggesting that chronic stress may induce corticosteroid resistance.140 AHR has also been demonstrated with psychological stress in other allergen-sensitized animal models.141 The fact that psychological factors may increase allergic inflammation suggests that there may be psychological approaches to alleviating allergic diseases. The interaction between the central nervous system and allergic inflammation suggests that psychological treatments or alternative therapies, such as hypnotism and acupuncture, may provide clinical benefit, although this has yet to be demonstrated convincingly in controlled trials.142

TRANSCRIPTION FACTORS The inflammation of asthma, allergic rhinitis, and atopic dermatitis is related to increased expression of multiple inflammatory proteins (cytokines, enzymes, receptors, adhesion molecules), which are regulated by transcription factors.143 Many transcription factors have now been implicated in the pathophysiology of allergic disease and have been considered as potential targets for new antiinflammatory therapies. Proinflammatory transcription factors are involved in the regulation of multiple inflammatory genes and are activated in all inflammatory diseases; they probably play an important role in amplifying and perpetuating inflammation.

NF-κB NF-κB can be activated by multiple stimuli, including allergens.144 NF-κB is activated in asthmatic airways, particularly in epithelial cells and macrophages, and regulates the expression of several key genes that are overexpressed in asthmatic airways, including proinflammatory cytokines (IL-1β, IL-6, TNF–α, GM-CSF), chemokines (CCL1, CCL5, CCL11, CCL17, CCL22), adhesion molecules (ICAM-1, VCAM-1), and inflammatory enzymes (cyclooxygenase-2 and iNOS). There is a common mechanism for inflammatory gene activation that involves activation of coactivator molecules by proinflammatory transcription factors, such as NF-κB, at the start site of transcription to induce acetylation of core histones, around which DNA is wound in the chromosome (Fig. 21.12). This unwinds DNA, opening up the chromatin structure, and allows gene transcription to proceed.145 Activated inflammatory genes are then switched off by the nuclear enzyme histone deacetylase-2 (HDAC2), which is recruited by activated glucocorticoid receptors to the activated inflammatory gene complex.

T Cell Transcription Other transcription factors have a more specific effect, because the transcript factor is restricted to specific cell types, such as T cells or epithelial cells. The transcription factor GATA3 (GATA-binding protein 3) is crucial for the differentiation of uncommitted naïve T cells into

Inflammatory stimuli (e.g., IL-1β, TNF-α)

IKK2 p65 p50 Inflammatory protein (e.g., GM-CSF) p65 p50

NF-κB IκB activation

CBP Coactivators HAT Acetylation

mRNA ↑ Inflammatory gene transcription

Gene activation

Gene repression

Fig. 21.12  Activation of NF-κB in allergic disease. Inflammatory stimuli activate the enzyme IKK2 (inhibitor of NF-κB kinase-2), which degrades inhibitor of NF-κB (IκB) so that the p65 and p50 subunits of nuclear factor-κB (NF-κB) translocate to the nucleus, where they bind to DNA recognition sites in the promoter regions of inflammatory and immune genes. This recruits coactivator molecules, such as CREB-binding protein (CBP), which has intrinsic histone acetyltransferase (HAT) activity, resulting in acetylation of core histones. This unravels the chromatin structure so that gene transcription may proceed with production of mRNA and translation to inflammatory proteins, such as granulocyte macrophage– colony stimulating factor (GM-CSF).

Th2 and ILC2 cells, and it also regulates the secretion of T2 cytokines.146 There is an increase in the number of GATA3+ T cells in the airways of asthmatic subjects compared with normal subjects.147 After simultaneous ligation of the T cell receptor (TCR) and coreceptor CD28 by antigen-presenting cells, GATA3 is phosphorylated and activated by p38 mitogen-activated protein kinase (MAPK). Activated GATA3 then translocates from cytoplasm to nucleus via the nuclear transporter protein importin-α, where it activates gene transcription (Fig. 21.13).148 GATA3 expression in T cells is regulated by the transcription factor STAT6 (signal transducer and activator of transcription 6) via IL-4 receptor activation. By contrast Th1 cells are regulated by T-bet, which regulates the expression of IFN-γ.149 T-bet expression is reduced in T cells from the airways of asthmatic patients compared with nonasthmatic patients. When phosphorylated, T-bet can associate with and inhibit the function of GATA3, by preventing it from binding to its DNA target sequences. T-bet-deficient mice show increased expression of GATA3 and production of Th2 cytokines, confirming that T-bet is an important regulator of GATA3. GATA3 expression is also regulated by IL-27, a member of the IL-12 family, which downregulates GATA3 expression and upregulates T-bet expression, thereby favoring the production of Th1-type cytokines, which then act to further inhibit GATA3 expression. In turn, GATA3 inhibits the production of T1 cytokines by inhibiting STAT4, the major transcription factor activated by the T-bet-inducing cytokine IL-12 (Fig. 21.14). Nuclear factor of activated T cells (NFAT) is another T-cell-specific transcription factor and appears to enhance the transcriptional activation of GATA3 at the IL4 promoter.150 GATA3 nuclear translocation is rapidly and potently inhibited by corticosteroids through competition of the activated glucocorticoid receptor for nuclear

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SECTION A  Basic Sciences Underlying Allergy and Immunology

Dendritic cell

CD3

CD28

p38 MAPK

Cytoplasm

P GATA3

GATA3

Jak-STAT

Importin-α

P GATA3 Th2 cytokine gene

Nucleus

IL-4, IL-5, IL-13

Fig. 21.13  Activation of GATA3 in allergic disease. In resting Th2 cells, GATA3 is localized to the cytoplasm. Interaction with antigen-presenting dendritic cells activates the T cell receptor (CD3) and costimulatory molecule CD28, which results in activation of p38 MAP kinase signal transduction, leading to phosphorylation of GATA3 and nuclear import via the nuclear import protein importin-α. Nuclear GATA3 then binds to the promoter region of Th2 cytokine genes to active gene expression, resulting in allergic inflammation.

IL-27

IL-12

Th17 cells are characterized by the expression of the transcription factor retinoic acid orphan receptor (ROR)-γt, whereas Tregs express FoxP3. However some T cells may express different T cell–regulating transcription factors. For example, a novel type of CD4+ cell that coexpresses GATA3 and ROR-γt and coproduces Th2 cytokines and IL-17 have been described in mice and may play a key role in exacerbations, when increased eosinophils and neutrophils are found.153 Interestingly, this new subtype can be induced by exposing Th2 cells to the proinflammatory cytokines IL-1β, IL-6, or IL-21, illustrating the plasticity of T cell populations.

IL-4 IL-33

Expression of many of the key cytokines in allergy is regulated by STAT transcription factors that are activated by Janus-activated tyrosine kinases (Jak).154 IL-4 and IL-13 stimulate Jak1 and Jak3 to phosphorylate STAT6, which activates GATA3, resulting in Th2 differentiation and cytokine expression.155 IL-12 activates Jak2 and Tyk2 to phosphorylate STAT4, which activates T-bet to drive differentiation into Th1 cells, so the balance between STAT4 and STAT6 play a key role in Th cell differentiation and function. In mice and human monocytes IFN-γ activates STAT1, which in turn regulates suppressor of cytokine synthesis (SOCS)-1 to suppress STAT6 and thus inhibit IL-4 expression. STAT5 appears to play an important signaling role in mast cells and mediates effects of IgE receptors and c-kit on proliferation, survival, and mediator release.156 The TSLP receptor signals through activation of STAT1, STAT3, and STAT5 via the activation of Jak1 and Jak2.157 Several Jak inhibitors are now in development as immunomodulators to interfere with these Jak-STAT signaling pathways in allergic diseases.158 JAK inhibitors inhibit the release of CXCR3 chemokines from airway epithelial cells.159

Epigenetic Regulation STAT1

STAT4

STAT6

+

–

Th1 cells

T-bet

Th1 cytokines (IL-2, IFN-γ)

–

GATA3

Th2 cells

Th2 cytokines (IL-4, IL-5, IL-9, IL-13) Allergic inflammation

Fig. 21.14  GATA3 interactions in allergic inflammation. The transcription factor GATA3 (GATA-binding protein 3) is regulated by interleukin-4 (IL-4) via STAT6 (signal transducer and activator of transcription 6) and regulates the expression of IL-4, IL-5, IL-9, and IL-13 from T helper 2 (Th2) cells and also inhibits the expression of T-bet via inhibition of STAT4. IL-33 enhances the actions of GATA3. T-bet regulates Th1-cell secretion of IL-2 and interferon-γ (IFN-γ) and also has an inhibitory action on GATA3. T-bet is regulated by IL-12 via STAT4 and by IL-27 via STAT1. This demonstrates the complex interplay of cytokines and transcription factors in asthma.

import via importin-α, as well as by induction of MAP kinase phosphatase-1, which inhibits the phosphorylation of GATA3 by p38 MAP kinase.151 An inhaled DNAzyme (SB010) that inhibits GATA3 reduces the early and late response to allergens in patients with mild asthma.152

Epigenetic changes include posttranslational modification of histones (acetylation, methylation, phosphorylation, ubiquitination), small RNAs, and DNA methylation, all of which may alter the expression of genes involved in allergic inflammation.160 Methylation of cytosine residues at CpG dinucleotide sequences in DNA may result in gene suppression, and these changes are long-lasting and involved in cell differentiation and may be heritable. Differentiation of naïve T cells into Th1 cells is associated with methylation of the promoter region of IL4, whereas Th2 differentiation is associated with demethylation of this region.161 Demethylation of IL4 and IL13 allows binding of STAT6 and GATA3, whereas Th1 differentiation is also associated with demethylation of the IFNG gene and the binding of T-bet. Similar mechanisms may regulate the expression of Th2 cytokine genes in other cells, such as mast cells. In Tregs the promoter region of FOXP3 becomes demethylated and therefore activated. DNA methylation may be affected by environmental influences, such as diet. In mice fed a diet rich in folate (a methyl donor) there was increased allergic inflammation, IgE, and AHR in the progeny associated with increased methylation and suppression of a RUNX3, a gene associated with suppression of IL4 and activation of FOXP3.162 Acetylation of core histones plays an important role in the regulation of inflammatory genes through the NF-κB-activated acetyltransferase activity of coactivator molecules such as CREB-binding protein, p300, and PCAF, which result in hyperacetylation of histones and unwinding of DNA so that RNA polymerase 2 binding may occur, leading to gene transcription.160 This is reversed by HDACs, particularly HDAC2. In bronchial biopsies of asthmatic patients there is evidence for increased histone acetyltransferase activity and reduced HDAC activity, thus favoring increased inflammatory gene transcription. HDAC activity is reduced in monocytes and macrophages from patients with severe asthma and associated with increased inflammatory gene expression.163

CHAPTER 21  Pathophysiology of Allergic Inflammation

Inflammatory Mediators Lipid mediators Cytokines Peptides Oxidants

Antiinflammatory Mediators IL-10, IL-1ra, IFN-γ, IL-12 Lipoxins, resolvins, protectins, PGE2

Fig. 21.15  Antiinflammatory mediators in asthma. There may be an imbalance between increased proinflammatory mediators and a deficiency in antiinflammatory mediators. IL, Interleukin; IL-1ra, interleukin-1 receptor antagonist; IFN-γ, interferon gamma; PGE2, prostaglandin E2.

MicroRNAs are small single-stranded noncoding RNAs that may influence the expression of groups of genes by repression of their translation and play a key role in the orchestration of allergic diseases.164 For example, selective blockade of miR-126 suppresses the Th2 response in sensitized mice by inhibiting GATA3 expression.165

ANTIINFLAMMATORY MECHANISMS IN ALLERGY Although most emphasis has been placed on inflammatory mechanisms in allergy, there are endogenous antiinflammatory mechanisms that may be defective in allergic diseases, resulting in increased inflammatory responses (Fig. 21.15).

Cortisol Endogenous cortisol may be important as a regulator of the allergic inflammatory response, and nocturnal exacerbation of asthma may be related to the circadian fall in plasma cortisol. Blockade of endogenous cortisol secretion by metyrapone results in an increase in the late response to allergen in the skin.166 Cortisol is converted to the inactive cortisone by the enzyme 11β-hydroxysteroid dehydrogenase-2, which is expressed in the epithelium and endothelium of asthmatic airways and may be a determinant of corticosteroid sensitivity.167 It is possible that this enzyme functions abnormally in asthma or may determine the severity of asthma.

Inhibitory Cytokines Various cytokines have antiinflammatory actions.168 IL-1 receptor antagonist (IL-1ra) inhibits the binding of IL-1 to its receptors and therefore has potential antiinflammatory effects. In a murine model of allergic asthma adenovirus expressing IL-1ra has a protective effect. There is increased expression of IL-1ra in airway epithelial cells in asthma.169 IL-12 promotes the differentiation of Th1 cells and thus the suppression of Th2 cells, resulting in a reduction in eosinophilic inflammation.170 IL-12 infusions in patients with asthma indeed inhibit peripheral blood eosinophilia.171 There is some evidence that IL-12 expression may be impaired in asthma.172 IL-27 is a member of the IL-12 family that promotes Th1 cell differentiation through a STAT1-dependent mechanism independently of IL-12.167 It is produced by activated APCs and enhances Th1 function by downregulating GATA3 expression and upregulating T-bet expression, thereby favoring the production Th1-type cytokines, which then act to further inhibit GATA3 expression.173 IL-10 is a potent antiinflammatory cytokine that inhibits the synthesis of many inflammatory proteins, including several cytokines (such as

325

TNF-α, GM-CSF, IL-5, and several chemokines) that are overexpressed in allergic disease and also inhibits antigen presentation. There is a reduction in IL10 transcription and secretion from macrophages in individuals who have asthma.174 IL-10 is produced by a subset of Tregs and by macrophages. Specific allergen immunotherapy results in increased production of IL-10-producing Tregs.175,176 Several cytokines belong to the IL-10 superfamily, including IL-19, IL-22, IL-24, IL-26, IL-28, and IL-29, and have immunomodulatory or antiinfective properties, but how they are involved in allergic inflammation has not yet been defined.177,178

Lipid Antiinflammatory Mediators Several lipid mediators may also have antiinflammatory and immunosuppressive effects. PGE2 has inhibitory effects on macrophages, epithelial cells, and eosinophils, and exogenous PGE2 inhibits allergen-induced airway responses and its endogenous generation may account for the refractory period after exercise challenge. However, it is unlikely that endogenous PGE2 is important in most asthmatics, because nonselective COX inhibitors only worsen asthma in a minority of patients (aspirinsensitive asthma). Lipoxins are generated from arachidonic acid through cell-cell interaction via 5- and 15-lipoxygenase and inhibit the action of cys-LTs.179 Lipoxins are generated in asthma but are reduced in aspirin-sensitive and severe asthma.180 Resolvins are related antiinflammatory lipid molecules that are generated from omega-3-fatty acids in the diet that are involved in the resolution of acute inflammation.181 In a murine model, resolvin E1 promotes the resolution of allergic inflammation through inhibiting the effects of IL-23 and IL-6, which drive Th17 cells,176 and promotes the resolution of allergic inflammation.182 Protectins are related lipid mediators that are involved in the resolution of inflammation. Protectin D1 accelerates the resolution of allergic inflammation in a murine model. Protectin D1 is found in the exhaled breath condensate of normal subjects but is reduced during exacerbations of asthma.

Indoleamine 2, 3-Dioxygenase Indoleamine 2, 3-dioxygenase (IDO), a tryptophan-degrading enzyme, is an important T cell immunomodulator and promotes immunologic tolerance.183 IDO expression by dendritic cells may suppress T cell responses and promote tolerance to allergens through direct effects on T cell function, which are mediated by tryptophan depletion, or toxic metabolites. IDO inhibits eosinophilic inflammation in murine models of asthma. IDO activity is reduced in asthma and restored by corticosteroid therapy through increased IL-10 secretion184 and is reduced in dendritic cells of house dust mite–sensitive subjects by the allergen Der p1.185

THERAPEUTIC IMPLICATIONS Allergic inflammation is complex, involving many cells and mediators, as discussed previously. This has identified many therapeutic targets. However, targeting individual mediators (such as individual cytokines) or cell types (such as eosinophils) is unlikely to produce very effective therapies for allergic diseases, suggesting that treatments with a broader spectrum of action are needed. Corticosteroids are highly effective in treating allergic diseases and when applied topically largely avoid systemic side effects. Corticosteroids are effective because they have multiple antiinflammatory effects, including recruitment of HDAC2 to switch off activated inflammatory genes and inhibition of GATA3 activity.186 It has proved very difficult to develop new therapies for asthma and other allergic diseases that are as effective as corticosteroids.187 Specific immunotherapy may be effective when specific allergens are involved, and major progress has been made in understanding the cellular and

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molecular mechanisms so that more effective immunotherapy may be available in the future through enhancing Treg function. The development of specific cytokine inhibitors has revealed that they are only effective in highly selected patients, necessitating the careful phenotyping of patients and the development of specific response biomarkers, such as blood or sputum eosinophils for anti-IL-5 therapies and FeNO for anti-IL-4/13 therapies. The recognition that epigenetic factors are likely to be important in regulating the inflammatory genes involved in allergic disease has also suggested that this may lead to new therapeutic approaches in the future.188,189

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CHAPTER 21  Pathophysiology of Allergic Inflammation 147. Caramori G, Lim S, Ito K, et al. Expression of GATA family of transcription factors in T-cells, monocytes and bronchial biopsies. Eur Respir J 2001;18:466–73. 148. Maneechotesuwan K, Xin Y, Ito K, et al. Regulation of Th2 cytokine genes by p38 MAPK-mediated phosphorylation of GATA-3. J Immunol 2007;178:2491–8. 149. Lazarevic V, Glimcher LH. T-bet in disease. Nat Immunol 2011;12:597–606. 150. Avni O, Lee D, Macian F, et al. T(H) cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat Immunol 2002;3:643–51. 151. Maneechotesuwan K, Yao X, Ito K, et al. Suppression of GATA-3 nuclear import and phosphorylation: a novel mechanism of corticosteroid action in allergic disease. PLoS Med 2009;6:e1000076. 152. Krug N, Hohlfeld JM, Kirsten AM, et al. Allergen-induced asthmatic responses modified by a GATA3-specific DNAzyme. N Engl J Med 2015;372:1987–95. 153. Wang YH, Voo KS, Liu B, et al. A novel subset of CD4(+) T(H)2 memory/effector cells that produce inflammatory IL-17 cytokine and promote the exacerbation of chronic allergic asthma. J Exp Med 2010;207:2479–91. 154. O’Shea JJ, Schwartz DM, Villarino AV, et al. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu Rev Med 2015;66:311–28. 155. Ashino S, Takeda K, Li H, et al. Janus kinase 1/3 signaling pathways are key initiators of TH2 differentiation and lung allergic responses. J Allergy Clin Immunol 2014;133:1162–74. 156. Morales JK, Falanga YT, Depcrynski A, et al. Mast cell homeostasis and the JAK-STAT pathway. Genes Immun 2010;11:599–608. 157. Wohlmann A, Sebastian K, Borowski A, et al. Signal transduction by the atopy-associated human thymic stromal lymphopoietin (TSLP) receptor depends on janus kinase function. Biol Chem 2010;391:181–6. 158. Kontzias A, Kotlyar A, Laurence A, et al. Jakinibs: a new class of kinase inhibitors in cancer and autoimmune disease. Curr Opin Pharmacol 2012;12:464–70. 159. Fenwick PS, Macedo P, Kilty IC, et al. Effect of JAK inhibitors on release of CXCL9, CXCL10 and CXCL11 from human airway epithelial cells. PLoS ONE 2015;10:e0128757. 160. Adcock IM, Ford P, Barnes PJ, et al. Epigenetics and airways disease. Respir Res 2006;7:21. 161. Tumes DJ, Papadopoulos M, Endo Y, et al. Epigenetic regulation of T-helper cell differentiation, memory, and plasticity in allergic asthma. Immunol Rev 2017;278:8–19. 162. Hollingsworth JW, Maruoka S, Boon K, et al. In utero supplementation with methyl donors enhances allergic airway disease in mice. J Clin Invest 2008;118:3462–9. 163. Cosio BG, Mann B, Ito K, et al. Histone acetylase and deacetylase activity in alveolar macrophages and blood monocytes in asthma. Am J Respir Crit Care Med 2004;170:141–7. 164. Dissanayake E, Inoue Y. MicroRNAs in allergic disease. Curr Allergy Asthma Rep 2016;16:67. 165. Mattes J, Collison A, Plank M, et al. Antagonism of microRNA-126 suppresses the effector function of TH2 cells and the development of allergic airways disease. Proc Natl Acad Sci USA 2009;106:18704–9. 166. Herrscher RF, Kasper C, Sullivan TJ. Endogenous cortisol regulates immunoglobulin E-dependent late phase reactions. J Clin Invest 1992;90:593–603. 167. Orsida BE, Krozowski ZS, Walters EH. Clinical relevance of airway 11beta-hydroxysteroid dehydrogenase type II enzyme in asthma. Am J Respir Crit Care Med 2002;165:1010–14. 168. Barnes PJ, Lim S. Inhibitory cytokines in asthma. Mol Med Today 1998;4:452–8.

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169. Sousa AR, Lane SJ, Nakhosteen JA, et al. Expression of interleukin-1 beta (IL-1b) and interleukin-1 receptor antagonist (IL-1ra) on asthmatic bronchial epithelium. Am J Respir Crit Care Med 1996;154:1061–6. 170. Wills-Karp M. IL-12/IL-13 axis in allergic asthma. J Allergy Clin Immunol 2001;107:9–18. 171. Bryan S, O’Connor BJ, Matti S, et al. Effects of recombinant human interleukin-12 on eosinophils, airway hyperreactivity and the late asthmatic response. Lancet 2000;356:2149–53. 172. Naseer T, Minshall EM, Leung DY, et al. Expression of IL-12 and IL-13 mRNA in asthma and their modulation in reponse to steroids. Am J Respir Crit Care Med 1997;155:845–51. 173. Yoshimoto T, Yoshimoto T, Yasuda K, et al. IL-27 suppresses Th2 cell development and Th2 cytokines production from polarized Th2 cells: a novel therapeutic way for Th2-mediated allergic inflammation. J Immunol 2007;179:4415–23. 174. John M, Lim S, Seybold J, et al. Inhaled corticosteroids increase IL-10 but reduce MIP-1a, GM-CSF and IFN-g release from alveolar macrophages in asthma. Am J Respir Crit Care Med 1998;157:256–62. 175. Bohle B, Kinaciyan T, Gerstmayr M, et al. Sublingual immunotherapy induces IL-10-producing T regulatory cells, allergen-specific T-cell tolerance, and immune deviation. J Allergy Clin Immunol 2007;120:707–13. 176. Campbell JD, Buckland KF, McMillan SJ, et al. Peptide immunotherapy in allergic asthma generates IL-10-dependent immunological tolerance associated with linked epitope suppression. J Exp Med 2009;206:1535–47. 177. Commins S, Steinke JW, Borish L. The extended IL-10 superfamily: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, and IL-29. J Allergy Clin Immunol 2008;121:1108–11. 178. Ouyang W, Rutz S, Crellin NK, et al. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol 2011;29:71–109. 179. Levy BD, Serhan CN. Resolution of acute inflammation in the lung. Annu Rev Physiol 2014;76:467–92. 180. Planaguma A, Kazani S, Marigowda G, et al. Airway lipoxin A4 generation and lipoxin A4 receptor expression are decreased in severe asthma. Am J Respir Crit Care Med 2008;178:574–82. 181. Serhan CN, Chiang N, Dalli J. New pro-resolving n-3 mediators bridge resolution of infectious inflammation to tissue regeneration. Mol Aspects Med 2018;64:1–17. 182. Aoki H, Hisada T, Ishizuka T, et al. Protective effect of resolvin E1 on the development of asthmatic airway inflammation. Biochem Biophys Res Commun 2010;400:128–33. 183. Le AV, Broide DH. Indoleamine-2,3-dioxygenase modulation of allergic immune responses. Curr Allergy Asthma Rep 2006;6:27–31. 184. Maneechotesuwan K, Supawita S, Kasetsinsombat K, et al. Sputum indoleamine-2, 3-dioxygenase activity is increased in asthmatic airways by using inhaled corticosteroids. J Allergy Clin Immunol 2008;121:43–50. 185. Maneechotesuwan K, Wamanuttajinda V, Kasetsinsombat K, et al. Der p 1 suppresses indoleamine 2, 3-dioxygenase in dendritic cells from house dust mite-sensitive patients with asthma. J Allergy Clin Immunol 2009;123:239–48. 186. Barnes PJ. Glucocorticosteroids. Handb Exp Pharmacol 2017;237:93–115. 187. Gross NJ, Barnes PJ. New therapies for asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2017;195:159–66. 188. Barnes PJ. Targeting the epigenome in the treatment of asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 2009;6:693–6. 189. Szyf M. Epigenetics, DNA methylation, and chromatin modifying drugs. Annu Rev Pharmacol Toxicol 2009;49:243–63.

CHAPTER 21  Pathophysiology of Allergic Inflammation

329.e1

SELF-ASSESSMENT QUESTIONS 1. GATA3 is a key transcription factor in which of the following cells? a. Airway epithelial cells b. ILC2 cells c. Macrophages d. Th17 cells 2. Which of the following cytokines function as an alarmin? a. IL-17 b. IL-10 c. GM-CSF d. IL-33 3. Which of the following lipid mediators has been shown to have antiinflammatory effects? a. Prostaglandin D2 b. Resolvin E2 c. Eoxin C4 d. 8-Isoprostane

4. Which chemokine receptor is involved in the recruitment of Th2 cells? a. CCR2 b. CCR3 c. CCR4 d. CCR5

22  Genetics and Epigenetics in Allergic Diseases and Asthma Ian Sayers, David J. Cousins, John W. Holloway

CONTENTS Introduction, 330 Phenotype Definition, 330 Heritability Studies, 331 Approaches to Study the Genetics of Common Disease, 331 Genetics of Self-Reported and Doctor-Diagnosed Allergic Disease, 334 Current Understanding of Allergic Disease Genetics, 341

Missing Heritability in Allergic Disease, 342 Functional Genomics Approaches, Translating Genetic Association Signals, 346 Epigenetics and Allergic Disease, 346 Pharmacogenetics of Asthma, 348 Summary, 348

SUMMARY OF IMPORTANT CONCEPTS

genetic susceptibility factors for allergic disease such as asthma and atopic dermatitis are not related to atopy susceptibility. In the main, susceptibility to allergic disease results from the inheritance of many genetic susceptibility factors, each of small effect. As for many common diseases, the specific biochemical defect(s) at the cellular level and environmental exposures that trigger initiation of allergic disease in an individual are unknown, even though considerable knowledge has accrued on the molecular pathways involved in pathogenesis. The study of the genetics of these conditions provides an opportunity to identify novel factors in allergic disease etiology, providing a greater understanding of the fundamental mechanisms of these disorders (Box 22.1 and Box 22.2). The focus of this chapter is to present the principles of approaches for identification of genetic factors underlying disease and to summarize the insights these have provided into disease pathogenesis. The advent of genome-wide association studies (GWAS) has seen considerable progress in the last decade in unravelling the contribution of specific genetic factors to an individual’s susceptibility to, and severity of, allergic disease. Furthermore, studies of gene-environment interaction have led to greater insight into the importance of environmental triggers for the initiation, exacerbation, and persistence of allergic diseases. Studies of the timing of action of genetic variants in determining disease susceptibility have highlighted the importance of in utero development and early life in determining susceptibility to allergic disease. More recently the study of epigenetic processes in allergic disease has provided understanding of the molecular processes that link both genetic variation and environmental exposure to disease pathogenesis.

• Susceptibility to and severity of allergic disease have a genetic basis. • Multiple genes, each with a modest effect, together with environmental influences combine to produce the phenotypes of allergic diseases. • Identification of genetic susceptibility factors through genome-wide association studies has provided novel insights into the pathogenesis of atopy and allergic disease. • Epigenetic processes that result from modifications of DNA structure without a change in the sequence, in response to environmental exposures, are important modifiers of disease. • Pharmacogenetic analysis of genes in pathways relevant to a given therapy allows treatment to be tailored to the patient.

INTRODUCTION It is now accepted that there is a genetic basis to susceptibility to most common diseases, and that individual susceptibility depends on the interaction between inherited factors and the environment. In addition, it is widely recognized that variation in individual response to therapy and risk of adverse reactions also has, in part, a genetic basis. Over the past three decades there have been significant advances in genetic studies of allergic diseases, which will be reviewed and discussed in this chapter. Classically, allergy has been defined as the result of immune reaction to antigens known as allergens. Atopy, the genetically mediated predisposition to produce specific IgE after exposure to allergens, is clinically defined as having evidence of allergic sensitization to at least one allergen.1 Atopy is fundamental to the pathogenesis of allergic disorders, which manifest as any combination of conjunctivitis, food intolerance, asthma, rhinitis, and atopic dermatitis. However, these clinical presentations can also appear in the absence of atopy, suggesting that there are likely to be factors, including genetic factors, that determine susceptibility to allergic disease that are independent of atopy.2 As will be outlined later, genetic studies have now confirmed that several

330

PHENOTYPE DEFINITION Because multiple genes are involved in their pathogenesis, allergic diseases such as asthma are considered complex genetic diseases. Complex disorders show a clear hereditary pattern, but the mode of inheritance does not follow any simple mendelian pattern such as autosomal dominant, recessive, or sex linked. Furthermore, in comparison to disorders caused by mutations in single genes (e.g. cystic fibrosis), they tend to

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

BOX 22.1  Benefits of Genetic Studies of

Allergic Diseases

• To understand the heritable component of diseases or traits • Explication of disease pathogenesis by identification of genes and molecular pathways, generating novel pharmacologic targets • Identification of environmental-genetic interactions and prevention of disease through environmental modification • Detection of susceptible individuals through screening early in life, allowing targeted interventions • Subclassification of disease by genetics, enabling therapies to be tailored to at-risk individuals • Determination of the likelihood of a therapeutic response or adverse response (i.e., pharmacogenetics) as the basis for individualized treatment plans

BOX 22.2  Genetic Roles in Asthma and

Allergic Disease

Susceptibility to Atopy Helper T cell type 2 (Th2) or immunoglobulin E (IgE) switch genes (e.g., α chain of the high-affinity IgE receptor associated with sensitization and serum IgE levels) Determination of Target-Organ Disease Asthma-susceptibility genes (e.g., OPN3, CHML, GSDMB) Genes that regulate propensity of lung epithelium and fibroblasts for remodeling in response to allergic inflammation (e.g., ADAM33) Atopic dermatitis–susceptibility genes (e.g., COL6A5, OVOL1) Genes that regulate dermal barrier function (e.g., FLG) Interaction of Environmental Factors With Disease Genes that determine responses to factors that drive Th1/Th2 skewing of the immune response (e.g., CD14 and TLR4 polymorphisms and early childhood infection) Genes that modulate the effect of exposures involving oxidant stresses such as tobacco smoke and air pollution on asthma susceptibility (e.g., glutathione S-transferase genes) Genes that alter interactions between environmental factors and established disease (e.g., CDHR3 genetic polymorphisms regulating responses to respiratory virus infection) Modification of Disease Severity Allele prevalence and risk of disease severity (e.g., TNFA, IL4RA) Pharmacogenetics Genetic variation and response to therapy (e.g., β2-adrenergic receptor polymorphism and response to β2-agonists or 5-lipoxygenase polymorphism and responses to leukotriene receptor antagonists or leukotriene synthesis inhibitors)

have much higher prevalence in the population. Asthma occurs in at least 10% of children in the United Kingdom, and atopy is as high as 40% in some population groups3 compared with a frequency of 1:2000 for cystic fibrosis in live Caucasian births. Characteristic features of mendelian diseases are that they are rare, involving mutations in a single gene, and the mutations are severe, resulting in a large phenotypic effect that may be independent of environmental influences. In contrast, complex disease traits are common and involve many genes, with “mild” mutations leading to small phenotypic effects with strong environmental interactions. Studies of a genetic disorder require that a phenotype be defined, to which genetic data are compared. Phenotypes can be classified in two ways;

331

they may be complex, such as asthma or atopy, and are likely to involve the interaction of many genetic and environmental factors. Alternatively, intermediate phenotypes may be used, such as lung function and bronchial hyperresponsiveness (BHR) for asthma, and serum total IgE levels and specific IgE responsiveness or positive skin prick tests to particular allergens for atopy. Most recently, cluster analysis has been used to identify individual phenotypic expressions of atopy and allergic disease.4,5 Provided these are stable over time and between patient groups, this may provide a powerful approach to genetic analysis in the future because it may overcome the issue of phenocopy, where individuals with differing causes of their disease (genetic or environmental) exhibit the same clinical phenotype. Investigators have used various approaches, including a reported physician diagnosis of asthma, questionnaire data on relevant symptoms, objective measures as discussed previously, or combinations to define asthma in genetic studies. Reliance on only historical information, such as a prior physician diagnosis of asthma, appears to underestimate the prevalence of asthma and allergy in family and epidemiologic studies; however, these definitions have still been very useful in large-scale association studies.6 In evaluating genetic studies and comparing results, it is important to be aware of differences in disease definition. Although performing well-standardized laboratory testing is time consuming and expensive, it is important to very carefully characterize individuals in genetic studies to avoid biases introduced by inconsistent or poorly defined disease status.

HERITABILITY STUDIES Heritability is the proportion of observed variation in a particular trait that can be attributed to inherited genetic factors in contrast to environmental ones. Like other complex genetic diseases, evidence for a heritable component in allergic disease has been confirmed by several types of studies including family studies, segregation analysis, twin and adoption studies, heritability studies, and population-based relative risk to relatives of probands. From familial aggregation and twin studies, a significant familial aggregation of atopy, allergic disease and related intermediate phenotypes such as BHR and total serum IgE levels has been described in numerous studies.7-10 For example, the presence of familial aggregation for asthma has been supported by studies reporting that an asthma phenotype is present in approximately 25% of the offspring of a parent with asthma.10 Higher concordance rates for a disease phenotype in monozygotic twins (who share 100% of their genes) compared with dizygotic twins (who share 50% of their genes identical by descent) also provides important evidence of a genetic component. An increased correlation of serum total IgE levels and a higher concordance of asthma is seen in monozygotic twins compared with dizygotic twins.7-9 A key observation from heritability studies of allergic disease is the issue of “end organ susceptibility”; that is, which allergic disease an atopic individual will develop is controlled by specific genetic factors, differing from those that determine susceptibility to atopy per se.11,12 For example, in a study of 176 normal families, Gerrard et al. found a striking association between asthma in the parent and asthma in the child, between allergic rhinitis in the parent and allergic rhinitis in the child, and between atopic dermatitis in the parent and atopic dermatitis in the child.11 Such observations from heritability studies have since been confirmed by molecular genetic studies of allergic disease.

APPROACHES TO STUDY THE GENETICS OF COMMON DISEASE Two general approaches have been widely used to study the genetics of allergic disease: candidate gene association studies usually performed

332

SECTION A  Basic Sciences Underlying Allergy and Immunology GWAS

Positional cloning

Families

Finer genetic mapping

Linkage

199

195

Asthma patients

Controls

Mapping Mutation Screen

Genotype for typically 500,000+ SNPs in large numbers of cases and controls using arrays

Genes in region tested for association

Compare differences to identify disease-specific SNPs using stringent correction for multiple testing

207

203

195

Physical

199

Genetic markers

Susceptibility genes

Fig. 22.1  Positional cloning involves linkage analyses, which follow the transmission of genetic information through families with multiple affected children followed by fine association mapping. Genome-wide association studies (GWAS) look at the frequency of a large number of common variants between cases and controls. Both approaches lead to novel gene discovery. SNPs, Single nucleotide polymorphisms. (Reproduced with permission from Shaw DE, Portelli MP, Sayers I. Asthma. In: Padmanabhan S, editor. Handbook of pharmacogenomics and stratified medicine. London: Elsevier; 2014. p. 617-51.)

in unrelated cases and controls and hypothesis-independent approaches (Fig. 22.1) that involve study of genetic variation genome-wide, either in family studies using genome-wide linkage approaches followed by positional cloning or, now more commonly, by genome-wide association studies in large case-control cohorts.13

Candidate Gene Association Studies Candidate gene association studies evaluate genetic variation in the region of genes that are physiologically suggested (candidates) to be involved in disease pathogenesis. For example, genes such as those encoding cytokines, chemokines, and their receptors as well as transcription factors, IgE receptor, etc., are plausible candidate genes for allergic disease. The data for this type of study are usually obtained from unrelated individuals (cases and controls). Polymorphisms within the gene that are believed to be functional (i.e., affecting gene expression or encoded protein function), or that are selected for maximal information on the basis of linkage disequilibrium patterns surrounding the gene (often termed tagging single nucleotide polymorphisms [SNPs]), are then tested for association with the disease or phenotype in question. The genetic variants that are usually used to conduct association studies are SNPs. These are the most common type of polymorphism, and it has been estimated that the human genome contains about 80 million SNPs. Compared with linkage studies, one advantage of association studies with candidate genes is their ability to identify genetic variations that have relatively small effects on susceptibility. Also, association studies are more efficient in terms of recruiting subjects and cost. However, the choice of controls can be difficult, because ideally subjects need to be matched for variables that may confound the results, for example, age, gender, and ancestry. The other problem is that interpretation of the results from association studies may be complicated, and there have been some efforts to implement stringent criteria for legitimacy of a candidate gene association study.14-16

The advantage of the candidate approach is that candidate genes have biologic plausibility and often display known functional consequences that have potentially important implications for the disease of interest. Disadvantages are the limitation to genes of known or postulated involvement in the disease, thereby excluding the discovery of novel genes that influence the disease.

Linkage Analysis in Families Genome-wide linkage studies use phenotypic data from all available members of a family (affected and unaffected) and DNA markers to examine whether the markers cosegregate with phenotypes of interest. Positional cloning is highly effective in discovering causal genes for monogenic disorders but less so for complex genetic diseases. However, before the technologic advancements that enabled GWAS of common disease, a number of genome-wide screens for atopy and allergic disease susceptibility genes were undertaken17,18 and a number of genes identified, for example ADAM3319, DPP1020, HLAG21, PHF1122, and PTGDR23 for asthma, PCDH1 for bronchial hyperresponsiveness,24 and COL29A125 for atopic dermatitis (Table 22.1). Despite the success of such positional cloning studies, linkage analysis for allergic disease phenotypes was slow and expensive because of the need to recruit and phenotype large cohorts of families. Thus despite recruiting several hundred families, the majority of linkage studies ended up being underpowered for identifying susceptibility genes for complex disease.

Genome-Wide Association Studies Genes have been identified for common diseases such as asthma and allergy from studies of candidate or pathway genes in cases and controls. Because of the mapping of polymorphisms in the genome and the advances in genotyping technology, it is now possible to scan the whole genome of cases and controls in a hypothesis-independent manner to identify multiple susceptibility genes, each of which contributes a small

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CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

TABLE 22.1  Positionally Cloned Genes for Asthma and Allergic Disease

Gene

Chromosomal Location

ADAM33

20p13

DPP10

Population/ Associated Phenotypes

Initial Study

Gene Product and Functional Role

Study Size

UK, US Asthma, BHR

ADAM metalloproteinase domain 33: involved in airway remodeling by fibroblasts and smooth muscle hyperactivity

460 families (Caucasian)

Van Eerdewegh et al, 200219

2q14.1

Australian, UK, German Asthma, atopy (by SPT), asthma severity

Dipeptidyl-peptidase 10: potassium channel regulator with no detectable protease activity; involved in cytokine processing, especially in T cells

244 families

Allen et al, 200320

PHF11

13q14.11

Australian, UK, European Total IgE, asthma, asthma severity

PHD finger protein 11: zinc finger transcription factor; may be involved in chromatinmediated transcription regulation; B cell clonal expansion and regulation of immunoglobulin expression may operate through shared mechanisms at this locus

230 families

Zhang et al, 200322

NPSR1 (formerly GPR154 or GPRA)

7p14.3

Finnish, Canadian, German, Italian, Chinese Asthma, total IgE, BHR, specific IgE, childhood asthma

Neuropeptide S receptor 1: G protein–coupled receptor on bronchial epithelial and smooth muscle surfaces; may modulate asthma, altering respiratory function90

86 families and 103 trios

Laitinen et al, 2004148

HLA-G

6p21.3

Caucasian, Hutterite, Dutch Asthma, BHR, atopy

MHC class I, type G: inhibits Th1-mediated inflammation; only soluble form (HLA-G5) is expressed in asthmatic bronchial epithelial cells

129 families

Nicolae et al, 200521

CYFIP2

5q34

Korean Atopic asthma, childhood asthma

Cytoplasmic FMR1-interacting protein 2: differentiation of T cells

155 families

Noguchi et al, 2005147

IRAK3 (formerly IRAK-M)

12q13.13

Sardinian, Italian Early onset, persistent asthma

IL-1 receptor–associated kinase 3: negative regulator of toll-like receptor/IL-1R pathways; master regulator of nuclear factor-κB and inflammation

100 families

Balaci et al, 2007149

COL6A5 (formerly COL29A1)

3q21.3

European Atopic dermatitis

Collagen, type VI, alpha 5: novel epidermal ECM collagen; expressed in skin, lung, small intestine, and colon; lacking in outer viable epidermal layers in atopic dermatitis patients

199 families (427 children) for discovery, 292 families (481 children) for replication

Soderhall et al, 200725

OPN3 and CHML

1q43 and 1q42-qter

Danish, UK, Norwegian Asthma, atopic asthma, BHR

Opsin 3 and choroideremia-like (RAB escort protein 2): regulation of peripheral circadian rhythms and Th1 and Th2 cell polarization

294 Danish families (1151 subjects), 442 UK or Norwegian families

White et al, 2008146

PLAUR (formerly UPAR)

19q13

UK, Dutch Asthma, lung function decline (FEV1), BHR

Plasminogen activator, urokinase receptor: key role in formation of serine protease plasmin; implicated in many processes, including cell differentiation, proliferation, migration, and fibrinolysis; may contribute to pathogenesis of asthma through airway remodeling

46 and 341 families, 200 families

Barton et al, 2009150

PCDH1

5q31.3

Dutch BHR

Protocadherin 1: the protocadherin subfamily is part of cadherin superfamily; membrane protein found at cell-cell boundaries; expressed in respiratory epithelial cells; potential role in wound repair

200 families in initial study; replication studies in seven Caucasian populations

Koppelman et al, 200924

ADAM, A disintegrin and metalloproteinase (family of peptidase proteins); BHR, bronchial hyperresponsiveness; ECM, extracellular matrix; FEV1, forced expiratory volume in 1 second; FMR1, fragile X mental retardation 1; IgE, immunoglobulin E; IL, interleukin; SPT, skin-prick test; Th1, helper T cell type 1; Th2, helper T cell type 2; UK, United Kingdom; US, United States.

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SECTION A  Basic Sciences Underlying Allergy and Immunology

effect (Fig. 22.1).26 Microarray chips are available for genotyping 500,000 to 2,500,000 SNPs per person. The cost has progressively decreased, and accuracy has increased, making this a powerful approach for studying the genetics of common diseases. This approach localizes the susceptibility locus to a much smaller region (10 to 500 kb) than is typically possible in a linkage study. More recently with the increased understanding of variation in the human genome (e.g., via the UK 10K genome project), arrays are designed to capture low-frequency variation (1% to 5% MAF), and it is possible to impute (or infer genotypes at specific SNPs that were not directly measured) for more than 28 million polymorphisms per individual.27 The first GWASs for complex diseases were reported in 2005 and have transformed the study of genetic factors in complex diseases.26,28 GWASs have provided compelling statistical associations for hundreds of loci in the human genome, giving insight into the physiologic parameters (e.g., body mass index) and biologic processes (e.g., blood eosinophil levels) that underlie these phenotypes and diseases.29 GWASs have proved successful in the identification of genetic factors underlying allergic disease.30 Unlike traditional candidate gene association studies, GWASs may identify novel genes and pathways, and unlike linkage studies, they can identify variants with small effects. GWASs of large populations of cases and controls have now become the standard approach to gene discovery. One problem with GWASs is the large number of false-positive results for the number of genotypes analyzed. False-positive results are a major problem in all association studies and even more of an issue in GWASs. Very stringent statistical significance (P < 5 × 10−8) and replication of positive findings in additional populations is crucial.

Interpreting Results of Genetic Studies The association between the phenotype and the allele is positive if the allele is the cause of, or contributes to, the phenotype. This association is expected to be replicated in other populations with the same phenotype, unless there are several different alleles at the same locus contributing to the same phenotype, in which case an association would be difficult to detect. Similarly, if the trait was predominantly the result of different genes in the other population (i.e., genetic heterogeneity) or depended on the interaction with an environmental exposure not present in the replication population, replication may not be achieved. Another reason for lack of replication may be different phenotype definitions used in different studies. For example, the atopy phenotype has been defined by a positive skin-prick test result, a positive radio­ allergosorbent test (RAST) result, a high serum level of total IgE, or a combination of these investigations. Although these phenotypes are clearly related, it is likely that some genes that influence total IgE levels do not influence specific IgE responses to allergens, and vice versa. Positive associations may not be replicated because the true model of genetic susceptibility for diseases such as asthma and atopy is complex. Any susceptibility variant may have a relatively minor effect on the phenotype, and the magnitude of its effect is influenced by genes at other loci (i.e., gene-gene interactions [epistasis]) and by environmental exposures (i.e., gene-environment interactions).31-34 Because background genes and environmental factors are different in disparate populations, it is not surprising that associations with single SNPs or haplotypes are dissimilar between populations. Associations between an allele and a phenotype are positive if that allele is in linkage disequilibrium with the phenotype-causing allele. Linkage disequilibrium is the correlation between nearby variants such that the alleles at neighboring polymorphisms (observed on the same chromosome) are associated within a population more often than if they were unlinked. For example, the SNP most strongly associated with the disease phenotype at a particular locus in a GWAS is unlikely

to be the true casual polymorphism; rather, it is marking a region of linkage disequilibrium containing one or more genes in which the causal polymorphism(s) lie. An association may not be replicated in subsequent studies because of different patterns of linkage disequilibrium in different populations. Differences in linkage disequilibrium patterns can be caused by variation in allele frequencies or the presence of more than one causal variant. This problem can be addressed by examining haplotypes instead of single SNPs. A haplotype is composed of alleles at different loci that are inherited together on the same chromosome. Even if the disease-causing variant is not identified, a shared haplotype that contains the disease variant occurs more commonly in cases than in controls, which can help to identify the true susceptibility variant. A positive association between an allele and a trait can be artefactual as a result of recent population admixture. In a population of mixed ancestry, any trait occurring at a higher frequency in a subgroup of the population (e.g., one ethnic group) produces a positive association with an allele that also happens to be more common in that population subgroup. To avoid a spurious association arising through admixture, studies should be performed in large, relatively homogeneous populations. In GWASs, population stratification is relatively easy to control for, because data from across the genome from each individual can be compared with reference data from individuals of different ancestry, and individuals who show mixed ancestry can be excluded from the analysis. Positive associations between polymorphisms and phenotype can reflect a type 1 error or false-positive results. Using a P value of .05 as the threshold for significance is equivalent to a 5% type 1 error rate, and it means that for every 20 tests, one finding by chance is expected. The main source for type 1 error is multiple comparisons. In these cases, P < .05 (i.e., 5% tolerance as the false-positive findings rate when the null hypothesis is not accepted) is no longer applicable because a 5% type 1 error rate is expected with each independent comparison (e.g., with polymorphisms in different genes). Another source of type 1 error is the large number of negative association studies with candidate genes that are never reported. Because the reported P values are rarely adjusted for the total number of studies performed (reported and unreported), the type 1 error rate for the reported studies is higher than the true level. Type 1 errors can also result from genotyping errors, such as overcalling one genotype over another.

GENETICS OF SELF-REPORTED AND DOCTOR-DIAGNOSED ALLERGIC DISEASE Asthma Asthma has been the most extensively studied allergic disease with respect to genetics. More than 1000 publications have examined polymorphisms in several hundred genes for association with asthma and related phenotypes such as airway hyperresponsiveness, bronchodilator response, and lung function. Increasing numbers of genes have been identified as asthma-susceptibility genes by the use of hypothesisindependent genome-wide linkage studies initially and then GWAS.

Hypothesis-Independent Approaches: Genome-Wide Linkage and Genome-Wide Association Studies Positional cloning using genome-wide scans for allergic disease phenotypes has identified many asthma-susceptibility genes, including ADAM33, DPP10, PHF11, HLA-G, OPN3, NPSR1 (formerly GPR154 or GPRA), PLAUR, and IRAK3 (formerly IRAK-M) for asthma and PCDH1 for BHR (Table 22.1). Identification of these genes, most of which had not been implicated in allergic disease previously and whose biologic functions in asthma were unknown, reveals the importance of using hypothesis-independent approaches to identify

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CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma susceptibility genes to understand disease pathogenesis. Despite these successes, linkage analysis for asthma has proved to be slow, expensive, and underpowered. Several GWASs of asthma have been performed with great success. Table 22.2 summarizes the findings of these studies. The first novel asthma-susceptibility locus to be identified by a GWAS was on chromosome 17q12-21.1.35 In this study, the genotypes were determined for 317,000 SNPs in 994 subjects with childhood-onset asthma and 1243 nonasthmatic controls. After adjustments for quality control, seven SNPs

remained above the 1% false discovery rate threshold, and all mapped to a region spanning 100,000 base pairs on chromosome 17. Replication of the findings was achieved by genotyping nine of the associations in 2320 subjects (200 asthmatic cases and 2120 controls), and five of the SNPs were significantly associated with disease. Analysis of the diseaseassociated SNPs with gene expression implicated the ORMDL3 and GSDMB genes. Many subsequent studies enrolling ethnically diverse populations have replicated the association between variation in this genetic region and childhood asthma.

TABLE 22.2  Selected Genome-Wide Association Studies for Asthma and Allergic Disease Population/ Study Size

Phenotype Analyzed

Genes or Loci*

Moffatt et al, 200735

European ancestry 994 cases and 1243 controls; replicated in 2320 cases and 3301 controls

Childhood-onset asthma

ORMDL3, GSDMA or GSDMB

Orosomucoid 1–like 3: transmembrane protein anchored in the endoplasmic reticulum, may have role in calcium homeostasis Gasdermin B: expressed in epithelium and gut, may have role in remodeling

Moffatt et al, 201036

European ancestry 10,365 cases and 16,110 nonasthmatic controls

Doctor-diagnosed asthma

IL18R, IL1RL1

IL-18 receptor 1: member of the IL-1 receptor family; DC-derived IL-18 drives Treg differentiation and plays a role in airway inflammation; IL-18 expression increased in asthma IL-1 receptor–like 1 (also called ST2): receptor elevated in human asthma, studies suggest receptor is induced by proinflammatory stimuli and may be involved in helper T cell function. MHC class II, DQ members: due to extensive linkage disequilibrium across the HLA region, difficult to identify specific genes that underlie association; extended haplotypes across the HLA region have been studied in relation to specific allergen sensitization and production of TNF-α, which is encoded in the HLA class III region IL-33: epithelial cell–derived IL-1–like cytokine ligand for the IL-1 receptor–related protein ST2; activates mast cells, ILC2, and Th2 lymphocytes SMAD family member 3: transcription modulator activated by TGF-β, a cytokine that controls proliferation, differentiation, and other functions in many cell types; Smad 3-deficient mice have increased inflammation in lungs IL-2 receptor, beta: IL-2 controls survival and proliferation of Tregs; implicated in differentiation and homeostasis of effector T cell subgroups, including Th1, Th2, Th17, and memory CD8+ T cells. Also involved in ILC2 function. IL-13: Th2 cytokine that drives IgE production by B cells and goblet cell differentiation and mucus production by airway epithelium. RAR-related orphan receptor A: encodes member of NR1 subfamily of nuclear hormone receptors; expressed with cluster of proteins that form the structural and innate immune defenses of the epithelial barrier Solute carrier family 22 (organic cation transporter), member 5: encodes a carnitine transporter See earlier See earlier

Study

HLA-DQ

IL33

SMAD3

IL2RB

IL13

RORA

SLC22A5 IgE levels

IL13 HLA locus

Gene Product and Functional Role of Selected Genes

Continued

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SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 22.2  Selected Genome-Wide Association Studies for Asthma and Allergic

Disease—cont’d Study

Population/ Study Size

Phenotype Analyzed

Genes or Loci* STAT6

IL4R, IL21R

Gene Product and Functional Role of Selected Genes Signal transducer and activator of transcription 6: transcription factor critical to IL-4 and IL-13 intracellular signaling regulating IgE and Th2 cytokine production IL-4 receptor: a subunit of the IL-4/IL-13 receptor IL-21 receptor: IL-21 and its receptor (IL-21R) are upregulated in skin lesions of patients with active atopic dermatitis; they play a critical role in sensitization and allergic inflammation in skin

Sleiman et al, 2010151

European ancestry 793 cases and 1988 matched controls (discovery set); 917 asthmatics and 1546 matched controls of European ancestry (replication 1); 1667 North American children of African ancestry who had asthma and 2045 ancestrally matched controls (replication 2)

Childhood asthma

DENND1B

DENN/MADD domain–containing 1B: gene is expressed by NK cells and DCs; DENND1B protein predicted to interact with the TNF-α receptor

Ferreira et al, 201138

European ancestry 2669 cases and 4528 controls combined with data from Moffatt et al, 2010 (n = 26,475); further replication in additional 25,358 independent samples

Doctor-diagnosed asthma

IL6R

IL-6 receptor: increased sIL-6R level in serum and airways of asthma patients; correlates with Th2 cytokine production in the lung; selective blockade of sIL-6R in mice suppresses IL-4, IL-5, and IL-13 production and decreases eosinophil numbers in the lung

11q13.5 near C11orf30, LRRC32

Leucine-rich repeat containing 32: this locus is also associated with atopic dermatitis (see later) and Crohn disease. C11orf30 regulates gene expression, epithelial barrier.

USP38 and GAB1

Ubiquitin-specific peptidase 38: function is unclear GRB2-associated binding protein 1: scaffolding adopter protein that plays a role in the signaling pathway activated by cytokine receptors for IL-3, IL-6, IFN-αI, IFNγ, and B cell and T cell receptors Region contains no reported genes but is located 1 Mb downstream of GATA3, a master regulator of Th2 cell differentiation Associated with type 1 diabetes and alopecia areata; strongest associated SNP is located 2 kb upstream from IKZF4 (i.e., EOS), which is involved in differentiation of T cells

Hirota T et al, 201139

Japanese 7171 cases and 27,912 controls

Adult asthma

A locus on chromosome 10p14 A gene-rich region on chromosome 12q13 Torgerson et al, 201137

Multiancestry (European, American, African American or African-Caribbean, and Latino)

Asthma

PYHIN1 ORMDL3, IL1RL1 TSLP IL33

Pyrin and HIN domain family member 1: asthma susceptibility specific to persons of African descent. TSLP, Thymic stromal lymphopoietin: epithelial cell-derived cytokine with a key role in induction of allergic inflammation

Wan et al, 201242

European ancestry 933 cases and 3346 nonasthmatic controls; replication 231 cases and 1345 nonasthmatic controls

Moderate–severe asthma (GINA 3 and above)

ORMDL3, GSDMA or GSDMB and IL1RL1/ IL18R1

See earlier

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CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

TABLE 22.2  Selected Genome-Wide Association Studies for Asthma and Allergic

Disease—cont’d

Population/ Study Size

Phenotype Analyzed

Ferreira et al, 2014152

European ancestry 6685 cases and 14,219 nonasthma controls, replication 878 cases and 2,455 controls

Asthma with allergic rhinitis

11 loci identified including HLA-DQB1, TLR1, WDR36, IL1RL1, LRRC32, GSDMA, TSLP, IL33, ZBTB10, SMAD3, CLEC16A/DEX1

TLR1, toll-like receptor 1, recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents. ZBTB10, transcriptional repressor that may regulate inflammatory genes. CLEC16A/DEX1, established locus for autoimmune disease, function unknown. WDR36, WD repeat domain 36: may facilitate formation of heterotrimeric or multiprotein complexes; family members involved in many cellular processes, including cell cycle progression, signal transduction, apoptosis, and gene regulation.

Bonnelykke et al, 201452

European ancestry 1173 cases and 2522 controls Replication 255 cases and 1927 controls

Asthma with recurrent acute hospitalizations (occurring 2–6 years of age)

Five loci identified including; GSDMB, IL33, RAD50, IL1RL1, CDHR3

RAD50 homolog (S. cerevisiae): important for DNA double-strand break repair, cell cycle checkpoint activation, telomere maintenance, and meiotic recombination; adjacent to the IL-4, IL-13 cytokine locus CDHR3, cadherin-related family member 3, coreceptor for RSV-C on airway epithelium

Pickrell et al, 201640

European ancestry 28,399 cases and 128,843 nonasthmatic controls

Self-reported asthma

27 loci identified

See main text

Demenais et al, 201841

Multiancestry (European, African, Japanese, Latino) 23,948 cases and 118,538 nonasthmatic controls

Doctor-diagnosed asthma

18 loci identified (22 independent signals)

See main text

EsparzaGordillo et al, 200972

European ancestry 939 cases, 975 controls; 270 complete nuclear families with two affected siblings

Atopic dermatitis

C11orf30 locus

See earlier

Paternoster et al, 201274

European ancestry 5606 cases and 20,565 controls Replication in 5419 cases and 19,833 controls

Atopic dermatitis

OVOL1

Ovo-like 1: belongs to highly conserved family of genes that regulate development and differentiation of epithelial tissues and germ cells; regulates epidermal proliferation and differentiation ADAM metallopeptidase with thrombospondin type 1 motif and actin-like 9: ADAMTS proteins are complex, secreted, zinc-dependent metalloproteinases that bind to and cleave extracellular matrix components and are involved in connective tissue remodeling and extracellular matrix turnover Kinesin family member 3A: located within the Th2 cytokine cluster at 5q31.1

Study

Genes or Loci*

Locus near ADAMTS10 and ACTL9

KIF3A Sun et al, 201173

Chinese Han 1012 cases and 1362 controls; replication in 3624 cases and 12,197 controls

Atopic dermatitis

TMEM232 and SLC25A46

TNFRSF6B and ZGPAT

Gene Product and Functional Role of Selected Genes

Transmembrane protein 232: encodes a protein belonging to the functional class of tetraspan transmembrane proteins Solute carrier family 25, member 46: this family encodes mitochondrial carrier proteins, which may shuttle metabolites across the inner mitochondrial membrane Tumor necrosis factor receptor superfamily, member 6B decoy: important role in adaptive immune responses Zinc finger, CCCH type with G patch domain: located at 20q13.3; limited analysis of variants found no significant association Continued

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SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 22.2  Selected Genome-Wide Association Studies for Asthma and Allergic

Disease—cont’d

Population/ Study Size

Phenotype Analyzed

Ellinghaus et al, 2013153

2425 cases and 5449 controls from Germany Replication 7196 cases and 15,480 controls from Germany, Ireland, Japan, and China

Atopic dermatitis

Identified nine loci; LCE3A(FLG), SLC9A4, IL13, C11orf30, TNFRSF6B, IL2-lL21, PRR5L, CLEC16A-DEXI, ZNF652

LCE3A, late cornified envelope gene 3A, barrier repair, psoriasis risk locus. FLG, filaggrin, epidermal barrier function (see main text). IL2-IL21, interleukins associated with immune-mediated disease. PRR5L, proline rich 5 like, subunit of mammalian target of rapamycin complex 2 (mTORC2), potential role in fibroblast migration. ZNF652, encodes a transcriptional repressor that is implicated in epithelial cancers.

Weidinger et al, 2013154

European ancestry 1563 cases and 4054 controls Replication; 2286 cases and 3160 controls

Atopic dermatitis

Identified four loci; FLG, RAD50/IL13, HLA-B, C11orf30

See earlier

Schaarschmidt et al, 2015155

European ancestry 870 cases and 5203 population controls Replication 1383 cases and 1728 controls

Atopic dermatitis

Identified five loci; EDC (FLG), XIRP2, RAD50/IL13/ KIF3A, DMRTA1, RNF111

EDC, epidermal differentiation complex/FLG. DMRTA1, doublesex and mab-3-related transcription factor-like family A1 implicated in sex differentiation. XIRP2, xin actin-binding repeat-containing protein, potential cross linking function for F-actin.

Paternoster et al, 201575

Multiancestry (European, African, Japanese, Latino) 21,300 cases and 95,464 controls Replication; 32,059 cases and 228,628 controls

Atopic dermatitis

27 loci identified

See main text

Ramasamy et al, 201276

European ancestry 3933 self-reported cases and 8965 controls

Allergic rhinitis

HLA-DRB4 C11orf30 locus TMEM232 and SLC25A46

See earlier

Weidinger et al, 200883

European ancestry 1530 individuals, replication in four independent samples (N = 9769)

IgE levels, allergic sensitization

FCER1A

Fc fragment of IgE high-affinity receptor 1, alpha polypeptide: IgE receptor α unit initiates inflammation and hypersensitivity responses to allergens

RAD50

See earlier

Gudbjartsson et al, 200987

Multiancestry (n = 9392 Icelandic), 12,118 (European), 5212 (East Asian), 7996 cases and 44,890 controls.

Blood eosinophil counts

IL1RL1, WDR36/ TSLP, RAD50, IL33, MYB

See earlier. MYB: Myeloblastosis viral oncogene homolog: nuclear transcription factor implicated in proliferation, survival, and differentiation of hematopoietic stem and progenitor cells

Hinds et al, 2013156

European ancestry 53,862 individuals

Self-reported allergy (cat, dust-mite, and pollen allergies)

16 loci identified; TLR1, WDR36, C11orf30, IL1RL1, HLA-DQA, HLA-C, PTGER4, PLCL1, LPP, IL33, NFATC2, GSDMB, SMAD3, GATA3, ADAD1(IL2-IL21), FOXA1, ZBTB10, ID2, CLEC16A, IL4R, PEX14, ETS1

PTGER4, prostaglandin E receptor 4, autoimmune/skin immune responses. PLCL1 phospholipase C–like 1, involved in inositol 1,4,5-triphosphate intracellular signaling, autoimmune diseases. LPP, lipoma-preferred partner, autoimmunity. NFATC2, nuclear factor of activated T cells, T cell differentiation FOXA1, forkhead-box transcription factor family A1, TH2-mediated inflammation and mucous production.

Study

Genes or Loci*

Gene Product and Functional Role of Selected Genes

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

339

TABLE 22.2  Selected Genome-Wide Association Studies for Asthma and Allergic

Disease—cont’d

Population/ Study Size

Phenotype Analyzed

Genes or Loci*

Marenholz et al, 201594

European ancestry 1151 cases and 10,030 controls Replication, 864 cases and 5346 controls

Atopic march defined as early onset atopic dermatitis (up to 3 years of age) followed by childhood asthma (up to 16 years of age)

Seven loci identified: FLG, IL4/KIF3A, EFHC1, OVOL1, C11orf30, TMTC2/SLC6A15 and IKZF3 (17q21).

EFHC1, EF-hand domain (C-terminal)-containing protein 1, mucociliary epithelium cilia function. TMTC2, transmembrane and tetratricopeptide repeat containing 2, calcium homeostasis. SLC6A15, solute carrier family 6 member 15, membrane transporter for neutral amino acids.

Ferreira et al, 201793

European ancestry 180,129 cases and 180,709 controls

Allergic disease phenotype (asthma, allergic rhinitis, or atopic dermatitis)

99 loci identified (136 independent signals)

See main text.

Study

Gene Product and Functional Role of Selected Genes

Studies focused to those with >500 cases. *Genes/loci identified are those reported by the original manuscript.

To date, GWASs have identified a large number of genetic susceptibility loci for asthma (Table 22.2). They included four large-scale analyses of European,36 American (including individuals of European American, African American, African Caribbean, and Latino ancestry),37 Australian,38 and Japanese39 populations. These studies identified several replicated loci, including IL6R, IL1RL1/IL18R1, TSLP, RAD50/IL13, HLA-DRA/DRQ, IL33, GATA3, LRRC32, SMAD3, 17q21 (ORMDL3/ GSDMB/ZPB2) and IL2RB (Table 22.2). To date, the largest GWAS of asthma was completed by the personal genomics company 23 & Me and included 28,399 cases of self-reported asthma and 128,843 nonasthmatic controls and identified 27 loci contributing to the susceptibility to develop asthma.40 This study identified many of the previous associations and identified new associations at ADAMTS4, D2HGDH, CLEC16A, LRP1, ADORA1, BACH2, PEX14, and STAT6.40 Several of these loci have now been replicated in a recent study using 23,948 self-reported asthma cases and 118,538 nonasthmatic controls from multiple ethnic groups that reported 18 loci (22 independent signals) and novel loci at NDFIP1, GPX5, and ZNF652.41 These GWAS demonstrate the power of the genome-wide approach for identifying complex disease-susceptibility variants, and the number is likely to increase with the use of additional metaanalyses that combine information from individual studies with analysis of other asthmarelated phenotypes such as disease severity42 and age of onset.43 For other complex, chronic inflammatory diseases such as Crohn disease and type 1 diabetes mellitus (which have been extensively studied using GWAS), the study results have not fully explained the heritability patterns. It is believed that the inability to find all of the genetic factors underlying disease susceptibility may be explained by limitations of GWAS, which include the presence of other variants in the genome not captured by the current generation of genome-wide genotyping platforms, analyses not being adjusted for gene-environment and gene-gene (i.e., epistasis) interactions, or epigenetic changes in gene expression.

Genetic Studies Explaining Asthma Pathogenesis The study of the genetic basis of asthma has revealed astonishing insights into the pathogenesis of this complex condition. Initially, most candidate gene studies of asthma were focused on association of functional polymorphisms in components of Th2-mediated immune responses. For example, the gene encoding IL-13, the Th2 effector cytokine, is one of the genes most consistently associated with asthma and related phenotypes.44 Because of the importance of Th2-mediated inflammation in

allergic disease and the biologic roles of IL-13 (e.g., switching B cells to produce IgE; wide-ranging effects on epithelial cells, fibroblasts, and smooth muscle; promoting airway remodeling; mucus production), IL13 is a strong candidate gene. IL13 is also a strong positional candidate, with linkage studies implicating the genetic region encompassing the Th2 cytokine gene cluster on chromosome 5q3145 and more recently in GWAS studies to date (Table 22.2). Several functional polymorphisms of IL13 have been characterized. They include promoter polymorphisms such as the −1112 C/T variant that appears to alter transcription factor binding and an amino acid polymorphism involving the substitution of glycine for arginine at amino acid 131 (residue 110 in the mature protein). This change alters the affinity of IL-13 for the decoy receptor IL13RA2, increases functional activity through IL13RA1, and enhances stability of the molecule in plasma. Polymorphisms in other genes that encode proteins regulating Th2 cell production, such as GATA3 (encoding GATA-binding protein 3), TBX21 (encoding T-bet, the transcription factor for Th1 cell development), IL4 (encoding the cytokine), IL4R (encoding the receptor for IL-4), and STAT6 (encoding the downstream signal transducer), have been repeatedly associated with increased susceptibility to asthma.46 Evidence indicates that there may be a synergistic effect on the risk of inheriting more than one of these variants across components of the same pathway.32 More recently GWAS has confirmed and significantly added to this list of immune-related genes, including CD247, BACH2, IL13, IL33, LRCC32, HLA, STAT6, CLEC16A, GATA3, TSLP, CAMK4, and TNFSF4, involved more broadly in innate in addition to adaptive immunity (Table 22.2). For example, CD247 encodes for T-cell receptor zeta and forms part of the T cell receptor-CD3 complex important in T cell activation,47 and BACH2 (BTB domain and CNC homolog 2) encodes a transcription factor that regulates CD8+ T cell development.48 Importantly, these recently identified signals (BACH2, STAT6) have now been replicated.41 Overall, the genes identified as contributing to asthma can be segregated into five broad groups: 1. Genes that can directly modulate the response to environmental exposures.49 These include genes that encode components of the innate immune system that interact with levels of microbial exposure to alter the risk of allergic immune responses—for example, the genes CD14 and TLR4 that encode components of the lipopolysaccharide

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(LPS) response pathway. Similarly, IL33 is an alarmin that is released from airway epithelial cells in response to rhinovirus and CDHR3, a receptor found on airway epithelial cells that can influence rhinovirus infection and disease exacerbation.50-52 Other environmental response genes include those encoding detoxifying enzymes (e.g., glutathione S-transferase [GST]), which modulate the effect of exposures involving oxidant stress, such as tobacco smoke and air pollution.53 2. Genes that maintain the integrity of the epithelial barrier at the mucosal surface and cause the epithelium to signal the immune system after environmental exposure.54,55 Like filaggrin in the epidermal barrier, genes encoding chitinases (e.g., CHIA [formerly AMCase], CHI3L1 [formerly YKL40]) appear to play an important role in modulating allergic inflammation and are produced in increased levels by the epithelium in patients with asthma. PCDH1, which encodes protocadherin 1, is a member of a family of cell adhesion molecules. It is expressed in the bronchial epithelium and has been identified as a susceptibility gene for BHR. IL-33 is produced by the airway epithelium in response to damage and, for example, virus (see earlier) and drives production of Th2-associated cytokines such as IL-4, IL-5, and IL-13. TSLP is also released by the airway epithelium and can drive induction of allergic responses by effects on several cell types, including dendritic cells.56 3. Immune responses can be regulated by genes, including those regulating T cell differentiation and effector function and others such as IL6R, which was identified in a GWAS of an Australian population38 and may regulate the level of inflammation that occurs in the lung. More recently a large number of genes relevant to T cell immunity have been identified in GWAS, including CD247, BACH2, LRCC32, HLA, CLEC16A, GATA3, and TSLP (see earlier). 4. Genes involved in determining the tissue response to chronic inflammation (e.g., airway remodeling) include ADAM33, which is expressed in fibroblasts and smooth muscle; PDE4D, which is expressed in smooth muscle and inflammatory cells; and SMAD3, an intracellular signaling protein that is activated by the profibrotic cytokine transforming growth factor-β (TGF-β). Recently it has been shown that GSDMB is elevated in the airway epithelium in asthma, and in mice increased expression led to spontaneous airway hyperresponsiveness.57 Similarly, ORMDL3 has been linked to calcium homeostasis in airway smooth muscle cells leading to airway hyperresponsiveness.58 5. Some disease-modifying genes, rather than directly determining susceptibility to asthma, alter phenotypes related to disease progression, such as exacerbation frequency, disease severity, and development of fixed (irreversible) airflow obstruction. For example, genetic factors can modify the effect of environmental exposures such as vitamin D59 or particulate air pollutants (e.g., particulate matter ≤10 µm in diameter [PM10])60 on exacerbation frequency. The positionally cloned asthma gene ADAM33 has been associated with accelerated lung function decline in patients with asthma.61 More recently several previously described asthma genes have also been shown to associate with disease exacerbation, suggesting a modification role also (i.e. GSDMB, IL33, RAD50, IL1RL1, and the novel gene CDHR3).52 The use of GWASs in populations of severe asthma and asthma exacerbations may improve prediction of exacerbation phenotypes and the classification of patients by subphenotypes that may reflect different levels of pathogenicity and responses to treatment, enabling better targeting of therapeutics.

Early Development and Asthma Susceptibility Genetic studies of asthma have reinforced observations from traditional epidemiologic assessments about the importance of early-life events in determining asthma susceptibility. For example, ADAM33 was identified

as an asthma-susceptibility gene using genome-wide positional cloning, and multiple ADAM33 protein isoforms exist in human embryonic lung when assessed at 8 to 12 weeks of development.62 A polymorphism in ADAM33 is associated with early-life measures of lung function (specific airway resistance at 3 years of age).63 Consistent with this observation, a GWAS of adult lung function64 found that alleles representing 11 of the 16 novel loci associated with FEV1 or forced vital capacity showed consistent effects on lung function in children (7 to 9 years of age), suggesting that genetic determination of lung function in adults may in part act through effects on lung development and/or early life events. Some genetic studies have shown that variations in genes regulating atopic immune responses do not contribute to susceptibility to asthma. GWASs such as the GABRIEL Consortium study36 have suggested this is because most of the asthma-susceptibility loci identified were not associated with serum total IgE levels, although serum IgE is not a specific marker of atopy. These findings have supported the importance of local tissue response factors and epithelial susceptibility factors in the pathogenesis of asthma and other allergic diseases. Although there is now a large number of loci identified using GWAS in asthma, the interpretation of findings and the relative contribution of genes/pathways to asthma pathogenesis requires further work; however, it is reassuring that the same genetic loci are being replicated (e.g., as described recently in the UK Biobank cohort, where 28 of 31 loci identified as genetic risk factors for asthma replicated using 380,000 individuals).65

Atopic Dermatitis As in asthma, atopic dermatitis is a complex trait with disease susceptibility involving interactions between multiple genes and environmental factors.66 Heritability studies support a role for genetic factors related to atopy in general and for disease-specific atopic dermatitis genes. The risk of atopic dermatitis in a child is much greater if one or both parents have atopic dermatitis than if one or both parents have asthma or allergic rhinitis.11 The genetic basis of atopic dermatitis has been investigated by many candidate gene association studies, hypothesis-independent positional cloning, and GWAS approaches. A comprehensive review of genetic studies of atopic dermatitis found more than 100 studies investigating 81 genes, and in 46 reports, at least one positive association with atopic dermatitis was demonstrated.67 This study was recently updated for the period 2009 to 2016 and identified a further 92 candidate gene studies, and overall loss of function variants in the filaggrin gene (FLG) remain the most robust replicated association for AD.68 Filaggrin, a filament-aggregating protein, is a major component of the protein-lipid cornified envelope of the epidermis, which is important for water permeability and for blocking the entry of microbes and allergens. FLG is located on chromosome 1q21 in the epidermal differentiation complex. In 2006 it was recognized that loss-of-function mutations in FLG caused ichthyosis vulgaris, a skin disorder characterized by dry, flaky skin and a predisposition to atopic dermatitis and associated asthma.69 The mutations in FLG appear to act in a semidominant fashion. Carriers of homozygous or compound heterozygous mutations (R501X and 2282del4) have severe ichthyosis vulgaris, and heterozygotes have milder disease. The combined carrier frequency of null filaggrin mutations is approximately 9% in Caucasian populations. Individuals who are heterozygous (i.e., carrying one copy) for these null alleles have a significantly increased risk of atopic dermatitis.70 Although FLG null alleles are relatively rare in the Caucasian population, they nonetheless account for up to 15% of the population’s attributable risk of atopic dermatitis, with penetrance estimated to be between 40% and 80%, meaning that 40% to 80% of subjects carrying one or more FLG null mutations will develop atopic dermatitis. Mechanistically, the role for FLG has been confirmed by analysis of the spontaneous,

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma recessive, mutant flaky tail gene (Flt) in mice, whose phenotype results from a frameshift mutation in the murine filaggrin gene (Flg) with enhanced cutaneous allergen priming and in allergen-specific IgE and IgG antibody responses.71 In family-based, genome-wide linkage scans the only gene identified by this approach has been COL6A5 (formerly COL29A1), which provides further support for a genetically determined deficit in epidermal barrier function underlying atopic dermatitis.25 Using a genome-wide association approach, a SNP adjacent to a locus of unknown function (C11orf30, which encodes a nuclear protein designated EMSY) on chromosome 11q13.5 was identified as being strongly associated with susceptibility to atopic dermatitis.72 This locus has previously been identified as conferring susceptibility to Crohn disease, which is another disease involving epithelial inflammation and defective barrier function. This suggests that the 11q13.5 locus represents another gene for an allergic disease that acts at the mucosal surface. In several GWASs, Sun and colleagues73 identified previously undescribed susceptibility loci at 5q22.1 (TMEM232 and SLC25A46) and 20q13.3 (TNFRSF6B and ZGPAT). In a metaanalysis Paternoster and coworkers74 identified three additional loci reaching genome-wide significance, including SNPs upstream of OVOL1 and near ACTL9; both were near genes that have been implicated in epidermal proliferation and differentiation (see Table 22.2 for details). Another SNP was identified in KIF3A within the Th2 cytokine cluster at 5q31.1. To date, the largest GWAS of AD was completed using a multiancestry cohort of 21,300 case and 95,464 controls in discovery and then 31,059 cases and 228,628 controls in replication, leading to the identification of 27 susceptibility loci.75 This study brought the number of AD loci to 31 and provided significant insight into AD, including confirming the role of genes in epidermal barrier function (e.g., FLG/LCE3A and adaptive immune responses) and autoimmunity (e.g., CD207, IL7R, STAT3, ETS1, IL2/IL21, IL6R, and CLEC16A [reference 75 and Table 22.2]).

Allergic Rhinitis Little is known about the genetics of allergic rhinitis. Familial aggregation has been observed in epidemiologic studies,8 but genetic studies have been limited. Genome-wide linkage studies have identified loci such as the HLA region and the C11orf30 or LRRC32 locus previously associated with atopic dermatitis in Caucasians.76 Several candidate gene studies for rhinitis have shown association with polymorphisms in inflammatory genes such as IL13,77 but most of these studies have been limited in size.

CURRENT UNDERSTANDING OF ALLERGIC DISEASE GENETICS Atopy Whereas the majority of studies of the genetics of allergic disease have focused on clinical manifestations of atopy such as asthma or atopic dermatitis, there have been many hundreds of candidate gene association studies undertaken examining association with phenotypes of atopy, specific-IgE responses and total serum IgE levels.13,78 For example, genes such as IL4, IL13, IL-4 receptor-α (IL4RA), and STAT6 that are known to encode proteins playing critical roles in the development of Th2-type immune responses have shown consistent association with atopy.32,33,79,80,81,82 More recently, the use of the genome-wide association approach has provided significant insights into the genetic basis of an atopic predisposition. For example, a GWAS analysis of 1530 individuals to identify loci associated with serum IgE levels and allergic sensitization showed strong association between functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 and both of these phenotypes.83 In addition this

341

study also confirmed previous candidate gene studies that implicated variants in both STAT6 and the genetic region on chromosome 5q31 that contains the genes encoding the typical Th2 cytokines IL-4 and IL-13. The exact causal polymorphism(s) at the locus are unclear, because there have been multiple polymorphisms identified in the promoters of both genes that regulate their transcriptional levels. In addition, both of these genes, together with the nearby cytokine gene IL5, appear to be coordinately regulated, through the actions of regulatory locus control elements extending into the adjacent RAD50 gene.84 Subsequent studies in other ethnic populations have identified further loci associated with serum IgE levels.85,86 Another atopy-related phenotype to be examined using a GWAS approach is blood eosinophil counts. In an Icelandic population, polymorphism in proinflammatory cytokine genes including IL1RL1 (IL33 receptor) and the gene encoding the Th2-promoting cytokine IL-33, alongside genes that encode molecules regulating hematopoietic progenitor cell differentiation and proliferation such as MYB, were shown to be associated with baseline blood eosinophil counts.87 Studies in Hispanic populations have also identified genes such as GATA2, a transcription factor with a key role in hematopoietic cell development, as associated with blood eosinophil counts.88 Using allergic sensitization defined as skin prick test positive to at least one allergen, Bønnelykke et al. meta-analyzed data from 10 studies in 5789 affected individuals and 10,056 controls and with replication in 6114 affected individuals and 9920 controls. They identified ten loci associated with sensitization including SNPs in or near TLR6, C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1, LPP, MYC, IL2, and HLA-B. Risk-associated variants at these ten loci were estimated to account for at least 25% of allergic sensitization and allergic rhinitis.89 As might be expected, loci identified in these studies are also associated with disease phenotypes involving Th2-mediated immunity or a role for eosinophils. For example, in the Icelandic population several of the loci associated with blood eosinophil levels were also associated with asthma and myocardial infarction.87 Variation within the IL4/IL13 locus has long been recognized as being associated with a wide range of atopic disease phenotypes such as asthma,90 and it is one of the few genetic loci identified as asthma susceptibility loci in candidate gene and linkage approaches to also show association with asthma in GWAS studies.36 This overlap between genetic variation identified as predisposing to atopy and that underlying asthma is not surprising given current understanding of the role played by IgE and Th2-mediated immune responses in the pathogenesis of allergic disease and studies of heritability that have suggested that genes that predispose to atopy overlap with those that predispose to asthma. However, what is remarkable is that the overlap between loci identified as predisposing to serum IgE levels and allergic disease is small. For example, in the large GABRIEL GWAS study of 10,365 subjects with physician-diagnosed asthma and 16,110 controls,36 loci strongly associated with IgE levels were not associated with asthma with the exception of IL-13 and the HLA region, supporting studies that have noted the absence of a relationship between atopic sensitization and asthma in some populations.91,92

Overlap in Genome-Wide Association Study Results of Allergic Disease There is accumulating evidence to suggest that allergic diseases and traits share a large degree of genetic susceptibility; for example the IL33/IL1RL1 axis appears to be particularly important, suggesting this pathway may represent a therapeutic opportunity across the allergic traits (Table 22.2). The “atopic march” (e.g., childhood AD) leads to an increased risk of developing asthma later in life, which may at least in part be explained by overlapping genetic susceptibility. Similarly, there is clearly concordance (e.g., C11orf30, SLC25A46, HLA-DQB1,

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SECTION A  Basic Sciences Underlying Allergy and Immunology

IL1RL1, MYC, IL2, and HLA-B) for, for example, specific IgE loci and self-reported allergic disease. However, it is important to note that there is also discordance for several loci (e.g., the FLG locus for AD). The concept of shared genetic origins for asthma, allergic rhinitis, and atopic dermatitis was recently formally tested by a large GWAS involving 180,129 cases (with asthma, allergic rhinitis, or atopic dermatitis) and 180,709 controls.93 This study identified 99 genetic susceptibility loci, including 136 independent signals spanning genes involved in predominantly immune functions that therefore represent potentially shared genetic susceptibility for allergic traits (Fig. 22.2). Interestingly, 130 of the 136 sentinel variants had similar allele frequencies in caseonly association analyses when compared using nonoverlapping groups with asthma, allergic rhinitis, and atopic dermatitis. The six variants showing more of an effect in a specific disease included, for example, FLG for AD (vs. asthma or AR) and GSDMB for asthma (vs. AD).93 Overall these data suggest asthma, atopic dermatitis, and allergic rhinitis coexist because they share common genetic risk factors influencing immune functions (Fig. 22.3). Interestingly, the potential genetic basis of the transition from infantile atopic dermatitis to childhood asthma has recently been studied using 1151 cases and 10,030 controls (stage 1) and 864 cases and 5346 controls (stage 2), with cases defined as early-onset atopic dermatitis (up to 3 years of age) followed by childhood asthma (up to 16 years of age).94 This study identified seven loci, with five loci previously identified in GWAS of allergic traits and two novel loci including on chromosome 6, EFHC1 (EF-hand domain (C-terminal)-containing protein 1) thought to be involved in epithelial functions and chromosome 12 between TMTC2 (transmembrane and tetratricopeptide repeat containing 2) involved in calcium homeostasis and SLC6A15 (solute carrier family 6 member 15), a membrane transporter for neutral amino acids (Table 22.2).

MISSING HERITABILITY IN ALLERGIC DISEASE Despite the advances made in the identification of allergic disease genes through the use of GWAS studies, it has been apparent that given the size of the effects of SNPs identified (ORs typically in the range 1.05 to 1.3) that there is a large proportion of heritability still to be accounted for. This also means that the use of genetic markers alone to predict disease susceptibility is not clinically useful. For example, in genomewide analysis of asthma in an Australian population by Ferreira et al,38 the use of multi-SNP risk scores, despite being significantly associated with asthma risk, provided low discrimination in disease status (i.e., low sensitivity and specificity, area under the curve = 0.565 for the 10 SNPs most significantly associated with asthma in the GABRIEL study) and as such have little or no diagnostic utility per se. The finding that loci identified through GWAS studies for common disease fail to account for all heritability of these conditions has been termed missing heritability. There are a number of reasons that may account for this observation. These include gene-gene interaction, gene-environment interaction, other types of genetic variation such as rare variants and structural variation, and epigenetic heritability.

Gene-Gene Interaction Similar to other complex genetic diseases, it has been shown that genes involved in the pathogenesis of allergic disease interplay with each other (Fig. 22.3). An example of gene-gene interaction in asthma includes genes in the IL-13/IL-4 cytokine pathway. Binding of IL-13 and IL-4 to their common receptor (IL-4 receptor α, IL-4Rα) induces the initial response for Th2 lymphocyte polarization. Both IL-13 and IL-4 are produced by Th2 cells and are capable of inducing isotype class switching of B cells to produce IgE after allergen exposure. In a case-control study design in a Dutch population, a significant gene-gene interaction

between the SNP S478P in IL4RA and the –1112C/T promoter polymorphism in IL13 was detected. Individuals with the risk genotype for both genes were at almost five times greater risk for the development of asthma compared with individuals with both nonrisk genotypes. These data suggest that an interaction between IL4RA and IL13 markedly increases an individual’s susceptibility to asthma.95 Another study on a large cross-sectional population of 1120 children aged 9 to 11 years indicated that the combinations of genetic variations in the IL4/ IL13 pathway are significantly related to the development of atopy and childhood asthma.32

Gene-Environment Interaction The evidence for the increased prevalence of allergic disease in the last decades is strongly suggestive of an important environmental component in its pathogenesis, with the onset of the disease and its clinical course determined by gene-environment interactions. Among affected individuals in the population, the relative influence of genetic and environmental factors probably varies, and individuals with different allergy-related genotypes have different sensitivities to environmental exposures. Several possible patterns for gene-environment interaction have been suggested.49 For example, both the presence of a given disease susceptibility gene and an environmental exposure may be necessary to produce excess risk of a disease. With regards to asthma, there are extensive data showing that passive tobacco smoke increases airway responsiveness and incidence of asthma, especially prenatal exposure,96 and that this interacts with genetic susceptibility to determine disease onset.97 Analysis of the effect of a number of asthma susceptibility genes has now shown interaction with tobacco smoke exposure and genetic variation to determine disease susceptibility. For example, the association between SNPs in the susceptibility locus on chromosome 17q21 encompassing the ORMDL3/GSDMB genes has been shown to be confined to early onset asthma, and in particular those who have been exposed to environmental tobacco smoke in early life.98 The association of these 17q21 variants is also enhanced in children who experience respiratory infections before the age of 2 years, with the strongest association in children exposed to both tobacco smoke and respiratory infections.99 Another example of gene-environment interaction is the interaction between polymorphisms in components of the innate immune response such as CD14 and TLR4 involved in the recognition and clearance of bacterial endotoxin (LPS), by activating a cascade of host innate immune responses. Single nucleotide polymorphisms that alter the biology of these receptors could influence the early life origins of allergic disease by modifying the effect of microbial exposure on the developing immune system. A number of studies have now shown interaction between polymorphism of CD14 and measures of microbial exposure such as living on a farm, consumption of farm milk, and household dust endotoxin levels in determining serum IgE levels, sensitization, and asthma34,100-102 (Fig. 22.4). Further supporting this concept of gene-environmental interactions being critical in asthma, a recent GWAS of severe asthma with exacerbation unmasked a potentially interesting virus-host interaction.52 In this study, 1173 cases and 2522 controls (stage 1) and 255 cases and 1927 controls (stage 2) identified five loci, with cases defined as asthma with recurrent acute hospitalizations (occurring at 2 to 6 years of age). Four loci had previously been identified in asthma GWAS, GSDMB, IL33, RAD50, and IL1RL1; however, the effect sizes were higher in this study and related to number of hospitalizations. Importantly, a novel signal involving polymorphisms spanning CDHR3 were associated with severe asthma with exacerbation, and a specific polymorphism (Cys529Tyr) was predicted to be functional.52 CDHR3 encodes cadherinrelated family member 3, with other family members being involved

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma 50 known loci

49 new loci STMN3 1 1

1 11 C22orf46, PMM1, NHP2L1, CCDC134, PHF5A, CSDC2, MEI1 X

[ZNF217] 0 1 [NFATC2] 0 1

1 0 [SIK1] 1 0 [RUNX1]

ORMDL3, CCR7, RP11-94L15.2, ZPBP2, GSDMB 7 4

22 21 20

CLEC16A-[]-RM12, RM12-[]--LITAF 0 2

19

1 1 DYNAP

18

1 2 MAP3K14-AS1, SPATA32

GNGT2 1 1

AAGAB 1 1 RP11-554D20.1 1 1

1 0 SLC7A10-[]-CEBPA 1 1 PIGN

1 0 [STAT5B]

17

[RAD51B] 0 1 FOXA1-[]--TTC6 0 1

1 1 ALOX15

16

NFKBIA, PPP2R3C, KIAA0391, FAM177A1 4 1

1 2 IQGAP1, CRTC3 1 2 RTF1, ITPKA

15

PITPNM2, OGFOD2, ARL6IP4, ABCB9, SBNO1 10 1 NAB2, STAT6, SUOX, RPS26, PA2G4, MYL6B, ERBB3 8 2

1 0 RCOR1-[]-TRAF3 1 0 JDP2-[]-BATF

14

KIRREL3-AS3---[]--ETS1 0 1

1 0 PIBF1-[]-KLF5

DDX6-[]-CXCR5 0 1

13

1 0 [FOXO1]

WNT11--[]-LRRC32 0 3

1 1 SPPL3

OVOL1, SNX32, RP11-770G2.5, EFEMP2 4 1

12

1 1 SH2B3

ACTRIA, TMEM180, ARL3 3 1

1 1 AQP5

[ZNF365] 0 1

1 1 HDAC7

11

GATA3---[]---SFTA1P, GATA3--[]SFTA1P 0 6

1 2 PPP2R1B, SIK2

IL2RA 1 2

1 0 SESN3--[]-FAM76B

10

C9orf114-[]-LRRC8A 0 1

1 1 FBXW2

ERMP1 1 3 [MYC] 0 1

1 1 GSAP 1 0 C7orf72-[]-IKZF1

9

MIR5708--[]--ZBTB10 0 1

1 1 JAZF1

ITGB8--[]-ABCB5, [ITGB8] 0 2

8

[BACH2] 0 1

1 0 RNASET2-[]-MIR3939 1 0 [ARID1B]

HLA-C, MCCD1P1, HLA-DQA1, GPANK1, PRRC2A 5 7

1 1 TNFAIP3

7

HDAC3, NDFIP1 2 2

1 0 [PTPRK]

SLC22A4, SLC22A5, C5orf56, IL13 4 3

1 0 [ATG5]

CAMK4, SLC25A46, TSLP, CTC-551A13.2, WDR36 5 4

6

PTGER4 1 1

1 2 RGS14, RAB24 1 0 MIR3142-[]-MIR146A

IL7R 1 1

1 0 [TNFAIP8]

FAM105A 1 1

5

KIAA1109 1 2

1 0 [MANBA] 1 0 STX18--[]-MSX1

TLR1 1 1

1 1 RASA2

FBXO45-[]-CEP19 0 1

4

RP11-132N15.2, RP11-132N15.1, BCL6 3 4

1 4 SLC15A2, EAF2, IQCB1, DC86 1 1 SENP7

[GLB1] 0 1

1 0 LINC00870--[]-RYBP

[D2HGDH] 0 1

3

BOLL, RFTN2, MARS2, PLCL1 4 1

1 1 INPP5D 1 0 CCL20-[]-DAW1

MFSD9, IL18R1, IL1RL1, IL18RAP 4 2

1 1 ARHGAP15

[LINC00299] 0 1 TNFSF18--[]-TNFSF4, FASLG-[]--TNFSF18 0 2

1 0 [IL1B]

2

CD247 1 1

2 0 BCL2L11--[]--ANAPC1 1 0 [LOC339807]

USF1, F11R, PPOX, ADAMTS4, B4GALT3, FCER1G 7 1

1 0 [ITPKB]

IL6R 1 1 THEM4, FLG 2 3

1 0 SFPQ-[]-ZMYM4

1

RPRD2, C10rf54, TARS2 3 1

1 1 RUNX3

18

15

12

9

–log10 (P value)

6

3

0

Target genes

n target genes

n sentinel variants

Target genes

21

n sentinel variants n target genes

1 1 TNFRSF14

RP5-1115A15.1, RERE 2 1

Fig. 22.2  Loci containing genetic variants associated with risk of allergic disease. Data highlights 136 risk variants located in 99 loci identified using 180,129 cases (asthma, allergic rhinitis, or atopic dermatitis) and 180,709 controls. The numbers of plausible target genes of sentinel risk variants identified for each locus are shown, with target gene names listed in blue font. For loci with many target genes, only a selection is listed. When no target gene was identified (black font), brackets are used to indicate the location of the sentinel risk variant relative to the nearest gene(s). The red vertical line in the Manhattan plot shows the genome-wide significance threshold used. (Reproduced with permission from Ferreira MA, Vonk JM, Baurecht H, Marenholz I, Tian C, Hoffman JD, et al. Shared genetic origin of asthma, hay fever and eczema elucidates allergic disease biology. Nat Genet 2017;49(12):1752-7.)

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JUN/JUNB/JUND

IL18RAP

IL 2R

BCL6

ILTR

IRAK

IFN type 1 Ilnar

IL18R1 Fcer IL2RA IL12 (complex) IL12 (1arnly)

TH2 Cytokine

CO3 CD3 group

STAT6

KIR FCER1G

L1RL1

STAT5a b TSLP

CD86 CD247 IL23

INTERLEUKIN Ige

ERK12 INPR50 Fogr3

Atox15

Gm-csf Fcer

SH283

EAF2 F11R

Top Canonical Pathways Name

p-value

T helper cell differentiation Th1 and Th2 activation pathway Th2 pathway iCOS-iCOSL signaling in T helper cells Nurr77 signaling in T lymphocytes

2.58E-10 6.67E-09 1.54E-07 5.55E-06 1.88E-05

Top Upstream Regulators Upstream regulator SPI1 IL21 ZBTB16 Phorbol myristate acetate E. coli B5 lipopolysaccharide

p-value 5.96E-07 6.60E-07 1.28E-06 1.31E-06 2.19E-06

Top Diseases and Bio Functions Name Inflammatory disease Respiratory disease Connective tissue disorders Immunological disease Inflammatory response

p-value 4.04E-03 - 6.15E-12 1.61E-03 - 6.15E-12 3.33E-03 - 3.40E-10 4.87E-03 - 3.40E-10 4.88E-03 - 3.40E-10

Fig. 22.3  This gene network illustrates how the results of genome-wide association studies (GWAS) can provide insight into the pathogenesis of allergic disease and identify potential new therapeutic targets. This network was constructed using Ingenuity Pathway Analysis (IPA) (Ingenuity Systems, www.qiagenbioinfor matics.com) when inputting the plausible target genes identified in a GWAS study of allergic disease.93 The network is the top interaction network from the IPA analysis as determined from the knowledge base using experimentally validated interactions between genes and proteins. These interactions are the edges of this network and the nodes represent genes or gene products. The brown coloring of the molecules in the network mean that they were in the list of plausible target genes. The tables illustrate the top five results from the enrichment categories of Canonical Pathways, Upstream Regulators and Diseases and Bio Functions from the list of genes identified from the GWAS study.

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma CDHR3/virus

CD14/bacteria

Rhinovirus Infection: Positive

1.0

Negative

CC CT TT

Predicted probability for sensitization

high

Acute Exacerbation Severity

low GG

A

AG CDHR3 Genotype

345

0.8

0.6

0.4

0.2

0.0

AA

B

1.0

7.4

54.6 403.4 2981 22000 162755 1.2 × 106 Endotoxin load (EU/m2)

Fig. 22.4  The effect of genotype on disease susceptibility may depend on environmental exposure. For example, (A) a polymorphism that changes Cys529Tyr in the CDHR3 protein (from G [Cys] to A [Tyr]) confers risk to be hospitalized for asthma exacerbation by age 6 years. The A (Tyr) risk allele increases surface expression of CDHR3 and influences binding and viral replication of rhinovirus-C in the airways, thus providing a putative mechanism. Note that the phenotype associated with the CDHR3 risk allele is expressed only when it occurs together with the triggering environmental exposure. (B) A promoter polymorphism of the CD14 gene can produce an opposing effect on allergic sensitization depending on the level of endotoxin exposure. The graph shows fitted predicted probability curves for allergic sensitization at 5 years of age in relation to environmental endotoxin load in children with CC, CT, and TT genotypes in the promoter region of the CD14 gene (CD14/−159 C to T). (A, Reprinted from Kantor DB, Phipatanakul W, Hirschhorn JN. GeneEnvironment Interactions Associated with the Severity of Acute Asthma Exacerbation in Children. Am J Respir Crit Care Med. 2018;197(5):545-7. B, Reprinted from Simpson A, John SL, Jury F, Niven R, Woodcock A, Ollier WE, et al. Endotoxin exposure, CD14, and allergic disease: an interaction between genes and the environment. Am J Respir Crit Care Med 2006;174(4):386-92.)

in epithelial polarity and cell-cell interactions. Recent data suggest that CDHR3 is the receptor for rhinovirus C, the most common respiratory virus associated with exacerbations in asthma, and that the key variant identified in GWAS modulates levels of CDHR3, providing a putative mechanism.51 In the future, the use of genome-wide gene-environment interaction studies (GWIS) may provide further important insights into the biologic mechanisms underlying the response to endotoxin exposure. For example, a GWIS investigating the effect of farming environments on the risk of childhood asthma has recently been undertaken.31 In this study data on 500,000 SNPs were assessed for interaction with seven farm-related exposures (living on a family-run farm, mother grew up on a farm, regular consumption of raw farm milk, regular contact with cows, regular contact with straw, regular contact with hay, coincidence of cow and straw exposure) in 1708 children that formed part of the larger GABRIEL study into the genetics of asthma.36 The GWIS did not reveal any significant interactions with common SNPs for which the study had more than 50% power. However, among rarer SNPs, 15 genes with crossover interactions or effect concentrations in the exposed group for asthma or atopy in relation to farming, consumption of farm milk, and contact with cows and straw were identified, many showing a “flip-flop” pattern of association, i.e. risk versus protective allele reversed. Although these interactions deserve further investigation, of more interest, no interactions were observed involving SNPs in genes previously identified as showing interactions with farming exposures such as CD14 and TLR4. This may reflect issues with exposure assessment, as endotoxin levels were not directly measured in the population, and farming exposure,

while correlated with endotoxin exposure, is nonetheless a surrogate measure of exposure. This highlights the critical need for accurate exposure assessment in such studies. Identification of the factors that influence variability in the responses to environmental exposure could improve allergic disease management in several ways. Interactions between SNPs in a causal pathway and a relevant environmental exposure, such as innate immunity SNPs and farm living, provide additional proof that the environmental exposure is truly causal and not a confounded or false-positive result. Better characterization of gene-environment interactions can help to identify at-risk groups (e.g. the virus-host interaction outlined with who could benefit from preventative strategies that include environmental modification). Identification of at-risk groups, the degree of their sensitivity to exposures, and their frequency in the population can aid the costbenefit analysis of safe exposure levels in the public health setting.

Other Sources of Genetic Variation: Copy Number Variants and Rare Variants A further source of heritability not fully explored is the contribution of genetic variants other than SNPs to allergic disease. Rare variants form the group of infrequent mutations that occur in less than 5% of the population. A large proportion of variants in this class occur at a much lower frequency (less than 0.1%), and many thousands are likely to be specific to ethnic groups, isolates, families, or even individuals. Nevertheless, this class of variation harbours multiple penetrant disease mutations, conferring medium to high risk. GWAS studies have typically focused on SNPs with allele frequency at 5% or above because

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of the need for sufficient statistical power coupled with the ease of genotyping and availability of high-density arrays. However, studies of other common inflammatory diseases and the example of filaggrin in atopic dermatitis susceptibility show that for the severe end of the phenotype spectrum, rarer variants in the population may play a significant role in individual susceptibility to disease. For example, a rare IL33 loss-of-function mutation present in the Icelandic population reduces blood eosinophil counts and protects from asthma.103 The results of high throughput sequencing studies to identify the contribution of rare variants to phenotypes such as severe asthma are awaited with interest.104,105 The advent of microarray-based comparative genomic-hybridization technologies and whole-genome sequencing has also revealed unexpectedly heterogeneous structural variation (deletions, duplications, inversions, and translocations) in the human genome, termed copy number variations (CNVs), and these have been shown to be associated with a range of disease phenotypes.106 Although large-scale genome-wide studies of CNVs in allergic disease have yet to be undertaken, examples of CNVs in candidate genes such as the GSTM1 and GSTT1 genes show that this class of genetic variant may also be relevant to allergic disease,107 and whole genome sequencing of individuals from a founder population identified an association between a 6kbp deletion in an intron of NEDD4L and increased risk of asthma.108

FUNCTIONAL GENOMICS APPROACHES, TRANSLATING GENETIC ASSOCIATION SIGNALS The use of hypothesis-independent approaches such as GWAS to the study of the genetics of allergic disease has resulted in identification of genes of unknown function as susceptibility factors for disease. Although we have focused this review to specific genes, it is important to note that the first stage from a GWAS association signal is trying to identify the causative gene(s) and variant(s) underlying the association signal. Several approaches can be used for this; however, the use of expression quantitative trait loci (eQTL) in conjunction with publically available data from the ENCylopedia Of DNA Elements (ENCODE) project can quickly facilitate this (Fig. 22.5). eQTL analyses relate the presence and absence of single nucleotide polymorphisms with the expression of mRNA levels of all 20,000 genes in the specific cell type or tissue under investigation and can identify a functional role—for example, the allele associated with asthma risk and elevated levels of genes close (cis-eQTL) or genes on other chromosomes (trans-eQTL) providing an initial insight into potential causative genes(s). ENCODE is a resource that can be examined to identify additional genetic variants that are inherited with the asthma risk variant identified in the GWAS and begin to identify which of the variants change specific motifs in the DNA and therefore may be functional (e.g., transcription binding sites) (Fig. 22.5). Animal models can provide insights into gene function when little is already known about the identified gene, such as by comparing physiologic responses in gene-knockout and wild-type mice. For example, the positionally cloned asthma gene NPSR1 (formerly GPR154) was shown to control respiratory function through a central nervous system– mediated pathway. This gene also highlights the importance of selecting the correct measurements and disease model. Measurement of inflammatory outcomes in a well-established ovalbumin challenge model failed to identify differences in Npsr1-deficient animals.109 Similarly, knockout or overexpression studies in mice have provided insight into other asthma genes including ORMDL3 and GSDMB, including identification of spontaneous airway hyperresponsiveness in these mice (see earlier). Disease gene biology can be investigated by identification of commonalities in genetic susceptibility and pathogenesis between complex diseases. For example, the 17q21 locus containing several genes, including

ORMDL3, has been associated with several inflammatory conditions, such as inflammatory bowel disease and rheumatoid arthritis in addition to asthma. ORMDL3 regulates endoplasmic reticulum (ER) stress, and several additional ER stress– and unfolded protein response (UPR)– associated genes, including XBP1, ORMDL3, AGR2, and MUC19, have been identified as risk factors for inflammatory bowel disease.110 The intestinal epithelium of these patients commonly exhibits marked ER stress.110 Because of the commonality in genetic association, ER stress may also be an important pathogenic factor in asthma. This overlap of asthma risk genotypes and other human conditions has more recently been investigated highlighting overlap in risk factors for cardiovascular disease, immune/autoimmune traits, and digestive system disorders, including Crohn disease, in addition to others.41 These and other functional studies of disease-susceptibility genes highlight the gap between gene identification and disease biology that will take considerable effort to close.

EPIGENETICS AND ALLERGIC DISEASE Epigenetics refers to biochemical changes to DNA that do not alter the DNA sequence but may be induced by environmental factors and transmitted mitotically and meiotically (i.e., through generations). Epigenetic factors include DNA methylation and modification of histones by acetylation and methylation. The modification of histones, around which DNA is coiled to make chromatin, regulates gene expression and alters the rate of transcription and hence alters protein production. DNA methylation, which involves adding a methyl group to specific cytosine bases at CpG dinucleotides in DNA, typically suppresses gene expression. Histone modifications and DNA methylation are developmentally regulated and cell-type specific but can also result from environmental exposures such as tobacco smoke, viral infection, and alterations in early-life environment such as maternal nutrition.111,112 Epigenetic regulation of gene expression relevant to allergic disease has been studied extensively in candidate genes implicated in Type 2 immune responses, in particular, the type 2 cytokine genes IL-4, IL-5 and IL-13. DNA methylation of the regulatory regions around the type 2 cytokine genes decreases during Th2 cell differentiation from naïve T cells, and this is associated with increased gene expression.113,114 The histone modification patterns also become more permissive for cytokine gene expression in both human and murine studies.115,116 The cell-type–specific nature of these epigenetic modifications requires intensive laboratory investigation to determine the importance of epigenetics in disease processes, and most other cell types involved in allergic disease have not been studied in detail. Several other lines of evidence support the importance of epigenetic factors in allergic disease. Studies have linked altered birth weight and head circumference at birth (i.e., proxy markers for maternal nutrition) with an increase in adult IgE levels and risk of allergic disease.117-119 One study found that increased environmental particulate exposure from traffic pollution resulted in a dose-dependent increase in DNA methylation in peripheral blood.120 Observations support the concept that transgenerational epigenetic effects mediated by DNA methylation may be operating in allergic disease. Examples include grandmaternal smoking increasing the risk of childhood asthma in their grandchildren,121 as does paternal smoking122 and sex-specific transmission of disease risk, such as paternal allergic disease predisposing male offspring to development of allergic disease and maternal disease predisposing female offspring.123 More support comes from the study of animal models. Mice exposed to in utero supplementation with methyl donors exhibit enhanced airway inflammation after allergen challenge, a phenotype that persists in the second generation despite the absence of further exposure.124 Studies

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CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

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Fig. 22.5  Approaches to move from a genome-wide association study (GWAS) signal to identify causative gene(s) and variant(s). (A) An illustrative hypothetical example of the cis-eQTL and trans-eQTL together with their associated per-genotype gene’s read counts. On the left side is the example of cis-eQTL, where allele C associates with low gene expression while allele G associates with high gene expression. A heterozygous individual with both alleles is showing allele-specific expression (ASE). On the right side is the example of trans-eQTL, where allele T associates with low gene expression while allele A associates with high gene expression. In contrast to cis-eQTL, trans-eQTL is not showing allele-specific expression (ASE) in a heterozygous individual. Note that total per gene read counts cannot distinguish between ASE and non-ASE, because reads have to be split depending on what paternal chromosome they align to. (B) Combining GWAS-associated locus with human genome regulatory annotation from the ENCylopedia Of DNA Elements (ENCODE) project. (A, Adapted from Sun W, Hu Y. eQTL Mapping Using RNA-seq Data. Stat Biosci 2013;5(1):198-219. B, Reproduced with permission from Schaub MA, Boyle AP, Kundaje A, Batzoglou S, Snyder M. Linking disease associations with regulatory information in the human genome. Genome Res 2012;22(9):1748-59.)

have begun to identify the effect of environmental exposures relevant to allergic disease, such as farm exposure125 on DNA methylation and its relation to disease susceptibility.126 Murine studies have also shown that the maternal microbiota can provide protective effects against the development of allergic airway inflammation in the progeny via epigenetic mechanisms.127-129 Prospective studies of large birth cohorts with information on maternal environmental exposures have identified numerous regions of differential methylation around genes with known function in the pathogenesis of allergic disease, including the Th2 cytokine genes. Further studies of multigenerational cohorts are likely to provide important insights into the role of epigenetic factors in the heritability of allergic disease.130 The development of microarray-based platforms to enable genomewide assessment of DNA methylation status has revolutionized epigenetics research with the capability to examine more than 450,000 individual CpG sites simultaneously. These tools have been used to perform

epigenome-wide association studies (EWAS) analogous to the GWAS described earlier in this chapter. EWAS should enable identification of regions of the genome that have a methylation state associated with altered risk of disease. The first large scale EWAS in allergy examined associations between serum IgE concentration and DNA methylation status of peripheral blood leukocytes in 355 subjects.131 The authors identified 36 loci with decreased DNA methylation that were associated with IgE levels. Many of the loci identified were associated with type 2 immunity with a particular enrichment of genes involved in eosinophil biology. A follow-up study in Hispanic children has highlighted the need to adjust the data from EWAS studies to consider the prevalence of different cell types in the sample used.132 When the data was adjusted for eosinophil levels there was a marked reduction in the number of methylation sites associated with IgE levels, indicating that eosinophils are responsible for most of the differential methylation observed. EWAS provide a powerful tool to examine the influence of epigenetics in

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disease development; however, future studies will need to be carefully designed to reveal the critical sites involved in allergic disease.112,133

PHARMACOGENETICS OF ASTHMA Pharmacogenetics refers to the relationship between genotype (i.e., genetic variation) and drug response. Pharmacogenetic studies are genetic association studies, but only patients with the disease of interest are studied, and the outcome is individual response to therapy or the development of adverse effects. The issues addressed in case-control association studies should be considered in pharmacogenetic studies, including sample size and statistical power, population stratification, quality and completeness of phenotyping, and appropriate adjustment for the number of SNPs and genes analyzed. There have been extensive reviews written on the candidate gene approaches in pharmacogenetics studies of asthma medication134 and more recently the application of GWAS.135 Overall the most robust associations include: (1) between variants of the β2adrenoceptor gene (ADRB2) and exacerbation frequency for patients carrying the Gly16>Arg16 polymorphism and taking long acting β2adrenergic receptor agonists; (2) between variants in the low-affinity IgE receptor (FCER2, intronic) and exacerbations in asthma patients taking inhaled corticosteroids (ICS); and (3) between 5 lipoxygenase (ALOX5, intronic SNP), an enzyme involved in leukotriene synthesis, and response to leukotriene receptor antagonists (LTRA) and leukotriene synthesis inhibitors (LTSI) in asthma.136-138 As with disease susceptibility analyses, GWASs are now being completed in pharmacogenetic studies in asthma (e.g., recently ASB3 [ankyrin repeat and SOCS box containing 3] that is thought to regulate smooth muscle differentiation was identified in a GWAS of response [change in FEV1] to short-acting β2-adrenergic receptor agonists).139 Though only covered briefly here, several genetic variants have been identified as predictors of response to or adverse effects of current asthma medications, including β2-adrenergic receptor agonists (e.g., salbutamol, salmeterol), inhaled corticosteroids such as beclomethasone, and leukotriene modifier and inhibitors such as zileuton and montelukast. With the use of approaches such as GWAS this is likely to increase. Similarly, genetic variants already discussed in current/ future targets (IL13, TSLP, IL33, or IL1RL1 [IL33 receptor]) may also be important in identifying responders and nonresponders in phase II trials for monoclonal antibodies targeting these mediators134 or indeed in clinical practice as monoclonal antibodies targeting the T2 pathway (e.g., anti-IL5 [mepolizumab]) are licensed.

SUMMARY The sometimes conflicting results of studies to identify susceptibility genes for allergic diseases reflect the genetic and environmental heterogeneity seen in these disorders. Despite the difficulty of identifying susceptibility genes for complex genetic diseases, there is a rapidly expanding list of genes robustly associated with a wide range of allergic disease phenotypes. It is currently not possible to predict whether an individual will develop allergic disease based on genetics alone. This reflects the complex interactions between genetic and environmental factors required to initiate disease and determine progression to a more severe phenotype. The predictive value of variation in any one gene is low, with a relative risk of 1.1 to 1.5 for a typical genotype.140 In the future, genetic studies combined with more sophisticated patient characterization may be used to define subphenotypes of allergic disease4,141 and may lead to genetic tests for predicting disease. Genetic studies of allergic disease are likely to have their greatest impact not in predicting risk but rather in revealing mechanisms of disease pathogenesis and the treatment and prevention of these

conditions. For example, the propensity to develop atopy is influenced by factors different from those that influence disease, but these disease factors require interaction with atopy or something else to trigger disease. This interaction highlights the importance of local tissue response factors and epithelial susceptibility factors in the pathogenesis of allergic disease.142 These contributions to the understanding of allergic disease are likely to promote development of therapeutics targeting novel pathways of disease pathogenesis. Although genetic studies of allergic disease have led to the identification of many loci that alter the susceptibility of an individual to allergic disease, further research is needed to understand how these genetic variants alter gene expression and protein function to contribute to the pathogenesis of disease. The focus of research going forward is likely to be the interaction between genes and environmental exposure in disease at the genetic and epigenetic levels for better prediction of disease susceptibility and progression and translation of insights gained into disease pathogenesis into clinical applications. However, it is of note that many of the new biologics in development, for example in asthma, anti-IL13, anti-TSLP, anti-IL33 and those recently brought into clinic such as anti-IL5, target genes and pathways that are genetically supported.

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65. Vicente CT, Revez JA, Ferreira MAR. Lessons from ten years of genome-wide association studies of asthma. Clin Transl Immunology 2017;6(12):e165. 66. Bussmann C, Weidinger S, Novak N. Genetics of atopic dermatitis. J Dtsch Dermatol Ges 2011;9(9):670–6. 67. Barnes KC. An update on the genetics of atopic dermatitis: scratching the surface in 2009. J Allergy Clin Immunol 2010;125(1):16–29.e1–11, quiz 30–1. 68. Bin L, Leung DY. Genetic and epigenetic studies of atopic dermatitis. Allergy Asthma Clin Immunol 2016;12:52. 69. Smith FJ, Irvine AD, Terron-Kwiatkowski A, et al. Loss-of-function mutations in the gene encoding filaggrin cause ichthyosis vulgaris. Nat Genet 2006;38(3):337–42. 70. Palmer CN, Irvine AD, Terron-Kwiatkowski A, et al. Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nat Genet 2006;38(4):441–6. 71. Fallon PG, Sasaki T, Sandilands A, et al. A homozygous frameshift mutation in the mouse Flg gene facilitates enhanced percutaneous allergen priming. Nat Genet 2009;41(5):602–8. 72. Esparza-Gordillo J, Weidinger S, Folster-Holst R, et al. A common variant on chromosome 11q13 is associated with atopic dermatitis. Nat Genet 2009;41(5):596–601. 73. Sun LD, Xiao FL, Li Y, et al. Genome-wide association study identifies two new susceptibility loci for atopic dermatitis in the Chinese Han population. Nat Genet 2011;43(7):690–4. 74. Paternoster L, Standl M, Chen CM, et al. Meta-analysis of genome-wide association studies identifies three new risk loci for atopic dermatitis. Nat Genet 2012;44(2):187–92. 75. Paternoster L, Standl M, Waage J, et al. Multi-ancestry genome-wide association study of 21,000 cases and 95,000 controls identifies new risk loci for atopic dermatitis. Nat Genet 2015;47(12):1449–56. 76. Ramasamy A, Curjuric I, Coin LJ, et al. A genome-wide meta-analysis of genetic variants associated with allergic rhinitis and grass sensitization and their interaction with birth order. J Allergy Clin Immunol 2011;128(5):996–1005. 77. Bunyavanich S, Shargorodsky J, Celedon JC. A meta-analysis of Th2 pathway genetic variants and risk for allergic rhinitis. Pediatr Allergy Immunol 2011;22(4):378–87. 78. Holloway JW, Yang IA, Holgate ST. Genetics of allergic disease. J Allergy Clin Immunol 2010;125(2 Suppl. 2):S81–94. 79. Graves PE, Kabesch M, Halonen M, et al. A cluster of seven tightly linked polymorphisms in the IL-13 gene is associated with total serum IgE levels in three populations of white children. J Allergy Clin Immunol 2000;105(3):506–13. 80. Heinzmann A, Mao XQ, Akaiwa M, et al. Genetic variants of IL-13 signalling and human asthma and atopy. Hum Mol Genet 2000;9(4):549–59. 81. Liu X, Nickel R, Beyer K, et al. An IL13 coding region variant is associated with a high total serum IgE level and atopic dermatitis in the German multicenter atopy study (MAS- 90). J Allergy Clin Immunol 2000;106(1 Pt 1):167–70. 82. Gao PS, Heller NM, Walker W, et al. Variation in dinucleotide (GT) repeat sequence in the first exon of the STAT6 gene is associated with atopic asthma and differentially regulates the promoter activity in vitro. J Med Genet 2004;41(7):535–9. 83. Weidinger S, Gieger C, Rodriguez E, et al. Genome-wide scan on total serum IgE levels identifies FCER1A as novel susceptibility locus. PLoS Genet 2008;4(8):e1000166. 84. Ansel KM, Djuretic I, Tanasa B, et al. Regulation of Th2 differentiation and Il4 locus accessibility. Annu Rev Immunol 2006;24:607–56. 85. Pino-Yanes M, Gignoux CR, Galanter JM, et al. Genome-wide association study and admixture mapping reveal new loci associated with total IgE levels in Latinos. J Allergy Clin Immunol 2015;135(6):1502–10. 86. Yatagai Y, Sakamoto T, Masuko H, et al. Genome-wide association study for levels of total serum IgE identifies HLA-C in a Japanese population. PLoS ONE 2013;8(12):e80941.

87. Gudbjartsson DF, Bjornsdottir US, Halapi E, et al. Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction. Nat Genet 2009;41(3):342–7. 88. Jain D, Hodonsky CJ, Schick UM, et al. Genome-wide association of white blood cell counts in Hispanic/Latino Americans: the Hispanic Community Health Study/Study of Latinos. Hum Mol Genet 2017;26(6):1193–204. 89. Bonnelykke K, Matheson MC, Pers TH, et al. Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization. Nat Genet 2013;45(8):902–6. 90. Holloway JW, Arshad SH, Holgate ST. Using genetics to predict the natural history of asthma? J Allergy Clin Immunol 2010;126(2):200–9, quiz 210–1. 91. Weinmayr G, Weiland SK, Bjorksten B, et al. Atopic sensitization and the international variation of asthma symptom prevalence in children. Am J Respir Crit Care Med 2007;176(6):565–74. 92. Pearce N, Pekkanen J, Beasley R. How much asthma is really attributable to atopy? Thorax 1999;54(3):268–72. 93. Ferreira MA, Vonk JM, Baurecht H, et al. Shared genetic origin of asthma, hay fever and eczema elucidates allergic disease biology. Nat Genet 2017;49(12):1752–7. 94. Marenholz I, Esparza-Gordillo J, Ruschendorf F, et al. Meta-analysis identifies seven susceptibility loci involved in the atopic march. Nat Commun 2015;6:8804. 95. Howard TD, Koppelman GH, Xu J, et al. Gene-gene interaction in asthma: IL4RA and IL13 in a Dutch population with asthma. Am J Hum Genet 2002;70(1):230–6. 96. Burke H, Leonardi-Bee J, Hashim A, et al. Prenatal and passive smoke exposure and incidence of asthma and wheeze: systematic review and meta-analysis. Pediatrics 2012;129(4):735–44. 97. Young S, Le Souef PN, Geelhoed GC, et al. The influence of a family history of asthma and parental smoking on airway responsiveness in early infancy. N Engl J Med 1991;324(17):1168–73. 98. Bouzigon E, Corda E, Aschard H, et al. Effect of 17q21 variants and smoking exposure in early-onset asthma. N Engl J Med 2008;359(19):1985–94. 99. Smit LA, Bouzigon E, Pin I, et al. 17q21 variants modify the association between early respiratory infections and asthma. Eur Respir J 2010;36(1):57–64. 100. Eder W, Klimecki W, Yu L, et al. Opposite effects of CD 14/-260 on serum IgE levels in children raised in different environments. J Allergy Clin Immunol 2005;116(3):601–7. 101. Smit LA, Siroux V, Bouzigon E, et al. CD14 and toll-like receptor gene polymorphisms, country living, and asthma in adults. Am J Respir Crit Care Med 2009;179(5):363–8. 102. Bieli C, Eder W, Frei R, et al. A polymorphism in CD14 modifies the effect of farm milk consumption on allergic diseases and CD14 gene expression. J Allergy Clin Immunol 2007;120(6):1308–15. 103. Smith D, Helgason H, Sulem P, et al. A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma. PLoS Genet 2017;13(3):e1006659. 104. Tennessen JA, Bigham AW, O’Connor TD, et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 2012;337(6090):64–9. 105. Torgerson DG, Capurso D, Mathias RA, et al. Resequencing candidate genes implicates rare variants in asthma susceptibility. Am J Hum Genet 2012;90(2):273–81. 106. Almal SH, Padh H. Implications of gene copy-number variation in health and diseases. J Hum Genet 2012;57(1):6–13. 107. Minelli C, Granell R, Newson R, et al. Glutathione-S-transferase genes and asthma phenotypes: a Human Genome Epidemiology (HuGE) systematic review and meta-analysis including unpublished data. Int J Epidemiol 2010;39(2):539–62. 108. Campbell CD, Mohajeri K, Malig M, et al. Whole-genome sequencing of individuals from a founder population identifies candidate genes for asthma. PLoS ONE 2014;9(8):e104396. 109. Allen IC, Pace AJ, Jania LA, et al. Expression and function of NPSR1/ GPRA in the lung before and after induction of asthma-like disease. Am J Physiol Lung Cell Mol Physiol 2006;291(5):L1005–17.

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma 110. Adolph TE, Niederreiter L, Blumberg RS, et al. Endoplasmic reticulum stress and inflammation. Dig Dis 2012;30(4):341–6. 111. Jirtle RL, Skinner MK. Environmental epigenomics and disease susceptibility. Nat Rev Genet 2007;8(4):253–62. 112. Potaczek DP, Harb H, Michel S, et al. Epigenetics and allergy: from basic mechanisms to clinical applications. Epigenomics 2017;9(4):539–71. 113. Santangelo S, Cousins DJ, Winkelmann N, et al. Chromatin structure and DNA methylation of the IL-4 gene in human T(H)2 cells. Chromosome Res 2009;17(4):485–96. 114. Tripathi SK, Lahesmaa R. Transcriptional and epigenetic regulation of T-helper lineage specification. Immunol Rev 2014;261(1):62–83. 115. Hawkins RD, Larjo A, Tripathi SK, et al. Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization. Immunity 2013;38(6):1271–84. 116. Rodriguez RM, Lopez-Larrea C, Suarez-Alvarez B. Epigenetic dynamics during CD4(+) T cells lineage commitment. Int J Biochem Cell Biol 2015;67:75–85. 117. Benn CSJDL, Hasselbalch H, Olesen AB, et al. Thymus size and head circumference at birth and the development of allergic disease. Clin Exp Allergy 2001;31(12):1862–6. 118. Fergusson DMCJ, Beasley R, Horwood LJ. Perinatal factors and atopic disease in childhood. Clin Exp Allergy 2006;27(12):1394–401. 119. Godfrey KMBDJP, Osmond C. Disproportionate fetal growth and raised IgE concentration in adult life. Clin Exp Allergy 2006;24(7):641–8. 120. Baccarelli A, Wright RO, Bollati V, et al. Rapid DNA methylation changes after exposure to traffic particles. Am J Respir Crit Care Med 2009;179(7):572–8. 121. Li YF, Langholz B, Salam MT, et al. Maternal and grandmaternal smoking patterns are associated with early childhood asthma. Chest 2005;127(4):1232–41. 122. Accordini S, Calciano L, Johannessen A, et al. A three-generation study on the association of tobacco smoking with asthma. Int J Epidemiol 2018;47(4):1106–17. 123. Arshad SH, Karmaus W, Raza A, et al. The effect of parental allergy on childhood allergic diseases depends on the sex of the child. J Allergy Clin Immunol 2012;130(2):427–34. 124. Hollingsworth JW, Maruoka S, Boon K, et al. In utero supplementation with methyl donors enhances allergic airway disease in mice. J Clin Invest 2008;118(10):3462–9. 125. Slaats GG, Reinius LE, Alm J, et al. DNA methylation levels within the CD14 promoter region are lower in placentas of mothers living on a farm. Allergy 2012;67(7):895–903. 126. Yang IV, Lozupone CA, Schwartz DA. The environment, epigenome, and asthma. J Allergy Clin Immunol 2017;140(1):14–23. 127. Conrad ML, Ferstl R, Teich R, et al. Maternal TLR signaling is required for prenatal asthma protection by the nonpathogenic microbe Acinetobacter Iwoffii F78. J Exp Med 2009;206(13):2869–77. 128. Brand S, Teich R, Dicke T, et al. Epigenetic regulation in murine offspring as a novel mechanism for transmaternal asthma protection induced by microbes. J Allergy Clin Immunol 2011;128(3):618–25.e1–7. 129. Brand S, Kesper DA, Teich R, et al. DNA methylation of TH1/TH2 cytokine genes affects sensitization and progress of experimental asthma. J Allergy Clin Immunol 2012;129(6):1602–10.e6. 130. Arshad SH, Karmaus W, Zhang H, et al. Multigenerational cohorts in patients with asthma and allergy. J Allergy Clin Immunol 2017;139(2):415–21. 131. Liang L, Willis-Owen SAG, Laprise C, et al. An epigenome-wide association study of total serum immunoglobulin E concentration. Nature 2015;520(7549):670–4. 132. Chen W, Wang T, Pino-Yanes M, et al. An epigenome-wide association study of total serum IgE in Hispanic children. J Allergy Clin Immunol 2017;140(2):571–7. 133. Miller RL, Ho SM. Environmental epigenetics and asthma: current concepts and call for studies. Am J Respir Crit Care Med 2008;177(6):567–73.

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134. Portelli M, Sayers I. Genetic basis for personalized medicine in asthma. Expert Rev Respir Med 2012;6(2):223–36. 135. Vijverberg SJH, Farzan N, Slob EMA, et al. Treatment response heterogeneity in asthma: the role of genetic variation. Expert Rev Respir Med 2018;12(1):55–65. 136. Turner S, Francis B, Vijverberg S, et al. Childhood asthma exacerbations and the Arg16 beta2-receptor polymorphism: a meta-analysis stratified by treatment. J Allergy Clin Immunol 2016;138(1):107–13.e5. 137. Tantisira KG, Lima J, Sylvia J, et al. 5-lipoxygenase pharmacogenetics in asthma: overlap with Cys-leukotriene receptor antagonist loci. Pharmacogenet Genomics 2009;19(3):244–7. 138. Tantisira KG, Silverman ES, Mariani TJ, et al. FCER2: a pharmacogenetic basis for severe exacerbations in children with asthma. J Allergy Clin Immunol 2007;120(6):1285–91. 139. Israel E, Lasky-Su J, Markezich A, et al. Genome-wide association study of short-acting beta2-agonists. A novel genome-wide significant locus on chromosome 2 near ASB3. Am J Respir Crit Care Med 2015;191(5):530–7. 140. Koppelman GH, te Meerman GJ, Postma DS. Genetic testing for asthma. Eur Respir J 2008;32(3):775–82. 141. Moore WC, Meyers DA, Wenzel SE, et al. Identification of asthma phenotypes using cluster analysis in the Severe Asthma Research Program. Am J Respir Crit Care Med 2010;181(4):315–23. 142. Holgate ST, Davies DE, Powell RM, et al. Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms. Eur Respir J 2007;29(4):793–803. 143. Shaw DE, Portelli MP, Sayers I. Asthma. In: Padmanabhan S, editor. Handbook of pharmacogenomics and stratified medicine. London: Elsevier; 2014. p. 617–51. 144. Sun W, Hu Y. eQTL mapping using RNA-seq data. Stat Biosci 2013;5(1):198–219. 145. Schaub MA, Boyle AP, Kundaje A, et al. Linking disease associations with regulatory information in the human genome. Genome Res 2012;22(9):1748–59. 146. White JH, Chiano M, Wigglesworth M, et al. Identification of a novel asthma susceptibility gene on chromosome 1qter and its functional evaluation. Hum Mol Genet 2008;17(13):1890–903. 147. Noguchi E, Yokouchi Y, Zhang J, et al. Positional identification of an asthma susceptibility gene on human chromosome 5q33. Am J Respir Crit Care Med 2005;172(2):183–8. 148. Laitinen T, Polvi A, Rydman P, et al. Characterization of a common susceptibility locus for asthma-related traits. Science 2004;304(5668):300–4. 149. Balaci L, Spada MC, Olla N, et al. IRAK-M is involved in the pathogenesis of early-onset persistent asthma. Am J Hum Genet 2007;80(6):1103–14. 150. Barton SJ, Koppelman GH, Vonk JM, et al. PLAUR polymorphisms are associated with asthma, PLAUR levels, and lung function decline. J Allergy Clin Immunol 2009;123(6):1391–400.e17. 151. Sleiman PM, Flory J, Imielinski M, et al. Variants of DENND1B associated with asthma in children. N Engl J Med 2010;362(1):36–44. 152. Ferreira MA, Matheson MC, Tang CS, et al. Genome-wide association analysis identifies 11 risk variants associated with the asthma with hay fever phenotype. J Allergy Clin Immunol 2014;133(6):1564–71. 153. Ellinghaus D, Baurecht H, Esparza-Gordillo J, et al. High-density genotyping study identifies four new susceptibility loci for atopic dermatitis. Nat Genet 2013;45(7):808–12. 154. Weidinger S, Willis-Owen SA, Kamatani Y, et al. A genome-wide association study of atopic dermatitis identifies loci with overlapping effects on asthma and psoriasis. Hum Mol Genet 2013;22(23):4841–56. 155. Schaarschmidt H, Ellinghaus D, Rodriguez E, et al. A genome-wide association study reveals 2 new susceptibility loci for atopic dermatitis. J Allergy Clin Immunol 2015;136(3):802–6. 156. Hinds DA, McMahon G, Kiefer AK, et al. A genome-wide association meta-analysis of self-reported allergy identifies shared and allergy-specific susceptibility loci. Nat Genet 2013;45(8):907–11.

CHAPTER 22  Genetics and Epigenetics in Allergic Diseases and Asthma

351.e1

SELF-ASSESSMENT QUESTIONS 1. Complex diseases such as asthma are common and involve: a. Many genes, with “severe” mutations leading to large phenotypic effects with strong environmental interactions. b. Few genes, with “mild” mutations leading to small phenotypic effects with strong environmental interactions. c. Many genes, with “mild” mutations leading to small phenotypic effects with strong environmental interactions. d. Many genes, with “mild” mutations leading to large phenotypic effects with limited environmental interactions. e. Few genes, with “mild severe” mutations leading to small large phenotypic effects with strong limited environmental interactions. 2. The first genome-wide association study (GWAS) in asthma was published in 2007 and identified which genomic region that has been replicated in all GWASs of asthma to date? a. IL6R b. TSLP c. GATA3

d. IL33 e. ORMDL3 3. Recently it has been shown that childhood-onset asthma, allergic rhinitis, and atopic dermatitis share a large number of genetic risk factors; how many risk factors from the 136 identified are believed to be disease specific? a. 6 b. 12 c. 21 d. 9 e. 2 4. Which one of the following changes is not an epigenetic change? a. DNA methylation b. Histone acetylation c. Histone methylation d. DNA sequence polymorphism e. Histone

23  Systems Biology Ariella T. Cohain, Eric E. Schadt, Supinda Bunyavanich

CONTENTS Introduction, 352 Technologic Advances, 353

SUMMARY OF IMPORTANT CONCEPTS • Systems biology applies algorithms and statistics to integrate multiple highdimensional data modalities to derive models and data-driven hypotheses representing complex living systems. • Big data and high-performance computing facilitate systems biology approaches. • A variety of computational and statistical methods, including artificial intelligence and machine learning algorithms, are available to address specific questions and improve certain aspects of biology. • A systems biology framework is important for holistically understanding complex diseases such as those in allergy and immunology. • Findings from systems biology include the identification of new genes involved in allergy and immunologic disorders, thus improving our understanding of underlying mechanisms and providing potentially novel therapeutic targets. • As technology continues to advance, more data will be generated, allowing the systems biology field to continue to grow and improve the current standard of care.

INTRODUCTION The field of systems biology began in the late 1990s as a framework for thinking about big data from biological and ‘omics profiling. Human beings are complex systems where multiple processes work in unison (Fig. 23.1). Systems biology embraces and encourages multilevel approaches for capturing this complexity. Instead of focusing on one aspect, the goal is to integrate data from multiple sources and model the human system as the true complex organism that it is. The activity of molecular constituents of a cell (DNA, RNA, proteins, metabolites and larger macromolecules) define the function and activity of cells, which in turn define the function and activity of tissues and organs, which in turn define the coherent operation of more complex living systems as well as the physiologic and pathophysiologic states associated with wellness and disease. This ideology has shifted how research is conducted and enabled data-driven hypotheses. Systems biology requires high throughput, high-dimensional data, typically at larger scales, to be systematically generated and collected, and the use of advanced computational approaches and higher ordered statistics and machine learning algorithms to understand and interpret these data. Systems

352

Methods for High-Dimensional Molecular Data Analysis, 355 Concluding Remarks, 360

biology aims to incorporate and integrate different data modalities to generate a comprehensive model to derive hypotheses. The results from system biology approaches are often predictive models depicted as a network. A commonly understood example of a network is a social network, where people are represented as nodes and their relationships or connections with others are shown as edges. Because of technologic advances that facilitate rapid and expansive connectivity among individuals, information from social networks is now easily and quickly accessible. These networks allow us to query and understand social structures and can inform not only on environmental and geographic features, but also on disease patterns and social behaviors.1 We can identify individuals who are super connectors globally and/or locally for a specific group of people (e.g., physicians working in health care), and the network also allows us to find novel connections (e.g., people highly connected with the medical field but not physicians themselves). Social networks can be based on different types of social data, such as friendships, employment, industry sector, and geographic distance. For biologic data, similar approaches can be employed to understand biologic processes on a more holistic scale. As we will show in this chapter, system biology approaches have been applied with much success to the field of biology, and increasingly to allergy and immunology. By integrating different types of data, we are able to create molecular networks of processes occurring within the cell, tissue, and across tissues to discover new interactions that are important for disease. In the first part of this chapter we will review technologies that have enabled the availability of many high-dimensional data types. We will then discuss analysis methods for systems biology and illustrate their implementation using examples from the allergy and immunology field where possible.

Rapid Cost Reduction of Sequencing Implementation of a systems biology approach has been facilitated by rapid cost reductions in genomic sequencing and ‘omics profiling. Sequencing a genome has gone from costing almost one billion dollars for the first human genome, to less than $1000 in 2015, the only technology known to exceed Moore’s law,2 as can be seen in Fig. 23.2. This has not only revolutionized the types of questions scientists can ask, but also required the creation of new methods and statistical approaches to analyze and store the vast oceans of data these technologies can produce. In the last two decades, we have gone from being limited to looking at small stretches of DNA or a handful of genes at a time in small numbers of individuals, to examining nearly all nucleic acids in

CHAPTER 23  Systems Biology

353

generation and integration of multiple types of ‘omics data (such as the proteome, epigenome, and microbiome) derived from populations of individuals. The breadth of our knowledge and understanding continues to expand and grow at an exponential pace.

TECHNOLOGIC ADVANCES RNA Microbiome

Environment H3C H3C

Epigenetics H3C

CH3 CH3

Metabolite CH2

HO

Protein

Major advances in technology have enabled us to continue to push the boundaries of systems biology. The first sequencing approach, Sanger sequencing, was built on the principle that multiple clonal copies of fragments of DNA could be sequenced, where genome scaffolds could be constructed to cover entire regions of interest (e.g., gene or genome). However, the incorporation of a region of interest into either a plasmid or phage that could then be expanded into many clonal copies was a laborious, time-intensive process, taking up to 1 week for the preparation alone. A quantum jump over this approach came with Next Generation Sequencing (NGS) technologies, which allowed for higher throughput sequencing, reducing both the cost and preparation time, and dramatically increasing the productivity and rate of data generation. In this section, we will briefly discuss the different high-throughput technologies and how they have enabled a more holistic view of disease, with a few brief examples on the impact these advances have had on allergy and immunology.

Array-Based DNA Genotyping DNA

Fig. 23.1  Overview of the different ‘omics present in an individual.

a cell, whether DNA or RNA, across populations of individuals over time. The resulting dramatic increase in dimensionality provides for a super exponential increase in the number of possible interactions that can be observed, resulting in a super exponential increase in the number of hypotheses that can be derived from such data. Clearly, without a way to organize this vast constellation of interactions and statistically filter and prioritize those hypotheses that are most strongly supported by the data, researchers would be at a loss to translate those vast data into meaningful knowledge and understanding. Systems biology approaches seek to do exactly this, improving our understanding of biology and shifting the traditional hypothesis testing paradigm of biology and medicine to a data-driven hypothesis generation paradigm, spanning genome-wide association studies (GWAS), large-scale transcriptomics sequencing projects and regulatory networks, and the

Genotyping array technologies work by designing probes to specifically target known DNA variants.3 Although the various companies that provide these arrays use different chemistries, they all generally work by hybridizing probes to targeted regions of DNA. The detection of hybridization events is achieved using color to detect different binding of A/T or G/C, and detecting whether the DNA binds with a perfect match or mismatch to the probe sequence.3 These arrays, commonly referred to as SNP arrays (or SNP chip), generally contain fewer than 1 million probes and cover roughly 0.03% of the genome. Despite this low coverage, SNP arrays have significant utility because most positions in the genome are not independent from one another in local neighborhoods, so that capturing information for one locus can provide information about others (linkage disequilibrium). Within allergy, these arrays have enabled genome-wide association studies (GWAS) through genotypic profiling of thousands of allergic individuals, resulting in the identification of loci in the genome that are associated with asthma, atopic dermatitis, allergen sensitization, eosinophilic esophagitis, and food allergy.4,5

RNA Microarrays Profiling RNA expression provides access to what is occurring within the cell in real time, as opposed to what is inherited. The first technologies to provide for comprehensive profiling of gene expression on a transcriptome-wide basis were again array-based, with microarray platforms using probes similar to those used in genotyping arrays, where hybridization of probe sequences to RNA can be detected and used to quantify the abundance of different genes being transcribed in the cell. This is accomplished by extracting RNA from a sample, converting the RNA to cDNA, and then using a probe-based array to measure the number of cDNA molecules hybridizing to the probe sequences.6 Many studies of asthma and allergic diseases have used this approach, resulting in the identification of differential gene expression signatures associated with disease in specific tissues.4,7 The identification of such signatures helps elucidate the pathways involved in disease by searching in those signatures for enrichments of different biologic processes that are differentially impacted, for example, between disease and control states.

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SECTION A  Basic Sciences Underlying Allergy and Immunology Costs of Genome

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1014

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$1M

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Ja Jan 19 Jan 1993 Jan 1994 Jan 1995 Jan 1996 Jan 1997 Jan 1998 Jan 2099 Jan 2000 Jan 2001 Jan 2002 Jan 2003 Jan 2004 Jan 2005 Jan 2006 Jan 2007 Jan 2008 Jan 2009 Jan 2010 Jan 2011 Jan 2012 Jan 2013 Jan 2014 Jan 2015 Jan 2016 n 2 17 01 8

$100

Date Fig. 23.2  Cost of sequencing and supercomputing growth versus Moore’s Law. The cost of sequencing a human genome is shown in purple on the right and on the left the growth of supercomputing in terms of floating point per second (a measurement of speed) plotted in blue. Both plots are on a log10 scale and in gray are the Moore’s Law equation lines depicting whether the cost would either double or halve every 2 years.

Read-Based Sequencing Sequencing can be applied to the genome (DNA) by whole genome sequencing (WGS), whole exome sequencing (WES), or targeted panels of genes or genome regions of interest. These methods not only enable capturing information other than common variation such as rare and novel mutations and structural variants, but they also do not depend on any a priori knowledge regarding the organism being sequenced, providing for a completely de novo, data-driven discovery. Rare mutations usually have a much larger effect size than common variants.8 WES involves first isolating the exomes, or coding regions of the genome, and then using read-based approaches to sequence these regions. WES is less costly than WGS because it targets a smaller fraction of the genome; however, given capture strategies, WES still results in sequencing 30% of the genome, even though the protein coding component of the genome is only 3%. By only sequencing a fraction of the genome, important regulatory and structural variations may be lost given such variants can exist in the noncoding portion of the genome. WES has been effectively used to identify genetic defects in patients with a variety of primary immunodeficiencies.9

RNA Sequencing (RNAseq) With the maturation of NGS technologies, RNA microarrays have rapidly given way to RNA sequencing (RNAseq), where RNA is converted to cDNA, and cDNA is then sequenced using a read-based approach, providing a more digital readout of gene expression than can be achieved using microarrays. RNAseq measures the expression levels of transcripts by enabling a direct counting of the number of RNA molecules for a given gene that have been sequenced. This provides for fewer technical problems and is not limited by a priori probe design (one does not need prior knowledge of the existence of a gene or particular isoform in a given organism to detect its expression profile). RNAseq produces

tens of millions of reads that are then assembled and aligned to the genome to determine expression levels for specific genes. This high throughput technology allows for easier detection of different isoforms or transcripts, as well as measurement of noncoding transcripts such as long noncoding RNA, circular RNA, and even bacterial genes.10 RNAseq is routinely used to identify differentially expressed genes between cases and controls (or other groupings of interest), such as looking for changes in transcripts in allergic individuals in response to an allergen challenge.4 For example, an RNAseq-based study of peanut-allergic children undergoing double blind placebo-controlled oral food challenges identified gene transcripts that changed due to acute peanut allergic reaction, providing insights into the biologic processes that were altered during food allergic reactions.11

Single-Cell Sequencing As recently as 2009, advances in NGS technology have enabled the direct sequencing of DNA and RNA from single cells (scRNA-seq).12 A major advantage of single-cell sequencing is that it enables examination of cell heterogeneity on an even more granular level.12 Multiple different methods can be used depending on the question of interest; the important step is to segregate individual cells before lysis and RNA extraction. Although to date single-cell sequencing has been mostly applied to cancer research, it has potential utility in allergy and immunology as a method to disaggregate the individual expression of disparate immune cells, particularly those that may be rare and elusive to study by traditional methods.

Proteome Although technologies for assaying nucleic acid sequences have facilitated dramatic biologic discoveries, nucleic acids are only part of the story. Assaying proteins is critical if we hope to fully characterize how cells function. Whereas mRNA sequencing may well capture upstream

CHAPTER 23  Systems Biology regulatory effects, proteins are the dominant functional units in the cell that also feedback onto the regulatory processes, and critical to assay to produce the best view of a living system. In proteomics, the peptide units comprising proteins can be profiled by mass spectrometry, which measures the mass of ionized biomolecules. Interestingly, the correlation between mRNA and protein levels are not as high as one might expect,13 demonstrating the importance of measuring protein states and levels alongside of RNA and DNA given they capture different aspects of the biology of living systems. Central to protein functions are proteinprotein interactions (PPI). PPI networks inform on how genes might interact with one another at the protein level, providing more direct, physical interaction-based insights into the more classic definition of causality in biology (one molecular entity physically interacting with another to alter functions that consequently lead to phenotypic changes of interest). Proteomic approaches have helped identify the structure of over 850 allergens, yielding information on epitopes.14

Epigenome NGS technology has also transformed our understanding of epigenomes and the critical role they play in regulating molecular and higher order processes. The epigenome is comprised of DNA modifications (methylation or acetylation) in the genome and transcriptome, informing on where and how DNA is packaged, which parts are open for binding, and which regions are transcriptionally active, thus playing a central role in the regulation of gene expression. Methods for profiling the epigenome include: (1) bisulfite sequencing, which targets CpG islands; (2) DNA methylation immunoprecipitation methods; (3) methylation arrays; and (4) detection of epigenetic modifications using kinetic information from single molecule sequencing technologies such as PacBio’s single molecule real time sequencer.15 Large-scale projects such as the Epigenome Roadmap project and ENCODE have produced extensive maps of epigenetic modifications present in various tissues and cell lines. DNA histone modifications can regulate cell differentiation and allergen-specific memory of T-helper (Th) cells.16 Epigenetic changes at specific genomic loci have also been associated with asthma, atopic dermatitis, allergic rhinitis, and food allergy.4

Metabolome Metabolites are the intermediary products of metabolism and again provide another fundamental molecular component of living systems. Profiling the metabolome can be done using either nuclear magnetic resonance (NMR) or mass spectrometry to identify and quantify different metabolites. Studies of metabolites measured from urine, serum, plasma, and exhaled breath condensate have shown that metabolites can discriminate between asthmatics and controls.4 Elevated levels of uric acid in serum have also been detected in metabolomic examination of mouse models of food allergy.17

Microbiome Human beings have more bacterial cells than human cells in their bodies. Therefore it is important to assess the role of the constellations of these bacteria (known as bacterial microbiota) in human health and disease. Given the difficulty in optimizing culture conditions for thousands of bacteria simultaneously, an alternative route is to sequence the 16S ribosomal RNA gene, a highly conserved region across bacteria that enables reasonable discrimination among different bacterial taxa, from biospecimens. The 16S ribosomal RNA is sequenced and can then be mapped to known bacterial reference sequences to yield the abundances of a myriad of bacterial taxa. NGS can also be used to sequence entire microbial genomes via shotgun metagenomics sequencing. The microbiome has been shown to interact with metabolites, nutrients and diet, and host’s genetics. The NIH-funded Human Microbiome Project, a

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resource of microbiota profiles from different anatomic sites from 250 normal individuals, found that samples are more similar based on the location sampled than on the individual person. The microbiome of the airway, skin, and gut have been associated with asthma, atopic dermatitis, and food allergy.18 It is possible that manipulation of the microbiome could be leveraged for prevention and treatment of allergic disorders.18

Exposome Consideration of environmental interactions with the immune systems is a cornerstone of allergy and immunology research, and characterization of environmental parameters or exposome can provide valuable information for systems biology-based analyses.4,19 High-throughput technologies can help to characterize environmental exposures, such as the microbial environment one is exposed to during a morning commute.20 Additionally, with technology enabling cheaper and smaller smartphones and wearables, we can collect increasing individually gathered information through these platforms. Integrating this information to better understand the effects of our environment is already happening. For example, the cell phone-based Apple Research Kit enables researchers to gather users’ locations and their exposures in real time as well as information input by users about their asthma control, offering a new way to conceive of studying asthma.21

High-Performance Computing Although the generation of data provides the raw material for systems biology, efficient storage and analysis of such high-dimensional data requires high-performance computing (or supercomputing) infrastructure. As can be seen in Fig. 23.2, although the cost of sequencing a genome was dropping dramatically, dropping faster than Moore’s law predicted, commoditized hardware that forms the core of most highperformance computing systems (including those at Google, Amazon, and Facebook) has kept the Moore’s law pace over a longer time scale than NGS technologies. Revolutionary sequencing technology needs to go hand in hand with advances in high-performance computing (HPC),22 and the more mature HPC field was ready for the big data challenge the NGS technologies provided. The demands to store, distribute, and analyze data is on par or projected to be more demanding than that of the fields of astronomy, quantitative finance, Twitter, or even YouTube.23

METHODS FOR HIGH-DIMENSIONAL MOLECULAR DATA ANALYSIS High-throughput technologies have enabled data collection on a wide array of different biologic modalities as well as on population-based scales, requiring statistical genetics techniques and systems biology approaches to understand them and derive and prioritize data-driven hypotheses. In this section, we will discuss some of the major statistical and systems biology approaches, the concepts underlying them, how they work, and highlight a few examples within allergy/immunology where these approaches have been successfully implemented. A highlevel summary of this section is provided in Table 23.1.

Statistical Genetics Approaches

Genome-Wide Association Studies.  Once populations of individuals could be efficiently genotyped at low cost, the first wave of GWAS stormed onto the scene.8 GWAS are generally conducted by recruiting hundreds to many tens of thousands of cases and controls (or cohorts for which a specific quantitative trait has been measured), genotyping each person, and then testing for association between genotype at each locus (or haplotype) and the phenotype of interest. For case-control constructions, the most general question asked is whether the proportion

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SECTION A  Basic Sciences Underlying Allergy and Immunology

TABLE 23.1  Summary of Statistical Genetics and Systems Biology Approaches Discussed in

This Chapter With Examples From the Field of Allergy and Immunology Analysis

Input

Output

Allergy Example

Notes

GWAS

DNA genotypes + phenotype

Loci associated with phenotype

Over 400 loci associated with allergy. Loci in HLA-DQB1 and HLA-DRB1 associated for food allergies.4,26,27

Positions in genome are not independent (LD blocks).

Mendelian randomization

Summary statistics from GWAS

Support or reject for causal interaction between two phenotypes

Increased BMI causes increased asthma32 and a lack of support for vitamin D deficiency being causal for asthma.33,34

Can also give the direction of causal interaction (e.g., increasing BMI causing increased asthma32).

Quantitative trait loci (QTL)

DNA + expression of genes, proteins, or metabolites

Loci associated with changes in expression of input (dosage effects)

eQTLs have been identified for peripheral blood CD4+ gene expression, suggesting functional context for GWAS loci.31

Cis and trans denote proximity of loci to gene. eQTL: expression QTL pQTL: protein QTL mQTL: metabolite QTL

Cell mixture deconvolution

Expression of genes and signature of cell-type specific gene signatures

Estimated fraction of cell types present in each sample

Macrophages M0 and neutrophils increase during peanut allergic reaction while naïve CD4+ T cells decrease.11

Requires a priori information on cell-type specific signatures. Can leverage publically available references.

Differential expression

Expression of genes, proteins, or metabolites + phenotypes

Identifies input that is significantly different between phenotypes of interest

Identification of genes that change during allergen response,11 and genes that differ in expression between cases and controls.4

If phenotype is continuous it will identify genes significantly correlated with phenotype.

Coexpression clustering

Expression genes, proteins, or metabolites

Module (cluster) that each input belongs to

Used to detect modules of coexpressed genes in peanut-allergic patients11 and allergic rhinitis.31

Genes are usually assigned to only one module.

Gene ontology enrichment

List of background genes (all that are expressed) + selected genes of interest

Gene sets or pathways significantly enriched for the genes of interest

Module associated with peanut allergy was enriched for acute phase response,11 and a module associated with allergic rhinitis was enriched for mitochondrial processes.31

Can be limited by the number of annotated (known) pathways or terms.

Classification approaches

Any data and outcome (e.g., case and control, severity, etc.)

List of classifiers and how well they predict the outcome in training and test sets

Used in asthma to distinguish cases from controls, or to predict severity of asthma.45

Two sets of data ideal: training and test set to ensure classifier is not overfitted.

Causal inference

DNA (or perturbation that must be the most upstream) and two downstream variables (e.g., two genes, gene + phenotype, etc.)

A p-value for the three objects supporting the causal model (DNA → object1 → object2)

Causal networks

Expression, optional: prior information (e.g., QTL, literature, etc.)

Directed network learned from the data

Causal networks constructed in peanut allergy research.11

Requires a large sample size (N > 300).

Key driver detection

Network + nodes of interest

Nodes in the network that are regulating more nodes of interest

Identified six genes as key drivers of acute peanut allergic reactions.11

Network can be directed or undirected.

of a given variant or genotype represented in each group differs between the groups (Fig. 23.3A). However, because the haploid human genome is roughly 3 billion bases long, the number of loci needed to tag all common variation in the genome is large (on the order of a million loci, a 3-order of magnitude reduction from all loci in the genome, which can be attributed to the linkage disequilibrium structure in human populations). Thus testing every locus means performing a million tests, which can lead to false positives because of the large number of tests being conducted and the by-chance probability of seeing highly

All three objects must be correlated with each other. If not using DNA, but using a different variable, it must always be the top node or most upstream.

significant associations at random. To carry out a robust analysis, correction for multiple testing must be done; the standard threshold for calling a locus as significantly associated with a trait at a genome-wide level is 5e-8 (roughly, think of a Bonferroni correction to the 0.05 significance levels in the context of 1,000,000 tests: 0.05/1,000,000 = 5e-8). Setting such a small p-value threshold has a significant effect on the power to identify valid associations. Although GWAS have elucidated numerous locations in the genome that are implicated in a variety of allergic diseases, the results from these studies tend to explain very little

CHAPTER 23  Systems Biology

A A

A A

A A

A G

G G

A G

G G

G G

G G

Cases

A

Density

Controls A A

Differential expression (DE)

SNP genotype

B

A A

A G

G G

Coexpression clustering

D

C

Controls Cases

Trait

eQTL Gene expression

GWAS

357

Gene expression

Gene expression

Probabilistic causal networks

E

Key drivers

F

Fig. 23.3  Selected systems biology analysis methods. (A) Genome-wide association study (GWAS) test for increased proportion of variant (genotype) between cases and controls. (B) Expression quantitative loci (eQTL) represent associations between loci and gene expression values. (C) Differential expression analyses test for significant changes in the distribution of a gene’s expression between case and control, or for a continuous trait, correlation of the trait to expression values. (D) Coexpression clustering takes genes (depicted in gray) to identify clusters of genes that are coexpressed. (E) Probabilistic causal networks learn the directed (causal) structure of the genes based on the data present (causal edges are depicted as directed black arrows). (F) Key driver analysis identifies the gene or genes that significantly regulate genes of interest. Here the key driver is outlined in black, and the genes of interest are represented by orange circles.

of the heritability of the disease24 (i.e., “missing heritability”). Furthermore, implicating a locus in the cause of a disease neither automatically gives the corresponding gene or pathway/network in which the implicated gene participates, nor does it generally indicate whether altering the functioning gene (increasing or decreasing) will treat or prevent disease. Nevertheless, GWAS findings do provide causal insights into disease, and when combined with other molecular data, can provide deeper insights into the mechanisms of disease, as described in the following sections. GWAS have been done in asthma, atopic dermatitis, allergic rhinitis, eosinophilic esophagitis, and food allergy, each finding significant disease-susceptibility loci. Perhaps not surprisingly, the majority of the identified GWAS loci fall within immune-related genes. The results from these GWAS have been summarized in review articles highlighting the major findings for asthma and allergy GWAS.4 Although many GWAS have been conducted, the loci reported explain very little of the full genetic risk for these diseases. For example, asthma is between 25% to 80% heritable, and to date only 26 loci have been identified as asthma loci, explaining only a small proportion of heritability.25 The National Human Genome Research Institute and the European Bioinformatics Institute catalog loci that have been associated with various diseases by GWAS at http://www.ebi.ac.uk/gwas, where they

list more than 400 loci identified for diseases such as allergic rhinitis, atopic dermatitis, asthma, allergic rhinitis, food allergy, IgE levels, and pulmonary function. More susceptibility loci have been reported for asthma relative to other allergic disorders.17 The C11orf30 locus has been associated with atopic dermatitis risk as well as allergic rhinitis and grass sensitization. For food allergy, two major genes found and replicated in multiple cohorts are HLA-DQB1 and HLA-DRB1, major histocompatibility complex (MHC) class II genes that are involved in antigen presentation to T cells. The studies delivering these results found SNPs and specific alleles that were associated with peanut allergy as well as differential DNA methylation patterns for these genes.26,27 Recently, a targeted analysis examining previous genes associated with related diseases (atopic dermatitis and eosinophilic esophagitis) found 14 loci associated with food allergy.28 Copy number variation (CNVs) studies identified CTNNA3 and RBFOX1 as harboring CNVs associated with food allergic patients.29 C11orf30 has also been found in connection with grass allergen sensitization.4 A metaanalysis GWAS for allergen sensitization identified another 10 candidate genes that were mostly inflammation responding genes.30 A major limitation with GWAS is that although they can suggest genes associated with disease, they do not necessarily indicate the causal gene or objectively implicate pathways or disease mechanisms.24 As

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such, it is important to think on a more global scale of what might be occurring in disease-relevant tissues using more integrative approaches.31

Mendelian Randomization.  Because DNA is a primary root of the information chain in complex living systems, and because the human genome undergoes natural randomization when we inherit alleles from our parents, we can use DNA as an instrumental variable and run mendelian randomization (MR) to determine causal relationships between traits. However, one major assumption is that there are no confounding variables that could explain the relationship between the instrumental variable (DNA) and the exposure. In allergy studies, MR has been used to show that increased BMI causes increased asthma, increased hay fever, and decreased lung function and has no causal interaction for hay fever or biomarkers of allergy.32 MR has also been used to show a lack of support for vitamin D deficiency as being causal for asthma.33,34 A major advantage of this method is that it does not require individual data, but rather information for each SNP at the population level (i.e. summary results). Expression Quantitative Trait Loci.  Access to multiple types of data on the same cohort improves systems biology implementation because these data can be integrated. One of the earliest integration methods was expression quantitative trait loci (eQTL), which looks for the significant associations between SNPs and gene expression35,36 (Fig. 23.3B). Because the genome is large and contains higher structure, this can be used to split eQTL analyses into cis and trans types. Cis eQTLs occur when the SNP associated with gene expression is within a predefined region near the gene (usually approximately +/- 1 MB), suggesting that functional regions in or adjacent to the gene, such as coding variation, variations affecting splicing sites, transcription factor binding sites, promoter sites, and enhancer regions, harbor variations that directly affect the regulation of the transcription of the gene or the stability of its transcripts or alter protein function or state in ways that ultimately feed back onto gene expression levels. eQTLs that are not cis are assumed to be trans, implying that variations in the genome are acting at a distance, perhaps via intermediary genes. For eQTL analysis, the use of surrogate or latent variables to detect all other sources of variation can improve the number of eQTLs detected. Because we know from the central dogma of biology that the DNA we inherit drives RNA, eQTLs can inform on the downstream interactions of SNPs and have been very helpful in understanding the genes identified from GWAS loci. Because this analysis is compute intensive and frequently done, numerous methods have been developed and optimized to run it quickly.37 The detection of cis eQTL is more powerful and straightforward compared with trans eQTL, given only variations in the neighborhood of a gene are considered for cis eQTL, whereas for trans eQTL variations across the genome must be tested. Given the number of tests needed for the detection of trans eQTL, after correcting for multiple testing the power to detect these is very low, a reflection that we still do not have the scale of data needed here to construct a full regulatory map of gene expression based on genetic variation. Reflecting this approach, researchers have studied a large cohort in which both genome-wide genotype data and peripheral blood CD4+ lymphocyte gene expression data were generated to identify GWAS loci and eQTLs corresponding to those loci, providing for a direct link between the GWAS loci, RNA from the same population, and disease context to provide a richer, functional context for the GWAS loci identified.31 Beyond associations between DNA loci and gene expression, epigenetic data (CpGs, histones, miRNA, etc.) can be analogously examined to identify epigenetic loci associated with genotypes or with gene expression. One example of using epigenetic and genotype data to detect QTLs is the examination of genotype and enhancer RNA regions to

identify enhancer RNA regions that are controlled by genotype (eeRNA).38 There can also be epigenetic modifications that can control gene expression, such as CpG islands that regulate gene expression independently from SNPs (eQTL), as was reported for the asthma locus ORMDL3.39

Association-Based Approaches

Cell Mixture Deconvolution.  A systems biology approach to transcriptomic data involves deconvolving gene expression information into signals from different cell types. This is based on the fact that most molecular profiling is carried out in complex tissue samples comprising many cell types. Different computational approaches have been developed to estimate the various cell types present in a heterogeneous sample or tissue.40 These methods can approximate the cells that are present, although they require a priori information about different cell types to do this accurately. There are even methods available to impute gene expression levels in different tissues based on genotype data. Leukocyte deconvolution has been implemented on whole blood RNA-seq profiles from peanut-allergic children where it was not possible to collect sufficient blood volumes for characterization of many types of immune cell subsets.11 The investigators were able to take the whole blood signature and approximate for each individual the fractions of leukocyte cell types that were present, finding that acute allergic reactions to peanut were linked to increases in macrophages M0 and neutrophils and a decrease in naïve CD4+ T cells.11 Differential Expression.  One type of analysis of RNA that is analogous to GWAS is differential expression analysis, where instead of looking for differing frequencies of alleles at a given SNP between two groups, gene expression changes between two conditions are assessed (Fig. 23.3C). Differential expression enables identifying groups of genes that are overexpressed or underexpressed between two or more conditions (or with respect to quantitative traits, genes that are significantly correlated with the trait), providing clues to what is occurring in certain cells and tissues under variable conditions. For allergy research, these tools have been used to identify sets of genes that are significantly different in response to allergens, between cases and controls, or associated with severity of disease.4 Although differential expression analysis was originally applied to microarray profiling data, with the advent of RNAseq, differential expression analysis is now able to capture more information than was possible with microarray data. Microarrays depend on probe sequences to identify the transcripts to detect, thus requiring a priori knowledge of the gene, whereas RNAseq provides exact estimates of the number of copies of transcripts in the tissue profiled and can more readily uncover completely novel coding aspects of the genome, novel isoforms, and even RNA modification and gene editing events. Upon detecting genes whose expression differs between conditions, it can be very helpful to annotate those genes to determine whether any known biologic processes or known functional groups are represented in the set of differentially expressed genes. There are a few very well-known and highly curated databases that contain annotations of genes: Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Molecular Signature Database (MSigDb). In addition, there are numerous publicly available tools and websites for enrichment analysis such as DAVID (https://david.ncifcrf.gov/), GOrilla (http:// cbl-gorilla.cs.technion.ac.il/), REVIGO (http://revigo.irb.hr/), and MSigDB (http://software.broadinstitute.org/gsea/msigdb/). In GO analyses, pathways or other functional gene sets are searched in the differentially expressed gene signatures to assess whether they are enriched more than we would expect by chance. Such enrichment analysis not only provides for interesting clues as to what pathways and functional processes are being differentially modulated, but also helps establish

CHAPTER 23  Systems Biology the degree of biologic coherence in the data, serving as a validation of the biologic signal represented in the differentially expressed gene sets of interest. Although these databases are excellent, they are often limited by processes that are actually annotated (i.e., known). Taking a recent study of the peripheral blood transcriptome in peanut-allergic children as an example, upon detecting genes that significantly changed during acute peanut allergic reactions, the investigators found that the majority of the genes were upregulated and were significantly enriched for gene ontology terms for acute phase responses.11

Data-Driven Clustering.  How genes cluster together based on their expression profiles can additionally inform on biologic processes that are altered between study groups (Fig. 23.3D). Although differential expression analysis and clustering procedures such as hierarchical clustering or K-means clustering provide useful insights into the data in the form of meaningful cluster analysis, they also result in destroying much richer components of the data by dramatically reducing the dimensionality of the data. In the case of differential expression, the expression data for each gene is reduced largely to a mean and variance estimate in each group, ignoring the rich correlation structure among genes. Clustering approaches such as hierarchical clustering consider correlation structure among the expression traits, but then project that onto an n-dimensional space, where n is the number of genes considered (that is, one goes from considering all n-squared correlations among all genes and reduce that to an n-dimensional dendrogram). More recent approaches for organizing the rich data structure that exists within high-dimensional molecular profiling data while preserving the dimensionality of the data include association-based network methods. Weighted gene coexpression network analysis (WGCNA),41 multiscale embedded gene coexpression network analysis (MEGENA),42 and algorithm for the reconstruction of accurate cellular networks (ARACNe)43 represent a few examples of such methods that enable the organization of the rich interaction structure that exists in molecular data generated on populations of individuals. The aim of these methods is to identify groups of molecular features (referred to as clusters or modules) such as genes that are more similarly related to each other than to any other features under consideration. For computational efficiency, most of these methods generally assume that a given gene can only be represented in one cluster across all the samples. WGCNA, a highly used method for clustering, builds on computing the correlation (or similarity measure) between all gene pairs, and then raising this similarity matrix to a given power (Beta), which results in an adjacency matrix that defines genes that are related to one another. The Beta is determined in such a way that the adjacency matrix is scale-free and thus the resulting network is scale-free, meaning that most nodes interact with only a few nodes, whereas a smaller proportion of nodes interact with many nodes (referred to as hub nodes), a feature that is representative of biologic and social networks alike. Once the adjacency matrix is defined, it can be transformed into a topologic overlap map (TOM), which captures both direct and indirect interactions. Hierarchical clustering is then applied to the TOM to organize the genes into clusters (or modules).41 WGCNA has been used in allergy research to identify modules of genes that are important to a given disease process, including detection of an acute phase response module for peanut-allergic patients,11 and a mitochondrial process module in allergic rhinitis.31 Clustering methods can also be applied to phenotype data. Clustering phenotypes using methods ranging from hierarchical clustering to coexpression modules enables the data-driven identification of broader phenotypic groups. This can help reduce the dimensionality of phenotype data and identify interesting groups of traits related to one another. Clustering has been used to identify subtypes and endotypes of asthma.44

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Classification Approaches. Advanced classification approaches developed in the machine learning field can also be applied to highdimensional biologic data to identify features (e.g., genes, biomarkers) that can best split a cohort into groups, moving beyond functional characterizations of expression changes to diagnostic potential with clinical impact. The general approach involves learning from one set of data (training data) a set of “classifiers” or traits that are capable of accurately predicting group membership. To assess the robustness of the classifiers identified in the training and to further tune the selection of classifiers, an independent set of data (validation or test data) are used to characterize the performance of the classifier using standard metrics such as precision and recall. In an ideal world, two completely different sets of data (training and test set) with the same groups of interest, the same feature sets scored in the same way, would be collected. The test set ideally is withheld from the beginning and only revealed after the classifier has been built, to assess objectively the accuracy of the classifier constructed on the first set. However, this is generally not the case, although methods have been developed to address this, most of which involve splitting a given data set into training and testing sets and iteratively repeating this process to both empirically estimate the performance of the classifier and to protect against classifier overfitting. There are numerous different machine learning algorithms that can and have been applied to biologic data to construct reliable biomarkers of disease. In the machine learning field one of the more successful approaches is advanced types of neural networks, such as convolution neural networks, to learn highly nonlinear patterns from large-scale data that can distinguish meaningful groups with high accuracy. In allergy, most of the research using classification methods has been applied to asthma to distinguish cases from controls, or to predict severity of asthma.45 Although machine learning has largely been applied to study asthma, these approaches can be extended to other allergic diseases as well.45

Causal Approaches

Causal Inference.  Because DNA is generally upstream of functional molecular changes, there are methods that take advantage of this to detect causal interactions between three correlated variables. One such method is the causal inference test, which was built to detect causal interactions between DNA, molecular trait, and phenotype.46 DNA as a systematic source of perturbation is a powerful concept for causal inference. If we did not start with the fact that DNA is upstream of molecular and higher-order phenotypes, the causal inference problem would be much harder, because there would be many more possible configurations to consider, some of which are statistically indistinguishable. This approach can also be used to detect trans eQTLs that are mediated by cis eQTLs (SNP → cis gene expression → trans gene expression).10 This method has not yet been applied directly to allergy research but could be applied to data sets that contain DNA, RNA gene expression, and clinical information to detect SNP → gene → clinical phenotype relationships, or if the DNA and RNA data set is large enough it could be used to detect SNP → cis eQTL → trans eQTL as we have previously shown in other diseases.10,47

Probabilistic Causal Networks.  Causality is an extremely important and useful piece of information to have, particularly if disease mechanisms and potential treatments in allergy and immunology are to be identified. Causality can inform whether states are not just correlated, but rather that one state causes the other to occur. Few methods exist that construct probabilistic causal networks to capture a more global view of the interactions present in data (Fig. 23.3E). Several of these methods take advantage of DNA being upstream and use eQTLs as perturbations to drive causal inference.48 One type of such network is

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Bayesian Networks (BN), which expand on Bayes’ principal of statistics to find conditional dependencies between the input nodes. This method has a few major limitations: (1) large samples sizes are required; (2) the amount of time it takes to run does not scale linearly with the number of genes or nodes (it is an NP-hard problem), so it requires many heuristics to constrain the size of the search space and thus more efficiently and find the networks that best fit the data; and, (3) a BN is by definition a graph that is directed and acyclic, containing no cycles, which biologically is not the case. Sample size is often a constraint for this type of analysis, and probabilistic causal networks have not been widely implemented in the field of allergy and immunology to date. However, researchers are beginning to leverage causal networks in allergy research.11

Key Drivers/Regulators.  Because not every node in a network is equal, it is important to leverage the structure of the network (i.e., its topology) to identify key driver nodes of the network that modulate sets of genes of interest49 (Fig. 23.3F). A number of published papers support the key driver (KD) approach as robust and leading to novel biologic discovery. It is also worth noting that KDs are often suggested as possible drug targets as they regulate or modulate groups of genes linked to a disease.50 For example, after detecting the genes that were differentially expressed because of acute peanut allergic reaction, investigators built coexpression modules, identified an acute phase response module significantly enriched with the differentially expressed genes, and then leveraged a Bayesian network to detect key drivers of the peanut-responding coexpression module.11 By doing this, the researchers identified six key drivers predicted to be most impactful, with a few already the focus of therapeutic interest.11 This is just one example of a systems biology work flow that can lead to translational results.

CONCLUDING REMARKS Computational Challenges.  As we generate more and more data, it is important to consider how to store data efficiently. Currently, supercomputers and massive data warehouses are used for storage, but group computing and sharing is starting to take a hold. An example of this is the World Community Grid, which asks individuals to offer extra compute power on their computers to help analyze the role of microbiomes in diseases such as asthma and Crohn disease. The idea of crowdsourcing and urging groups to work together and compete against one another is not new; one of the most successful instances of this are the DREAM challenges run by Sage Bionetworks. Here, people from all over the world compete with different methods on how to best analyze the data made available for a given challenge, and they are constantly challenged with new data. Another example of crowdsourcing is the Human Diagnosis Project (https://www.humandx.org/), which tries to help individuals and clinicians provide better care to patients using big data and machine learning algorithms. To fully understand what is occurring in an individual and to accurately predict the best therapies for an individual’s condition, it is imperative to use systems biology approaches to integrate the different strata of data available and to capture more and more “missing information” (Fig. 23.4). As technology continues to improve for sequencing and compute power, we will continue to not only gather more data, but to also integrate it and make sense of it. It is through this more extensive integration that we will be able to derive data-driven hypotheses, improve our understanding of diseases, and ultimately be able to have a larger impact on an individual’s health and wellness. Through an integrative systems biology framework, the field of allergy and immunology research will change both in biologic understanding and in translational clinical applications. It is only a matter of time

Microbiome Environment Epigenetics Protein

RNA

DNA

Metabolomics

Nutrition

Fig. 23.4  Schematic depicting the known and possible interactions between different biologic data. The directed arrows represent the central dogma of biology.

until these approaches will become standard practice and translated to improve patient outcomes in clinical settings. As our understanding of what causes allergy and immunologic processes deepens, better therapeutics will become clearer and drugs currently available may even be repurposed. New therapeutics might not be drugs in the traditional sense but could be individualized microbiota tailored to an individual’s bacterial composition, for example. Other translational examples are accurately predicting an individual’s severity of allergic reaction, or which individuals will respond positively toward allergen immunotherapy. Ultimately, systems biology approaches will lead to better treatment of the individual.

REFERENCES 1. Charles-Smith LE, Reynolds TL, Cameron MA, et al. Using social media for actionable disease surveillance and outbreak management: a systematic literature review. PLoS ONE 2015;10(10):e0139701. 2. NHGRI. The Cost of Sequencing a Human Genome: NHGRI; 2017 [updated July 6, 2016]. Available from: https://www.genome.gov/ sequencingcosts/. 3. van Dijk EL, Auger H, Jaszczyszyn Y, et al. Ten years of next-generation sequencing technology. Trends Genet 2014;30(9):418–26. 4. Bunyavanich S, Schadt EE. Systems biology of asthma and allergic diseases: a multiscale approach. J Allergy Clin Immunol 2015;135(1):31–42. 5. Marenholz I, Grosche S, Kalb B, et al. Genome-wide association study identifies the SERPINB gene cluster as a susceptibility locus for food allergy. Nat Commun 2017;8(1):1056. 6. Goodwin S, McPherson JD, McCombie WR. Coming of age: ten years of next-generation sequencing technologies. Nat Rev Genet 2016;17(6):333–51. 7. Sordillo J, Raby BA. Gene expression profiling in asthma. Adv Exp Med Biol 2014;795:157–81. 8. Purcell S. Genetic Methodologies and Applications. New Methods and New Technologies for Preclinical and Clinical Neurobiology; 2013. p. 160-71. 9. Maffucci P, Filion CA, Boisson B, et al. Genetic diagnosis using whole exome sequencing in common variable immunodeficiency. Front Immunol 2016;7:220. 10. Franzen O, Ermel R, Cohain A, et al. Cardiometabolic risk loci share downstream cis- and trans-gene regulation across tissues and diseases. Science 2016;353(6301):827–30. 11. Watson CT, Cohain AT, Griffin RS, et al. Integrative transcriptomic analysis reveals key drivers of acute peanut allergic reactions. Nat Commun 2017;8(1):1943.

CHAPTER 23  Systems Biology 12. Papalexi E, Satija R. Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol 2018;18(1):35–45. 13. Vogel C, Marcotte EM. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet 2012;13(4):227–32. 14. Nony E, Le Mignon M, Brier S, et al. Proteomics for allergy: from proteins to the patients. Curr Allergy Asthma Rep 2016;16(9):64. 15. Beaulaurier J, Zhang XS, Zhu S, et al. Single molecule-level detection and long read-based phasing of epigenetic variations in bacterial methylomes. Nat Commun 2015;6:7438. 16. Tumes DJ, Papadopoulos M, Endo Y, et al. Epigenetic regulation of T-helper cell differentiation, memory, and plasticity in allergic asthma. Immunol Rev 2017;278(1):8–19. 17. Kong J, Chalcraft K, Mandur TS, et al. Comprehensive metabolomics identifies the alarmin uric acid as a critical signal for the induction of peanut allergy. Allergy 2015;70(5):495–505. 18. Huang YJ, Marsland BJ, Bunyavanich S, et al. The microbiome in allergic disease: current understanding and future opportunities - 2017 PRACTALL document of the American Academy of Allergy, Asthma & Immunology and the European Academy of Allergy and Clinical Immunology. J Allergy Clin Immunol 2017;139(4):1099–110. 19. Renz H, Holt PG, Inouye M, et al. An exposome perspective: early-life events and immune development in a changing world. J Allergy Clin Immunol 2017;140(1):24–40. 20. Afshinnekoo E, Meydan C, Chowdhury S, et al. Geospatial resolution of human and bacterial diversity with city-scale metagenomics. Cell Syst 2015;1(1):72–87. 21. Chan YY, Wang P, Rogers L, et al. The Asthma Mobile Health Study, a large-scale clinical observational study using ResearchKit. Nat Biotechnol 2017;35(4):354–62. 22. Schadt EE, Linderman MD, Sorenson J, et al. Computational solutions to large-scale data management and analysis. Nat Rev Genet 2010;11(9): 647–57. 23. Stephens ZD, Lee SY, Faghri F, et al. Big data: astronomical or genomical? PLoS Biol 2015;13(7):e1002195. 24. Hasin Y, Seldin M, Lusis A. Multi-omics approaches to disease. Genome Biol 2017;18(1):83. 25. Demenais F, Margaritte-Jeannin P, Barnes KC, et al. Multiancestry association study identifies new asthma risk loci that colocalize with immune-cell enhancer marks. Nat Genet 2018;50(1):42–53. 26. Hong X, Hao K, Ladd-Acosta C, et al. Genome-wide association study identifies peanut allergy-specific loci and evidence of epigenetic mediation in US children. Nat Commun 2015;6:6304. 27. Madore AM, Vaillancourt VT, Asai Y, et al. HLA-DQB1*02 and DQB1*06:03P are associated with peanut allergy. Eur J Hum Genet 2013;21(10):1181–4. 28. Hirota T, Nakayama T, Sato S, et al. Association study of childhood food allergy with genome-wide association studies-discovered loci of atopic dermatitis and eosinophilic esophagitis. J Allergy Clin Immunol 2017;140(6):1713–16. 29. Li J, Fung I, Glessner JT, et al. Copy number variations in CTNNA3 and RBFOX1 associate with pediatric food allergy. J Immunol 2015;195(4):1599–607. 30. Bonnelykke K, Matheson MC, Pers TH, et al. Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization. Nat Genet 2013;45(8):902–6.

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31. Bunyavanich S, Schadt EE, Himes BE, et al. Integrated genome-wide association, coexpression network, and expression single nucleotide polymorphism analysis identifies novel pathway in allergic rhinitis. BMC Med Genomics 2014;7(1):48. 32. Skaaby T, Taylor AE, Thuesen BH, et al. Estimating the causal effect of body mass index on hay fever, asthma and lung function using Mendelian randomization. Allergy 2018;73(1):153–64. 33. Mao Y, Zhan Y, Huang Y. Vitamin D and asthma: a Mendelian randomization study. Ann Allergy Asthma Immunol 2017;119(1): 95–7.e1. 34. Hysinger EB, Roizen JD, Mentch FD, et al. Mendelian randomization analysis demonstrates that low vitamin D is unlikely causative for pediatric asthma. J Allergy Clin Immunol 2016;138(6):1747–9.e4. 35. Schadt EE, Monks SA, Drake TA, et al. Genetics of gene expression surveyed in maize, mouse and man. Nature 2003;422(6929):297–302. 36. Zhu Z, Zhang F, Hu H, et al. Integration of summary data from GWAS and eQTL studies predicts complex trait gene targets. Nat Genet 2016;48(5):481–7. 37. Ongen H, Buil A, Brown AA, et al. Fast and efficient QTL mapper for thousands of molecular phenotypes. Bioinformatics 2016;32(10): 1479–85. 38. Hauberg ME, Fullard JF, Zhu L, et al. Differential activity of transcribed enhancers in the prefrontal cortex of 537 cases with schizophrenia and controls. Mol Psychiatry 2018. doi: 10.1038/s41380-018-0059-8. 39. Acevedo N, Reinius LE, Greco D, et al. Risk of childhood asthma is associated with CpG-site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus. Hum Mol Genet 2015;24(3):875–90. 40. Newman AM, Liu CL, Green MR, et al. Robust enumeration of cell subsets from tissue expression profiles. Nat Methods 2015;12(5): 453–7. 41. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008;9(1):559. 42. Song WM, Zhang B. Multiscale embedded gene co-expression network analysis. PLoS Comput Biol 2015;11(11):e1004574. 43. Margolin AA, Wang K, Lim WK, et al. Reverse engineering cellular networks. Nat Protoc 2006;1(2):662–71. 44. Deliu M, Sperrin M, Belgrave D, et al. Identification of asthma subtypes using clustering methodologies. Pulm Ther 2016;2:19–41. 45. Tartarisco G, Tonacci A, Minciullo PL, et al. The soft computing-based approach to investigate allergic diseases: a systematic review. Clin Mol Allergy 2017;15:10. 46. Millstein J, Zhang B, Zhu J, et al. Disentangling molecular relationships with a causal inference test. BMC Genet 2009;10:23. 47. Peters LA, Perrigoue J, Mortha A, et al. A functional genomics predictive network model identifies regulators of inflammatory bowel disease. Nat Genet 2017;49(10):1437–49. 48. Zhu J, Wiener MC, Zhang C, et al. Increasing the power to detect causal associations by combining genotypic and expression data in segregating populations. PLoS Comput Biol 2007;3(4):e69. 49. Cohain A, Divaraniya AA, Zhu K, et al. Exploring the reproducibility of probabilistic causal molecular network models. Pac Symp Biocomput 2017;22:120–31. 50. Zhang B, Gaiteri C, Bodea L-G, et al. Integrated systems approach identifies genetic nodes and networks in late-onset Alzheimer’s disease. Cell 2013;153(3):707–20.

CHAPTER 23  Systems Biology

361.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following statements aligns with the systems biology approach? a. Develop a hypothesis and run experiments to test it b. Gather as much information as possible and use a data-driven approach to generate hypotheses c. Reliance on extensive literature review only to develop a hypothesis d. None of the above 2. Which analysis incorporates genomic and transcriptomic data, incorporating the information of gene locations on chromosomes? a. Genome-wide association study (GWAS) b. Expression quantitative trail loci (eQTL) mapping c. Differential gene expression d. Gene coexpression network analysis

3. What has systems biology helped to discover? a. Genetic variants that contribute to disease risk b. Transcriptomic changes and regulatory mechanisms of disease c. Potential allergy biomarkers d. All of the above 4. What is a key driver gene? a. Gene that significantly changes between two conditions b. Gene that regulates a significant proportion of genes in a given state c. Membrane receptor gene activity in a given state d. Transcription factor

24  Immunobiology of IgE and IgE Receptors Hans C. Oettgen

CONTENTS Introduction, 362 IgE Structure and Mechanisms of IgE Isotype Switching, 363 FcεRI, the High-Affinity IgE Receptor, 368

SUMMARY OF IMPORTANT CONCEPTS • IgE antibody production is regulated by Th2 cells. Activated Th2 cells trigger IgE production in B cells via a combination of signals including secreted cytokines (IL-4 or IL-13) and cell-surface molecules (CD40L). • IgE-producing B cells are the progeny of bone marrow emigrant IgM+ B cells. The process of class switch recombination (CSR) occurs via somatic gene rearrangements leading to the assembly of a gene encoding the εheavy chain. Class switching to IgE is sequential, with an IgG+ intermediate B cell stage in which IgE memory resides. • The major IgE functions are mediated by two receptors, FcεRI (the highaffinity IgE receptor) and CD23 (low-affinity IgE receptor), found on mast cells and basophils, but also other cells. • Aggregation of FcεRI-bound IgE on mast cells and basophils by polyvalent antigen leads to the activation of a complex array of signaling pathways resulting in the release of preformed and newly synthesized mediators of immediate hypersensitivity. • IgE binding to CD23 is important in IgE-facilitated antigen uptake by antigenpresenting cells, transcellular allergen transport in gastrointestinal and respiratory epithelium, and regulation of IgE production. • IgE antibodies exert important immunomodulatory functions including regulation of IgE-receptor density, promotion of mast cell survival, suppression of innate antiviral immune responses, and enhancement of Th2 responses to allergens.

INTRODUCTON IgE antibodies are infamous as instigators of the immediate hypersensitivity reactions that trouble patients with allergies. Testing for their presence is a cornerstone of allergy evaluation. As implied in the terms immediate and hypersensitivity, IgE is unique among immunoglobulin isotypes in its capacity both to induce extremely rapid biologic responses, including potentially fatal anaphylaxis, and to act as an extremely sensitive immunologic amplifier, triggering reactions upon exposure to minute quantities of antigen. IgE, along with its receptors, FcεRI and CD23, and effector cell lineages, mast cells and basophils, has persisted through vertebrate evolution, suggesting a significant adaptive advantage. This arm of the immune response is likely to be important in controlling infestation with helminths in parasite endemic regions. In high-income countries, however,

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CD23, the Low-Affinity IgE Receptor, 371 Relationships with Other Systems, 372 Conclusion, 373

parasites are not prevalent, and IgE production occurs in individuals with the atopic conditions: eczema, allergic rhinitis, and asthma. During the past two decades, treatment of allergic individuals with the humanized monoclonal anti-IgE antibody, omalizumab, has become an important part of the therapeutic armamentarium. Observations on the effects of IgE blockade in patients, as well as the results of animal research, have shed light on an array of previously unknown functions of IgE. It is now clear that in addition to triggering acute allergic reactions, IgE antibodies mediate a number of critical tasks related to immune homeostasis. IgE antibodies regulate IgE receptor expression, enhance allergen uptake by antigen-presenting cells (thereby amplifying cellular immune responses), modulate mast cell survival, and facilitate antigen transport across intestinal and respiratory epithelia. Their effects extend beyond the realm of hypersensitivity. IgE antibodies are now known to suppress innate immune responses to respiratory viruses in asthmatics and to amplify the induction of effector T cell responses to allergens while dampening the emergence of regulatory T cells. This chapter provides an overview of the structure of the IgE protein and the Cε gene, the factors that govern IgE production, the receptors mediating IgE responses, and our expanding understanding of the range of responses that can be triggered in the cell types bearing those receptors.

Structure and Function (Pathophysiology)

The Discovery of IgE.  The vanishingly low amounts of IgE normally present in serum delayed its discovery for decades after the IgM, IgG, IgA, and IgD isotypes had been characterized. Early in the 20th century, a component of the γ-globulin fraction of plasma, referred to as reagin, had been shown to be capable of transferring allergic sensitivity from a sensitized subject to a naïve host in the passive cutaneous anaphylaxis assay, also known as the Prausnitz and Küstner reaction after its discoverers. Unlike known antibody isotypes, reagin neither fixed complement nor produced precipitin lines in agar diffusion reactions with antigen. Using the passive cutaneous anaphylaxis model, it was established that reagin was heat-labile and did not cross the placenta. Eventually, myelomas were discovered that produced an unknown antibody isotype. Antibodies raised against that isotype were shown to inhibit the PrausnitzKüstner test. Characterization of the reaginic isotype by the converging efforts of the Ishizakas in the United States and Bennich and Johansson in Sweden in 1967 revealed a new immunoglobulin class named IgE (E for erythema).1

CHAPTER 24  Immunobiology of IgE and IgE Receptors Antigen

IgE in Parasitic Immunity IgE antibodies, along with an intricate system of IgE receptors and effector cell lineages, have persisted through evolution, suggesting an important adaptive advantage. The nature of the evolutionary pressure maintaining this system is suggested by the presence of high IgE levels in populations residing in helminth-endemic regions, indicating that IgE might be important in controlling the host–parasite interaction. In areas where infection with the blood fluke, Schistosoma mansoni, is common, IgE levels correlate with resistance to reinfection. Direct evidence for a role of IgE in parasite immunity was first provided by reports that IgE could opsonize S. mansoni for killing by human eosinophils and even platelets.2,3 In animal models it has been shown that IgE enhances granuloma formation in the liver while assisting in the clearance of adult worms during primary infection. Effective host responses to another parasite, Trichinella spiralis, are also mediated by IgE antibodies. IgE accelerates the removal of adult T. spiralis worms from the intestine and mediates the destruction of tissue cysts.4 Parasites and humans have evolved together and are closely coadapted so that parasitic infestation can occur even in subjects with functional IgE systems. However epidemiologic studies and animal models provide evidence that IgE is important in controlling effector immune responses and parasite burden, and this may account for the evolutionary persistence of the IgE system.

IgE STRUCTURE AND MECHANISMS OF IgE ISOTYPE SWITCHING Structure of IgE The serum concentration of IgE in normal individuals is typically around 50 to 100 ng/mL, logs less than IgG, which is present at concentrations on the order of 5 to 10 mg/mL. The low levels of IgE and its very short half-life in circulation are accounted for by the exceedingly small number of B cells committed to IgE synthesis and by its localized production at tissue sites of allergen exposure, where it is rapidly and tightly bound to FcεRI on mast cells, on which it can persist for months. Like other immunoglobulins, the IgE protein is a tetramer composed of two ε-heavy and two κ or λ light chains. N-terminal variable regions of the heavy (VH) and light (VL) chains create unique antigenspecific binding pockets (see Chapter 3). The C-terminal constant regions of the ε-heavy chains are made up of four Cε domains, each encoded by the Cε1-4 exons near the 3’ end of the immunoglobulin heavy chain locus (IgH). This is in contrast to the three Cγ domains contained in the γ-heavy chains of IgG molecules. The four Cε domains confer the isotype-specific functions of IgE, including binding to its receptors FcεRI and CD23. Cε2 occupies the position comparable to the flexible “hinge” region contained in Cγ of γ-heavy chains. X-ray crystallography of IgE-Fc fragments has revealed a very sharp backward turn in the molecule in the Cε2-3 region so that the native conformation of IgE is compact and bent, unlike the more stretched out and flexible structure of IgG (Fig. 24.1). In IgE-committed B cells, mRNA splice variants containing M1 and M2 exons that encode hydrophobic sequences give rise to transmembrane IgE. IgE antibodies are heavily glycosylated, a fact that may account for their affinity for galectins, lectin-type proteins that can interact with IgE both in solution and at cell surfaces. One glycan, present in the Cε3 domain that interacts with FcεRI, is critical for IgE binding and effector cell activation.5 M1 and M2 exons at the 3’ end of the Cε locus encode hydrophobic sequences, which are incorporated into ε-heavy chain mRNA encoding the transmembrane form of IgE expressed on the surface of IgE+ B cells.

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VH

Vκ/λ

Cε1

Cκ/λ

IgE

Cε4

Cε2 Cε3 α

α β

γ

Tetrameric FcεRI αβγ2 Mast cells, basophils

γ

Trimeric FcεRI αγ2 Langerhans cells, dendritic cells, monocytes, eosinophils, platelets

Fig. 24.1  IgE structure and interaction with FcεRI. Unlike IgG antibodies, whose γ-heavy chains have three constant region domains and a flexible hinge region, IgE antibodies have four constant region domains, Cε1-4 in their ε-heavy chains. A sharp backward bend is conferred by Cε2 and Cε3, and Cε3 interacts with the IgE-binding α-chain of FcεRI. FcεRI exists in two conformations. The classical tetrameric form expressed on mast cells and basophils contains the IgE-binding α chain in association with the β chain, a member of the tetraspanin family of four-transmembrane domain proteins, thought to amplify signal transduction, and a disulfide linked dimer of γ chains important for signal transduction. A trimeric form of FcεRI lacking the β chain is present on a number of other cell types including Langerhans cells and dendritic cells.

Regulation of IgE Synthesis: Cellular Interactions and Secreted Signals The molecular pathways responsible for deletional switch recombination in B cells are tightly regulated and depend both on secreted cytokines provided by T-helper 2 (Th2) cells and on the interaction of cell-surface activation molecules during cell-cell contact. A back-and-forth signaling conversation occurs between a B cell or antigen-presenting cell (APC) and Th cells, each step providing both an amplification circuit for the switch signal and a check on uncontrolled responses. The process is initiated when antigen is taken up and processed into MHC II–associated peptides by APC residing in skin, mucosal tissues, or lymphoid organs. Although “professional” APCs, including dendritic cells residing at these sites, are the most effective in driving T cell responses, B cells can also provide an antigen-presenting function. In situations of recurrent seasonal allergen exposure, preexisting B cell clones of the relevant specificity can serve as antigen-focused APCs, taking up antigen specifically via their membrane-associated immunoglobulin (or alternatively via allergen specific IgE bound to their cell surface IgE receptor, CD23). Processed antigenic peptides are displayed at the APC surface in association with class II MHC proteins and recognized by Th2 cells bearing a T cell receptor (TCR) of appropriate specificity. TCR ligation results in the activation of a specific transcriptional program, driving both production of IL-4 and the induction of CD40ligand (CD40L), also known as CD154, which is not normally present on resting T cells (Fig. 24.2). These signals engage their constitutively expressed counterparts on the adjacent B cell, the IL-4 receptor, and the tumor necrosis factor (TNF) receptor superfamily member, CD40. In humans, either IL-4 or IL-13, another Th2 cytokine, can provide the cytokine signal. This sets into motion parallel signaling cascades that trigger the two key early steps in IgE isotype switching: (1) germline

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Fig. 24.2  T/B cell interactions leading to IgE isotype switching. The signals driving isotype switching are provided during an ordered series of interactions between an antigen-specific Th2 cell and a B cell expressing immunoglobulin of the same antigenic specificity. An antigen-specific B cell (1) binds the antigen via surface immunoglobulins (sIgM), internalizes and processes it, and presents it to an allergen-specific Th2 cell as an MHC II–associated peptide fragment (2). Engagement of the T cell receptor by the MHC class II–antigen complex results in the initiation of cytokine transcription as well as the rapid expression of CD40L (CD154), which is not expressed on resting T cells (3). This in turn engages CD40, which is constitutively expressed on B cells (4). T/B cell signals mediated via CD40/CD40L are amplified by interactions between costimulatory molecules, particularly the CD28/CD80-CD86 ligand/receptor pair. Engagement of CD40 upregulates CD80CD86 expression on B cells (5). CD80-CD86 then engage CD28 back on the T cell (6), strongly enhancing the cytokine transcription induced by T cell receptor signaling (7) resulting in secretion (8) of IL-4 and/or IL-13. Engagement of their receptors (9) provides the “cytokine signal” for isotype switching and results in the activation of the transcription factor, STAT-6, which drives ε-germline transcription (εGLT) at the IgH ε-locus (10). At the same time, CD40 signaling activates NF-κB transcription factors, which induce expression of the enzyme activation induced cytidine deaminase (AID), which is recruited to the transcriptionally active ε-locus and initiates the DNA recombination events (11) that lead to class switching and IgE secretion (12).

transcription at the Cε locus, and (2) expression of activation-induced cytidine deaminase (AID). In addition, the IL-4/CD40L signals trigger an amplifying loop leading to B cell upregulation of CD80/86 (B7.1 and B7.2), which, in turn, engage CD28, expressed on the neighboring T cell, leading to potent amplification of cytokine transcription.

The Generation of IgE+ B Cells: a Multistep Process of Somatic Gene Rearrangements

A fully functional ε-heavy chain gene is the product of two sequential sets of somatic DNA rearrangement occurring in pre-B and B cells (Fig. 24.3). In the first of these, VH, D and JH exons are randomly combined in pre-B cells in the bone marrow to generate a VHDJH cassette encoding variable region domain of the heavy chain responsible for determining the structure and hence antigenic specificity of the resultant antibody (see Chapter 3). The second process, class switch recombination (CSR), occurs in peripheral IgM+ or IgG+ B cells of a committed antigenic specificity.6 In this rearrangement, which is stimulated by cytokine signals and T cell contact, the VHDJH cassette 5’ of the Cµ or Cγ exons is spliced to a location just upstream of the Cε exons. The resulting locus encodes an ε-heavy chain, which retains the antigenic specificity of the IgM+ or IgG+ B cell parent, while acquiring the biologic functions of IgE. These rearrangements are irreversible and tightly regulated. As large sections of germline DNA between the µ or γ and ε loci are excised in the process, it has been referred to as deletional switch recombination. Although direct switching from IgM to IgE production can occur in B cells, it has been shown that sequential switch recombination occurs commonly, meaning that B cells are first induced to change from µ to γ heavy chain production and in a later independent event switch from γ to ε. The key cytokine regulators of IgE switching are IL-4 and IL-13. The receptors for these two cytokines are dimers sharing the same

IL-4R-α chain (Fig. 24.4). The type I IL-4 receptor, which binds IL-4, is composed of ligand-binding IL-4Rα and the signal-transducing common cytokine receptor γ-chain, γc. The type II receptor can bind either IL-4 or IL-13. It contains the IL-4R-α chain paired with an IL-13 binding chain, IL-13Rα. IL-4 receptor signaling triggers the activation of Janus family tyrosine kinases (JAK-1 via IL-4Rα, JAK-3 via γc) and TYK2 (via IL-13Rα).7 These in turn phosphorylate tyrosine residues in the intracellular domains of the receptor chains. The phosphotyrosines serve as docking sites for STAT-6, which is phosphorylated next, leading to its dimerization and translocation to the nucleus. In addition to cytokine signals, the activation of IgE class switching requires costimulation provided by direct T-B cell contact and engagement of the B cell surface receptor, CD40, by its ligand, CD40L (CD154). CD40L is transiently expressed on antigen-stimulated T cells and upon binding to CD40 activates signal transduction via intracellular proteins of the TRAF family of TNF-receptor associated factors. TRAF-2, -5, and -6 trigger the dissociation of NF-κB dimers from their inhibitor, IκB, allowing NF-κB to translocate to the nucleus. CD40L is encoded on the X chromosome. Males with X-linked immunodeficiency with hyper-IgM (XHIM) are deficient in CD40L. Consequently, their B cells are unable to produce IgG, IgA, or IgE. The molecular mechanisms mediating deletional switch recombination have been dissected to a great level of detail and include three critical steps: (1) germline RNA transcription; (2) introduction of double-strand DNA breaks; and (3) DNA repair (Fig. 24.5). In the germline configuration of the IgH locus, the four Cε exons which encode the ε-heavy chain constant region domains are preceded by a switch region (Sε), the I exon (Iε), and the Iε promoter. The Iε promoter contains binding sites for transcription factors induced by activation of both the IL-4 receptor and CD40, the two critical signals arising

CHAPTER 24  Immunobiology of IgE and IgE Receptors

2 IL-4/IL-13 IS VDJ

IS Cµ

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Switch excision circle 6

Cα2

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4 CD40

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365

Cγ2

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Fig. 24.3  Molecular events in IgE isotype switching. Before switching occurs in a B cell, the immunoglobulin heavy chain (IgH) locus is in its germline configuration with exons encoding the heavy chain constant region domains distributed over about 150 kb of genomic DNA. However, VDJ recombination has already occurred, thus determining the antigen specificity of the antibody (1). Stimulation with IL-4 or IL-13 (2) results in the transcriptional activation of the IgE locus (3). The process of ε-germline transcription targets the locus for recruitment of the enzyme AID, induced by CD40 engagement (4), and this activates class switch recombination. DNA double-strand breaks followed by ligation of the targeted S regions generates chimeric Sµ/Sε regions composed of the 5′ Sµ joined to the 3′ portion of the targeted Sε region (5). During recombination, the intervening genomic DNA between Sµ and Sε is deleted as a switch excision circle (6).

IL-4

γc

IL-13

IL-13Rα1

IL-4Rα

JAK-3

JAK-1

IL-4Rα

JAK-1

TYK-2

STAT-6

STAT-6 Nucleus

Fig. 24.4  Structure of the receptors for IL-4 and IL-13. The chains that form the heterodimeric IL-4R and IL-13R are shown, together with the associated JAK kinases specific to each receptor complex. Engagement of either receptor results in STAT6 phosphorylation and nuclear translocation.

during T-B cell interaction in the induction of IgE switching. Translocation of activated STAT-6 to the nucleus is triggered by IL-4 and IL-13 signaling, and STAT-6 activation appears to be the key regulator of ε-germline transcription. Nuclear translocation of activated NF-κB family dimers and binding to their promoter elements is also required to activate transcription. The NF-κB pathway is activated by CD40 signaling. Negative regulatory elements are present in the Iε promoter.

Bcl-6 binding to its target in the Iε promoter appears to inhibit transcription, and Bcl-6-/- mice have elevated IgE levels. Activation of the Iε promoter results in the production of RNA transcripts containing Iε and Cε1-4, so-called germline transcripts. These RNAs are normally spliced, polyadenylated, and exported to the cytosol. Based on the results of targeted gene disruption studies, it has long been known that the process of germline transcription is absolutely indispensable in IgE production; B cells from mice lacking either the Iε exon or its promoter are incapable of switching to IgE. Conversely, introduction of a constitutively active promoter (one that drives transcription regardless of the presence of STAT-6 or NF-κB) drives very high levels of IgE switching.8 Paradoxically, however, the transcripts produced in this process do not encode a functional protein. The Iε exon contains stop codons, giving rise to the designation sterile transcripts for these RNAs. It has recently become clear that the process of transcription per se is critical in driving switch recombination to the Cε locus.9 Transcriptionally active sites in the IgH locus attract the enzyme, AID. AID, in turn, deaminates deoxycytidine residues contained in GAGCT repeats within the Sε region, converting them to deoxyuracils. The newly introduced U residues serve as substrates for the enzyme uracil N-glycosylase (UNG), which introduces abasic sites (apurinic/apyrimidinic site) into the double-stranded DNA. When present at high density, such abasic regions lead to double-strand DNA breaks, the first step in the excisionrecombination reaction of CSR. The critical contribution of the enzymes AID and UNG to this process is underscored by the existence of patients with hyper-IgM syndromes in whom homozygous mutations of the genes encoding either protein result in a complete block in immunoglobulin isotype switching and, hence, high levels of IgM but no detectable IgG, IgA, or IgE.

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V

D J

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U Base-excision/mismatch repair

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Nicks ( ) on opposite strands → double-stranded breaks

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Synapsis

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D J

Cε Productive IgE class switching

Fig. 24.5  Molecular events leading to somatic DNA recombination in immunoglobulin class switching. Cytokineinduced transcription through two participating switch (S) regions leads to the recruitment of activation-induced cytidine deaminase (AID). AID converts cytidines to uracils. The subsequent action of uracil-DNA glycosylase (UNG) introduces abasic sites, and apurinic/apyrimidinic endonuclease 1 (APE1) can introduce high-density nicks on the nontemplate strand. These nicks, combined with closely spaced corresponding nicks on the opposite strand, can lead to the generation of double-stranded breaks in the S regions. Isotype switching is completed by fusion of two S regions’ double-stranded breaks, by the nonhomologous end-joining pathway.

Double-strand DNA breaks introduced into Sε in this process are accompanied by breaks simultaneously being created at the upstream locus (Sµ or Sγ) from which switching is being driven. Together these activate a series of steps mediated by the DNA repair mechanism of the cell. This leads to processing and ultimately to physical juxtaposition and then joining of DNA breaks by classical and alternative nonhomologous end-joining pathways with ligation of Sµ or Sγ sequences (along with the VHDJH cassette residing upstream thereof) to Sε and its downstream Cε1-4 exons.10 The product of this excision-repair reaction is a complete ε-heavy chain gene in which the VHDJH cassette encoding the variable region domain is followed by the four Cε exons encoding the constant region domains (Fig. 24.6). Studies of mouse mutants and of humans with inherited immunodeficiencies have implicated the apurinic apyrimidinic endonuclease 1 (APE 1), Artemis, DNA ligase 4, and ATM DNA repair enzymes as critical mediators of this process. This somatic recombination process not only leads to the construction of a brand-new gene encoding an ε-heavy chain, which can be indefinitely propagated through chromosomal replication during B cell divisions, but also to a second episomal product, the so-called switch excision circle. This circle is formed by ligation of the ends of the large germline DNA segment, which is excised between Sµ or Sγ and Sε.

Switch excision circles are not replicated during B cell divisions and during clonal expansion are gradually diluted. Both switch excision circles and ε-germline transcripts are excellent markers of ongoing B cell switching to IgE. Their presence can be probed in tissue samples, and they have been used to demonstrate ongoing IgE switching in the respiratory mucosa of allergic subjects during seasons of exposure.11 Such observations provide evidence for the importance of local IgE production, outside the lymphoid organs and directly in allergen-exposed tissues.

Sites of IgE Production and IgE Memory The generation of high-affinity IgG responses and B cell memory is known to be dependent on the “germinal center reaction” in secondary lymphoid tissues. Antigens are transported to these tissues by dendritic cells (DC). DC residing in the respiratory and gastrointestinal mucosa or in the skin serve as sentinels, extending their dendritic processes into surrounding tissues to probe for allergen. Using innate immune system receptors that recognize a wide range of conserved structures on pathogens and allergens, they are the first to recognize antigen. Dust mite allergen, for example, has been shown to activate DC by interactions with toll-like receptors (TLR), C-type lectin receptors and

CHAPTER 24  Immunobiology of IgE and IgE Receptors VH

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Cε4

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Membrane IgE protein Fig. 24.6  Schematic structures of the mature ε-heavy chain gene along with secreted and transmembrane forms of the IgE protein. The ε-heavy chain gene does not exist in its functional form in genomic DNA but rather is constructed via a somatic DNA recombination process occurring in B cells. The product of the process is a gene in which a V-D-J cassette of exons encodes the N-terminal VH (heavy chain variable) domain of the ε-heavy chain, and four Cε exons encode the constant region domains. A hybrid Sµ/Sε sequence remains between the VH and Cε exons, a byproduct of the isotype switching process. B cell clones that have arisen from sequential switching from IgM+ to IgG+ to IgE+ can be identified by the presence of a hybrid Sµ/ Sγ/Sε sequence. IgE, with four constant regions, has one more of these domains than IgG. Intrachain disulfide bonds (indicated by single or double lines) are contained within each of the immunoglobulin domains. IgE molecules are heavily glycosylated, as indicated by the circles. The M1’ and M2 exons are retained in ε-mRNA splice isoforms encoding the transmembrane form of IgE expressed at the surface of IgE+ B cells. The transmembrane form of IgE is unique among immunoglobulin isotypes in having a relatively long spacer stretch of amino acids, as part of the M1’ domain, between the cell membrane and Cε4 (indicated with **). This unusual proline-rich sequence has been considered as a target for therapeutics directed against IgEproducing B cells.

protease-activated receptor 2 (PAR-2). As discussed later, DC express the IgE receptors, FcεRI and CD23, and allergen uptake is facilitated in the presence of specific IgE antibodies. Once activated, DC display increased MHC class II molecules at the cell surface and express the chemokine receptor, CCR7, which directs them to regional lymph nodes, where they provide a source of antigen, costimulatory molecules and Th2-inducing cytokines to T cells of appropriate specificity (see also Chapter 13). In the lymph nodes B cells residing in follicles engage in cognate interactions with T cells that have been activated by these antigen-bearing DCs in the surrounding T cell zones. This results in the activation of IL-4:IL-4R and CD40L(CD154):CD40 signaling leading to germinal center formation. Specialized follicular T helper cells (TFH) which are CXCR5+ and ICOS+ are important in this process, and Th2-like TFH can provide help for IgE responses.12 Within germinal centers, B cells expand and are induced to activate somatic hypermutation, an AID (activation-induced cytidine deaminase)-dependent genetic process in which mutations are introduced into the variable domain complementarity determining regions of the B cell clones (see also Chapter 3). B cell clones in which these mutations confer higher antigen affinity are selectively expanded in a micro-darwinian process known as affinity

maturation. Specific cytokine and costimulatory signals drive isotype switching in the germinal centers. In contrast to IgG antibodies, IgE circulates at very low levels and is present predominantly affixed to mast cells in tissues. This observation has stimulated interest in the possibility of IgE switching and IgE+ B cell formation occurring not in the germinal center reaction of specialized lymphoid organs but directly in mucosal sites. One clinical observation often cited as evidence for such a process is the known presence of allergen-specific IgE antibodies in the nasal secretions of patients with seasonal rhinitis but negative skin tests for the same allergen. A number of additional findings have provided strong evidence for the occurrence of IgE switching in situ in the mucosa. These include the detection of the essential components of isotype switch machinery (IL-4, CD40L and/or BAFF, another TNF family member capable of driving switching in B cells, and AID) as well as demonstration of the presence of ε-germline transcripts, switch excision circles and IgE+ plasma cells. The processes of efficient B cell isotype class switching to IgG from IgM+ precursors, generation of high-affinity IgG antibody responses through affinity maturation, and creation of memory B cell clones all occur in germinal centers. The IgE response may be very different. In contrast to IgG+ B cells, germinal center IgE+ B cells are short-lived

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and exhibit a tendency for rapid transition to plasma cells as well as a susceptibility to apoptotic cell death. These properties may reflect a unique fate of B cells expressing surface IgE (sIgE), which appears to be present at low levels and signals poorly compared with sIgM or sIgG.13-16 Both IgE affinity maturation and memory appear to reside in an intermediate IgG+ stage. Evidence for this progression is provided by the observations that high-affinity IgE B cell clones tend to have hybrid switch sequences, Sµ-Sγ-Sε, a footprint of previous existence as IgG clones, and the IgE responses of mice lacking the Cγ locus fail to exhibit affinity maturation.17 Sequence analysis of millions of peripheral blood IgH genes in normal and atopic patients indicated a lineage progression in which all somatically mutated IgE sequences are derived from an identically mutated IgG parent clone, consistent with a pathway in which IgG+ B cells provide the IgE memory pool and mediate affinity maturation.18 It is possible that some IgE production is actually T cell–independent, as is suggested by the observation that levels of “natural” IgE, which appears to lack classic allergen specificities, are increased in patients with T cell deficiencies, and in Th-deficient mice and mice with mutations (such as lck-/-) that lack germinal center responses.19

FCεRI, THE HIGH-AFFINITY IgE RECEPTOR Unlike IgG antibodies, which are present at high levels in plasma and exert their effects by binding to circulating antigens and forming immune complexes that in turn activate the complement system or interact with Fcγ receptors, circulating IgE antibody levels are very low. The vast majority of the systemic pool of IgE exists already tightly affixed to effector cells in tissues via IgE receptors. Consequently, antigen–antibody interactions involving IgE occur almost exclusively via cell-bound IgE. IgE antibodies mediate their biologic effects via two receptors, FcεRI (the high-affinity receptor) and CD23 (also referred to as the low-affinity IgE receptor). FcεRI is expressed in one of two forms, an αβγ2 tetramer, found on mast cells and basophils, and a trimeric, αγ2 isoform present on Langerhans cells of the skin and several other dendritic cell types (Fig. 24.1). The tetrameric form functions in the activation of mast cells and basophils in immediate hypersensitivity, whereas the trimeric form probably plays a greater role in FcεRI-mediated antigen uptake by APCs. Murine APCs do not constitutively express the trimeric form. However, induction of trimeric αγ2 receptors on APCs has been reported in mice challenged with viral infection or allergen. The α chain of FcεRI is responsible for IgE binding (Fig. 24.1). It contains two extracellular immunoglobulin domains and interacts with the Cε2-3 regions of the ε-heavy chain as well as a transmembrane and short cytosolic domain. This is a very high affinity interaction with an association constant (Ka) of 1010 M-1 and, in contrast to Fcγ receptors, FcεRI is fully occupied by ligand (IgE) at physiologic IgE levels. Signal transduction by the receptor is mediated in large part by the γ-chain, which contains tandem repeats of immunoreceptor tyrosine-based activation motifs (ITAMs), which are targets for phosphorylation by receptor-associated tyrosine kinases (see later). The FcεRI β chain, present in the tetrameric receptor found on mast cells and basophils, also contains ITAMs and belongs to a tetraspanner family (MS4A) of proteins, which cross the cytoplasm four times with both N- and C-termini residing in the cytosol. The β chain may serve as a chaperone for the α chain and has been shown to amplify some of the early events in FcεRI signaling. The best-known function of FcεRI is triggering classic immediate hypersensitivity reactions, whereupon exposure and cross-linking of IgE bound to the receptor to the offending allergen very rapidly elicits symptoms of allergy. Depending on the organ where sensitization and subsequent exposure have occurred, immediate hypersensitivity might

manifest as sneezing, urticaria, acute bronchospasm, food-induced intestinal smooth muscle constriction, and secretory diarrhea in the gut or cardiovascular collapse, leading to shock in systemic anaphylaxis. Cross-linking of FcεRI-bound IgE by polyvalent allergens triggers the membrane fusion of mast cell granules with resultant exocytosis of preformed mediators of immediate hypersensitivity, such as histamine, and also induces rapid de novo synthesis of lipid mediators such as prostaglandins and leukotrienes. Receptor activation also induces a specific program of gene transcription leading to cytokine and chemokine secretion several hours later.

FcεRI Signaling Pathways Although none of the component chains of FcεRI has inherent enzymatic activity capable of inducing tyrosine phosphorylation or cleaving membrane phospholipids, activation of the receptor is known to activate a number of SRC-family kinases (including LYN, FYN, ZAP-70, and SYK), all of which have been shown to be required for receptor function (Fig. 24.7).20 In addition, FcεRI activates phosphatidylinositol 3-kinase, which acts on membrane phospholipids to produce products capable of regulating cytosolic calcium ([Ca2+]i) concentration and activating protein kinase family enzymes and their downstream signaling intermediates.

Protein Tyrosine Kinase Activation Early in FcεRI Signaling The SRC kinase family has nine members that share a number of domains (SH domains) responsible for kinase activity, protein-protein interactions, and enzyme regulation. The SRC kinase, LYN, is loosely associated with FcεRI-β, and aggregation of FcεRI in the membrane by extracellular receptor-bound IgE with polyvalent allergens favors LYN-mediated phosphorylation of the cytosolic ITAMs residing on an adjacent FcεRI-β or -γ (“transphosphorylation”). The association of LYN with FcεRI appears to be enhanced in lipid rafts, membrane domains enriched in cholesterol and sphingolipids into which FcεRI is shifted upon allergen-mediated aggregation. This early event sets into motion a cascade of protein tyrosine phosphorylation events leading to the assembly of a complex signaling structure activating several parallel pathways. On a side note, there is evidence that LYN not only serves to mediate this critical early activation event but also drives pathways that ultimately serve to keep FcεRI signaling in check, including phosphorylation of the C-terminal SRC kinase (CSK)-binding protein (CBP), which in turn inactivates SRC family kinases by phosphorylation of their C-terminal negative regulatory tyrosines and also inactivates the inositol phosphatases, SHIP-1 and SHIP-2. The phosphorylated ITAMs of the FcεRI-β and -γ chains in turn, serve as docking sites for the SH2-domain containing tyrosine kinase, SYK. SYK phosphorylates a series of scaffolding and adapter molecules, leading to the assembly of a supramolecular signaling complex, focused around transmembrane (LAT1/2) and cytosolic (Gads, Grb2, SHC and SLP-76) adapter molecules. SYK levels are downregulated during chronic IgE-driven signaling via FcεRI in basophils, suggesting a possible mechanism whereby drug desensitization might attenuate mast cell activation. Inhibitors of SYK are very effective blockers of signaling both by FcεRI and by the B cell receptor (BCR) for antigen and are currently in clinical trials for asthma, rheumatoid arthritis, systemic lupus erythematosus (SLE), and B cell lymphoma. The inhibitory effects of currently available compounds are not entirely restricted to SYK, however, and more specifically SYK-targeted drugs are being actively pursued.

Ca2+ and Diacylglycerol (DAG) in FcεRI Signaling The protein scaffold assembled after SYK activation serves to recruit a series of enzymatically active intermediates that further propagate the FcεRI signal via several parallel signaling pathways.21 Among the

CHAPTER 24  Immunobiology of IgE and IgE Receptors

Allergen

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RAS

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Arp2/3 9 Actin polymerization Degranulation Gene expression

Gene transcription Nucleus Fig. 24.7  Early events in FcεRI signaling. The Cε2 and Cε3 domains of the IgE ε-heavy chain are bound to the α-chain of FcεRI. Upon interaction of receptor-bound IgE with polyvalent allergen, aggregation of the receptor allows the protein tyrosine kinase, LYN (which is constitutively associated), to transphosphorylate tyrosine residues in the immunoreceptor tyrosine-based activation motifs (ITAMs) on the β and γ chains of neighboring FcεRI molecules (1). This generates docking sites for SYK, the key early kinase of FcεRI signaling (2). SYK is then activated by phosphorylation and, in turn, phosphorylates the membrane-associated scaffold protein, LAT, leading to the recruitment of a number of signaling intermediates and assembly of a macromolecular signaling complex (3). One of these intermediates, phospholipase Cγ (PLCγ), hydrolyzes the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2), to generate inositol 3,4,5-trisphosphate (IP3) and diacylglycerol (DAG) (4). DAG activates protein kinase Cβ (PKCβ) (5), an enzyme that triggers signaling cascades leading to degranulation and to transcriptional activation. IP3 acts on the endoplasmic reticulum (ER), leading to the release of Ca2+ stores into the cytosol (6). Once ER stores are depleted, the ER protein STIM1 interacts with calcium-regulated calcium channels (CRAC) in the plasma membrane to induce the influx of extracellular Ca2+ (7). Recruitment of the GTP exchange factors, VAV and SOS, to the signaling complex (via Grb2) activates Ras and its downstream pathways, driving cytoskeletal changes, degranulation, and gene expression (8). Activation of the rho family GTPase, Cdc42, by VAV (associated via the linkers Gads and SLP76, which also recruit Bruton’s tyrosine kinase [BTK]), induces activation of the Arp2/3 complex by the Wiskott-Aldrich Syndrome Protein (WASP) with resultant actin polymerization and cytoskeletal changes (9).

molecules assembled in this complex are phospholipases-Cγ (PLCγ1 and PLCγ2), which hydrolyze the membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 3,4,5-trisphosphate (IP3), and diacylglycerol (DAG). IP3 is one of several small molecule second messengers that can mediate the release of Ca2+ from endoplasmic reticulum stores, resulting in increased intracellular calcium ([Ca2+]i) levels. The other product of PIP2 hydrolysis, DAG, is a potent activator of protein kinase C enzymes, so the action of the phospholipases-Cγ is critical in inducing two parallel signaling pathways after FcεRI activation. Several intracellular processes, including those related to secretion and degranulation as well as key enzymes in arachidonate metabolism, are sensitive to [Ca2+]i, and the DAG liberated early in FcεRI signaling sets into motion a cascade of events that leads to a biphasic increase in [Ca2+]i. The first phase results from the liberation of intracellular stores of Ca2+ from the endoplasmic reticulum (ER) into the cytosol, with the second phase driven by an influx of extracellular Ca2+ into the cell. The ER has an IP3-sensitive Ca2+ channel that

mediates the first phase. Depletion of intracellular ER Ca2+ stores triggers the extracellular influx in a process whereby the ER membrane protein, STIM1, when it senses low Ca2+ levels, interacts with calciumrelease-activated (CRAC) Ca2+ channels leading to their opening. One of these channels is ORAI (Orai is the Greek mythological gatekeeper of heaven). This pathway of Ca2+ influx is shared by numerous other signaling pathways, and it is interesting that ORAI mutations have been found to give rise to a form of autosomal recessive severe combined immunodeficiency.22 Although a number of cellular enzymatic functions are regulated by [Ca2+]i, one of the key processes activated in mast cells and basophils after FcεRI signaling is granule exocytosis. Increases in [Ca2+]i alter the conformation of the SNARE (SNAP [soluble NSF attachment protein] receptor) family integral membrane proteins that restrain the granules tethered to the inside of the cell, allowing granule fusion with the plasma membrane and the expulsion of preformed mediators (histamine, heparin, serotonin, and mast cell proteases) into the extracellular space.

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Negative Feedback in FcεRI Signaling The mind-boggling intricacy of the activating pathways triggered by FcεRI signaling seems to be rivaled only by the complexity of the inhibitory mechanisms that are triggered in parallel. Analogous to the activating scaffold proteins that lead to the assembly of a supramolecular activation complex, FcεRI activation drives construction of a parallel regulatory complex. The downstream of kinase (DOK) family proteins assemble to recruit inhibitory elements including RASGAP (a Ras GTPase), SHIP1 (SH2 inositol 5’-phosphatase), and CSK (c-SRC tyrosine kinase), which phosphorylates the inhibitory/regulatory site on LYN and other SRCfamily kinases. The activity of CSK is balanced by the membrane-associate protein tyrosine phosphatase, CD45, which, upon cell activation, cleaves the negative regulatory phosphates from SRC-family kinases. In addition, the protein tyrosine phosphatases SHP-1 and SHP-2 act as negative regulators for early tyrosine phosphorylation events, apparently by targeting the ITAM phosphotyrosines of the FcεRI γ-chain.23

Antigen-Independent IgE-Mediated FcεRI Activation As described previously, the inciting event in FcεRI signaling has long been considered to be receptor aggregation driven by interaction of bound IgE antibodies with polyvalent allergens. Under this paradigm, an FcεRI occupied by IgE might be considered a “loaded gun” ready to fire but essentially inert before allergen encounter. Under some circumstances, however, the mere occupancy of FcεRI by IgE is sufficient to induce signaling. This has been best described in cultured rodent mast cells where exposure to purified monoclonal IgE antibodies triggers tyrosine phosphorylation of signaling intermediates. IgE antibodies that can stimulate cytokine release by mast cells and have been designated “cytokinergic” IgEs. A potential explanation for the apparent ability of some IgE antibodies to activate FcεRI independent of antigen encounter is suggested by the x-ray crystallographic solution of the structures of antigen–antibody complexes of one of the cytokinergic clones, SPE-7, which showed that the antigen-binding domains of the ε-heavy chain V-regions exist in an equilibrium and that although one binds tightly to the nominal antigen for which the IgE is known to be specific, others may have significant affinity for unrelated ubiquitously expressed autoantigens. Although a low-frequency interaction of an antibody binding site with an autoantigen might not result in biologic consequences for other antibody isotypes, the IgE:FcεRI system is so intensely bioamplified that this could account for the apparent antigen-independent signaling observed in many studies. Another possible explanation for antigen-independent effector cell activation by IgE antibodies such as SPE-7 is that homotypic interactions result in IgE multimer formation and that these multimers can cross-link FcεRI.24

IgE-Independent Immediate Hypersensitivity In allergy practice, demonstration of allergen-specific IgE is considered the sine qua non for diagnosis of immediate hypersensitivity. However, a number of historical and recent observations challenge the view that immediate hypersensitivity reactions are triggered exclusively by IgE. The earliest evidence for IgE-independent pathways of hypersensitivity came from observations by Prausnitz in Germany almost a century ago that “reagin,” a heat-stable component of the γ-globulin fraction of serum could passively transfer cutaneous sensitivity. This was in contrast to the classic reagin, subsequently identified as IgE, whose activity was destroyed by heating for 1 hour at 56° C. The heat-stable fraction was ultimately identified as IgG and, in contrast to IgE-mediated reactions, the IgG response was short in latency. IgG injected into the skin is not as tightly bound to FcγRs as IgE is to FcεRI, which has much greater affinity. Hence IgG diffuses away with resultant loss of sensitivity, whereas IgE is retained and its sensitizing effect persists for weeks.

Animal models have provided additional evidence for IgEindependent, IgG-mediated immediate hypersensitivity. Experiments using IgE-deficient (IgE-/-) mice generated by gene targeting showed definitively that active systemic anaphylaxis in rodents can arise by IgE-independent mechanisms.25 Further studies have shown that IgG1 can mediate anaphylaxis in mice, and that this can occur both via mast cells and via mast cell–independent pathways. IgG-mediated anaphylaxis is complement-independent but requires the presence of the IgG receptor FcγRIII. The interaction between IgG and FcγRIII is so much weaker than IgE and FcεRI that the concentrations of IgG and antigen required for IgE-independent anaphylaxis are not normally observed outside the laboratory, and so the presence of IgG-dependent anaphylaxis has never been definitively established in humans. Nevertheless, all the requisite effector mechanisms for IgG-mediated anaphylaxis exist in humans, and the presence of immediate allergic reactions in subjects in whom allergen-specific IgE cannot be detected by sensitive methods suggests that IgE-independent pathways might be operative. Efforts to define biomarkers distinguishing IgE- from IgG-mediated anaphylaxis are underway and could lead to a better understanding of the importance of IgG-mediated reactions in human allergic hypersensitivity reactions.26

IgE Levels Regulate FcεRI One of the most striking phenotypes initially observed in IgE “knockout” (IgE-/-) mice was the very low density of FcεRI present on their mast cells and basophils.27 Exposure of these cells to physiologic levels of IgE either in culture or by intravenous injection of the antibody in vivo rapidly restored strong surface expression of FcεRI. Binding of IgE to FcεRI stabilizes the receptor, preventing internalization and protease degradation.28 Thus as FcεRI continues to be synthesized within a cell, the presence of ambient IgE favors the capture and accumulation of FcεRI at the cell surface. The same phenomenon is observed in humans. Cell surface density of FcεRI tracks with IgE levels across a number of clinical conditions including atopy, parasite infestation, and hyper-IgE syndromes.29 Conversely, depletion of circulating free IgE by treatment with the anti-IgE therapeutic omalizumab results in decreased FcεRI levels on a number of cell types, including mast cells, basophils, and dendritic cells.30-32 As might be expected, this modulation of cellsurface FcεRI levels by anti-IgE has direct physiologic consequences. The allergen activation threshold for basophils with reduced FcεRI levels from omalizumab-treated subjects with dust mite allergy is significantly higher than for those from placebo-treated controls. Thus the efficacy of anti-IgE therapy results not only from the depletion of allergen-specific IgE but also from a significant reduction in FcεRI levels and consequently allergen sensitivity of the effector cells of immediate hypersensitivity.

IgE Antibodies and Mast Cell Homeostasis In addition to activating the release of preformed and newly synthesized mediators of allergy and regulating surface levels of FcεRI, IgE antibodies have been shown to modulate mast cell proliferation and survival. Rodent mast cells can be cultured from bone marrow using medium containing IL-3 and stem cell factor (SCF). The addition of IgE antibodies to such cultures accelerates the proliferation of mast cells. When cultured mast cells are deprived of their obligate growth factors, they undergo apoptosis.33 IgE antibodies can protect these cells from cell death by inducing the expression of antiapoptotic factors. Although these observations on cultured mast cells have been reproduced by many groups, their relevance to mast cell homeostasis in vivo has not yet been fully explored. One study, using a murine model of asthma induced by inhalation of the mold allergen Aspergillus fumigatus, showed that allergen-driven mast cell expansion in the airways is indeed

CHAPTER 24  Immunobiology of IgE and IgE Receptors dependent on the presence of IgE antibodies.34 Another investigation in mice infested with Trichinella spiralis showed that the parasite-induced intestinal mastocytosis was markedly attenuated in IgE-/- mice.4 Taken together these observations suggest a significant role for IgE–FcεRI interactions in regulating tissue levels of mast cells under conditions known to provoke mast cell expansion. Several reports have provided evidence that monomeric IgE antibodies have effects on human mast cells (either isolated from lung tissue or cultured from umbilical cord blood), including induction of cytokine and chemokine production,35,36 but effects on survival and/or proliferation have not been described. Thus the relevance of the findings regarding IgE effects on mast cell homeostasis in murine models of allergy and parasitic infection to human mast cell biology remains to be seen and will likely become clear as additional mechanistic studies emerge from trials of omalizumab.

CD23, THE LOW-AFFINITY IgE RECEPTOR Although its common designation as the “low-affinity” IgE receptor implies differently, CD23 in its monomeric form binds to IgE with a Ka of about 106 to 107 M-1, with its affinity increasing to 108 to 109 M-1 in its oligomeric form (compared with 1010 M-1 for FcεRI). CD23 has a very different structure and cellular expression pattern from FcεRI and mediates a distinct repertoire of biologic effector functions (Fig. 24.8).37 Unlike all the other immunoglobulin receptors, it is not a member of

the Ig superfamily. Instead it belongs to the C-type lectin family and is a type II transmembrane protein, meaning that its N-terminus is intracellular. CD23 is assembled as an oligomeric structure with each monomer consisting of a long extracellular α-helical coiled-coil stalk, which is abundantly N-glycosylated terminating in a globular head domain that binds to IgE. Only oligomeric CD23 will bind to IgE. CD23 has homology to receptors, including the asialoglycoprotein receptor, known to have functions in endocytosis. In addition to binding IgE, CD23 has a second ligand, the B cell surface molecule CD21. CD21 is a complement receptor (CR2) and also is the binding site for EpsteinBarr virus (EBV). The IgE-binding heads of CD23 can be cleaved from the protein by a variety of proteases, including some present in allergens (like the Der p 1 protease of dust mites) and the endogenous ADAM (A disintegrin and metalloproteinase) proteases, ADAM-8, -10, and -33. ADAM-10 appears to be the most important sCD23 enzyme in B cells.38 However, the high levels of expression of ADAM8 and ADAM33 in inflamed lung tissue suggest a possible role for these enzymes in CD23 cleavage in the setting of airway allergy. These proteases target several cleavage sites, giving rise to heterogeneous products retaining stalk remnants of varying lengths. The liberated CD23 heads, referred to as soluble CD23 (sCD23), retain their IgE-binding capacity. CD23 expression, like FcεRI, correlates with ambient IgE levels.39 Occupancy of the receptor by its ligand, IgE, protects it from shedding by inducing a conformational

Allergen CD23 structure

IgE Cε4 Lectin domain CD23

α-helix coiledcoiled stalk

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IgE B cell Regulation of IgE production

Fig. 24.8  CD23 structure and function. CD23 is a member of the C-type lectin family of proteins and is a type II transmembrane protein with an intracellular N-terminus. It is expressed as an oligomer of coiled stalks bearing lectin domain heads, which bind to the Cε3 and Cε4 domains of IgE. Protease sites in the stalks can be accessed by both endogenous (ADAM) and allergen (Der p 1) proteases to give rise to soluble CD23 fragments (sCD23), which retain their IgE-binding properties. Functions of CD23 are illustrated in the lower panels. CD23 serves to facilitate transepithelial allergen transport in gastrointestinal and respiratory epithelium. CD23 on B cells and APCs can mediate antigen uptake for more efficient processing and presentation of antigenic peptides complexed to MHC class II molecules to the TCR of specific T cells. CD23 regulates IgE production in B cells, with the transmembrane form suppressing IgE production (red arrows) and soluble CD23 enhancing IgE responses (green arrows), perhaps by coligating membrane IgE on IgE+ B cells with CD21.

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change affecting protease accessibility. Receptor expression is also stimulated by a number of cytokines (IL-4, IL-5, IL-13, GM-CSF), CD40L (CD154), and TLR ligands, including LPS. Alternative mRNA splice variants give rise to two isoforms of the protein, CD23a, which is present predominantly on B cells, and CD23b, which is expressed on a wide range of cells including monocytes, dendritic cells, Langerhans cells, and perhaps eosinophils as well as gastrointestinal and respiratory epithelium. A variety of functions have been attributed to membrane-bound and soluble CD23. These involve both facilitated uptake by various cell types and regulation of B cell function. One important pathway whereby CD23 can enhance antigen uptake at mucosal sites is by mediating the transport across the epithelium. In this mechanism, antigen-specific IgE antibodies bound to CD23 on the luminal surface of the gut epithelium promote antigen uptake into the cell. Antigen is then transported in endosomes to the basal surface, where it gains access to the APC and lymphocytes residing in the mucosa. CD23-mediated allergen transport in the gut is dependent on IL-4, and there is evidence that this mechanism of transport protects food allergens from degradation before their interaction with mucosal immune cells. CD23 appears to have regulatory influences on IgE synthesis and allergic inflammation. Engagement of the membrane form of CD23 on B cells suppresses IgE production. CD23 transgenic mice, over­ expressing the receptor on their B cells, have suppressed IgE responses and, conversely, B cells from CD23-/- mice exhibit enhanced IgE production.40 The increased IgE levels observed in CD23-/- mice are accompanied by the development of more severe airway inflammation and bronchial hyperresponsiveness in a murine asthma model.41 In contrast, there are some findings that suggest that sCD23, which has been proteolytically cleaved from the cell surface but retains its IgE binding properties, enhances IgE. It has been suggested that sCD23 might compete for IgE, preventing its suppressive effect on IgE production mediated by binding to transmembrane CD23. Alternatively, as CD23 is known to bind both IgE and CD21, it could be that the interaction of sCD23 mediated coligation of transmembrane IgE and CD21 on IgE+ B cells exerts an IgE-inducing effect. Reports on the ability of sCD23 to induce IgE production in B cells have been somewhat inconsistent, and it is possible that the exact conditions of proteolysis are important, with sCD23 fragments retaining greater stalk lengths (and hence greater ability to oligomerize) having the most activity. It is interesting that in a clinical trial of anti-CD23 in patients with asthma, a dose-dependent decrease in IgE levels was reported.42 Such a finding could be explained either by exertion of a suppressive effect by activating membrane CD23 on B cells and/or by blocking any activating effects exerted by sCD23.

IgE Receptors and Antigen-Presenting Cell Function Both FcεRI and CD23 are expressed on APC and can mediate the uptake of allergen bound to IgE.43 Although rodent APC are FcεRI-negative at baseline, human APC constitutively express FcεRI and can use the receptor to internalize IgE-bound allergens for processing and presentation to CD4+ T-helper cells. Studies of cells in the skin of patients affected by atopic dermatitis have revealed the presence of several FcεRI+ APC populations, including Langerhans cells and inflammatory dendritic epidermal cells (which unlike Langerhans cells do not contain Birbeck granules) in the epidermis and dermal dendritic cells, which comprise 2% to 5% of cells in the dermis. FcεRI is markedly upregulated on these cell types during allergic flares. Recently a murine transgenic model in which DC constitutively express human FcεRI was used to show that FcεRI+ DC can take up allergen and instruct Th cells to differentiate toward a Th2 phenotype.44 Basophils, which are strongly FcεRI positive, have also been implicated in murine studies as early IL-4 producers and Th2 inducers in allergic responses.45,46

Similarly, one of the best studied functions of CD23 is “facilitated antigen presentation,” whereby existing IgE antibodies, generated in response to a previous allergen encounter, can amplify Th2 responses upon reexposure to the same allergen. The process involves both B cells and CD11c+ dendritic cells (DC). Recent data suggest a process whereby allergen-specific IgE-allergen complexes, bound to B cells in the periphery via CD23, are transported to B cell follicles, where B cells both transfer antigen to resident DC and activate them for efficient antigen presenting function.47 Consistent with this model, exogenous IgE does not augment immune responses in CD23–/– mice, but will enhance humoral and cellular immunity after reconstitution with cells from CD23+ donors. The efficiency of IgE-mediated facilitated antigen presentation is related both to the diversity of the IgE repertoire for a specific allergen (the diversity of recognized epitopes) and to the avidity of the pooled IgE for antigen.48

RELATIONSHIPS WITH OTHER SYSTEMS Although IgE is perhaps best recognized for its dominant role in triggering immediate hypersensitivity reactions along with related functions in regulating expression of its receptors and mast cell homeostasis, there is growing evidence that IgE effects can cross over into the realms of innate and adaptive immune responses. IgE effects on innate antiviral responses were suggested by observations that virus-induced asthma exacerbations are reduced in subjects being treated with omalizumab, for instance in the Inner-City AntiIgE Therapy for Asthma49 trial. Although this may relate in part to the overall reduction in airway inflammation that results from IgE blockade, there is additional evidence pointing to a beneficial effect on the response of plasmacytoid dendritic cells (pDC) to viruses. Gill and others have demonstrated that cross-linking of IgE bound to pDC via FcεRI suppresses the TLR-mediated induction of type-I interferons (α and β).50 Clinically this has been shown to translate to decreased duration of rhinovirus infections, viral shedding, and risk of RV illnesses in asthmatics treated with omalizumab.51 These clinical and mechanistic observations of effects of IgE on viral immune responses indicate that the IgE-mediated process contributes to pathogenesis in atopic diseases in ways that extend beyond hypersensitivity and into the suppression of effector immune mechanisms. It has been speculated that suppression of innate viral immune responses may contribute to the susceptibility of patients with atopic dermatitis to cutaneous viral infections including herpes simplex (giving rise to “eczema herpeticum”) and molluscum contagiosum. Subjects who generate allergen-specific IgE responses often reencounter those allergens, for instance during seasonal pollen exposures in allergic rhinitis or after repeated ingestions in the setting of food allergy. Emerging evidence suggests that allergen–IgE interactions in these situations not only lead to mast cell– and basophil-mediated hypersensitivity reactions, but also promote the induction and consolidation of T helper 2 (Th2) responses while suppressing the induction of regulatory T cells. In animal models, the Treg effect extends to inducting a “pathogenic” Treg phenotype in which T cells with the classic FoxP3+ Treg phenotype begin to produce the proallergic cytokine IL-4, and by so doing contribute to local allergic responses rather than suppress them.52,53 Suppression of Th2 responses has similarly been reported in omalizumab-treated patients with atopic dermatitis.54 Such observations provide further rationale for the use of IgE blockade in clinical settings, suggesting that blocking IgE in the course of seasonal allergen exposures in asthma or during food ingestions in oral immunotherapy might help to suppress established Th2 responses and facilitate the emergence of suppressive Treg cells.

CHAPTER 24  Immunobiology of IgE and IgE Receptors

CONCLUSION IgE is unique among immunoglobulin isotypes by virtue of possessing an extra Fc domain. Residing primarily in tissues bound to cell surface receptors, it is capable of using the extremely sensitive FcεRI highaffinity receptor and its bioamplified signaling pathways to elicit intense hypersensitivity reactions to minute amounts of antigen. At the same time, it serves to regulate the induction of immune responses to antigens at mucosal surfaces, using the low-affinity receptor, CD23, to facilitate their transepithelial transport and their uptake and processing by APC. The coevolution of IgE antibodies with its receptors, as well as several committed effector cell lineages responding to IgE with distinct functional programs, tell us that this intricate and tightly orchestrated system confers a selective advantage. It is likely that IgE may have developed to control host–parasite interactions. However, studies performed over the past decade have made it increasingly clear that in addition to activating mechanisms of host defense, IgE antibodies regulate immune homeostasis at a number of levels, modulating expression of their own receptors, enhancing antigen uptake and presentation, and sustaining mast cell survival in mucosal tissues. As the use of anti-IgE therapies continues to expand, there is no doubt that mechanistic studies will provide additional insights on IgE biology in the years to come.

REFERENCES Introduction: Discovery of IgE, Function in Parasitic Immunity, Sites of Production 1. Platts-Mills TA, Heymann PW, Commins SP, et al. The discovery of IgE 50 years later. Ann Allergy Asthma Immunol 2016;116(3):179–82. 2. Joseph M, Auriault C, Capron A, et al. A new function for platelets: IgE-dependent killing of schistosomes. Nature 1983;303(5920):810–12. 3. Gounni AS, Lamkhioued B, Ochiai K, et al. High-affinity IgE receptor on eosinophils is involved in defence against parasites. Nature 1994;367(6459):183–6. 4. Gurish MF, Bryce PJ, Tao H, et al. IgE enhances parasite clearance and regulates mast cell responses in mice infected with Trichinella spiralis. J Immunol 2004;172(2):1139–45.

IgE Structure and Mechanisms of IgE Isotype Switching and IgE Memory 5. Shade KT, Platzer B, Washburn N, et al. A single glycan on IgE is indispensable for initiation of anaphylaxis. J Exp Med 2015;212(4):457–67. 6. Methot SP, Di Noia JM. Molecular mechanisms of somatic hypermutation and class switch recombination. Adv Immunol 2017;133:37–87. 7. Chatila TA. Interleukin-4 receptor signaling pathways in asthma pathogenesis. Trends Mol Med 2004;10(10):493–9. 8. Bottaro A, Lansford R, Xu L, et al. S region transcription per se promotes basal IgE class switch recombination but additional factors regulate the efficiency of the process. EMBO J 1994;13(3):665–74. 9. Xu Z, Zan H, Pone EJ, et al. Immunoglobulin class-switch DNA recombination: induction, targeting and beyond. Nat Rev Immunol 2012;12(7):517–31. 10. Boboila C, Alt FW, Schwer B. Classical and alternative end-joining pathways for repair of lymphocyte-specific and general DNA double-strand breaks. Adv Immunol 2012;116:1–49. 11. Cameron L, Gounni AS, Frenkiel S, et al. S epsilon S mu and S epsilon S gamma switch circles in human nasal mucosa following ex vivo allergen challenge: evidence for direct as well as sequential class switch recombination. J Immunol 2003;171(7):3816–22. 12. Morita R, Schmitt N, Bentebibel SE, et al. Human blood CXCR5(+) CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 2011;34(1):108–21.

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13. Yang Z, Sullivan BM, Allen CD. Fluorescent in vivo detection reveals that IgE(+) B cells are restrained by an intrinsic cell fate predisposition. Immunity 2012;36(5):857–72. 14. Laffleur B, Duchez S, Tarte K, et al. Self-restrained B cells arise following membrane IgE expression. Cell Rep 2015;10(6):900–909. 15. Haniuda K, Fukao S, Kodama T, et al. Autonomous membrane IgE signaling prevents IgE-memory formation. Nat Immunol 2016;17(9):1109–17. 16. Tong P, Granato A, Zuo T, et al. IgH isotype-specific B cell receptor expression influences B cell fate. Proc Natl Acad Sci USA 2017;114(40):E8411–20. 17. Xiong H, Dolpady J, Wabl M, et al. Sequential class switching is required for the generation of high affinity IgE antibodies. J Exp Med 2012;209(2):353–64. 18. Looney TJ, Lee JY, Roskin KM, et al. Human B-cell isotype switching origins of IgE. J Allergy Clin Immunol 2016;137(2):579–86.e7. 19. McCoy KD, Harris NL, Diener P, et al. Natural IgE production in the absence of MHC class II cognate help. Immunity 2006;24(3):329–39.

FcεRI, the High-Affinity IgE Receptor 20. Dema B, Suzuki R, Rivera J. Rethinking the role of immunoglobulin E and its high-affinity receptor: new insights into allergy and beyond. Int Arch Allergy Immunol 2014;164(4):271–9. 21. Gilfillan AM, Beaven MA. Regulation of mast cell responses in health and disease. Crit Rev Immunol 2011;31(6):475–529. 22. Feske S, Wulff H, Skolnik EY. Ion channels in innate and adaptive immunity. Annu Rev Immunol 2015;33:291–353. 23. Zhu Z, Oh SY, Cho YS, et al. Tyrosine phosphatase SHP-1 in allergic and anaphylactic inflammation. Immunol Res 2010;47(1–3):3–13. 24. Bax HJ, Bowen H, Beavil RL, et al. IgE trimers drive SPE-7 cytokinergic activity. Sci Rep 2017;7(1):8164. 25. Oettgen HC, Martin TR, Wynshaw-Boris A, et al. Active anaphylaxis in IgE-deficient mice. Nature 1994;370(6488):367–70. 26. Finkelman FD, Khodoun MV, Strait R. Human IgE-independent systemic anaphylaxis. J Allergy Clin Immunol 2016;137(6):1674–80. 27. Yamaguchi M, Lantz CS, Oettgen HC, et al. IgE enhances mouse mast cell Fc(epsilon)RI expression in vitro and in vivo: evidence for a novel amplification mechanism in IgE-dependent reactions. J Exp Med 1997;185(4):663–72. 28. Borkowski TA, Jouvin MH, Lin SY, et al. Minimal requirements for IgE-mediated regulation of surface FC epsilon RI. J Immunol 2001;167(3):1290–6. 29. Saini SS, Klion AD, Holland SM, et al. The relationship between serum IgE and surface levels of FcepsilonR on human leukocytes in various diseases: correlation of expression with FcepsilonRI on basophils but not on monocytes or eosinophils. J Allergy Clin Immunol 2000;106(3):514–20. 30. Beck LA, Marcotte GV, MacGlashan D, et al. Omalizumab-induced reductions in mast cell Fcepsilon RI expression and function. J Allergy Clin Immunol 2004;114(3):527–30. 31. MacGlashan DW Jr, 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 1997;158(3):1438–45. 32. Prussin C, Griffith DT, Boesel KM, et al. Omalizumab treatment downregulates dendritic cell FcepsilonRI expression. J Allergy Clin Immunol 2003;112(6):1147–54. 33. Kawakami T, Kitaura J. Mast cell survival and activation by IgE in the absence of antigen: a consideration of the biologic mechanisms and relevance. J Immunol 2005;175(7):4167–73. 34. Mathias CB, Freyschmidt EJ, Caplan B, et al. IgE influences the number and function of mature mast cells, but not progenitor recruitment in allergic pulmonary inflammation. J Immunol 2009;182(4):2416–24. 35. Cruse G, Kaur D, Yang W, et al. Activation of human lung mast cells by monomeric immunoglobulin E. Eur Respir J 2005;25(5):858–63. 36. Matsuda K, Piliponsky AM, Iikura M, et al. Monomeric IgE enhances human mast cell chemokine production: IL-4 augments and dexamethasone suppresses the response. J Allergy Clin Immunol 2005;116(6):1357–63.

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CD23, the Low-Affinity IgE Receptor 37. Sutton BJ, Davies AM. Structure and dynamics of IgE-receptor interactions: FcepsilonRI and CD23/FcepsilonRII. Immunol Rev 2015;268(1):222–35. 38. Gibb DR, El Shikh M, Kang DJ, et al. ADAM10 is essential for Notch2-dependent marginal zone B cell development and CD23 cleavage in vivo. J Exp Med 2010;207(3):623–35. 39. Selb R, Eckl-Dorna J, Neunkirchner A, et al. CD23 surface density on B cells is associated with IgE levels and determines IgE-facilitated allergen uptake, as well as activation of allergen-specific T cells. J Allergy Clin Immunol 2017;139(1):290–9.e4. 40. Payet ME, Woodward EC, Conrad DH. Humoral response suppression observed with CD23 transgenics. J Immunol 1999;163(1):217–23. 41. Haczku A, Takeda K, Hamelmann E, et al. CD23 deficient mice develop allergic airway hyperresponsiveness following sensitization with ovalbumin. Am J Respir Crit Care Med 1997;156(6):1945–55. 42. Rosenwasser LJ, Busse WW, Lizambri RG, et al. Allergic asthma and an anti-CD23 mAb (IDEC-152): results of a phase I, single-dose, dose-escalating clinical trial. J Allergy Clin Immunol 2003;112(3):563–70. 43. Rosenwasser LJ. Mechanisms of IgE inflammation. Curr Allergy Asthma Rep 2011;11(2):178–83. 44. Sallmann E, Reininger B, Brandt S, et al. High-affinity IgE receptors on dendritic cells exacerbate Th2-dependent inflammation. J Immunol 2011;187(1):164–71. 45. Sokol CL, Chu NQ, Yu S, et al. Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response. Nat Immunol 2009;10(7):713–20. 46. Yoshimoto T, Yasuda K, Tanaka H, et al. Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells. Nat Immunol 2009;10(7):706–12.

47. Engeroff P, Fellmann M, Yerly D, et al. A novel recycling mechanism of native IgE-antigen complexes in human B cells facilitates transfer of antigen to dendritic cells for antigen presentation. J Allergy Clin Immunol 2018;142(2):557–68.e6. 48. Holm J, Willumsen N, Wurtzen PA, et al. Facilitated antigen presentation and its inhibition by blocking IgG antibodies depends on IgE repertoire complexity. J Allergy Clin Immunol 2011;127(4):1029–37.

Relationships Between IgE and Other Systems 49. Busse WW, Morgan WJ, Gergen PJ, et al. Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children. N Engl J Med 2011;364(11):1005–15. 50. Durrani SR, Montville DJ, Pratt AS, et al. Innate immune responses to rhinovirus are reduced by the high-affinity IgE receptor in allergic asthmatic children. J Allergy Clin Immunol 2012;130(2):489–95. 51. Esquivel A, Busse WW, Calatroni A, et al. Effects of omalizumab on rhinovirus infections, illnesses, and exacerbations of asthma. Am J Respir Crit Care Med 2017;196(8):985–92. 52. Burton OT, Noval Rivas M, Zhou JS, et al. Signal inhibition during allergen ingestion leads to reversal of established food allergy and induction of regulatory T cells. Immunity 2014;41(1):141–51. 53. Noval Rivas M, Burton OT, Wise P, et al. Regulatory T cell reprogramming toward a Th2-cell-like lineage impairs oral tolerance and promotes food allergy. Immunity 2015;42(3):512–23. 54. Iyengar SR, Hoyte EG, Loza A, et al. Immunologic effects of omalizumab in children with severe refractory atopic dermatitis: a randomized, placebo-controlled clinical trial. Int Arch Allergy Immunol 2013;162(1):89–93.

CHAPTER 24  Immunobiology of IgE and IgE Receptors

374.e1

SELF-ASSESSMENT QUESTIONS 1. An immunologist in the laboratory adds IL-4 to a suspension of highly purified B cells in an effort to induce IgE production but is surprised to find no IgE in the culture supernatant 8 days later. How could she get this to work better next time? a. She should add IL-13, which is a better inducer of IgE production. b. Including antibiotics in the medium would prevent bacterial contamination and give a better result. c. Adding some activated T cells would help push the B cells into IgE production. d. Antigen-presenting cells should be included in the culture to get the B cells to switch. 2. Cross-linking of the high-affinity IgE receptor, FcεRI, by IgE binding to polyvalent allergens initiates a series of intracellular signaling events. Which of the following statements is correct with regard to those signaling pathways? a. FcεRI is a multisubunit ion channel that, upon ligand binding, allows Ca2+ ions to flow into mast cells or basophils.

b. Activation of the receptor leads to phosphorylation of ITAM motifs in the b and g chains with subsequent recruitment of the signaling kinase, ZAP-70. c. Constitutively receptor-associated kinases, including LYN, are important both in initiating signaling and in bringing it under control. d. In the presence of its ligand, IgE, levels of FcεRI are downregulated on the cell surface. 3. IgE antibodies affect antiviral immune responses in which of the following ways? a. IgE, bound to viruses, activates complement, leading to opsonization for phagocytosis by macrophages. b. IgE, signaling via FceRI, suppresses the production of type I interferons by plasmacytoid dendritic cells. c. IgE antibodies bound to viral coat proteins prevents viral binding to target cells. d. IgE-mediated mast cell activation induces the production of peptides that disrupt the viral coat proteins.

25  Neuronal Control of Airway Function in Allergy Bradley J. Undem, Brendan J. Canning

CONTENTS Introduction, 375 Lower Airway Innervation, 375 Allergen-Induced Airway Neuromodulation, 379

SUMMARY OF IMPORTANT CONCEPTS • Symptoms of allergic airway disease, such as sneezing, rhinorrhea, unproductive coughing, episodic bronchospasm, and sensations of breathlessness, are neuronally mediated in part or in total. • The vagus nerves supply the lower respiratory tract with the vast majority of its afferent (sensory) innervation and all its preganglionic parasympathetic innervation. The innervation from spinal afferent nerves or sympathetic nerves is relatively sparse by comparison. • The afferent nerves provide the communication pathway between the lungs and central nervous system. Their activity initiates protective and autonomic reflexes, provides some control over the rate and depth of breathing, and can cause or contribute to the sensation of urge to cough or dyspnea. • The parasympathetic branch of the autonomic nervous system controls airway smooth muscle tone through cholinergic contractile innervation and noncholinergic relaxant innervation. The autonomic nervous system (ANS) also regulates glandular secretion and blood flow through the bronchial and pulmonary vasculature. • Allergic inflammation can modulate airway innervation by stimulating or influencing the excitability of primary afferent terminals, altering gene expression in sensory ganglion neurons, increasing synaptic transmission within the central nervous system (CNS) and autonomic ganglia, and increasing transmitter secretion at the level of the nerve-effector junction.

Clinical Allergy and the Neural Hypersensitive State, 382 Conclusion, 383

released during immune reactions can act on the nervous system to modulate its activity. These nerve-immune interactions can be beneficial to the host. Experimentally interrupting these interactions can be detrimental; in an animal model, chemical denervation of airway C fiber sensory nerves substantially decreases the host’s ability to clear Mycoplasma infection of the airways.1 Nerve-immune interactions can also be inappropriate and deleterious, as with allergy; the immune response triggered by allergen exposure can recruit the nervous system in a way that is not beneficial to the host and causes or exacerbates the symptoms of allergic disease: irritation, pruritus, sneezing, coughing, hypersecretion, reversible bronchospasm, and dyspnea. In patients with asthma only poorly controlled with an inhaled corticosteroid, simply blocking neuronally mediated bronchoconstriction with tiotropium, a pseudo-irreversible anticholinergic drug, is as effective as the β-adrenoceptor agonist salmeterol in controlling symptoms and is arguably more effective than salmeterol in improving lung function.2 This chapter provides an overview of the neurophysiology of the lower airway wall and a review of the mechanisms by which this neuro­ physiology is altered during allergic reactions. Although this discussion focuses on the lower airways, the fundamental principles extend to upper airways and other organs in which allergic reactions take place.

LOWER AIRWAY INNERVATION Extrinsic Innervation

INTRODUCTION Both the immune system and the nervous system are critical to host defense within 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 two systems act in a complementary, non­ redundant manner; however, they can be viewed as integrated systems (in addition to the endocrine system). Immune tissues are directly innervated by the autonomic nervous system (ANS) and provide pathways for the brain to influence immune function. Stimulation of nerves can also indirectly augment immune function by stimulating inflammation in so-called neurogenic inflammatory reactions. Mediators

Vagus Nerves.  The airways and lungs are innervated bilaterally by the vagus nerves (cranial nerve X). The vagi are mixed nerves, and the majority of vagal fibers are afferent (or sensory) in nature.3 Vagal afferent nerve fibers have their cell bodies in one of two ganglia: the jugular (superior vagal) or nodose (inferior vagal) ganglia.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.6 The remaining vagal nerves are preganglionic parasympathetic nerves, which innervate parasympathetic ganglia, and motor nerve fibers, which innervate 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) 375

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and the nucleus ambiguus (nA). Although these brainstem structures have viscerotropic organization, the sites of afferent nerve subtype termination and efferent projection overlap considerably. This overlap contributes in part to the nonselective clustering of autonomic reflexes (e.g., effects on heart rate, respiratory pattern, and airway caliber) initiated by selective activation of specific afferent nerve subtypes.7

Spinal Nerves.  The majority of postganglionic sympathetic nerves projecting to the airways arise bilaterally from the superior cervical ganglia and the stellate ganglia.4 Spinal afferent nerves also project to the airways. Studies in animals suggest that spinal afferent nerves regulate airway sympathetic reflexes and perhaps respiratory pattern.8,9 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. There is no evidence that postganglionic sympathetic nerves have contralateral projections.

Intrinsic Innervation Afferent and efferent nerve fibers occupy multiple nerve plexuses in the airway wall from the larynx to the terminal bronchioles.10,11 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 innervation of the epithelium has been described.12 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 (Fig. 25.1).13,14

Airway parasympathetic ganglia occupy a serosal nerve plexus of the extrapulmonary airways, a plexus that merges with the lamina propria plexus in the intrapulmonary airways. Occasionally, ganglia may also be found elsewhere in the airway wall. Parasympathetic ganglia containing as few as 1 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. Airway afferent nerves form receptive fields, often nonspecialized, 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.15 Afferent nerve fibers may also innervate other effector tissues in the airway wall, including glands, airway smooth muscle, blood vessels, and airway parasympathetic ganglia. Postganglionic autonomic nerves innervate structures throughout the airway wall, including glands, blood vessels, airway smooth muscle, and adjacent airway parasympathetic ganglia.16 Morphologic analyses reveal little change in nerve fiber densities in the smooth muscle from the large bronchi to the bronchioles.17

Airway Parasympathetic Ganglia.  Postganglionic parasympathetic nerve terminals are found throughout the airways. Parasympathetic ganglia, however, are few in number and contain only a handful of neurons. Airway parasympathetic tone is thus determined by the actions of relatively few ganglia neurons in fewer still ganglia.18 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 serve an important integrative role (Fig. 25.2, A). This function is facilitated by the complex morphology of the ganglia neurons and by the many biophysical properties of the neurons that facilitate integration of synaptic input.18,19 Synaptic transmission between preganglionic and postganglionic parasympathetic nerves in the bronchi is mediated primarily, if not exclusively, by acetylcholine acting on nicotinic receptors.18 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 generation20 (Fig. 25.2, B and C). The filtering capacity of airway ganglia neurons can be modulated by a number of mechanisms, either through modulatory effects of non­ cholinergic 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.18 Airway ganglia neurons may also innervate adjacent ganglia neurons.21 Collaterals of afferent nerves containing neuropeptides are also found in airway ganglia and can regulate synaptic transmission in the ganglia through peripheral reflexes.22,23

Reflex Regulation of Airways

Fig. 25.1  Fluorescence photomicrograph of confocal microscope image stacks showing dense neural network in a biopsy specimen from an asthmatic airway. (Nerves stained with protein gene product 9.5; calibration bar: 50 µm.) (From Goldie RG, Fernandes L, Rigby P. Airway nerves: detection and visualisation. Curr Opin Pharmacol 2002;2:273–7.)

Afferent Nerve Subtypes.  Multiple afferent nerve subtypes innervate the airways; a comprehensive review of airway afferent nerves has recently been written.24 These nerve subtypes can be subclassified based on their neurochemistry, responsiveness to physical and chemical stimuli, myelination, conduction velocity, sites of termination in the CNS, and ganglionic origin.3,25–27 Airway low-threshold mechanoreceptors have at least two

CHAPTER 25  Neuronal Control of Airway Function in Allergy

377

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 fibers, are generally unresponsive to mechanical stimuli and are thus essentially quiescent during tidal breathing33 (Fig. 25.3). These nociceptors, however, are activated by myriad inflammatory mediators and an acidic environment that may accompany allergic reactions.25 When activated, airway nociceptors initiate increases in airway parasympathetic drive, sensation of urge to cough, and dyspnea.34

Autonomic Nerve Subtypes.  Both sympathetic and parasympathetic A

10 mV 10 ms

B

nerves innervate the airways. Sympathetic nerves primarily innervate the bronchial vasculature, whereas airway parasympathetic nerves innervate the vasculature but also the glands and airway smooth muscle.16 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, autonomic control is much more complex. 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.16

Autonomic Regulation of Airway Smooth Muscle Tone.  The 10 mV 10 ms

C Fig. 25.2  (A) Drawing of single neuron injected with Neurobiotin and processed for peroxidase histochemistry; asterisk (*) indicates axon (calibration bar: 20 µm). (B) Fast excitatory postsynaptic potentials (fEPSPs) in human bronchial ganglia. Overlay traces show responses by human bronchial ganglia neuron to 10 consecutive peribronchial nerve stimulations (shock artifact at vertical arrow; stimulus = 1.0 ms, 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. The integrative membrane properties of human bronchial parasympathetic ganglia neurons. Am J Respir Crit Care Med 2001;164:1927–32.)

subtypes: rapidly adapting receptors (RARs), which respond to the dynamic physical effects of lung inflation, and slowly adapting receptors (SARs), which respond to the sustained physical effects of lung inflation (see Allergic Modulation of Afferent Nerves). Some airway mechanoreceptors can also be activated indirectly by bronchoconstrictors, such as histamine, acetylcholine, and leukotrienes.28,29 When activated, airway mechanoreceptors may initiate alterations in autonomic nerve activity and cough and may play an integral role in controlling respiratory rate and tidal volume.3,30 Therefore it is surprising that many airway mechanoreceptors are active during the respiratory cycle31 (Fig. 25.3). This continuous activity of airway mechanoreceptors may be of fundamental importance to maintaining baseline autonomic tone and respiratory pattern and to how subsequent reflexes proceed.30,32

autonomic control of airway function has been comprehensively reviewed.16 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.35 Although other species have a relative paucity of sympathetic innervation of the intrapulmonary airways, human airway smooth muscle expresses abundant beta adrenoceptors, primarily β2-adrenoceptors. It seems likely, therefore, that hormonal catecholamines are the primary ligand for the β-adrenoceptors 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 terminus and methionine at C terminus (PHM, human form of PHI, with isoleucine at C terminus), as well as the gaseous transmitter nitric oxide (NO, synthesized from arginine by neuronal NO synthase). These nonadrenergic, noncholinergic (NANC) relaxant responses can be evoked in airways from the trachea to the small bronchi.36,37 It has been assumed that the neurotransmitters mediating NANC relaxation of airway smooth muscle are coreleased with acetylcholine (ACh) from a single population of postganglionic parasympathetic nerves. It was further hypothesized that the function of these neurotransmitters was to prevent excessive constriction during periods of elevated parasympathetic nerve activity. Studies in guinea pigs, however, reveal that at least in this species, noncholinergic parasympathetic neuro­ transmitters are not necessarily coreleased with ACh from postganglionic parasympathetic nerves. Rather, data from studies with several laboratory animals support the hypothesis that an entirely distinct parasympathetic pathway regulates noncholinergic nerve activity in the airways.38–41 Separate cholinergic and noncholinergic parasympathetic ganglion neurons have also been identified in human airways.42

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SECTION A  Basic Sciences Underlying Allergy and Immunology Cap

Cap

10 0 150 0

AP

ABP Pt (mm Hg) (cmH2O)

AP

Cap

ABP (mm Hg)

Pt (cmH2O)

10

0 150 10 s

0

A

B

C

Fig. 25.3  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. ABP, Arterial blood pressure. (A) Pulmonary C fiber arising from an ending in right upper lobe of anesthetized, open-chest rat (conduction velocity of fiber = 1.05 m/s). 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/s). 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/s). 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 H2O for 10 seconds while the respirator was turned off. (From Ho CY, Gu Q, Lin YS, et al. Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respir Physiol 2001;127:113–24.)

Many stimuli initiate reflex alterations in airway parasympathetic nerve activity.36 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 histamine, prostaglandin D2 (PGD2), leukotrienes, and even methacholine initiate both cough and reflex bronchospasm.43 Vagal C fiber stimulants such as capsaicin, bradykinin, hypertonic saline, and acidic solutions also initiate reflex bronchospasm.27 Other stimuli initiating reflex bronchospasm include chemoreceptor stimulation, esophageal afferent nerve stimulation, and nasal airway afferent nerve stimulation.44,45 Stimuli initiating reflex parasympathetic bronchodilation include stimulants that activate airway mechanoreceptors and airway nociceptors.43 By contrast, chemoreceptor stimulation has no apparent effect on noncholinergic parasympathetic nerve activity.46,47 Activation of skeletal muscle afferent nerves, as might occur during exercise, also initiates reflex bronchodilation, primarily through withdrawal of baseline cholinergic tone.48 Direct evidence for airway sympathetic reflexes following spinal or vagal afferent nerve activation has been reported in guinea pigs.8 β-Adrenoceptor antagonists can lead to bronchoconstriction in laboratory animals and in humans with reactive airways disease, implicating endogenous catecholamines as regulators of airway caliber and responsiveness. As discussed earlier, circulating catecholamines are likely more

important in regulating human airway function than the sparse sympathetic adrenergic innervation of the human airways.

Autonomic Regulation of Glands.  Airway glands are regulated primarily by the parasympathetic nervous system. Acetylcholine is the primary neurotransmitter regulating airway glandular secretion, but other peptide neurotransmitters (e.g., VIP) may also play a role in mucus secretion.16,49 Sympathetic nerves play relatively little or no role in mucus secretion. Reflexes initiating parasympathetic nerve–dependent mucus secretion are induced by many of the same stimuli that initiate reflex bronchospasm. Therefore 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, however, that the neurochemistry of the postganglionic parasympathetic nerves differs depending on whether glands or airway smooth muscles are innervated. Autonomic Regulation of Bronchial Vasculature.  Sympathetic and parasympathetic nerves regulate bronchial vascular tone.16,50,51 Sympathetic nerves mediate vasoconstriction through the actions of norepinephrine and neuropeptide Y, whereas parasympathetic nerves mediate vasodilation through the actions of ACh, NO, and perhaps peptides (e.g., VIP). Reflex regulation of bronchial vascular tone is poorly

CHAPTER 25  Neuronal Control of Airway Function in Allergy described, largely because of the difficulty in studying the bronchial vasculature. Airway nociceptor stimulation, however, is known to initiate parasympathetic reflex dilation of the bronchial vasculature.52

Axon Reflexes.  Activation of some sensory nerves, primarily nociceptors, causes the release of proinflammatory transmitters, including substance P and perhaps adenosine triphosphate (ATP), from their peripheral endings, and these transmitters act on the tissue of innervation to initiate neurogenic inflammation.53 This peripheral release of neurotransmitters from sensory nerve collaterals and the resulting endorgan effects is the axon reflex. In most species, including humans, afferent nerves innervating the airway mucosa 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 and initiate bronchospasm, mucus secretion, vasodilation, plasma exudation, and inflammatory cell recruitment. These observations have led to the hypothesis that axonal reflexes contribute to the pathogenesis of inflammatory airway disease.54 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.45 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, but this is likely a nonneural effect.55 Bronchospasm initiated by inhalation of putative C fiber stimulants in human subjects (but not in rats or guinea pigs) is caused primarily by CNS-dependent parasympathetic reflexes.43

Cough and Dyspnea.  Cough plays an essential role in clearing inhaled pathogens, aeroallergens, irritants, particulate matter, secretions, and aspirate and thus protects the 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 airway nociceptors, which are activated by irritants such as acid (including aspirate), bradykinin, and capsaicin.43 Dyspnea is associated with airway obstruction and other respiratory reflexes and sensations, but the relationships are complex. The exact mechanisms underlying the sensation of dyspnea are unclear but likely involve the afferent innervation of the airways and lungs.56 Much of our understanding of this respiratory sensation comes from clinical studies. In general, more severe asthma (based on basal percentage predicted FEV1) is associated with a blunted sensation of dyspnea or airway obstruction.57 Allergic inflammation and mediators associated with allergic inflammation may, however, acutely alter the sensations of dyspnea. Doses of histamine and methacholine that are equally effective at inducing airway obstruction produce differing levels of dyspnea, with histamine producing more symptoms than methacholine.58 These results imply that histamine, but not methacholine, has a direct effect on airway sensory nerve activity and excitability. In healthy adults, prostaglandin E2 (PGE2) inhalation does not induce dyspnea or airways obstruction but worsens the sensations of dyspnea associated with exercise; these observations are consistent with the belief that mediators of allergic inflammation enhance the excitability of vagal afferent nerves

379

and trigger the sensation of dyspnea.59 Intravenous adenosine also causes dyspnea but does not induce airway obstruction in nonasthmatic patients.60 Inhalation of ATP (and to a lesser extent AMP) induces coughing, dyspnea, and airway obstruction in patients with asthma but does not induce dyspnea or airway obstruction in nonasthmatic individuals.61 Recent clinical studies performed in patients with chronic, refractory cough suggest that ATP may be a primary regulator of cough via activation of P2X3 or P2X2,3 receptors.62

ALLERGEN-INDUCED AIRWAY NEUROMODULATION The symptoms of allergic disease are largely the result of altered neuronal activity. The nervous system serves as the principal transducer between immunologic aspects of allergic inflammation and the symptomatology of immediate hypersensitivity, which can include pruritus (itchy airways), excessive coughing, eye irritation, gastrointestinal irritation, runny nose, or the rapid and reversible bronchospasm that leads to dyspneic sensations.63 Symptoms likely occur because 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-immune-nerve interactions, but the mediators likely include histamine, arachidonic acid metabolites, tryptase, neurotrophins, chemokines, and cytokines.34,64–68 Reflexes can be conceptually subdivided into four components (Fig. 25.4). First, a 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 influences reflex physiology in the airways by modifying each of the four steps in the pathway of reflex action.

Allergic Modulation of Afferent Nerves Many airway afferent nerves are sensitive to mechanical perturbation. Allergic reactions can lead indirectly to mechanosensitive afferent nerve activation by releasing mediators that cause bronchial smooth muscle contraction. For example, histamine leads to activation of afferent RARs and SARs by a mechanism that can be inhibited by bronchodilators.25 This finding may explain the AP discharge in RARs observed after allergen challenge in rabbit airways.69 In various experimental models of allergy, allergen provocation can also directly influence the activity of afferent nerve fibers. With nociceptive-like fibers, chemical mediators such as histamine, leukotriene D4 (LTD4), serotonin (5-hydroxytryptamine), tryptase, bradykinin, PGD2, and PGE2 metabolites directly evoke AP discharge or increase their electrical excitability to other activating stimuli to cause allergic reactions in airways.25,70,71 Fig. 25.5, A, shows an example of allergen-induced activation of an airway vagal C fiber. The direct activation of vagal C fibers by inflammatory mediators often involves the gating of transient receptor potential (TRP) channels, in particular TRPV1 and TRPA1.25,71 An increase in excitability is also illustrated (Fig. 25.5, B), in which allergen challenge did not overtly evoke AP discharge in specialized cough receptor A fiber nerves in the trachea but did cause a fourfold decrease in the mechanical force required to activate the nerve fibers. Mediators

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(3)

(2)

CNS

Sensory ganglion

Autonomic ganglion

(4)

(1) Airways

(5)

Fig. 25.4  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 and lead 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 brainstem, 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 parasympathetic transmitters vasoactive intestinal peptide and nitric oxide. CNS, Central nervous system.

associated with allergic reactions can also increase airway C fiber afferent nerve responsiveness to mechanical stimulation in vivo.72 This may lead to a scenario in which the normally quiescent nociceptive C fibers become responsive to the tissue distention occurring during simple eupneic respiration. Regardless of the mediators and ionic mechanisms involved, an increase in AP-discharge vagal nociceptors in the airways will lead to increases in parasympathetic secretion, bronchoconstriction, and sensations such as urge to cough. In addition to causing an increase in AP discharge, allergic reactions can also modify neuropeptide release from the peripheral nerve terminals of nociceptive-type afferent nerves. As discussed earlier, inflammationstimulating neuropeptides may also be released from peripheral terminals as a consequence of axon reflexes. Allergen provocation can augment this process by stimulating vagal C fibers and by increasing the amount of neuropeptide secretion per impulse.73 This effect occurs at very low concentrations of antigen and persists for more than 2 hours. Histamine and cysteinyl leukotrienes (cysLTs) are the mediators responsible for these effects. In fact, cysLTs are more potent at affecting sensory

neuropeptide secretion than contracting airway smooth muscle. Both histamine and cysLTs interact directly with sensory nerves by decreasing resting potassium currents, which leads to membrane depolarizations.68 Substance P and related sensory neuropeptides are metabolized in the airways by neutral endopeptidase. Allergic inflammation may also increase neurogenic inflammatory reactions by decreasing airway neutral endopeptidase activity.74 Either by overtly activating or by increasing excitability, mediators associated with the allergic reaction in the airways will lead to increases in the number and frequency of APs arising in the CNS from nerve terminals in the respiratory tract. This quantitative increase likely explains many of the acute symptoms of the allergic response (sneezing, coughing, reflex bronchoconstriction, secretion). The allergic reaction can also lead to changes in gene expression in the afferent neurons. This neuroplasticity can qualitatively change the communication between the airways and brainstem, an effect that may outlast the acute inflammatory response. In some patients the allergic response leads to a phenotypic switch in the afferent neurons such that low-threshold mechanosensitive A fibers (responsive to mechanical effects of respiration) take on characteristics of nociceptive C fibers with respect to both neuropeptide production and ion channel expression.75–77

Allergen and Central Nervous System Integration Increased activity of primary afferent nerves results in increased activity of the secondary neurons to which the primary nerves project. Because most vagal afferent nerves project to neurons in the nucleus tractus solitarius, allergen-induced activation of vagal afferent nerves will lead to increases in the activity of neurons within the nTS.78 In various pain syndromes, peripheral inflammation not only leads to synaptic activation of secondary neurons but also can lead to increased efficacy of synaptic transmission such that neurotransmission is magnified within the spinal cord. This process is referred to as central sensitization.79 Given the similarities between visceral hyperreflexia and somatosensory hyperalgesia, allergic inflammation may also lead to central sensitization. Evidence shows that stimulation of airway C fibers can augment reflex bronchospasm and cough through central sensitizing mechanisms.80,81 Likewise, in an allergic monkey model, sensitization to and repeated inhalation exposure with house dust mite allergen leads to sensitization of the electrophysiologic properties of secondary neurons in the brainstem (nTS neurons).82 The nTS neurons from control monkeys respond to prolonged suprathreshold current pulses with about 20 APs, whereas nTS neurons isolated from allergen-exposed monkeys responded to the depolarizing current pulse with more than 100 APs (Fig. 25.6). 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. Such afferent nerve interactions and sensitizations within the CNS may explain in part how extrapulmonary disorders such as gastroesophageal reflux disease,83 allergic rhinitis,84 and upper respiratory tract infections85 initiate pulmonary symptoms such as cough and reflex bronchospasm and perhaps airway hyperreactivity.

Allergenic Modulation of Airway Ganglionic Transmission By increasing primary afferent nerve activity and increasing synaptic transmission in the CNS, allergic inflammation will likely lead to an increase in activity of autonomic preganglionic nerves. 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 central sensitization.18 As previously described, airway parasympathetic ganglia filter or integrate input from

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CHAPTER 25  Neuronal Control of Airway Function in Allergy Recording electrode

CNS Nodose ganglion

SLN

+

–

ES Vagus

Recurrent laryngeal nerve

OVA 10 µg/mL

A

B

Ovalbumin 10 µg/mL

0.35

0.8

1.7

0.35

0.35

mN

Fig. 25.5  (A) Allergen-induced overt activation of a vagal C fiber nerve ending in a mouse lung. The mouse was previously sensitized to ovalbumin (OVA); at the arrow, ovalbumin was applied to the nerve’s receptive field in an ex vivo, vagally innervated mouse lung preparation. The length of the horizontal arrow represents 15 minutes. (B) Mechanical sensitivity of an Aδ cough nerve in isolated guinea pig trachea before and after allergen challenge. Mechanical sensitivity was determined using von Frey filaments and expressed in milli­ newtons (mN) before and 15 minutes after allergen challenge in trachea isolated from actively sensitized guinea pigs. Unlike C fibers, allergen (OVA) exposure did not overtly evoke action potential discharge in the Aδ cough fibers (not shown) but did cause a substantial increase in the nerve’s excitability, as manifested in the decrease in force required to activate the mechanical receptive field. CNS, Central nervous system; ES, electrical stimulation; SLN, superior laryngeal nerve. (From Riccio MM, Myers AC, Undem BJ. Immunomodulation of afferent neurons in guinea-pig isolated airways. J Physiol 1996;491:499.)

40 Control

mV –60 40 mV

Allergen

–60 40 pA

60 pA

80 pA

100 pA

Fig. 25.6  Current-clamp recordings of neurons in nucleus tractus solitarius (nTS) of isolated monkey brainstem from control animals and animals chronically exposed to inhalation of house-dust mite allergen. Membrane potential is recorded in response to 500-ms injections of increasing amounts of current (40 to 100 pico­ amperes, pA). Note that neurons in nTS of the 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. Extended allergen exposure in asthmatic monkeys induces neuroplasticity in nucleus tractus solitarius. J Allergy Clin Immunol 2001;108:557.)

the CNS. Any process that increases the efficacy of synaptic transmission, which results in increases in EPSP amplitude, will cause a decrease in filtering and a generalized increase in airway parasympathetic tone. Allergen challenge to bronchi isolated from actively sensitized guinea pigs or mice increases the excitability of bronchial ganglion neurons.86,87

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

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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 mast cell activation and a pronounced increase in synaptic neurotransmission.88–90 The antigen-induced effect is persistent; a 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 called “antigen-induced long-term potentiation.”88

Allergenic Modulation of Postganglionic Transmission 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 and cause neurotransmitter release and activation of effector cells. In the airways, allergen challenge is associated with an elevation in the amount of ACh released from the airway postganglionic nerve varicosities. Several mediators associated with allergic reactions interact with postganglionic nerve fibers to enhance transmitter release.91 In addition to having conventional autacoid effects, allergen-induced eosinophilic inflammation may augment ACh release by inhibiting the function of cholinergic muscarinic M2 receptors.92–94 The prejunctional M2 receptors serve as negative-feedback autoreceptors; when the released ACh acts on these receptors, signals are produced that lead to an inhibition of further ACh release. Allergen challenge inhibits this process, apparently through an inhibitory effect of eosinophil-derived major basic protein (MBP) on M2 receptor function.95 Few studies have examined the effect of allergen challenge on the release of noncholinergic transmitters from postganglionic parasympathetic nerve endings. In animals, allergen challenge attenuates subsequently evoked noncholinergic relaxant responses.96,97 Elevated levels of superoxide, perhaps derived from infiltrating eosinophils, may attenuate relaxant responses mediated by neuronal NO.97,98 Arginase released during allergic inflammation may also reduce arginine levels in the airways, thus limiting the substrate required for NO formation by airway autonomic nerves.99 Alternatively, mast cell tryptase or other peptidases may attenuate relaxant responses mediated by VIP and other relaxant neuropeptides.74 Allergen challenge has been associated with phenotypic changes in airway parasympathetic neurons. In the guinea pig, allergic airway inflammation is followed by a phenotypic switch in the relaxant (VIP/ NO) parasympathetic neurons such that they begin lose their expression of relaxant transmitter (VIP) and begin to express de novo choline acetyltransferase, the rate-limiting enzyme for ACh synthesis.100 Such changes could lead to an overall increase in the cholinergic contractile versus relaxant innervation to the smooth muscle.

Role of Nerve in Animal Models of Allergic Asthma The most frequently studied laboratory animal model of allergic asthma over the past decades is the allergen challenged mouse. Typically, the mouse is actively sensitized to an allergen, commonly ovalbumin or house dust mite extract, then repetitively challenged with the allergen via airway inhalation or instillation. This inevitably leads to a classically defined TH2-driven airway inflammatory response with many similarities to the inflammation noted in human asthma. These mice develop an airway hyperreactivity to methacholine, and thus it is considered a model of asthma. Studies have found that the airway hyperreactivity to methacholine is prevented if before the methacholine challenge the vagus nerves are severed.101 This is also the case for the allergen-induced hyperreactivity to histamine noted in the allergically inflamed guinea pigs.93 Tränkner et al.102 recently took this further by using genetic strategies to selectively eliminate only the sensory C-fiber neurons from the vagal sensory ganglia (nodose/jugular), while leaving the other vagal

afferent nerves as well as the parasympathetic nerves in the vagus intact. Removing the C-fiber neurons did not influence the markers of inflammation that they measured, but virtually abolished the hyperreactivity to methacholine. Talbot et al.103 used a pharmacologic strategy in which the capsaicin-sensitive vagal afferent nerves in the airways were selectively anesthetized and also noted that the hyperreactivity to methacholine was abrogated. These data, collectively, demonstrate that in the laboratory animal much, if not all, of the hyperreactivity is related to an airway hyperreflexivity.

CLINICAL ALLERGY AND THE NEURAL HYPERSENSITIVE STATE As reviewed earlier, the allergic reaction in the respiratory tract is associated with overt activation; increases in electrical excitability; and phenotypic changes in sensory, central, and autonomic neurons. Much of the literature reviewed in previous sections is based on studies with experimental animals and isolated neurons. It is more difficult to characterize allergic neuronal hypersensitivity in humans. Although much of the activation of sensory nerves in the lower airways is not associated with corresponding conscious sensations, studies of human sensations are instructive in addressing hypotheses relating to allergy and neural hypersensitivity. Patients with allergic rhinitis typically complain of irritating nasal sensations and symptoms induced by various perfumes and other strong odors, smoke, or changes in atmospheric conditions. Of 350 participants with allergic rhinitis who completed a protocol screening questionnaire, 60% listed smoke as one of the triggers of their nasal symptoms, 58% listed other irritants, and 47%, cold air.104 These percentages were even higher in the volunteers with perennial versus seasonal allergic rhinitis. In addition, nasal exposure to low levels of chlorine gas leads to stronger responses in those with seasonal allergic rhinitis versus no allergy.105 As outlined earlier, the peripheral nervous system functions in terms of reflex arcs. Although an increase in airway reflex activity is self-evident to persons coughing and sneezing during their allergy season, it is difficult to measure hyperreflexia. Studies in the human nose have provided a clear-cut example of upregulation in reflex physiology by allergy. A sensory nerve irritant (bradykinin) was applied to one nostril of subjects before and during their allergy season. When subjects were not experiencing allergies, bradykinin had negligible effect on airway physiology. Applying bradykinin to the nose of the same subjects during their allergy season, however, led to excessive sneezing and parasympathetic reflex cholinergic secretions (Fig. 25.7).106 This observation has been repeated in other laboratories and may be observed with the use of other types of sensory nerve stimulants.107,108 In the lower respiratory tract, where conscious sensations are not as acute as in the nose, the same principle is observed. The most prominent triggers of asthma attacks are those associated directly or indirectly with sensory nerve stimulation.109 Inhalation of an innocuous concentration of sulfur dioxide into the lower airways produces minimal effect in subjects with healthy airways but leads to strong reflex bronchoconstriction in those with asthma.110 This same type of hyperreflexia occurs in the lower airways when cough is the outcome variable.111 In many subjects the classic airways hyperreactivity that typifies allergic asthma may be in fact a hyperreflexivity.34 Methacholine, often thought to act only as a direct bronchial smooth muscle agonist, can also act as an initiator of parasympathetic cholinergic reflex contractions.112 In moderate to severe asthma, virtually all the increase in airway resistance that can be reversed with a β-adrenoceptor agonist is cholinergic in nature, which means that an antimuscarinic drug such as tiotropium is as effective as a β-agonist in reducing airway resistance.2

CHAPTER 25  Neuronal Control of Airway Function in Allergy Contralateral

Ipsilateral

Secretion weight (mg)

383

50

50

40

40

30

30

20

20

10

10

0

0 Dil

1 10 Bradykinin (µg)

100

Dil

1 10 Bradykinin (µg)

100

Fig. 25.7  Effect of nasal provocation with bradykinin in nine patients with seasonal allergy challenged in season (open circles) and out of season (closed circles). Significant increase in contralateral secretion weights was seen in subjects challenged in season (P 10%). Removal of the allergen from the source material greatly reduces the biologic and immunochemical (IgE) activity of the extract. The allergen represents a significant proportion of the total extractable protein in the extract. The allergen may be used as a marker of environmental exposure. The allergen, its cDNA or its constituent peptides can be shown to be effective in an allergy vaccine. Both humoral (IgE) and cellular (T cell/basophil) responses to the allergen can be measured in a high proportion of a sensitized population.

Table modified from Chapman MD. Allergen Nomenclature. In Lockey RF, Ledford DK, editors. Allergens and Allergen Immunotherapy, 2008. Boca Raton: CRC Press; p. 47-58.

are the tropomyosins in arthropods (mites, snails, cockroaches, and crustaceans) and mollusks, the parvalbumins in different fish species, and the profilins from physiologically dissimilar tissues such as pollens and more distantly related fruits. These allergens are known as panallergens,9 and respiratory sensitization can give rise to oral allergy syndrome (OAS) in patients eating food containing them. It is generally thought that for cross-reactivity to be manifested serologically and clinically, amino acid sequence identity should be more than 50%. Finally, the availability of defined recombinant allergens has enabled allergy diagnosis to be performed using a panel of source-related allergens rather than a crude extract, a process known as component resolved diagnosis.10 Such panels can now use marker allergens, which can categorically identify the primary sensitizing source rather than signifying a cross-reactive pan-allergen present in a number of diverse sources. In addition, used in combination with birth cohorts, these panels identify risk allergens. For example, IgE to three or more of Ara h1, Bet v1, Fel d 1, or Phl p 1, or three or more of Der p 1, Der f 2, Phl p 1, Phl p 5, or Fel d 1 specific allergens, respectively, at age 4 years has made it possible to predict asthma and/or rhinitis at age 16 years and, therefore, risk.11 These studies also demonstrate that allergic children produce IgE to just one or two immunodominant allergens early in sensitization, giving rise to the term initiator allergens, whereas broadening develops later.

AEROALLERGENS Exposure to aeroallergens (e.g., pollens, fungal spores, insect and mite feces, animal danders, and dusts) may be perennial, as with HDM, or seasonal, as with tree pollen in spring and grasses and herbaceous dicotyledons in summer through autumn. Symptoms generally correspond to atmospheric allergen concentrations, although perennial exposure may occur to seasonal allergens such as pollens through their accumulation in house dust. Aeroallergen production and exposure can also be influenced by atmospheric pollutants, climatic changes, and severe weather (Table 26.4). Fungal and mite allergen exposure may also be seasonal because of domestic microclimate effects on reproductive processes and diet but often is perennial as allergens accumulate in house dust. Exposure to dander allergens, as well as occupational aeroallergens, also can be perennial unless the precipitating source is removed.

Pollen Aeroallergens Pollens (ca 5 - >200 µm) contain all the biochemical material necessary to deliver the male gamete to the ovum. Pollen allergy arises from exposure to pollen released from wind-pollinated (anemophilous) angiosperms and gymnosperms, including trees, herbaceous dicotyledons (weeds), and grasses, and 20 to 100 pollen grains/m3 are sufficient to provoke symptoms. For any individual, pollen allergy will reflect the flora in a particular location, as well as pollen- and pollen allergen– specific characteristics such as releasability, buoyant density, dispersibility, abundance, and profusion. Pollen from angiosperms hydrate on contact with the stigma, and pollen constituents either diffuse out or are expelled from the grains onto the stigma surface (a process mirrored when pollens impact moist respiratory epithelia). The expulsion of pollen contents to the exterior may occur by osmotic shock through apertures in the pollen wall, with some pores possessing a lid-type structure (opercula). The exudate comprises cytoplasm and cytoplasmic particles such as starch granules (amyloplasts, 3 µm in diameter; approximately 700 per pollen grain), and polysaccharide (pectin)-containing wall precursor bodies, termed P-particles. Once a pollen grain has landed on the stigma, the pollen tube emerges and grows toward the ovum. However, in gymnosperms, which lack stigma and possess naked ovules, the fertilization process is different because pollen is either trapped by a pollination drop that emanates from the exposed ovula and then absorbed into the micropyle chamber, or else engulfed by the ovula extensions. Only after this does the pollen tube develop through the pollen wall. Individuals may also be exposed to pollen allergens released into the atmosphere by other means.12 For example, pollens may release allergenic material through osmotic shock on contact with rainwater, giving rise to respirable submicronic or paucimicronic material as drying occurs. Additionally, such material may be released from rupturing pollen tubes per se as they abortively develop when landing on wet surfaces13 or as actively secreted in pollensomes, which are thought to be released as part of the natural growth of the tube. Specific allergens associate with distinct parts of the pollen grain or pollen tube. For example, the olive tree pollen allergens Ole e 1, 11, and 12 are found in pollensomes, and Cyn d CP and Cyn d EXY are found in pollen wall exudates. Finally, allergens (e.g., Cry j 1) may be present in anther-associated submicronic orbicules (Ubisch bodies) rather than in pollen per se.

CHAPTER 26  The Structure and Function of Allergens

393

TABLE 26.4  Effects of Pollution and Climate Change on Allergens and Allergenicity of Pollen Pollutantsa

Climate Changeb

Enhanced allergenicity due to adjuvant properties of particulates

Extended pollen seasons—earlier start, later finish

Differential expression of allergens in pollen grains

Increased pollen production

Increased allergen content in pollen grains

Increased allergen expression in pollen grains

Increase in pollen protein expression with possibility of creating new allergens

Increased allergen content in pollen grains

Increased releasability of cytoplasmic allergens and allergen-laden granules from pollen grains

Increase in pollen protein expression with possibility of creating new allergens

Posttranslational modification of pollen allergens

Change in distribution of pollen-producing plants

Enhanced antigen presentation

Induction of fungal sporulation and therefore allergen release

Alteration in pollen germination rate a

Particulates, heavy metals, diesel exhaust particles (DEP), environmental tobacco smoke (ETS), NO2, SO2, O3. Temperature, CO2. Adapted from Stewart GA, Peden DP, Thompson PJ, Ludwig M. Allergens and air pollutants. In: Holgate ST, Church MK, Broide DH, Martinez FD, editors. Allergy. 4th ed. Edinburgh: Saunders; 2012. For a review, see Ziska L H, Beggs PJ. Anthropogenic climate change and allergen exposure: The role of plant biology. J Allergy Clin Immunol 2012;129:27-32. b

Grass Pollen Aeroallergens Unlike trees and herbaceous dicotyledons, grasses belong to a single family (Poaceae), and the majority of allergenic grasses (temperate and tropical) belong to the subfamilies Pooideae (e.g., rye grass, Lolium perenne; timothy, Phleum pretense; and Kentucky blue grass, Poa pra­ tensis), Chloridoideae (e.g., Bermuda grass, Cynodon dactylon) and Panicoideae (e.g., Bahia grass, Paspalum notatum).14 Many allergens have been described, and significant sequence similarities have made it possible to collate them into about 15 groups (Table 26.5) with most, but not all, represented in the various subfamilies. Notably, the group 6 allergens are restricted to Anthoxanthum, Phleum, and Poa species, whereas the group 5 allergens are found only in Pooideae.14 The group 1 allergens are found in all the grass subfamilies and are related to the lower molecular weight group 2 and 3 allergens through sequence identity in their C-terminal halves. The group 1 allergens are β-expansins involved in the cell wall–loosening process, which enables pollen tube growth. They are the most important grass allergen group and represent a marker allergen for grass pollen sensitization. The Group 1 allergens comprise 2 domains; an N-terminal EG45 domain, which is homologous to the family 45 endoglucanases (although inactive), and a C-terminal cellulose binding domain (CBD), whereas the group 2 and 3 allergens comprise just the CBD domain. These allergens solubilize glucuronoarabinoxylan and homogalacturonan components of the matrix present in the middle lamella (outer wall) of cells forming the transmitting tract through which the pollen tube grows. These polymers are absent in herbaceous dicotyledons and trees and probably explains why equivalent group 1 proteins are not present in these species. Other significant allergens include a number of carbohydratedegrading enzymes likely involved in pollen wall and pollen tube growth, including the group 13 allergenic polygalacturonases and endoxylanases. Other grass pollen major allergens have been characterized and include the group 4 berberine bridge enzymes involved in alkaloid synthesis, the group 5 allergens, which are single-stranded nucleases with topoisomerase-like activity, and the group 11 allergens, which share sequence similarity with the Lamiales group 1 allergens (e.g., Ole e 1) and soybean trypsin inhibitor, suggesting a protease inhibitory role. In addition, cysteine protease allergens have been identified. Several minor grass pollen allergens exist, although the function of some remains to be determined. They include the polcalcins, which

modulate Ca2+ during pollen tube growth by binding it to an ion-binding peptide loop sequence flanked by a small helical sequence (an “EF” hand).15 They are also common in herbaceous dicotyledon and tree pollens and are described as pan-allergens because of their sequence similarity (60% to 90% identity) across the three pollen types. Similarly, the pan-allergen profilins are involved in pollen tube growth, where they are essential to cytoskeletal actin streaming. All of these allergens may be distributed in specific locations within pollen, for example, between anther cuticexine and locules, intine and exine pollen wall layers, pollen cytoplasm, and orbicules. With regard to timothy, Phl p 1, 3 to 6, and 12 are present in both pollen and cytoplasmic granules; Phl p 11 is present in granules; and Phl p 2 and 13 are present in pollen but not granules.16 In addition, two previously unrecognized major allergens, Cyn d CP (a cysteine protease) and Cyn d EXY (endoxylanase), have been isolated from the pollen coats of untreated Bermuda grass.

Herbaceous Dicotyledon (Weed) Species–Derived Pollen Aeroallergens.  Pollens from herbaceous dicotyledon species, often referred to as weeds, may also be allergenic, particularly in species of the Amaranthaceae (Russian thistle, goosefoot, or lamb’s quarters), Asteraceae (ragweed, mugwort, feverfew, sunflower), Brassicaceae (oilseed rape and turnip), Euphorbiaceae (annual mercury), Plantaginaceae (English plantain), and Urticaceae (wall pellitory) families.17 A range of allergen groups have been identified (Table 26.6), but unlike the unifamiliar grasses, the immunodominant groups vary both within and between families. However, allergenic cross-reactivity is high between biochemically identical allergens from different families, and marker allergens for some species have been identified (e.g., Amb a 1, Art v 1, Pla l 1, and Par j 2). Major allergens in Ambrosia species include the group 1 pectate lyases, the group 3 plastocyanins, and the group 11 cysteine proteases. The lyases are also found in ragweed, sunflower, mugwort, and feverfew, but not in species from the other allergenic orders. Other pectinassociated enzymes such as polygalacturonase or pectate methylesterase have yet to be described in Ambrosia species, although both have been described as allergenic in Russian thistle. The cysteine protease allergens are found in both ragweed and sunflower pollen as well as in the grass pollens described earlier but remain to be described in tree pollens.

394

SECTION B  Aerobiology and Allergens

TABLE 26.5  Physicochemical and Biochemical Characteristics of Grass Pollen Aeroallergens Allergen

Frequency of Reactivitya (%)

Mol. Sizeb (kDa)

Function

c

Poaceae and Panicoideae Example Species: Phleum Pratense, Lolium Perenne, Cynodon Dactylon, Holcus lanatus, Dactylis Glomerata, Agrostis Elongata, Paspalum Notatum, Oryza Sativa, Sorghum Halpense, Secale Cereale, Triticum Aestivum Group 1 (e.g., Lol p 1) >90 30 β-Expansin; involved in cell wall loosening; shows C-terminal sequence similarity with group 2 and 3 allergens and the mite group 2 allergen; C-terminal domain demonstrates oxidized cellulose binding activity, present in all Poaceae subfamilies >60

11

Shows sequence similarity with the C-terminal half of group 1 allergens; shows sequence similarity with group 3 allergens

Group 3 (e.g., Lol p 3)

70

11

Shows sequence similarity with group 1 and group 2 allergens

Group 4 (e.g., Phl p 4)

22-92

57

Berberine bridge enzyme, member of flavoprotein oxidoreductase superfamily

Group 5 (e.g., Lol p 5)

62-80

29-31

Group 6 (e.g., Phl p 6)

14-64

11

Group 7 (e.g., Phl p 7)

>10

6

Group 10 (e.g., Lol p 10)

?

12

Cytochrome c

Group 11 (e.g., Lol p 11)

66

16

Function unknown; shows sequence similarity with tree allergen Ole e 1, lamb’s quarters allergen Che a 1, and soybean trypsin inhibitor

Group 12 (e.g., Phl p 12)

20-36

14

Profilin

Group 13 (e.g., Phl p 13)

50

55-60

Group 15 (e.g., Cyn d 15)

?

9

Group 22 (e.g., Cyn d 22)

?

48

Group 23 (e.g., Cyn d 23)

?

9

Group 24 (e.g., Cyn d 24)

?

21

Pathogenesis-related protein; PR-1

Cyn d CP

63

23

Cysteine protease; shows sequence similarity with enzymes from maize and rice, enzyme also found in timothy and Johnson grass pollen

Phl p CP

?

23

Cysteine protease

Cyn d EXY

75

30

Endoxylanase; shows sequence similarity with enzymes from maize and rice

Lol p Cyp

42

26

Cytophilin

Lol p FT

16

71.3

Fructosyltransferase

Lol p Legumin

21

38

11S globulin

Group 2 (e.g., Lol p 2)

Single-stranded nuclease with topoisomerase-like activity; shows sequence similarity with the group 6 allergens; present in Pooideae subfamily only Shows sequence similarity with group 5 allergens; associated with P-particles, may be restricted to timothy grass pollen Polcalcin, 2EF-hand calcium binding protein; shows sequence similarity with Bet v 4, Ole e 3, Aln g 4, Jun o 2

Polygalacturonase; degrades pectin, a major plant cell wall polymer of α-linked galacturonic acid β-Expansin, shows sequence similarity with other grass groups 1, 2, and 3 Enolase, may be restricted to Bermuda grass pollen Function unknown

Data obtained from and http://www.allergen.org and http://www.allergome.org or from original references. a Frequency data presented in these tables are indications only, because they will vary with the population studied and their geographic location. In addition, the data presented may reflect immediate hypersensitivity diseases, including atopic dermatitis and allergic bronchopulmonary aspergillosis, as well as delayed-type hypersensitivity disease. “?” Indicates lack of data at the time of the publication, although IgE binding has been demonstrated. When frequency data are shown for allergens described in groups, the data refer to the examples shown in parentheses. Allergens yet to be denominated by the IUIS Allergen Nomenclature Subcommittee are indicated by reference to their biochemical properties as described in the Allergome database or in specific publications yet to be curated. b Molecular sizes obtained from allergen databases or calculated from mature protein amino acid sequence data. c Classification of species throughout tables is derived from the Catalog of Life (www.catalogueoflife.org). Adapted from Stewart GA, Peden DP, Thompson PJ, Ludwig M. Allergens and air pollutants. In: Holgate ST, Church MK, Broide DH, Martinez FD, editors. Allergy. 4th ed. Edinburgh: Saunders; 2012.

In Asteraceae other than Ambrosia species, the major allergen groups include PR pathogenesis-related (PR)-12 defensin-like group 1 proteins and the PR-1-like proteins as well as a protein of unknown function (Hel a 1) (Table 26.6). The pathogenesis-related proteins play an important role in plant innate immune systems,18 where they defend against various pathogens and their virulence factors, as well as abiotic stressors such as mechanical wounding. There are 17 groups of proteins (Table 26.7), which are constitutively expressed or upregulated in response to insults.

Many are hydrolytic enzymes capable of digesting cell walls of fungi, whereas others are lipid transfer proteins (LTPs). In the Urticaceae, Brassicaceae, Euphorbiaceae, and Amaranthaceae families, the major allergens include the nonspecific LTP (nsLTPs), polcalcins, profilins, and an Ole e 1–related allergen (Che 1). The nsLTP allergens (PR-14) are a widespread (but absent in grass pollen) group of low molecular weight proteins possessing four disulfide bonds and a hydrophobic core, conferring stability and allergenicity after cooking.

395

CHAPTER 26  The Structure and Function of Allergens

TABLE 26.6  Physicochemical and Biochemical Characteristics of Pollen-Derived Aeroallergens

from Herbaceous Dicotyledon Species Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Asterales: Asteraceae Short Ragweed (Ambrosia Artemisiifolia, Ambrosia Elatior, Ambrosia Psilostachya, Ambrosia Trifida) Group 1 (e.g., Amb a 1) >90 38 Pectate lyase, cleaved into two chains by pollen trypsin-like protease; shows sequence similarity with Art v 6; several isoforms exist; cleave pectin by eliminative cleavage rather than by hydrolysis Group 3 (e.g., Amb a 3)

51

11

Plastocyanin, a copper-containing protein

Group 4 (e.g., Amb a 4)

20-39

30

Defensin-like protein with a proline-rich C-terminal domain; shows sequence similarity with Art v 1

Group 5 (e.g., Amb a 5)

10-15

5

Group 6 (e.g., Amb a 6)

21

11

Nonspecific lipid transfer protein type 1

Group 7 (e.g., Amb a 7)

15-20

12

Plastocyanin, possible isoallergen of Amb a 3

Group 8 (e.g., Amb a 8)

25-56

14

Profilin

Group 9 (e.g., Amb a 9)

10-15

10

Polcalcin, 2EF-hand calcium binding protein

Group 10 (e.g., Amb a 10)

9-26

10

Polcalcin, 3EF-hand binding protein

Group 11 (e.g., Amb a 11)

53

37

Cysteine protease

Group 12 (e.g., Amb a 12)

66

48

Enolase

Mugwort (Artemisia Vulgaris, A. Annua) Art v 1 95

28

Plant defensin-like domain and a proline-rich domain (PR-12); the defensin-like domain shows sequence similarity with Amb a 4, Par h 1

Art v 2

58-63

20

Pathogenesis-related protein PR-1 like

Art v 3

25-56

12

Nonspecific lipid transfer protein type 1

Art v 4

36

14

Profilin

Art v 5

10

10

Polcalcin, 2EF-hand calcium binding protein

Art v 6

89

44

Pectate lyase; shows sequence similarity with Amb a 1

Art an 7

94

62

Galactose oxidase

Feverfew (Parthenium Hysterophorus) Par h 1 >90

31

Defensin-like protein; contains a defensin-like domain fused to a proline-rich region (PR-12); the defensin-like domain shows sequence similarity with Amb a 4 and Art v 1

Asteraceae: Sunflower (Helianthus Annuus) Hel a 1 57

34

Function unknown

Hel a 2

31

14.7

Profilin

Hel a 3

?

9

Nonspecific lipid transfer protein type 1 Art v 1-like defensin

Secreted basic protein

Hel a 4

?

?

Hel a 6

37

42

Pectate lyase

?

34

Cysteine protease

15

Nonspecific lipid transfer protein type 1, Par j 1.0101 isoform with a 37 amino acid extension possess LPS binding activity

Hel a Cys

Rosales: Urticaceae Wall Pellitory (Parietaria Judaica/Officinalis) Group 1 (e.g., Par o 1) 95 Group 2 (e.g., Par o 2)

82

10-14

Group 3 (e.g., Par j 3)

100

14

Group 4 (e.g., Par j 4)

6

9

Malpighiales: Euphorbiaceae Annual Mercury (Mercurialis Annua) Mer a 1 59

14

Nonspecific lipid transfer protein type 1 Profilin Polcalcin, 2EF-hand calcium binding protein

Profilin Continued

396

SECTION B  Aerobiology and Allergens

TABLE 26.6  Physicochemical and Biochemical Characteristics of Pollen-Derived Aeroallergens

from Herbaceous Dicotyledon Species—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Caryophiyllales: Amaranthaceae Lamb’s Quarters or Goosefoot (Chenopodium Album) Che a 1 77 17

Ole e 1-related protein

Che a 2

50-60

14

Profilin

Che a 3

46

10

Polcalcin, 2EF-hand calcium binding protein

Russian Thistle (Salsola Kali) Sal k 1 65

38

Pectin methylesterase

Sal k 2

?

36

Protein kinase homolog

Sal k 3

67

45

Methione synthase

Sal k 4

46

14.4

Profilin

Sal k 5

34-64

18.2

Ole e 1-like protein

Sal k 6

32

47

Polygalacturonase

Sal k 7

40

8.5

Polycalcin

Lamiales: Plantaginaceae English Plantain (Plantago Lanceolata) Pla l 1 86

18

Ole e 1-related protein

Pla l 2

15

Profilin

71-86

Brassicales: Brassicaceae Oilseed Rape (Brassica Napus, Brassica Rapa) Group 3 (e.g., Bra r 3) >30

7

Nonspecific lipid transfer protein type 1

Group 5 (e.g., Bra r 5)

9

Polcalcin, 2EF-hand calcium binding protein Polcalcin, 2EF-hand calcium binding protein

100

Group 7 (e.g., Bra n 7)

?

9

Group 8 (e.g., Bra n 8)

34

14

Profilin

Bra n PG

28

43

Polygalacturonase

See Table 26.16 for ingested Brassica spp. allergens. Both taxonomic Order and Family are indicated.

Two main forms (Types 1, 9 kDa and 2, 13 kDa) exist, and most allergenic nsLTPs are type 1 proteins and are prominent also in seeds, latex, fruits (in skin), and vegetables.19 In contrast, type 2 nsLTP allergens have been described in tomato, peanut, and celery. In the Plantainaceae, the major allergen is the Ole e 1–related allergen of unknown function. Diverse minor allergens have also been described (Table 26.6), including cystatin, profilins, polcalcins, a putative electron transfer–associated protein, and proteins of unknown function. In contrast to the Urticaceae, nsLTPs from Asteraceae and Brassicaceae are minor allergens.

Tree Pollen–Derived Aeroallergens.  Clinically important tree pollen allergens are derived from angiosperms (flowering trees) as well as from the gymnosperms (nonflowering conifers).20 The most important angiosperm families include the Fagales (e.g., birch, hazel, oak), the Lamiales (e.g., olive, privet, ash) and the Proteales (e.g., London plane tree), and different groups of allergens appear to be immunodominant (Table 26.8). The major allergen group in Fagales comprises the plant steroid carrier protein belonging to the PR-10 family, which also possesses ribonuclease activity. Bet v 1 is considered a marker allergen for sensitization to Fagales pollens given its high sequence similarity across species within this family and its absence from other allergenic angiosperm or gymnosperm trees. The remaining Fagales allergens are minor and include profilin, polcalcins, isoflavone reductase-like proteins, cyclophilin (a peptidyl-prolyl cis-trans isomerase), and glutathione

transferase groups. In the Lamiales, the group 1 allergen of unknown function (although showing sequence similarity with soybean trypsin inhibitor) has been identified as a marker allergen. Other major allergens (e.g., group 2 profilins, group 4 and 9 glucanases, the group 11 pectate methylesterases, and the group 7 nsLTPs) have been identified as well as several minor. In the Proteales, three major allergens have been identified, including an invertase inhibitor, polygalacturonase, and profilin. Regarding gymnosperm allergenic species (cedar, juniper, and cypress), there are allergenic similarities with angiosperm tree pollen proteins, with distinctions reflecting differences in fertilization processes. Similarities include the presence of group 1 pectate lyases and the group 2 polygalacturonases as well as pan-allergens such as profilins, nsLPTs, and the thaumatin-like proteins. Unlike the angiosperm tree pollens, pectate methylesterase allergens are absent in the gymnosperm species analyzed. Other major allergens include the yet to be denominated class 4 chitinases, the isoflavone reductases, and the subtilisin proteases. Several intermediate or minor allergen groups have been described and include thaumatins (depending on species), calmodulins, and nsLTPs.

Fungi-Derived Aeroallergens Of the more than 180 fungal species shown to produce allergenic proteins, those of the Ascomycota and Basidiomycota phyla are clinically most important (Table 26.9).21 All use airborne conidia (spore) dispersal for reproduction. In addition to spores, allergens are found in mycelia,

397

CHAPTER 26  The Structure and Function of Allergens

TABLE 26.7  Relationship between Plant Pathogenesis-Related Proteins and Allergens ALLERGEN EXAMPLE Family

Description or Characteristics

Pollen

Fruit/Seeds/Leaf

PR-1

Antifungal, mechanism unknown

Typical Size (kDa) 15

Phl p 24, Art v 2

Cum c 3

PR-2

Endo-β-1,3-glucanases

30

Ole e 4/9

Mus a 5

Hev b 2

PR-3

Chitinases (types I, II, IV, V, VI, VIII)

25-30

Per s a 1, Cas a 5

Hev b 11

PR-4

Chitinases (types I, II)

15-20

Bra r 2

Hev b 6

PR-5

Thaumatin-like proteins; antifungal; may have endo-β-1,3-glucanase activity

PR-6

Protease inhibitors

PR-7

Proteases

75

PR-8

Chitinase (type III)

28

PR-9

Peroxidases

35

PR-10

Plant steroid carrier (ribonuclease-like)

17

PR-11

Chitinase (type I)

40

PR-12

Defensins

5

PR-13

Thionins

5

PR-14

Nonspecific lipid transfer proteins type 1

9

25

Cry j 3, Jun a 3 Cup a 3

8

Oxalate oxidase (germins)

20

PR-16

Oxalate oxidase–like (germin-like)

20

PR-17

Unknown

27

Pru av 2, Mal d 2 Tri a SPI Hev b 14 Tri a Peroxidase

Bet v 1, Cor a 1, Aln g 1

Mal d 1, Pru av 1, Pyr c 1, Api g 1, Dau c 1, Act c 8, Gly m 4, Ara h 8, Cas s 1 Pers a 1

Art v 1, Amb a 4

Gly m 2 Tri a 37

Art v 3, Amb a 6, Par j 1, 2, Ole e 7, Amb a 6, Art v 3

Nonspecific lipid transfer proteins type 2

PR-15

Latex

Pru p 3, Mal d 3, Gly m 1, Jug r 3, Tri a 14, Cor a 8, Ara h 17, Ara h Sola I 6 Api g 6 Ara h 16

Hev b 12

TABLE 26.8  Physicochemical and Biochemical Characteristics of Tree Pollen Aeroallergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Angiosperms Fagales Birch (Betula Verrucosa), Alder (Alnus Glutinosa), Hazel (Corylus Avellana), Hornbeam (Carpinus Betulus), Oak (Quercus Alba), Chestnut (Castanea Sativa) Group 1 (e.g., Bet v1) >95 17 Plant steroid carrier; shows sequence similarity with pathogenesis-related proteins (e.g., PR-10) Group 2 (e.g., Bet v 2)

5-37

15

Profilin

Group 3 (e.g., Bet v 3)

10

24

Polcalcin, 3EF-hand calcium binding protein

Group 4 (e.g., Bet v 4)

20

9

Group 6 (e.g., Bet v 6)

32

35

Isoflavone reductase-like proteins, belongs to the NmrA-like family

Group 7 (e.g., Bet v 7)

21

18

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

Group 8 (e.g., Bet v 8)

13

?

Bet v Glucanase

12

35

Glucanase

3

25

Thaumatin-like protein

Bet v TLP

Polcalcin, 2EF-hand calcium binding protein; shows sequence similarity with Aln g 4, Ole e 3, Syr v 3

Glutathione S transferase

Lamiales Olive (Olea Europaea), Lilac (Syringa Vulgaris), Privet (Ligustrum Vulgare), Ash (Fraxinus Excelsior) Group 1 (e.g., Ole e 1) >90 20 Shows limited sequence similarity with soybean trypsin inhibitor and Lol p 11 Continued

398

SECTION B  Aerobiology and Allergens

TABLE 26.8  Physicochemical and Biochemical Characteristics of Tree Pollen

Aeroallergens—cont’d

Frequency of Reactivity (%)

Mol. Size (kDa)

Group 2 (e.g., Ole e 2)

16-70

15

Group 3 (e.g., Ole e 3)

20->50

9

Group 4 (e.g., Ole e 4)

80

32

Glucanase

Group 5 (e.g., Ole e 5)

35

16

Cu/Zn superoxide dismutase

Group 6 (e.g., Ole e 6)

5-20

10

Cysteine-rich protein

Group 7 (e.g., Ole e 7)

47-100

10

Nonspecific lipid transfer protein type 1

Group 8 (e.g., Ole e 8)

13

21

Polcalcin, 4EF-hand calcium binding protein

Group 9 (e.g., Ole e 9)

65

45

β-1,3-Glucanase (Family 17), contains a carbohydrate binding domain; shows sequence similarity with peptide originally designated Ole e 10

Group 10 (e.g., Ole e 10)

55

11

Shows sequence similarity with the C-terminal domain of Ole e 9, carbohydrate-binding module CBM 43

Group 11 (e.g., Ole e 11)

Allergen

Function Profilin Polcalcin, 2EF-hand calcium binding protein

62-65

39

Pectate methylesterase

Group 13

2-7

23

Thaumatin-like protein

Group 14 (e.g., Ole e 11)

13

47

Polygalacturonase

Proteales London Plane Tree (Platanus Acerifolia) Pla a 1 87

18

Invertase inhibitor

Pla a 2

83

43

Polygalacturonase

Pla a 3

61

10

Nonspecific lipid transfer protein type 1

Pla a 8

90

15

Profilin

7

25

Thaumatin-like protein

Pla a TLP

Gymnosperms (Conifers) Cupressaceae Japanese Cedar (Cryptomeria Japonica) Cry j 1 >85

41-45

Pectate lyase; shows sequence similarity with bacterial pectate lyase and Amb a 1 and 2

Cry j 2

76

45

Polygalacturonase

Cry j 3

27

27

Thaumatin-like protein, osmotin, and amylase/trypsin inhibitor; PR-5–related

Cry j 4

?

?

Cry j AP

58

52

Aspartate protease

100

34

Chitinase, class 4

89

90

Plant subtilisin-like serine protease

Cry j IFR

67-72

34

Isoflavone reductase-like protein; shows sequence similarity with Bet v 6

Cry j LPT

37

10

Nonspecific lipid transfer protein type 1

Cry j Chitinase Cry j CPA9

Polycalcin

Juniper Species (e.g., Juniperus Ashei, Juniperus Rigida, Juniperus Virginiana, Juniperus Oxycedrus, Juniperus Communis) Group 1 (e.g., Jun a 1) 71 43 Pectate lyase Group 2 (e.g., Jun a 2)

100

43

Polygalacturonase

Group 3 (e.g., Jun a 3)

33

30

Thaumatin-like protein, osmotin, and amylase/trypsin inhibitor; PR-5–related

Group 4 (e.g., Jun o 4)

15

29

Polcalcin, 4EF-hand calcium binding protein

Cypress (e.g., Cupressus Sempervirens, Cupressus Arizonica, Chamaecyparis Obtusa) Group 1 (e.g., Cup s 1) 50-81 38-42 Pectate lyase Group 2 (e.g., Cha o 2)

83

45

Polygalacturonase

Group 3 (e.g., Cup a 3)

63

34

Thaumatin-like protein, osmotin, and amylase/trypsin inhibitor; PR-5–related

Group 8 (e.g., Cup s 8)

?

14

Profilin

399

CHAPTER 26  The Structure and Function of Allergens fragmented hyphae, and yeast forms, and such sources are relevant in conditions such as allergic bronchopulmonary aspergillosis (ABPA) and immediate and delayed-type dermal infections involving Aspergil­ lus fumigatus, Candida albicans, Trichophyton species, and Malassezia furfur. Most allergens are proteins or glycoproteins, but mannans from C. albicans and M. furfur also may be allergenic. It is not clear whether

atopic individuals are initially sensitized to spore- or to mycelium-derived allergens, but sensitization may occur with exposure to fragmented spores or hyphae rather than intact structures.22 Clinically important fungi include Aspergillus, Penicillium, Clado­ sporium, and Alternaria species (Table 26.9), with allergens that may be cell wall– or cytoplasm-derived. Many are involved in protein synthesis

TABLE 26.9  Physicochemical and Biochemical Characteristics of Fungi-Derived Aeroallergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Ascomycota Alternaria Alternata Alt a 1

>80

14

Secreted protein, function unknown

Alt a 2

0-61

20

EIF-2α kinase

Alt a 3

5

70

HSP70

Alt a 4

42

57

Protein disulfide isomerase

Alt a 5

8

11

Ribosomal P2 protein; shows sequence similarity with Cla h 4

Alt a 6

50

45

Enolase

Alt a 7

7

22

1,4-Benzoquinone reductase; shows sequence similarity Cla h 5

Alt a 8

41

29

Mannitol dehydrogenase

Alt a 10

2

54

Aldehyde dehydrogenase; shows sequence similarity with Cla h 3

Alt a 12

?

11

Acid ribosomal P1 protein

Alt a 13

100

26

Glutathione S-transferase

Alt a 14

?

24

Mn superoxide dismutase

Alt a 15

6

58

Serine protease

Aspergillus Fumigatus Asp f 1 85

17

Ribonuclease; ribotoxin shows sequence similarity with mitogillin

Asp f 2

96

37

Shows sequence similarity with Candida albicans fibrinogen-binding protein

Asp f 3

84

19

Peroxisomal membrane protein; belongs to the peroxiredoxin family; thiol-dependent peroxidase

Asp f 4

78-83a

30

Shows sequence similarity with bacterial ABC transporter–binding protein; associated with peroxisome

Asp f 5

74

40

Metalloprotease

27

Manganese superoxide dismutase; shows sequence similarity with Mal s 11 and Hev b 10

a

Asp f 6

42-56

Asp f 7

29

12

Shows sequence similarity with fungal riboflavin, aldehyde-forming enzyme

Asp f 8

8-15

11

Ribosomal P2 protein

Asp f 9

31

34

Shows sequence similarity with plant and bacterial endo-β1,3;1,4-glucanases

a

Asp f 10

3-28

34

Aspartic protease

Asp f 11

90

24

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

Asp f 12

?

90

Heat shock protein P90

Asp f 13

79

34

Alkaline serine protease

Asp f 15

?

16

Shows sequence similarity with a serine protease antigen from Coccidioides immitis; also designated Asp f 13

Asp f 16

70

43

Shows sequence similarity with Asp f 9

Asp f 17

?

19

Galactomannoprotein

Asp f 18

79

34

Vacuolar serine protease

Asp f 22

30

46

Enolase, shows sequence similarity with Pen c 22

Asp f 23

27

44

L3 ribosomal protein

Asp f 27

75

18

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

Asp f 28

30

13

Thioredoxin

Asp f 29

50

13

Thioredoxin

Asp f 34

93

20

Phi A cell wall protein Continued

400

SECTION B  Aerobiology and Allergens

TABLE 26.9  Physicochemical and Biochemical Characteristics of Fungi-Derived

Aeroallergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Cladosporium Herbarum Cla h 2 43

45

Function unknown

Cla h 3

36

53

Aldehyde dehydrogenase

Cla h 5

22

11

Acidic ribosomal protein P2 (previously Cla h 4)

Cla h 6

20

46

Enolase

Cla h 7

22

22

Flavodoxin (previously Cla h 5)

Cla h 8

57

28

NaDP-dependent mannitol dehydrogenase

Cla h 9

16

38

Vacuolar serine protease; shows sequence similarity with Pen ch 18 and Asp f 18

Cla h 10

36

53

Aldehyde dehydrogenase

Cla h 12

?

11

Acidic ribosomal P1 protein

Cla h HSP70

?

70

HSP, previously denominated Cla h 4

Cla h TCTP

50

19

Shows sequence similarity with human translationally controlled tumor protein (TCTP)

Penicillium Brevicompactum Pen b 13 91

33

Alkaline serine protease

Pen b 26

11

Acidic ribosomal protein P1

87

Penicillium Chrysogenum (Formally Notatum) Pen ch 13 >80 34

Alkaline serine protease

Pen ch 18

77

32

Vacuolar serine protease

Pen ch 20

56

68

β-N-acetylglucosaminidase from Candida albicans

Pen ch 31

?

?

Calreticulin

Pen ch 35

?

37

Transaldolase

Penicillium Citrinum Pen c 1

?

33

Serine protease

34

Serine protease, shares sequence similarity with Asp f 18, Pen ch 13

Pen c 2 Pen c 3

46

18

Peroxisomal membrane protein; belongs to the peroxiredoxin family; thiol-dependent peroxidase

Pen c 13

100

33

Alkaline serine protease

Pen c 19

41

70

Heat shock protein P70

Pen c 22

30

46

Enolase

25

Elongation factor 1β

Pen c 24

7.6

Pen c 30

48

97

Catalase

Pen c 32

?

40

Pectate lyase

Penicillium Oxalicum Pen a 18 89

34

Vacuolar serine protease

Candida Albicans/Boidinii Cand a 1 77

40

Alcohol dehydrogenase

Cand b 2

100

20

Peroxisomal membrane protein A

Cand a 3

56

20

Peroxisomal protein

Cand a FPA

?

37

Aldolase

Cand a PGK

?

43

Phosphoglycerate kinase

Cand a Enolase

50

46

Enolase

Cand a CAAP

75

35

Aspartate protease

Trichophyton Tonsurans Tri t 1 54

30

Glycosyl hydrolase family 1 member

Tri t 2

42

30

Subtilisin-like protease; shows sequence similarity with Pen ch 13, Pen c 13

Tri t 4

61

83

Dipeptidyl peptidase

CHAPTER 26  The Structure and Function of Allergens

401

TABLE 26.9  Physicochemical and Biochemical Characteristics of Fungi-Derived

Aeroallergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Trichophyton Rubrum Tri r 1/2 43

30

Subtilisin-like protease; shows sequence similarity with Pen ch 13, Pen c 13

Tri r 4

44

83

Dipeptidyl peptidase

Curvularia Lunata Cur l 1

80

31

Serine protease

Cur l 2

75

48

Enolase

Cur l 3

?

54

Cytochrome c

Cur l 4

81

54

Vacuolar serine protease

Basidiomycota Malassezia Furfur Mala f 2

72

21

Peroxisomal membrane protein; belongs to the peroxiredoxin family, thiol-dependent peroxidase; shows sequence similarity with Asp f 3

Mala f 3

70

20

Peroxisomal membrane protein; belongs to the peroxiredoxin family, thiol-dependent peroxidase; shows sequence similarity with Asp f 3, Mala f 2

Mala f 4

83

35

Mitochondrial malate dehydrogenase

Malassezia Sympodialis Mala s 1 61

36

Function unknown; cell wall protein

Mala f 5

19-35

18

Oxidoreductase

Mala f 6

21-92

19

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

Mala s 7

60

19

Function unknown

Mala s 8

72

26

Function unknown

Mala s 9

36

17

Function unknown

Mala s 10

69

86

Heat shock protein P70

Mala s 11

75

23

Manganese superoxide dismutase; shows sequence similarity with Asp f 6

Mala s 12

?

67

Glucose-methanol-choline (GMC) oxidoreductase

Mala s 13

50

13

Thioredoxin

Coprinus Comatus Cop c 1

34

11

Leucine zipper protein

Cop c 2

19

12

Thioredoxin

Cop c 3

?

37

Nucleotide binding protein

Cop c 5

?

16

Function unknown

Cop c 7

?

16

Function unknown

Psilocybe Cubensis Psi c 1

>50

46

Function unknown

Psi c 2

>50

16

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

HSP, Heat shock protein; NaDP, nicotinamide-adenine dinucleotide phosphate. a Higher frequency determined in patients with allergic bronchopulmonary aspergillosis.

or energy metabolism, although secretory (e.g., proteases) allergens may also be involved. As with pollens, many allergens are common across allergenic fungal species of both Ascomycota and Basidiomycota. For example, the ribosomal P2 proteins, enolases, heat shock proteins (HSPs), aldehyde dehydrogenases, thioredoxins, proteases, and cyclophilins are common minor or major homologous allergens. Specific allergens are described, but excepting Alt a 1, it is unclear whether any are marker allergens. The major allergens from clinically important species in respiratory disease include Alt a 1, 2, and 13; Asp f 1, 2, and 4; and Cla h 1. The

functions of Alt a 1 and Cla h 1 are unknown, but Alt a 1 possesses a unique β-barrel dimeric structure restricted to fungi.23 Alt a 2 is a low molecular weight alkaline protein with eukaryote-2 initiation factor (EIF) α kinase activity and probably plays a role in protein synthesis, and Alt a 13 is a glutathione S-transferase. Asp f 1 is homologous with the fungal cytotoxin mitogillin from Aspergillus restrictus and α-sarcin from Aspergillus gigantus. These are cytotoxic, low molecular weight, nonglycosylated purine-specific ribonucleases found in both spores and mycelium. The Asp f 2 allergen is homologous with the C. albicans 54 kDa mannoprotein, which binds human fibrinogen. The Asp f 4 is a

402

SECTION B  Aerobiology and Allergens

binding protein associated with peroxisomes, self-replicating organelles that undertake metabolic detoxification in cells. Major allergens from Penicillium, Candida, and Trichophyton species include serine proteases, dipeptidyl peptidases, aspartic proteases, enolases, and peroxisomal membrane proteins, or have unknown function. Generally, fungal proteases are like bacterial subtilisins, and two types have been identified, namely, the group 13 alkaline proteases and the group 18 vacuolar proteases.24 The dipeptidyl peptidase allergen from Trichophyton species also is a secretory protein and is similar to enzymes from Aspergillus species implicated in aspergilloma. The function of the 3 Trichophyton allergen is unknown, although it may be an exo-β-1,3-glucanase. The peroxisomal membrane protein allergens possess thiol-dependent peroxidase activity. The group 6 enolases, glycolytic enzymes involved in the dehydration of glycerate-2-phosphate to produce phosphoenolpyruvate, represent a major group of cross-reacting allergens from a variety of fungal species. Several minor

allergens have been identified and are associated with specific cellular functions. Some are molecular chaperones involved in protein assembly and include protein disulfide isomerases, cyclophilins, and HSP. These proteins are found in spores from Aspergillus and Alternaria species, and cognate molecules are expected to be allergenic in other fungal species.

Animal Dander–Derived Aeroallergens Clinically important allergen-producing animals have been identified in both domestic and occupational settings (e.g., cats, dogs, cows, rats, mice, horses, rabbits, mice, gerbils, and guinea pigs) (Table 26.10), with their danders, epithelia, fur, urine, or saliva being the main sources. Many dander allergens are low molecular mass (approximately 20 kDa) lipocalins of the calycine superfamily and may be monomers or homodimers. They all possess a characteristic β-barrel fold with 8 antiparallel strands, which form a hydrophobic cavity to support their role in the

TABLE 26.10  Physicochemical and Biochemical Characterization of Dander-Derived

Aeroallergens Allergen

Frequency of Reactivity (%)

Cat (Felix Domesticus) Fel d 1 95

Mol. Size (kDa) 33-39a

Function Secretoglobin family member, tetramer of two heterodimers (chains 1 and 2), a possible ligand-binding molecule; chain 1 shows sequence similarity with 10-kDa secretory protein from human Clara cells, mouse salivary androgen-binding protein subunit, rabbit uteroglobin, and a Syrian hamster protein

Fel d 2

20-35

69

Serum albumin: food allergen, cross reacts with pork, beef albumin

Fel d 3

10

11

Cystatin

Fel d 4

60

20

Fel d 5

38

400

Lipocalin, shows sequence similarity with other mammalian lipocalin allergens Immunoglobulin A; food allergen, IgE is directed against the galactose β-1,3-galactose moiety, also found on the heavy chain of immunoglobulin M; present in pork, beef, and lamb

Fel d 6

?

900

Fel d 7

38

18

Von Ebner gland protein, cysteine protease inhibitor

Fel d 8

19

24

Latherin, surfactant protein

Dog (Canis Familiaris) Can f 1 50

Immunoglobulin M

19-25

Lipocalin; shows sequence similarity with Von Ebner gland protein, which has cysteine protease inhibitory activity

Can f 2

20-22

27

Lipocalin; shows sequence similarity with Can f 1 and Fel d 4, and with other lipocalin allergens

Can f 3

16-40

69

Serum albumin

Can f 4

35

23

Lipocalin, shows sequence similarity with bovine odorant-binding protein

Can f 5

70

28

Prostatic kallikrein; shows sequence similarity with human prostate-specific antigen (PSA), which is also allergenic

Can f 6

38

27, 29

Can f 7

17

16

Epidydimal secretory protein, member of the NPC2 family

Can f Fel d 1-like

?

20

Shows high sequence similarity with Fel d 1

Lipocalin

Horse (Equus Caballus) Equ c 1 100

25

Lipocalin; shows sequence similarity with rodent urinary proteins

Equ c 2

100

17

Lipocalin; shows sequence similarity with rodent urinary proteins

Equ c 3

50

67

Serum albumin

Equ c 4

77

17, 20.5

Cow (Bos Taurus) Bos d 2

97

20

Latherin, surfactant protein Lipocalin

Bos d 3

?

11

S100 calcium-binding protein

Bos d OSCP

31

21

Oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex

CHAPTER 26  The Structure and Function of Allergens

403

TABLE 26.10  Physicochemical and Biochemical Characterization of Dander-Derived

Aeroallergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Guinea Pig (Cavia Porcellus) Cav p 1 70

20

Function Lipocalin; shows sequence similarity with Cav p 2

Cav p 2

65

17

Lipocalin; shows sequence similarity with Bos d 2

Cav p 3

54

19

Lipocalin

Cav p 4

53

66

Serum albumin

Cav p 6

53

18

Lipocalin; shows sequence similarity with various other mammalian lipocalin allergens

Mouse (Mus Musculus) Mus m 1 >80

17

Major urinary protein; shows sequence similarity with lipocalins such as β-lactoglobulin, odorantbinding proteins, Rat n 2 Rat (Rattus norvegicus)

Rat (Rattus Norvegicus) Rat n 1 >90

17

Lipocalin; shows sequence similarity with lipocalins such as β-lactoglobulin Bos d 5, odorant-binding proteins, Mus m 1

Rat n 4

100

69

Rat n 7

47

150

Rabbit (Oryctolagus Cuniculus) Ory c 1 100

18 19-21

Serum albumin Immunoglobulin G Lipocalin

Ory c 3

77-100

Ory c 4

46

24

Lipocalin

Lipophilin, glycosylated heterodimer and similar to Fel d 1

Ory c 6

6

69

Serum albumin

a

Molecular size given represents dimer form, with two chains of approximately 18 kDa each. Note that for NAC (nascent polypeptide-associated complex alpha subunit) and keratin, deduced molecular masses are given.

binding and transport of small hydrophobic molecules such as pheromones, histamine, vitamins, and steroids.25 Significant similarity exists between the animal lipocalin allergens (approximate 60% identity), accounting for their high allergenic cross-reactivity. The Bos d 5 lipocalin allergen (β-lactoglobulin), like nonallergenic lipocalins, has nonspecific endonuclease activity. The catalytically important Glu134 in Bos d 5, necessary for phosphodiester bond cleavage, is conserved in Bos d 2, Equ c 1, Can f 1 (but not Can f 2), and Mus m 1, as well as the cockroach Bla g 4 lipocalin allergen, suggesting they are similarly active. In contrast with other danders, the predominant allergen in cat is Fel d 1, a heterodimeric protein. Fel d 1 comprises two distinct disulfidebonded peptides, designated chains 1 and 2, and chain 1 shows sequence similarity with antiinflammatory uteroglobins. The next most prominent animal allergens are the serum albumins, immunoglobulins, cystatins, kallikrein, and latherins. The latter are surfactants and major components of sweat, where they play a role in evaporative cooling. Others include an oligomycin sensitivity-conferring protein of the mitochondrial adenosine triphosphate synthase complex, a calcium-binding psoriasin-like allergen, a horse allergen homologous with rat mandibular gland protein A, and a protein of unknown function. Concerning IgA and IgM allergens found in cat dander, it is likely that the epitope recognized by IgE from patients is the cross-reactive alpha gal epitope.

Arthropod-Derived Aeroallergens Arthropod allergens are mainly derived from insect and arachnid species, and allergy may arise domestically or in places where arthropods are reared. HDM and cockroaches are the most important species, with allergens being derived from whole bodies and fecal pellets, which

accumulate in dusts. Many allergens are digestive enzymes or proteins involved in metabolite uptake and are, therefore, present in fecal pellets, whereas others are associated with muscle activity or ligand transport and accumulate in body debris. Several allergenic proteins in each of these arthropods are similar, as they are to biochemically related allergens from other arthropods such as crustaceans. However, certain allergens are restricted to one or other arthropod type and can thus be considered marker allergens.

Insect-Derived Aeroallergens The main insect groups producing allergens are the midges, moths, silk worm larvae, and cockroaches. In midges only hemoglobins and tropomyosins (Table 26.11) have thus far been shown to be allergenic, whereas tropomyosins, arginine kinases, and thioredoxin are allergenically important in Pyralidae and Bombycidae. In contrast, 15 allergen groups have been described in cockroaches,26 at least 6 of which are gut-associated proteins involved in either digestion, the transfer of ligands, or innate immunity. Remaining groups occur elsewhere in the body and arise in dusts when insects die, for example, the muscle-associated proteins (groups 6, 7, 8, and 9), the group 3 arylphorins, the group 4 lipocalins (from the male reproductive tract), the group 5 glutathione S-transferases, enolase, and vitellogenin (a hemolymph protein predominantly found in females). The cockroach group 1 allergen comprises 3 “insect allergen–related repeat, nitrile-specifier detoxification” domains, each comprising 6 helices. This domain is a paralog of the nitrile specifier domain protecting butterflies against toxic plants, but it lacks this function in cockroaches. However, it possesses two domains creating a hydrophobic cavity, suggesting that it facilitates lipid absorption from the gut,

404

SECTION B  Aerobiology and Allergens

TABLE 26.11  Physicochemical and Biochemical Characteristics of Arthropod-Derived Indoor

Aeroallergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Insecta: Diptera Chironomidae (Midges), Chironomus Thummi, Cladotanytarsus Lewisi, Polypedilum Nubifer, Chironomus Kiiensis Groups 1-9 (e.g., Chi t l to 9) >50 15 Hemoglobins Group 10 (e.g., Chi k 10)

81

33

Tropomyosin

Blattodea or Blattaria Ectobiidae and Blattidae, German Cockroach (Blattella Germanica) and American Cockroach (Periplaneta Americana) Group 1 (e.g., Bla g 1) 1-77 46 Function unknown, may be involved in lipid transport; contains three insect allergen– related repeat, nitrile-specifier detoxification domains, although such a detoxification role has not been demonstrated Group 2 (e.g., Bla g 2)

7-62

36

Aspartate protease (pseudoprotease); shows sequence similarity with pepsin

Group 3 (e.g., Per a 3)

26-95

79

Arthropod hemocyanin; hexameric copper containing proteins involved in oxygen transport in hemolymph

Group 4 (e.g., Bla g 4)

5-53

21

Lipocalin, found only in the male reproductive tract, binds tyramine and octopamine, involved in pheromone transport

Group 5 (e.g., Bla g 5)

7-72

23

Glutathione S-transferase (delta class); the equivalent allergen from American cockroach is a sigma class enzyme with low sequence similarity to Bla g 5

Group 6 (e.g., Bla g 6)

50

21

Troponin C; a muscle associated calcium binding protein

Group 7 (e.g., Bla g 7)

2-31

31

Tropomyosin

Group 8 (e.g., Bla g 8)

14

20

Myosin light chain

Group 9 (e.g., Per a 9)

34-100

43

Arginine kinase; shows sequence similarity with mite group 20 allergens, the meal moth allergen Plo i 1 and the shell B group 2 allergens such as Pen m 2

Group 10 (e.g., Per a 10)

82

28

Trypsin

Group 11 (e.g., Bla g 11

41

57

Amylase

Group 12 (e.g., Per a 12)

45

60-64

Chitinase

Bla g Chymotrypsin

29

23

Chymotrypsin

Bla g Enolase

25

45

Enolase

Bla g Vitellogenin

47

50

Shows sequence similarity with Der p 14

Lepidoptera Pyralidae, Indianmeal Moth (Plodia Interpunctella) Plo i 1 25 40 Plo i 2

8

Bombycidae Silkworm Larvae (Bombyx Mori) Bomb m 1 >90

Arginine kinase; shows sequence similarity with mite group 20 allergens and the cockroach group 9 allergens

12

Thioredoxin

42

Arginine kinase; shows sequence similarity with mite group 20 allergens and the cockroach group 9 allergens

Bomb m 7

Tropomyosin

Chelicerata: Arachnida Pyroglyphidae/Glycyphagidae/Acaridae/Echimyopodidae Mites, Dermatophagoides Pteronyssinus, D. Farinae, Euroglyphus Maynei, Blomia Tropicalis, Tyrophagus Putrescentiae, Lepidoglyphus Destructor Group 1 (e.g., Der p 1) >90 25 Cysteine protease Group 2 (e.g., Der p 2)

>90

14

MD-2–related protein, lipid binding, binds LPS

Group 3 (e.g., Der p 3)

90

25

Trypsin

Group 4 (e.g., Der p 4)

25-46

60

Amylase

Group 5 (e.g., Der p 5)

9-70

14

Function unknown; possible ligand-binding protein

CHAPTER 26  The Structure and Function of Allergens

405

TABLE 26.11  Physicochemical and Biochemical Characteristics of Arthropod-Derived Indoor

Aeroallergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Group 6 (e.g., Der p 6)

39

25

Group 7 (e.g., Der p 7)

24-53

26-31

Chymotrypsin

Group 8 (e.g., Der p 8)

40

27

Glutathione S-transferase

Group 9 (e.g., Der p 9)

>90

29

Collagenase-like serine protease

Group 10 (e.g., Der p 10)

81

36

Tropomyosin

Group 11 (e.g., Der f 11)

82

103

Paramyosin

Group 12 (e.g., Blo t 12)

50

16

May be a chitinase; shows sequence similarity with Der f 15 due to chitin-binding domain

Group 13 (e.g., Lep d 13)

11-23

15

Fatty acid–binding protein

Group 14 (e.g., Der f 14)

84

177

Vitellogenin or lipophorin

Group 15 (e.g., Der f 15)

95

98, 109*

Group 16 (e.g., Der f 16)

50-62

53

Gelsolin/villin

Group 17 (e.g., Der f 17)

35

30

Calcium-binding protein

Group 18 (e.g., Der f 18)

63

60

Chitinase; shows sequence similarity with the cockroach group 12 allergen and mite group 15; contains a chitin binding peritrophin-A domain (CBM-14) characteristic of peritrophic membranes of insects and a glyco 18 chitinase domain; is a GH 18 chitinase superfamily member

Group 19 (e.g., Blo t 19)

10

7

Group 20 (e.g., Der p 20)

0-44

40

Arginine kinase

Group 21 (e.g., Der p 21)

26

15

Function unknown; shows sequence similarity with group 5 allergens

Group 22 (e.g., Der f 22)

?

17

Shows sequence similarity with group 2 mite allergen; belongs to MD-2-related lipid recognition (ML) domain family; implicated in lipid binding

Group 23 (e.g., Der p 23)

74

14

Unknown function; shows sequence similarity with peritrophin-A domain and contains a chitin-binding domain

Group 24 (e.g., Der f 24)

100

13

Ubiquinol-cytochrome c reductase binding protein like protein

Group 25 (e.g., Der f 25)

76

34

Triosephosphate isomerase

Group 26 (e.g., Der f 26)

62

14

Myosin alkali light chain

Group 27 (e.g., Der f 27)

35-100

48

Serpin-trypsin inhibitor

Group 28 (e.g., Der f 28)

68

70

Heat shock protein

Group 29 (e.g., Der f 29)

70-86

16

Peptidyl-prolyl cis-trans isomerase (cyclophilin)

Group 30 (e.g. Der f 30)

63

16

Ferritin

Group 31 (e.g., Der f 30)

31-100

15

Cofilin, actin-binding protein

Group 32 (e.g., Der f 32)

15-100

35

Secreted inorganic pyrophosphatase

Group 33 (e.g., Der f 33)

25-100

52

Alpha tubulin

Group 34 (e.g., Der f 34)

68

16

Enamine/immine deaminase

Group 35 (e.g., Der f 35)

78

14

MD-2-related protein, shows sequence similarity with mite group 2 allergens

Group 36 (e.g., Der f 36)

42

23

Function unknown, contains a C-terminal C2 domain (pfam00168), which is associated with signal transduction enzymes

Group 37 (e.g., Der f 37)

21

29

Chitin binding protein

Group 38 (e.g., Der p 38)

78

15

Bacteriolytic enzyme belonging to the NlpC/P60 family

Group 39 (e.g. Der f 39)

9

18

Troponin C; a muscle associated calcium binding protein

Bactericidal permeability-increasing-like protein Function unknown; belongs to the juvenile hormone-binding family of proteins found in insects; may have lipid-binding properties; binds the lipopeptide polymyxin B

Chitinase; shows sequence similarity with the cockroach group 12 allergen and mite group 18; contains a chitin binding peritrophin-A domain (CBM-14) characteristic of peritrophic membranes of insects and belongs to the GH 18 chitinase family

Function unknown; shows high sequence similarity with putative antibacterial peptides from helminthic worms

HSP, Heat shock protein; MD-2, myeloid differentiation factor-2. *Glycosylated forms, DNA sequence indicates a nonglycosylated protein of 63 KDa. Frequency determined in dogs with atopic dermatitis.

406

SECTION B  Aerobiology and Allergens

consistent with its upregulation on feeding. Analogously, Bla g 2 shows sequence similarity with aspartate proteases such as pepsin, but it is proteolytically inactive, and its true function within the cockroach is unclear.

House Dust Mite–Derived Aeroallergens HDMs are ubiquitous, but the most clinically important species belong to the Pyroglyphidae, Acaridae, Glycyphagidae, and Echimyopodidae families (Table 26.11). They include Dermatophagoides pteronyssinus, D. farinae, and Euroglyphus maynei found in temperate climates; Blomia tropicalis, found in subtropical and tropical climates; and the storage mites such as Lepidoglyphus destructor, Tyrophagus putrescentiae, Acarus siro, and Glycyphagus domesticus that infest stored foods such as grains. Thus far, 39 allergen groups have been described, and although certain proteins are allergenic in both mites and cockroaches (albeit denominated differently), mite allergens equivalent to cockroach groups 1 to 4 and enolase are absent and, conversely, several mite groups are absent from cockroaches. Illustrating similarities, trypsin, chymotrypsin, amylase, vitellogenins, arginine kinases, and chitinases are allergenic in both, as are the muscle or cytoskeletal associated tropomyosins, troponins and myosins. Mites produce several allergens not produced by cockroaches, for example the group 1 cysteine proteases, the group 9 collagenolytic serine proteases and the group 38 peptidoglycan D,L endopetidases. In addition, mites produce allergens that bind various ligands in the gut that are absent from cockroaches. For example, mites produce three groups of allergens (2, 22, and 35) having approximately 40% sequence identity and which possess an MD-2-related lipid-recognition domain, which may interact with toll-like receptor 4 (TLR4). Similarly, the group 5, 7, and 21 allergens possess cavities that may bind ligands, and Der p 7 shares sequence similarity with proteins such as bactericidal permeabilityincreasing (BPI) proteins and the odorant-binding protein. In addition, the group 13 allergens are designated as fatty acid binding proteins, and the high molecular weight group 14 allergens are characterized as lipidtransporting proteins. Mites also produce two allergens associated with the mite peritrophic membrane that contain chitin binding domains, but the group 12, in contrast to the group 23 allergen, is restricted to B. tropicalis and L. destructor. The immunodominant mite allergens are the groups 1 and 2 proteins, as evidenced by the number of individuals sensitized to them as well as the proportion of total serum IgE accounted for by allergenspecific IgE (50% to 70%, for group 1 and 2).27 However, a number of other allergens such as the groups 10-12, 14, 15, 18, 24, 25, 28-30, 34, 35 and 38 are major allergens based on frequency of reactivity. Other mite allergens have an intermediate, minor, or undetermined role. However, immunodominant allergens may vary by geographic territory and mite population, as suggested by the D. farinae group 24 ubiquinol-cytochrome c reductase binding-like protein shown to be immunodominant in a Chinese community, in contrast with data from elsewhere. Similarly, amylase is a more prominent allergen in atopic North Western Australian aboriginal people compared with atopic, non-aboriginal Australians living in metropolitan Perth. Interestingly, as with other clinically important allergen sources, susceptible children are initially sensitized to a limited number of mite allergens (groups 1, 2 and 23) and either remain paucisensitized or become polysensitized as they develop.

Occupation-Associated Aeroallergens Occupational allergens are proteins used industrially that sensitize through unprotected exposure to environmental dusts.28 The occupation involved usually describes the resulting condition—for example, baker’s asthma with flour allergens. The main allergens include various

hydrolytic enzymes, egg powder, latex products, and flours derived from various seeds (Table 26.12). Exposure may be experienced outside occupational settings, an example being in port cities where the environmental release of soybean husks occurring during transfer between silos and cargo ships resulted in soybean allergy. Although inhalation is the major route of occupational exposure, sensitization and provocation may also occur percutaneously, as, for example, with natural rubber allergens present in latex gloves.

Enzyme Aeroallergens Derived from Fungal, Bacterial, and Mammalian Sources.  Hydrolytic enzymes used industrially to facilitate the breakdown of biologic polymers such as proteins and polysaccharides may be allergenic. The clinically important examples include bacterial subtilisins and amylases used in the detergent industry, the mammalian serine proteases (e.g., trypsin, chymotrypsin, pepsin) used in pancreatic supplements in the treatment of cystic fibrosis, the fruit cysteine proteases papain and bromelain used in the pharmaceutical industry (and which may also be ingested allergens), and the fungal amylases and various other carbohydrases such as β-xylosidase, cellulase, and glucoamylase used in the baking industry. The major egg aeroallergens in egg powder are derived from either the white or the yolk.29 Egg white allergens include ovomucoid, ovalbumin, conalbumin, and lysozyme, whereas that in yolk is α-livetin (chicken albumin). The most commonly recognized egg allergens, however, are Gal d 3 and Gal d 5.

Seed-Derived Aeroallergens.  The major seed-derived aeroallergens are proteins involved in defense, storage, or metabolism18 (Table 26.12), and allergy arises from exposure to the flours produced from them, particularly wheat, barley, and rye belonging to the Triticeae tribe of the Pooideae subfamily. Several allergens from wheat and rye flour are characterized, none being major. Those sensitizing bakers include major defense-related allergens such as the 12 to 15 kDa amylase/trypsin inhibitory albumin proteins (e.g., Poaceae group 15), several α-amylase inhibitors (e.g., Poaceae groups 28, 29, 30), the nsLTP allergens, defensins, and trypsin inhibitors from soybean husks. The abundant hydrophobic and cysteine-rich nsLTPs from soybeans are seed surface proteins that reduce wettability and are, therefore, thought to play a role in defense. Several minor allergens have been described, particularly from the Poaceae family, and include gliadins and thioredoxin. Natural Rubber Latex Aeroallergens.  Allergy to proteins present in natural rubber latex from the lactiferous rubber tree (Hevea brasil­ iensis) arises through airborne, percutaneous, or parenteral routes and is prevalent among individuals in frequent contact with natural rubber latex materials (e.g., gloves and surgical materials).28 In health care workers airborne exposure is prominent and results from the attachment of latex proteins to dry lubricants that are used to facilitate donning of gloves, although sensitization rates have decreased with new manufacturing techniques. For patients, exposure is parenteral and caused by leaching from latex materials used surgically. Fifteen allergen groups have been delineated (Table 26.13). In patients with spina bifida, the immunodominant allergens are Hev b 1, 3, and 5, whereas in health professionals they are Heb v 4, 5, 6. The remaining allergens are considered to be minor, of which some are pathogenesis-related proteins (Table 26.7).

INGESTED ALLERGENS Seven sources of ingested allergens (eggs, peanut, milk, nuts, soy, fish, wheat) are generally considered to account for more than 90%

407

CHAPTER 26  The Structure and Function of Allergens

TABLE 26.12  Physicochemical and Biochemical Characteristics of Occupation-Associated

Aeroallergens

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Fungi-Derived Aspergillus Niger Asp n 14

88

105

Xylosidase

Asp n 18

?

34

Vacuolar serine protease

Asp n 25

>50

66-100

Asp n Cellulase

8

26

Asp n Glucoamylase

5

Asp n Pectinase

40

35

Poly(1,4)-α-D-galacturonidase

71

34

Alkaline serine protease; belongs to subtilase family

Allergen

Aspergillus Oryzae Asp o 13

Histidine acid phosphatase (phytase) 1,4-(1,3;1,4)-β-D-Glucan glucanhydrolase 1,4-α-D-Glucan glucanhydrolase

Asp o 21

56

53

α-Amylase

Asp o Lactase

29-31

135

1,4-β-D-Galactoside galactohydrolase

?

34

Aspartate protease; shows sequence similarity with mammalian and cockroach pepsins

Bacteria-Derived Bacillus Subtilis Alcalase

>50

28

Subtilisin serine protease

Bacillus Licheniformis Esperase

>50

28

Subtilisin serine protease

Clostridium Histolyticum Collagenasea

>50

68-125

Metalloprotease

19-32

20-60

Pronase B, a mixture of proteases

Vertebrate-Derived Trypsin (porcine)

?

24

Serine protease; shows sequence similarity with mite groups 3, 6, and 9 allergens

Chymotrypsin (bovine)

?

25

Serine protease; shows sequence similarity with mite groups 3, 6, and 9 allergens

Pepsin (porcine)

?

35

Aspartate protease; shows sequence similarity with Bla g 2 and renin

Chicken (Gallus Domesticus) - Egg White Gal d 1 34-38

20

Ovomucoid, protease inhibitor

Gal d 2

32

43

Ovalbumin, function unknown but protein shows sequence similarity 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)

Cryphonectria Parasitica Renin

Streptomyces Griseus Empynase

Chicken (Gallus Domesticus) - Egg Yolk Gal d 5 >50

65-70

Plant Seed–Derived Angiosperms: Fabaceae Seed Husk or Flour Allergens, Soybean (Glycine Max) Gly m 1 90 8

Serum albumin (α-livetin)

Cysteine-rich, hydrophobic seed protein Continued

408

SECTION B  Aerobiology and Allergens

TABLE 26.12  Physicochemical and Biochemical Characteristics of Occupation-Associated

Aeroallergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Gly m 2

100

8

Defensin

Gly m Agglutinin

31

31

Agglutinin

6-86

22

Trypsin inhibitor

Gly m TI

Angiosperms: Pooideae Triticeae: Barley (Hordeum vulgare), Wheat (Triticum Aestivum, T. Turgidum), Rye (Secale Cereale) b Group 14 (e.g., Tri a 14) 11 9 Nonspecific lipid transfer protein type 1, exists as isoforms (9 and 9.7 kDa) Group 15 (e.g., Tri a 15)

9

13

α-Amylase/trypsin inhibitor; shows sequence similarity with 2S albumin allergens

Group 19 (e.g., Tri a 19)

1

?

Omega 5 gliadin

Group 21 (e.g., Tri a 21)

11

?

Alpha, beta gliadin

Group 25 (e.g., Tri a 25)

18

13

Thioredoxin

Group 27 (e.g., Tri a 27)

27

27

Interferon-inducible thiol reductase

Group 28 (e.g., Tri a 28)

24

13

α-Amylase inhibitor

Group 29 (e.g., Tri a 29)

20

15

α-Amylase inhibitor CM2

Group 30 (e.g., Tri a 30)

10

16

Tetrameric α-amylase inhibitor CM3

Group 31 (e.g., Tri a 31)

10

?

Triose phosphate isomerase

Group 32 (e.g., Tri a 32)

14

?

Peroxiredoxin

Group 33 (e.g., Tri a 33)

8

43

Trypsin inhibitor

Group 34 (e.g., Tri a 34)

5

?

Glyceraldehyde 3-phosphate dehydrogenase

Group 35 (e.g., Tri a 35)

2

?

Dehydrin

Group 39 (e.g., Tri a 39)

18

?

Serine protease inhibitor

36-73

23

Papain, cysteine protease

Angiosperms: Bromeliaceae Pineapple (Ananas Comosus) Ana c 1 42

15

Profilin

Ana c 2

23

Bromelain, cysteine protease

Plant Fruit–Derived Angiosperms: Annonaceae Pawpaw (Carica Papaya) Car p 1

2-75

a

Represents a mixture of proteases. Frequency data are the mean response of all bakers studied from Spain, Netherlands, and Germany obtained using the same panel of recombinant wheat allergens. (Sander I, Rihs HP, Doekes G, et al. Component-resolved diagnosis of baker’s allergy based on specific IgE to recombinant wheat flour proteins. J Allergy Clin Immunol 2015;135:1529-37.) b

of food-induced allergy (Table 26.14). Ingested food proteins can evoke a variety of symptoms, with certain sources often linked with specific allergic manifestations. For example, peanuts, fish, and crustaceans are often associated with anaphylaxis, eggs and milk are associated with atopic dermatitis, and wheat allergens may be associated with exercise-induced anaphylaxis. Food allergens tend to be stable when exposed to heat and acid, and more resistant to proteases than nonfood allergens, thereby facilitating their entry through the gut mucosa.30

Animal-Derived Ingested Allergens The major allergens in cow’s milk include α-lactalbumin, β-lactoglobulin, and casein, and there is a high degree of cross-reactivity with sheep and goat milk proteins, and several minor allergens have been identified (e.g., serum albumin, immunoglobulin, and transferrin).31 The other

main animal-derived allergens include various muscle proteins, as well as the red meat–associated alpha gal epitope associated where the sensitizing allergen is not red meat, but rather exposure to the tick Ambly­ omma americanum. The major allergen group in fish is the muscle-derived calciumbinding parvalbumin (a 3 EF-hand calcium binding protein32) (Table 26.14). Despite the significant global consumption of cartilaginous fish (Chondrichthyes), only bony fish (Osteichthyes) cause problems. This distinction appears related in some way to the presence of the αbut not the β-parvalbumin lineage, in cartilaginous fish. The sequence identity between them is approximately 50% and likely explains the lack of reported allergenic cross-reactivity, but this does not account for the lack of sensitization to Chondrichthyes α-parvalbumin. The other fish allergens include β-enolases and aldolases. All three proteins are allergens in chicken meat.

CHAPTER 26  The Structure and Function of Allergens

409

TABLE 26.13  Physicochemical and Biochemical Characteristics of Natural Rubber Latex Aeroallergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Euphorbiaceae Rubber Tree (Latex) (Hevea Brasiliensis) Hev b 1 50-82 14 Rubber elongation factor; exists as 58-kD homotetramer with pI of 8.5 Hev b 2

20-61

34

Endo-1,3-β-glucosidase (PR-2) Shows some sequence similarity with rubber elongation factor, Hev b 1

Hev b 3

79

24

Hev b 4

25-89

53-55

Hev b 5

56-92

16a

Shows sequence similarity with an acidic protein from kiwifruit and potato

Hev b 6

83

20

Prohevein; chitin-binding lectin; causes latex agglutination; native hevein exists as 5-kDa protein (PR-6)

Microhelix component, lecithinase homolog

Hev b 7

8-49

42

A patatin-like protein with lipid acyl-hydrolase and phospholipase A2 activity; shows cross-reactivity with Sol t 1

Hev b 8

24

15

Profilin, may exist in dimeric form

Hev b 9

15

51

Enolase

Hev b 10

4

26

Manganese superoxide dismutase; shows sequence similarity with Asp f 6

Hev b 11

3

30

Class I chitinase (PR-3)

Hev b 12

24

9

Nonspecific lipid transfer protein type 1

Hev b 13

78

42-46

Hev b 14

3-67

30

Chitinase

Hev b 15

?

7.5

Serine protease inhibitor

Esterase; shows sequence similarity with early nodule-specific protein from legumes

pI, Isoelectric point; PR-2, PR-3, PR-6, pathogenesis-related protein groups 2, 3, 6. a Hevein is a 4.7-kDa chitin-binding domain from this precursor.

TABLE 26.14  Physicochemical and Biochemical Characteristics of Animal-Derived

Ingested Allergens Allergen Mammalian-Derived Cow (Bos Taurus) Bos d 4

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

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

>90

20-30

Caseins (see Bos d 9-12)

Bos d 9

98

20-30

Alpha S1-casein

Bos d 10

95

20-30

Alpha S2-casein

Bos d 11

95

20-30

Beta-casein

Bos d 12

95

20-30

Kappa-casein

75-kDa allergen

16

75

Transferrin

Bird-Derived Chicken (Gallus Domesticus) - Egg White Gal d 1 34-38

28

Ovomucoid, a Kazal-type serine protease inhibitor

Gal d 2

32

43

Ovalbumin, serine protease inhibitor

Gal d 3

47-53

78

Ovotransferin

Gal d 4

15

14

Lysozyme

Chicken (Gallus Domesticus) - Egg Yolk Gal d 5 >50 Gal d 6

18

64-70 35

α-Livetin, a serum albumin YGP42, fragment of vitellogenin-1 precursor Continued

410

SECTION B  Aerobiology and Allergens

TABLE 26.14  Physicochemical and Biochemical Characteristics of Animal-Derived

Ingested Allergens—cont’d

Mol. Size (kDa)

Function

Chicken (Gallus Domesticus) - Meat Gal d 8 61

12

Parvalbumin

Gal d 9

75

40

Enolase

Gal d 10

83

50

Aldolase

Allergen

Frequency of Reactivity (%)

Fish/Amphibians/Crustaceans/Mollusks Bony Fish, e.g., Atlantic Salmon (Salmo Salar), Cod (Gadus Callarias), Tuna (Thunnus Albacares) Group 1 (e.g., Gad c 1) 100 12 Beta-parvalbumin, calcium-binding protein Group 2 (e.g., Gad c 2)

59

47

Beta-enolase

Group 3 (e.g., Gad c 3)

39

40

Aldolase

Edible Frog (Rana Esculenta) Rana e 1 ?

12

Alpha-parvalbumin

Rana e 2

12

Beta-parvalbumin

?

Crustaceans (Shrimps, Prawns, Crabs, Lobsters) Shrimps/Prawns (Metapenaeus, Penaeus, Litopenaeus, Artemia, and Crangon Species) Group 1 (e.g., Met p 1) >50 34-36 Tropomyosin Group 2 (e.g., Pen m 2)

70

39

Arginine kinase

Group 3 (e.g., Lit v 3)

55

20

Myosin light chain 2

Group 4 (e.g., Lit v 4)

38

22

Sarcoplasmic EF-hand calcium binding protein

Group 5 (e.g., Cra c 5)

20

17.5

Myosin light chain 1

Group 6 (e.g., Cra c 6)

24

21

Troponin C; a muscle associated calcium binding protein

Group 8 (e.g., Arc s 8)

44

28

Triosephosphate isomerase

Crabs (Charybdis Feriatus, Scylla Serrata, S. Paramamosain, Portunus Pelagicus, Eriocheir Sinensis) Group 1 (e.g., Cha f 1) >50 34 Tropomyosin Group 2 (e.g., Scy s 2) Eri s 2

100

40

Arginine kinase

66

28

Ovary development-related protein, shows sequence similarity to Panulirus stimpsoni mannose-binding lectin

Lobsters (Homarus Americanus), Spiny Lobster (Panulirus Stimpsoni) Hom a 1 ? 37 Tropomyosin Hom a 3

8

23

Myosin light chain

Hom a 6

24

20

Troponin C; a muscle associated calcium binding protein

Mollusks (Oysters, Mussels, Snails, Squid, Octopus) Squid (Todarodes Pacificus) Group 1 (e.g., Tod p 1) >50 38

Tropomyosin

Octopus (Octopus Fangsiao, O. Vulgaris, O. Luteus, Enteroctopus Dofleini) Group 1 (e.g., Oct f 1) ? 35 Tropomyosin Group 2 (e.g., Oct f 2)

?

38

Arginine kinase

Oct v PM

?

100

Paramyosin

Oct f TPI

?

28

Triosephosphate isomerase

Snail (Helix Aspersa) Group 1

18

36

Tropomyosin

CHAPTER 26  The Structure and Function of Allergens In crustaceans (shrimps, prawns, crabs, lobster), the major allergen groups are the tropomyosins, the actin-associated arginine kinases, the myosin light chains (1 and 2), the sarcoplasmic calcium binding proteins, the troponins C, and the triosephosphate isomerases (Table 26.14). Mollusks, (octopus, squid, snails) share a more limited but similar spectrum of allergen groups (Table 26.14). Several crustacean protein groups are heat stable (tropomyosins, myosins) and remain allergenic when ingested. All are muscle-associated but, recently, a crab ovary–specific allergen has been described (Eri s 2).33 Because arthropods evolved from crustaceans, they share similar structural properties, resulting in allergenic cross-reactivity that is often manifested as OAS (Table 26.15).34

Seed-Derived Ingested Allergens The clinically important seed-derived allergens are found in peanuts and include the cupin and conglutin storage proteins, PR-10 protein, an nsLTP and peanut lectin, (Table 26.16), whereas the minor ones include profilins, glycinins, and conglutins. The most important ingested allergens from soybean include Gly m 1, Gly Bd 28K, and Gly m 3. Gly m 1 is an nsLTP, whereas Gly Bd 30K/P34 is a cysteine protease with sequence similarity to members of the papain family including the HDM allergen Der p 1. In contrast, Gly m 3 is a profilin, and Gly m Bd 28K is similar to a range of vicilin-related storage proteins, including

411

Ara h 1, and may possess chitin-binding properties. Allergens belonging to the vicilin family are also present in walnut and coconuts. Profilins are usually minor allergens, but in both soybean and Mercurialis annua pollen, they are major ones. The soybean minor allergens include members of the glycinin family that share sequence similarity with Ara h 3 and the α-subunits of β-conglycinin. Seed proteins belonging to a group of methionine-rich 2S albumins have been described in walnut and brazil nuts, as well as sunflower and sesame seeds.

Fruit- and Vegetable-Derived Ingested Allergens Sensitivity to allergens from fruits and vegetables probably arises from prior sensitization to pollen allergens and manifests as OAS or pollenfood syndrome and is related to cross-reactivity between proteins in the respiratory allergen source and those in food, usually pan-allergens in fruit (Table 26.15). The condition is associated with uncooked food, because cooking and processing results in protein denaturation, destroying conformational epitopes. Clinical manifestations range from mild oropharyngeal symptoms to severe systemic reactions. OAS reactions are also classified by reference to the respiratory sensitizer and oral elicitors other than fruit and vegetables (Table 26.15). The allergens associated with latex–fruit allergies are endo-1,3 β-glucosidases, patatin-like proteins, profilins, and nonspecific lipid transfer proteins;

TABLE 26.15  Allergens Involved in Cross-Reactivity Syndromes Syndrome/ Sensitizing Source

Provoking Source

Cross-Reacting Allergen(s) in Sensitizing Source

Invertebrate- and Vertebrate-Derived Arthropod–Shellfish Mites Crustaceans and mollusks

Tropomyosin (Der p 10)

Mites

Anisakis simplex

Tropomyosin (Der p 10)

Cockroach

Crustaceans and mollusks

Tropomyosin (Per a 7)

Bird–Egg Bird material

Egg yolk

Serum albumin (Gal d 5)

Egg–Egg Egg white powder

Egg-containing foods

Lysozyme (Gal d 4)

Fish–Chicken Chicken meat

Fish

Parvalbumin (Gal d 8), aldolase (Gal d 9), enolase (Gal d 10)

Pork–Cat Animal meat

Animal danders

Serum albumin (Bos d 6)

Plant-Derived Latex–Fruit Latex

Avocado, potato, banana, tomato, chestnut, kiwifruit, herbs, carrot

Endo-1,3 beta glucosidase (Hev b 2), patatin-like proteins (Hev b 7), profilins (Hev b 8), and nonspecific lipid transfer proteins type 1 (Hev b 12)

Apple, carrot, cherry, pear, peach, plum, fennel, walnut, potato, spinach, wheat, buckwheat, peanut, honey, celery, kiwifruit, persimmon, strawberry, peanut, soybean, almond, hazelnut

Pathogenesis-related protein PR-10 (Bet v 1), profilin (Bet v 2), and Bet v 6 analogs

Grass pollen

Melon, tomato, orange, cherry, potato, peach

Profilin (grass group 12)

Japanese cedar pollen

Melon, apple, peach, kiwifruit

Pectate lyase (Cry j 1), thaumatin-like proteins (Cry j 3)

Mugwort pollen

Celery, carrot, spices, melon, apple, chestnut, camomile, watermelon, hazelnut

Nonspecific lipid transfer protein (Art v 3), profilin (Art v 4), PR-12 (Art v 1)

Ragweed pollen

Melon, camomile, honey, banana, sunflower seeds, potato, zucchini

Pectate lyase (Amb a 1)

Pollen–Fruit Birch pollen

412

SECTION B  Aerobiology and Allergens

TABLE 26.16  Physicochemical and Biochemical Characteristics of Seed- and Fruit-Derived

Ingested Allergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Angiosperms: Fabaceae Peanut (Arachis Hypogaea) Ara h 1 >90

64

Cupin (vicilin type), a7S globulin, seed storage protein

Ara h 2

>90

17

Conglutin, 2S albumin, seed storage protein

Ara h 3

35-53

60

Cupin (legumin type), 11S globulin, seed storage protein

Ara h 5

16

15

Profilin

Ara h 6

38

15

Conglutin (2S albumin)

Ara h 7

43

15

Conglutin (2S albumin)

Ara h 8

85

17

Pathogenesis-related protein PR-10; shows sequence similarity with Bet v 1

Ara h 9

91

10

Nonspecific lipid transfer protein, type 1

Ara h 10

?

16

Oleosin

Ara h 11

?

14

Oleosin

Ara h 12

?

8

Defensin

Ara h 13

?

8

Defensin

Ara h 14

45

17.5

Oleosin

Ara h 15

45

17

Oleosin

Ara h 16

16

8.5

Nonspecific lipid transfer protein type 2

Ara h 17

16

11

Nonspecific lipid transfer protein type 1

Ara a Agglutinin

50

27

Lectin

Soybean (Glycine Max) Gly m 1

95

7

Hydrophobic seed protein

Gly m 2

90

8

Defensin

Gly m 3

69

14

Profilin

Gly m 4

5-100

17

Pathogenesis-related protein PR-10, Bet v 1–like

Gly m 5

3-54

48-65

β-Conglycinin, various subunits

Gly m 6

12-20

52-61

Member of the G1 glycinin family; shows sequence similarity with Ara h 3; exists as subunits

Gly m 7

78

76.2

Seed-specific biotinylated protein

Gly m 8

55

12

2S seed protein

Gly m Bd 28K Gly m Bd 30K/P34

>50

22

Vicilin-like protein, shows sequence similarity with Ara h 1

90

34

Syringolide receptor, seed vacuolar protein; shows sequence similarity with mite group 1 allergen, papain, and bromelain but not active

Gly m Agglutinin

31

30

Lectin

Gly m CPI

67

25

Cysteine protease inhibitor

Gly m EAP

67

60

Embryonic abundant protein

Gly m TI

10

20

Trypsin inhibitor

Angiosperms: Lecythidaceae Brazil Nut (Bertholletia Excelsa) Ber e 1 100 Ber e 2

?

9

2S Albumin

29

11S Globulin

Angiosperms: Juglandaceae English Walnut (Juglans Regia), Black Walnut (Juglans Nigra) Group 1 (e.g., Jug r 1) 75 15-16 2S Albumin Group 2 (e.g., Jug r 2)

60

44

Group 13 (e.g., Jug r 3)

80

9

Vicilin-like glycoprotein

Group 4 (e.g., Jug r 4)

57

50-60

11S globulin seed storage protein

Group 5 (e.g., Jug r 5)

?

20

PR-10 related protein

Nonspecific lipid transfer protein type 1

Group 6 (e.g., Jug r 6)

25

47

Vicilin-like cupin

Group 7 (e.g., Jug r 7)

22

13

Profilin

413

CHAPTER 26  The Structure and Function of Allergens

TABLE 26.16  Physicochemical and Biochemical Characteristics of Seed- and Fruit-Derived

Ingested Allergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Angiosperms: Musaceae Banana (Musa Acuminata, M. Cavendishii) Mus a 1 44 15

Profilin

Mus a 2

Chitinase - hevein-like

34

33

Mus a 3

?

9

Mus a 4

72

20

Thaumatin-like protein

Mus a 5

84

30

β1-3-glucanse; show sequence similarity with Ole 9

Mus a 6

?

27

Ascorbate peroxidase

Angiosperms: Asteraceae Sunflower (Helianthus Annuus) Hel a 3 ? Hel a 2S Albumin

66

Angiosperms: Apiaceae Celery (Apium Graveolens) Api g 1 100

9

Nonspecific lipid transfer protein type 1

Nonspecific lipid transfer protein type 1

16/17

2S Albumin

15

Pathogenesis-related protein PR-10

Api g 2

26-58

9

Api g 3

?

28

Nonspecific lipid transfer protein type 1 Chlorophyll Ab binding protein, chloroplast

Api g 4

23

14

Profilin

Api g 5

100

58

Flavine adenine dinucleotide–dependent oxidase

Api g 6

38

7

Nonspecific lipid transfer protein type 2

Angiosperms: Rosaceae Apple (Malus Domestica), Cherry (Prunus Avium), Peach (Prunus Persica), Plum (Prunus Cerasifera), Almond (Prunus Dulcis), Apricot (Prunus Armeniaca), Strawberry (Fragaria Ananassa) Group 1 (e.g., Pru av 1) 89 9 Pathogenesis-related protein PR-10, related to Bet v 1, strawberry allergen (Fra a 1) involved in flavonoid pigment biosynthesis Group 2 (e.g., Pru av 2)

100

23-30

Thaumatin-like protein

Group 3 (e.g., Pru av 3)

3-100

10

Nonspecific lipid transfer protein type 1

Group 4 (e.g., Pru av 4)

16-17

14

Profilin

Group 7 (e.g., Pru p 7)

42

7

Angiosperms: Solanaceae Potato (Solanum Tuberosum) Sol t 1 74

43

Patatin, defense-related storage protein; has phospholipase A2 activity

Sol t 2

51

21

Cathepsin D protease inhibitor

Sol t 3

43

21

Cysteine protease inhibitor

Sol t 4

58

16

Aspartate protease inhibitor

Tomato (Solanum Lycopersicum) Sola l 1 32

14

Profilin

Sola l 2

40

50

Beta-fructofuranosidase

Sola l 3

?

9

Sola l 4

?

?

Gibberellin-regulated protein

Nonspecific lipid transfer protein type 1 Pathogenesis-related protein PR-10, related to Bet v 1

Sola l 5

?

19

Sola l 6

>50

7

Peptidyl-prolyl cis-trans isomerase (cyclophilin) Nonspecific lipid transfer protein type 2

Sola l 7

71

12.5

Nonspecific lipid transfer protein type 1 Continued

414

SECTION B  Aerobiology and Allergens

TABLE 26.16  Physicochemical and Biochemical Characteristics of Seed- and Fruit-Derived

Ingested Allergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Angiosperms: Cucurbitaceae Melon (Cucumis Melo) Cuc m 1 100

67

Cucumisin; subtilisin serine protease

Cuc m 2

65-100

14

Profilin

Cuc m 3

71

17

Pathogenesis-related protein PR-1; shows sequence similarity with the vespid group 5 allergens

Angiosperms: Actinidiaceae Kiwifruit (Actinidia Chinensis/Deliciosa) Group 1 (e.g., Act d 1) 100

30

Cysteine protease, actinidin

Group 2 (e.g., Act d 2)

10

24

Thaumatin-like protein

Group 3 (e.g., Act d 3)

33

40

Unknown function

Group 4 (e.g., Act d 4)

20

11

Phytocystatin

Group 5 (e.g., Act c 5)

2-100

26

Kiwellin

Group 6 (e.g., Act c 6)

72

18

Pectin methylesterase inhibitor

Group 7 (e.g., Act d 7)

32

50

Pectin methylesterase

Group 8 (e.g., Act c 8)

43

17

Pathogenesis-related protein PR-10

Group 9 (e.g., Act d 9)

20

14

Profilin

Group 10 (e.g., Act c 10)

33

10

Nonspecific lipid transfer protein type 1

Group 11 (e.g., Act d 11)

22

17

Latex protein

Group 12 (e.g., Act d 12)

71

50

Cupin, 11S globulin

Group 13 (e.g., Act d 13)

18

11

2S Albumin

Angiosperms: Brassicaceae Oilseed Rape (Brassica Napus), Turnip (Brassica Rapa), Cabbage (Brassica Oleracea) Group 1 (e.g., Bra n 1) 38-74 10-14 2S Albumin Group 2 (e.g., Bra r 2)

82

25

Group 3 (e.g., Bra o 3)

86-94

9

Chitinase, Prohevein homolog; shows sequence similarity with Heb b 6

Mustard Seed (Sinapis Alba L.) Group 1 (e.g., Sin a 1) 50-78

14

2S Albumin

Group 2 (e.g., Sin a 2)

47-59

51

11S Globulin

Group 3 (e.g., Sin a 3)

41-100

12

Nonspecific lipid transfer protein type 1

Group 4 (e.g., Sin a 4)

24-27

13

Profilin

Angiosperms: Euphorbiaceae Castor Bean (Ricinus Communis) Ric c 1 96

11

2S Albumin

Ric c 2

?

47

11S Globulin

Ric c 3

85

12

2S Albumin; shows sequence similarity with Ric 1 and similar seed allergens from sources such as peanut and mustard (e.g., Ara h 2)

Nonspecific lipid transfer protein type 1

Angiosperms: Polygonaceae Buckwheat (Fagopyrum Esculentum) Moench (F. Tataricum) Group 2 (e.g., Fag e 2) 78 16 2S Albumin Group 3 (e.g., Fag e 3)

?

19

7S Vicilin

Group 4 (e.g., Fag e 4)

71

12

Antimicrobial peptide

Group 5 (e.g., Fag e 5)

86

55

8S Globulin

Angiosperms: Poaceae Wheat (Triticum Aestivum) Tri a 14 60-100 Tri a 18

?

9 21

Nonspecific lipid transfer protein type 1 Agglutinin isolectin 1

CHAPTER 26  The Structure and Function of Allergens

415

TABLE 26.16  Physicochemical and Biochemical Characteristics of Seed- and Fruit-Derived

Ingested Allergens—cont’d Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Tri a 19

2-100

65

ω-Gliadin; shows sequence similarity with rye secalins and barley hordein

Tri a 20

?

33

Gamma gliadin

Tri a 25

13-47

13

Thioredoxin

Tri a 26

17-59

88

High molecular weight glutenin

Tri a 36

57

46

Low molecular weight glutenin

Tri a 37

11-23

13

Alpha purothionin, PRP-13

20

Mitochondrial ubiquitin ligase activator of NF-κB 1

Tri a 41

5

Tri a 42

13

31

Hypothetical protein from cDNA

Tri a 43

13

36

Hypothetical protein from cDNA

Tri a 44

4

36

Endosperm transfer specific PR60 precursor

Tri a 45

13

34

EF-1, elongation factor 1

Gymnosperms: Pinaceae Stone Pine (Pinus Pinea), Korean Pine (P. Koraiensis) Pin p 1 75 15-17 2S Albumin Pin k 2

4/14

48

Vicilin

See also previous tables for inhaled allergens derived from these sources. a Many seed-derived proteins were originally classified based on their sedimentation coefficient, which is expressed in nonmetric Svedberg units (S).

Injected allergens typically are arthropod-derived. Venoms and saliva from biting and stinging insects are complex and serve defensive and nutritional functions for the host such as inhibition of blood clotting (thrombin inhibition, inhibition of agonist-induced platelet aggregation), kinin liberation, neurotoxicity, immunomodulation, and tissue permeability. The most important allergens derive from the stinging or biting hymenopteran, dipteran, and hemipteran insects and include venoms from bees, wasps, hornets, paper wasps, and ants, and salivary proteins from mosquitoes, flies, ticks, kissing bugs, and fleas (Table 26.17).

(Table 26.17). Melittin accounts for approximately 50% of the injected venom in this species (although absent from wasps and ants) and causes pain by activation of transient receptor potential channels and nociceptive sodium channels. Allergenic enzymes present in bees include phospholipase A2, the group 2 hyaluronidases, the group 3 acid phosphatases, and various peptidases. Some enzymes are common to wasps, such as the hyaluronidases and acid phosphatases, but the phospholipase present in wasp venom is type A1, which cleaves phospholipid differently to type A2.35 The phosphatases are likely to be involved in inhibition of platelet aggregation, whereas the hyaluronidases facilitate venom spread by cleaving extracellular matrix. Group 5 allergens, absent from bee venom, are major allergens in wasps and belong to a family of cysteine-rich secretory proteins homologous with PR-1 proteins and those associated with mammalian reproduction. Additionally, other venom enzymes show sequence similarity with sperm hyaluronidases and prostatic-like acid phosphatases, suggesting they probably evolved from proteins associated with insect reproduction. Attempts have been made to determine venom-associated marker allergens, but cross-reactivity between certain groups of allergens is high.36 Several venom allergens have also been described in the fire and jumper ants,37 some of which correspond to those in wasp or bee venoms such as the vespid group 5 allergen and phospholipase A1, although minor. The major but not immunodominant allergen in the jumper ant is pilosulin, a basic, low molecular weight peptide,38 whereas the major fire ant allergen has unknown function. The major ant venom allergens are the group 2 proteins of unknown function.

Venom-Derived Allergens

Saliva-Derived Allergens

Venoms of bees, wasps, and hornets contain various bioactive components, several of which are allergenic, with the potential to cause anaphylaxis in sensitized patients. At least 12 allergen groups have been delineated for honeybee, including various enzymes and melittin

Saliva from hematophagous insects such as fleas, mosquitos, and horse flies contains several minor and major allergens (Table 26.17).39 Most extensively studied is the mosquito, in which at least 15 allergen groups have been delineated. Compared with other allergen sources, no allergen

those associated with arthropod crustaceans are tropomyosins; with egg–egg, lysozymes are involved. In northern and central Europe, the important respiratory sensitizers are the Fagales tree pollen groups 1 and 2 allergens, whereas in the Mediterranean region, the nsLTP allergens from mugwort, the 34 kDa and 60 kDa allergens of unknown function from pellitory, the thaumatin-like allergens, and endochitinases are prominent. Because of their lability, the Fagales tree pollen groups 1 and 2 allergens evoke mild oral symptoms, in contrast with the stable nsLTP allergens, which may evoke anaphylaxis. The allergens associated with exposure to kiwifruit and fig include cysteine proteases and uncharacterized proteins. Allergens from potato include patatin and several protease inhibitors that can inhibit the activities of three of the major classes of protease (Table 26.15).

INJECTED ALLERGENS

416

SECTION B  Aerobiology and Allergens

TABLE 26.17  Physicochemical and Biochemical Characteristics of Injected

Arthropod-Derived Allergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Envenomating Insects Apidae Honey Bee (Apis Mellifera, A. Cerana, A. Dorsata, A. Florea) Group 1 (e.g., Api m 1) 12-91 16 Phospholipase A2 Group 2 (e.g., Api m 2)

48-100

39

Hyaluronidase; shows sequence similarity with yellow jacket wasp allergen Ves v 2

Group 3 (e.g., Api m 3)

38-50

43

Acid phosphatase

Group 4 (e.g., Api m 4)

23

3

Group 5 (e.g., Api m 5)

58

100

Group 6 (e.g., Api m 6)

42

8

Group 7 (e.g., Api m 7)

?

39

CUB serine protease

Group 8 (e.g., Api m 8)

46

70

Carboxylesterase

Group 9 (e.g., Api m 9)

80

60

Serine carboxypeptidase

Group 10 (e.g., Api m 10)

51-61

50-55

Icarapin variant 2

Group 11 (e.g., Api m 11)

15-34

50

Major jelly protein

Group 12 (e.g., Api m 12)

47

200

Melittin Dipeptidyl peptidase IV; shows sequence similarity with Ves v 3 Serine protease inhibitor

Vitellogenin; shows sequence similarity with Ves v 6

Bumble Bee (Bombus Pennsylvanicus/Terrestris) Group 1 (e.g., Bom p 1) 82 16

Phospholipase A2

Group 2 (e.g., Bom p 2)

82

39

Hyaluronidase

Group 3 (e.g., Bom p 3)

82

49

Acid phosphatase

Group 4 (e.g., Bom p 4)

82

27

Protease

Vespidae White-Faced and Yellow Hornets (Dolichovespula Species), Paper Wasps (Polistes Species), and Yellow Jackets (Vespula Species) Group 1 (e.g., Pol a 1) 46 34 Phospholipase A1 Group 2 (e.g., Ves v 2)

50-76

39

Group 3 (e.g., Ves v 3)

50

100

Hyaluronidase Dipeptidyl peptidase IV

Group 4 (e.g., Pol d 4)

?

32-34

Serine protease

Group 5 (e.g., Ves v 5)

51-90

23

Group 6 (e.g., Ves v 6)

39

200

Belongs to the SCP family of proteins; shows sequence similarity with PR-1 proteins, may possess protease activity Vitellogenin

Formicidae Fire Ant (Solenopsis Invicta, S. Geminata, S. Richteri, S. Saevissima) Group 1 (e.g., Sol i 1) 87 18 Phospholipase A1 Group 2 (e.g., Sol i 2)

61

14

Function unknown

Group 3 (e.g., Sol i 3)

61

26

Shows sequence similarity with the vespid group 5 allergens

Group 4 (e.g., Sol i 4)

74

12

Shows sequence similarity with Sol i 2

Australian Jumper Ant (Myrmecia Pilosula) Myr p 1 52

9

Pilosulin 1, histamine-releasing protein

Myr p 2

35

5

Pilosulin 3

Myr p 3

?

9

Pilosulin 4.1

Hematophagous Insects Culicidae Mosquito (Aedes Aegypti, A. Albopictus, Anopheles Darlingi, Culex Aegypti) Aed a 1 29-65 68 Apyrase Aed a 2

11-32

37

Female specific protein D7

Aed a 3

32

30

Function unknown

CHAPTER 26  The Structure and Function of Allergens

417

TABLE 26.17  Physicochemical and Biochemical Characteristics of Injected

Arthropod-Derived Allergens—cont’d Frequency of Reactivity (%)

Mol. Size (kDa)

Aed a 4

47

67

α-Glucosidase

Aed a 5

67

22

Sarcoplasmic calcium binding protein

Aed a 6

33

31

Porin 3

Aed a 7

50

24

Function unknown

Aed a 8

83

70

Heat shock protein 70

Aed a 10

60

32

Tropomyosin

Aed a 11

50

42

Lysosomal aspartate protease

Pulicidae Cat Flea (Ctenocephalides Felis) Cte f 1 80a

18

Function unknown

Cte f 2

?

27

Salivary protein; shows sequence similarity with ant Sol i 3 allergen and vespid group 5 allergens

Cte f 3

40a

25

Function unknown

Tabaninae Horse Fly (Tabanus Yao) Tab y 1

81

70

Apyrase

Tab y 2

91

35

Hyaluronidase

Tab y 5

86

26

Shows sequence similarity with wasp venom antigen 5

Argasidae Pidgeon Tick (Argasidae Reflexus) Arg r 1 100

17

Lipocalin

Reduviidae Kissing Bug (Triatoma Protracta) Tria p 1 89

20

Procalin, a member of the lipocalin family; shows sequence similarity with triabin, a thrombin inhibitor

Allergen

Function

a

Frequency data based on flea-allergic dogs.

dominates, but a mixture of Aed a 6, Aed a 8, and Aed a 10 identifies more than 80% of mosquito-allergic individuals.39 Major mosquito allergens include apyrase, an enzyme that catalyzes the breakdown of ATP (an important platelet agonist) to release phosphate, sarcoplasmic calcium binding protein, α-glucosidase, HSP 70, and tropomyosin. A high degree of cross-reactivity exists between mosquito tropomyosin and those from mites, cockroaches, and shrimp.40 Apyrase is also a major allergen in the horse fly (as are venom allergen 5–like protein and hyaluronidase). In the other hematophagous insects, lipocalins are prominent but the functions of several allergens remain to be determined. Although a venom allergen 5–like protein has yet to be been demonstrated in A. aegypti, it is present in other mosquito species.

PATHOGEN-DERIVED ALLERGENS AND AUTOALLERGENS Pathogenic organisms associated with allergic diseases include fungi (which may colonize patients with preexisting conditions including asthma and cystic fibrosis), bacteria associated with atopic dermatitis, and helminthic parasites ingested in raw seafood. Besides allergens associated with bacteria and parasites, certain host-derived proteins may stimulate IgE production, particularly in atopic dermatitis.

Helminth-Derived Allergens Various intestinal and blood fluke parasitic infections trigger the production of specific IgE. From the human host’s perspective, this represents an active part of the immune response to the parasite. Some helminthic parasite allergens have been characterized41 and some identified as homologs of plant, fungal, and nonhelminthic animal allergens such as proteases, the muscle-associated allergens tropomyosin, paramyosin and troponin C, glutathione S-transferases, cyclophilins, and protease inhibitors (Table 26.18). However, functions of others remain unknown. Many, if not all, are found in secretions or excretions from either living or dead parasites. Illustratively, Asc as 1 is a major allergen found in Ascaris body fluid and is a polyprotein, which is cleaved into smaller subunits capable of binding host fatty acids.42 Of all the parasites studied, the greatest number of allergens has been described in the herring worm Anisakis simplex, which can act as both an intestinal parasite allergen source or as an allergen source from ingested, parasitized fish. At least 15 allergens have been described. Some, particularly the tropomyosins and glutathione S-transferases, are structurally similar to aeroallergens from HDM and cockroaches.43

Bacteria-Derived Allergens In addition to developing allergy to bacterial enzymes used in the occupational setting, production of IgE to Staphylococcus aureus has been

418

SECTION B  Aerobiology and Allergens

TABLE 26.18  Physicochemical and Biochemical Characteristics of Helminth-Derived Allergens Allergen

Frequency of Reactivity (%)

Mol. Size (kDa)

Function

Ascaridida Herring Worm (Anisakis Simplex) Ani s 1 14-86

24

Shows sequence similarity with Kunitz-type serine protease inhibitors

Ani s 2

88

97

Paramyosin

Ani s 3

13

41

Tropomyosin

Ani s 4

30-75

9

Ani s 5

25-49

15

Ani s 6

18

8

Ani s 7

100

139

Ani s 8

25

15

SXP/RAL family

Ani s 9

14

14

SXP/RAL family

Ani s 10

39

21

Function unknown

Ani s 11

47

27

Function unknown; shows sequence similarity with Ani s 10

Ani s 12

57

31

Function unknown; shows sequence similarity with aggrecans; shows sequence similarity with Ani s 7

Ani s 13

63

37

Hemoglobin

Ani s 14

54

24

Third stage larval protein, function unknown; shows sequence similarity with Ani s 7, 12 allergens

Ani s Troponin

20

21

Troponin C; a muscle associated calcium binding protein

Roundworm (Ascaris Suum, A. Lumbricoides) Group 1 (e.g., Asc s 1) 75

10

Polyprotein, lipid-binding protein

Group 3 (e.g., Asc l 3)

42-78

40

Tropomyosin

Group 13 (e.g., Asc s 13)

42-90

23

Glutathione S transferase

Cyclophyllidae Tape Worm (Echinococcus Granulosus) EA21 80

17

Peptidyl-prolyl cis-trans isomerase (cyclophilin); shows sequence similarity with Mal f 6 and Asp f 11

EgEF-1 beta/delta

Cysteine protease inhibitor Member of the SXP/RAL family Serine protease inhibitor Function unknown

56-90

14

Translation elongation factor

EgHSP70

57

40

Heat shock protein 70

AgB

?

12

Protease inhibitor

Antigen 5

?

67

Dimer: 22-kDa chain and 38-kDa chain with trypsin-like similarity, although not active

Spirurida Filarial Nematode Worm (Brugia Malayi) Bru m 3 ?

35

Tropomyosin; show sequence similarity with arthropod and shell fish allergens

Bru m 13

?

24

Glutathione S transferase

Bru m Bm33

?

33

Aspartate protease inhibitor

Strongylida Hookworm (Necator Americanus) Nec a ASP-2 ?

21

Ancystoma-secreted protein 2

Nec a Calreticulin

60

Calreticulin

?

Strigeatida Blood Flukes (Schistosoma Japonicum, S. Mansoni, S. Haematobium) Group 13 (e.g., Sch j 13) ? ? Glutathione S transferase Group PM (e.g., Sch ma PM)

40

84

Paramyosin

Sch j Sj67

43

67

Ezrin/radixin/moesin family protein, actin binding

Sch ma Sm20

?

20

Calcium ion binding protein

Sch ma Sm21

?

21

Calcium ion binding protein

Group 22 (e.g., Sch ma Sm22)

38

22

Calcium ion binding protein

Group 31 (e.g., Sch ma Sm31)

?

31

Cathepsin B-like cysteine protease

CHAPTER 26  The Structure and Function of Allergens

419

TABLE 26.19  Physicochemical and Biochemical Characterization of Human Autoallergens and

Bacterial Allergens Allergena

Frequency of Reactivity (%)

Humans (Homo Sapiens) Hom s 1 ?

Mol. Size (kDa) 55-60

Function Squamous cell carcinoma antigen SART-1

Hom s 2

?

10

Hom s 3

40

22-23

Hom s 4

18

36

Calcium-binding protein; shows sequence similarity with Phl p 7 and Cyp c 1 (carp parvalbumin)

Hom s 5

20

43

Cytokeratin, type II cytoskeletal 6A

Hom s Cyclophilin A-C

?

17-23

Hom s IL-24

80

24

Hom s MnSOD

43

27

Manganese superoxide dismutase; shows sequence similarity with Asp f 6, Hev b 10, and Mala s 11

Hom s P2 protein

?

11

Ribosomal P2 protein; shows sequence similarity with Asp f 8

Hom s Profilin

?

14

Shows sequence similarity with Bet v 2

Hom s PSA

?

33

Prostate-specific antigen, chymotrypsin-like protease; shows sequence similarity with Can f 5

Hom s Thioredoxin

?

12

Thioredoxin; shows sequence similarity with Asp f 29, Mala s 13

Bacteria (Staphylococcus Aureus) SEA 8-70

24

Superantigen, enterotoxin A

SEB

4-70

28

Superantigen, enterotoxin B

SEC

4-28

24

Superantigen, enterotoxin C

SED

4-35

28

Superantigen, enterotoxin D

TSST-1

9-75

22

Toxic shock syndrome toxin-1

36

65

Fibronectin-binding protein

FBP

Nascent polypeptide-associated complex alpha subunit (NAC) BCL7B protein

Peptidyl-prolyl cis-trans isomerase (cyclophilin); shows sequence similarity with Asp f 11, Mala s 6 Interleukin 24

a

Allergens associated with clinical atopic dermatitis, except for Hom s PSA.

reported in the context of atopic dermatitis, asthma, and rhinitis. Among allergens from S. aureus are enterotoxins and toxic shock syndrome toxin (TSST-1), as well as fibronectin-binding protein, flagellin, ribosomal proteins, DNA binding proteins, and proteases. The staphylococcal enterotoxins A and B (SEA, SEB) and TSST-1 are most prominent (Table 26.19),44 but in contrast to all the allergenic proteins described previously, they are inherently potent T cell mitogens. They act as super­ antigens that cross-link MHC class II molecules and the variable region domain of the T cell receptor β chain and stimulate T cell proliferation in a non–antigen-specific manner. They promote a TH2 environment favoring eosinophilic inflammation, which may influence the severity of asthma. In addition, current smoking is also associated with increased IgE production to the enterotoxins, particularly SEA.45

Human Autoallergens The study of human autoallergens arose from observations that human dander caused wheal and flare reactions in allergic individuals.46 Subsequent molecular studies defined several autoallergens (Table 26.19), revealing that they arose primarily in patients with atopic dermatitis or ABPA. They are divided into two categories, namely those sharing significant sequence similarity with allergens from environmental sources such as pollen and fungi, and are thus classified as cross-reactive allergens (e.g., Hom s 4), and those that do not (e.g., Hom s 1),47 indicating that they may be genuine autoallergens. Most autoallergens are intracellular proteins, although Hom s PSA is secreted and plays a role in females sensitized to seminal fluid48 and shares sequence similarity with the dog allergen Can f 5. A majority of autoallergens are restricted to skin but can be found in sera complexed with IgE, suggesting that they may be released, presumably because of tissue damage in disease.

ALLERGENS AND ALLERGENICITY The development of an appreciation for an allergen’s functional bioactivity as a pivotal event in allergy has mirrored the renaissance of interest in innate immunity as an “engine” of disease. Thus understanding how allergens activate or inactivate innate immune mechanisms is revealing new insights into how allergy is initiated and how their persistent operation contributes to the allergic march. It is also clear that although protein structure does not encrypt a universal signature of allergenicity, certain structural features reveal explanations of cross-reactivity (e.g., similarity in protein folds) or properties that affect stability (Table 26.20).

Allergens and Epithelial Transcriptomics Casting new light on the problem of allergenicity is no longer the exclusive domain of allergy specialists. Arguably, greatest progress has come from a cross-disciplinary adoption of a variety of techniques. For example, systems biology and transcriptomics have enabled broad, hypothesisindependent surveys of gene expression with the potential to identify previously unrecognized events, as well as confirming existing knowledge. Philosophically, the polar opposite tactic is a hypothesis-driven examination of events and/or pathways that are already suspected to be important in allergy. Self-evidently, a convergence of these different approaches creates the greatest opportunities for progress. Regarding the latter approach, extracts derived from several allergen sources such as HDM, grass pollens, cockroach, and fungi, as well as purified allergens such as the mite allergens Der p 1 and Der p 9, have been examined. Exposure to these stimulates the production of various cytokines and chemokines in airway epithelial cells, in addition to initiating the IgE-independent release of histamine and IL-4 in mast cells,

420

SECTION B  Aerobiology and Allergens

suggesting similar effector pathways are activated by allergens from diverse sources. The pleiotropic nature of events activated by allergen contact with epithelial cells or antigen presenting cells (APC) and immune response cells makes them ideal candidates for transcriptomics, and several studies have been reported. For example, significant increases in expression levels of various genes have been reported after exposure to allergen extracts or purified allergens.49,50

Allergenicity and Innate Immunity Transcriptomics neither reveals how allergens stimulate epithelial cells, nor indicates their impact on determining allergenicity. Although no peptide motif provides a simple universal signature of this property, molecular structure does determine the functional and molecular recognition characteristics of proteins. Straightforwardly, molecules whose

TABLE 26.20  Allergy-Related Biologic Effects of Protease Allergens or Allergen-Associated

Protease Adjuvantsa Effect

Allergen or Extract

Epithelial Cell Activation – Protease Activated and Mas-Related G-Protein-Coupled Receptors PAR-1 and PAR-4 activation Der p 1 (indirectly as prothrombinase) PAR-2 activation

Der p 1, 2 and 3, cockroach extract

PAR-2 inactivation

Der p 1

PAR-1 and PAR-2 upregulated expression

Der p 1

Mrpgr activation

Der p 1

Epithelial Cell Activation – Cytokine, Chemokine and Growth Factor Release EGFR activation Der p 1 (indirectly as prothrombinase) Pannexon gating

Der p 1 (indirectly as prothrombinase)

VEGF production

Cockroach extract

MMP-9 upregulation

Cockroach extract

Activation of NLRP3 inflammasome

Der f 1

Cytokine/chemokine expression and release

HDM extract, Der p 1, Der p 3, Der p 6, Der p 9, fungal extracts, Asp f 13, cockroach extract

Epithelial Danger-Associated Molecular Patterns/Alarmin Release ATP release Der p 1 (indirectly as prothrombinase), fungal, weed and birch pollen extracts Uric acid

HDM extract

Epithelial Oxidative Stress Response ADAM 17 activation

Der p 1 (indirectly as prothrombinase)

ADAM 10 activation

Der p 1 (indirectly as prothrombinase)

ROS generation

HDM fecal pellet extract, Der p 1

PAR-2-associated dual oxidase-2 upregulation

Cockroach extract

Transductional convergence with signaling from TLR3 and TLR7

HDM fecal pellet extract, Der p 1

Epithelial Cleavage – Tight Junction Proteins Cleavage of occludin and claudin

HDM fecal pellet extract, Der p 1, Der p 3, Der p 6, Der p 9, Pen ch13, miscellaneous pollen extracts

Disruption of epithelial adherens junctions

Der p 1 (directly and indirectly via ADAM 10)

Cleavage and/or Inactivation of Host Proteins DC-SIGN/DC-SIGNR

Der p 1

Low affinity IgE receptor (CD23)

Der p 1 (directly and indirectly via ADAM 10)

IL-2R (CD25)

Der p 1

Surfactant proteins

Der p 1

Airway antiproteases

Der p 1

Fibrinogen

Aspergillus protease

Inflammatory Cell Recruitment and Cell Activation Inflammatory cell recruitment

Der p 1, HDM extract

Epithelial-mesenchymal transition

HDM extract

Breaking of immune tolerance

Der p 1, Asp f 13, papain

Activation of mast cells

Der p 1

CHAPTER 26  The Structure and Function of Allergens

421

TABLE 26.20  Allergy-Related Biologic Effects of Protease Allergens or Allergen-Associated

Protease Adjuvantsa—cont’d Effect

Allergen or Extract

IgE-independent mast cell stimulation

Der p 1, HDM extract

ENA-78/CXCL5 inactivation and reduction in neutrophil migration

HDM extract

Kininogen/Kallikrein Pathways Kinin release from low molecular weight kininogen and high molecular weight kininogen

D. farinae group 3 serine protease

Decreased thiol protease (cathepsins) inhibitory activity of kininogens

D. farinae group 3 serine protease

Prothrombinase activity

HDM fecal pellet extract, Der p 1

Complement Activation Anaphylatoxin release from C3 and C5

D. farinae group 3 serine protease

ADAM 10, ADAM 17, A disintegrin and metalloprotease 10 and 17; DC-SIGN/DC-SIGNR, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin and its homolog; DUOX-2, dual oxidase-2; EGFR, epidermal growth factor receptor; HDM, house dust mite; IgE, immunoglobulin E; IL-2R, interleukin-2 receptor; MMP-9, matrix metalloprotease 9; PAR-1, PAR2, PAR-4, protease-activated receptors 1, 2 and 4; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor. a Here the term adjuvant is applied to extracts for which the causative proteolytic activity was not confirmed as being a known allergen.

TABLE 26.21  Pattern Recognition Receptors Potentially Capable of Binding Glycosylated Allergens Receptor

Specificity

Allergen Interaction

C-Type Lectins: Membrane-Bound DC-SIGN (CD209) Mannose

Der p 1, Ara h 1, Can f 1

DC-SIGNR (CD299)

Mannose

Der p 1

Dectin-1

β-Glucan

Fungal β-glucans

Dectin-2

Mannose, fucose

Dermatophagoides pteronyssinus, Dermatophagoides farinae

Mannose receptor (CD206)

Mannose, fucose, N-acetylglucosamine

Der p 1, Der p 2, Ara h 1, Bla g 2, Fel d 1, Can f 1

C-Type Lectins: Soluble Mannose-binding lectin

Mannose, glucose, fucose, N-acetylmannosamine

Der p 1

Surfactant protein A

N-acetylmannosamine, maltose, glucose

Der p 1

Surfactant protein D

Mannose, glucose, inositol

Der p 1

bioactivities enable them to interact with and/or activate the human immune system more effectively than others are more significant allergenic threats because they are intrinsically equipped to activate signaling events necessary for allergy development. Thus competency in triggering innate immune signaling differentiates immunodominant allergens from minor allergens lacking these properties, which must rely on the bioactivities of other allergens, or adjuvants, to become allergenic threats. An exemplar is ovalbumin, which is normally tolerogenic in the absence of adjuvant, when delivered to experimental animals via the airways. Similarly, in animal models some major allergens demonstrate weaker allergenicity after purification compared with crude extracts containing the same allergen. However, immunization of animals with admixtures of enzyme-rich allergen extracts from sources such as HDM, cockroach, and Aspergillus species or biochemically active allergens (e.g., Der p 1, Asp f 13, Per a 10) with ovalbumin or bystander allergens converts a tolerogenic response to a robust allergic reaction.51,52 Direct or indirect ligation of sentinel cell surface and intracellular recognition receptors (PRRs) such as TLRs and C-type lectins (collectins etc.) (Table 26.21) by allergens is a fundamental, perhaps indispensable, component linking innate and acquired immunity. Binding to sentinel

receptors on epithelial cells and on dendritic cells (DCs) residing below the epithelium, and subsequent activation, is associated with the induction of a TH2 immune response. Allergens do not appear to have equal ability to sustain the progression from innate to acquired immunity; in fact the ability to do this resides within a small cadre. In this regard, allergens possessing protease activity are preeminent (and the most studied), because proteolytic events foster allergen delivery and the breaking of immune tolerance in a Th2-directed manner. Tables 26.20 and 26.22 list functional activities contributing toward enhanced allergenicity.

Functional Bioactivities of Allergens Important to the Development and Persistence of Allergy

Mucosal Defenses.  For an immune response to be initiated to a protein, it must be detected by a network of APCs (e.g., DCs in the airways or Langerhans cells in skin). Each cell in an APC network possesses an array of pattern recognition receptors (PRR), which may exhibit a polarized or otherwise protected distribution (e.g., localization of TLRs to epithelial crypts in the gut) to prevent continual activation of innate responses by the host mucosal microbiome or “nuisance calls”

422

SECTION B  Aerobiology and Allergens

TABLE 26.22  Summary of Potential Consequences of Intrinsic and Extrinsic Factors and

Properties of Allergens and Allergen Sources Property

Cell/Molecular Interaction

Possible Consequence(s)

Extrinsic Host DAMPs (e.g., HSP, uric acid, ADP)

Epithelium, corneum, DCs

Increased allergen presentation

Genetic factors (e.g., MHC class I and II, filaggrin, TLR)

Epithelium, corneum, DCs, lymphocytes

Increased allergen exposure, presentation

Reactive oxygen species

Epithelium

Increased permeability, influencing allergen entry

Respiratory viral infection

Epithelium, DCs, macrophages

Increased permeability, upregulation of carbohydrate-recognizing receptors

Tight junction protein dysregulation

Epithelium

Increased permeability

Environment Environment (e.g., rural/urban)

Various

Increased allergen exposure, immunomodulation

Pollutants (e.g., diesel exhaust particulates, environmental tobacco smoke)

Epithelium, DCs

Carrier of allergen into lower lung, epigenetic effects

Climate (temperature, CO2)

Epithelium, DCs

Increase in production of specific allergens

Allergen-associated High focal allergen

Epithelium

Increased permeability due to osmotic shock, influencing allergen entry and presentation

PAMPs (e.g., LPS, prostaglandins, CpG, mannose)

Epithelium, DCs

Increased allergen presentation

Pollen lipid mediators

Epithelium, DCs, polymorphonuclear cells

Increased allergen

Intrinsic Allergen Glycosylation

DCs, macrophages

Increased antigen presentation due to opsonization or binding cell surface receptors (e.g., surfactant proteins A and D, and CTLR), DC activation and T cell polarization

Oligomerization

DCs, macrophages

Increased allergenicity

MD-2 surrogacy by Group 2 HDM allergens

TLR4 activation

Promotion of allergic sensitization

Hydrolytic Enzyme Activity Protease

Epithelium

Various; see Table 26.20

Hyaluronidase

Hyaluronic acid (HA)

Low molecular weight proinflammatory HA derivatives

Phospholipase A2

Mast cells

Cytokine release, T cell polarization

Cytolytic activity

Keratinocytes

Release of DAMPs; stimulation of cytokines

Resistance to heat and digestion

Epithelium

Increased allergen uptake and processing

ADP, Adenosine monophosphate; CTLR, C-type lectin receptor; DAMPs, danger-associated molecular patterns; DC, dendritic cell; HSP, heat shock protein; LPS, lipopolysaccharide; MHC, major histocompatibility complex; PAMPs, pathogen-associated molecular patterns; TLR, toll-like receptor.

of low threat value to the host. The first step is, therefore, to understand how allergens engage with these protected sensors. For some allergens, such as those present in venoms, the engagement is likely to be a consequence of the physical process of stinging or biting that directly circumvents barriers. For others, the process is more convoluted and may involve impairment of the biochemical and biophysical barriers at mucosal surfaces. For example, in the airway lumen, alveolar macrophages will be capable of phagocytosing particulate allergen sources such as HDM fecal pellets, fungal spores, and subpollen particles, which would tend to suppress allergic responses, including those mediated by DC. Similarly, the

alveolar- and Clara cell–derived collectin CTLR lung surfactant proteins SP-A and SP-D attenuate allergic inflammation by promoting phagocytosis of allergens and preventing their interaction with IgE. However, protease allergens from mites (e.g., Der p 1 and Der f 1) and cockroach (serine proteases) have been shown to inactivate surfactant proteins, as well as altering alveolar macrophage phenotype. This inactivation capacity is not restricted to surfactant proteins, because the same allergens attenuate lung-associated protease inhibitors (α1-antitrypsin, the elastase inhibitor, elafin, and secretory leukocyte protease inhibitor) with the consequence that both protease and bystander allergens are potentiated.

423

CHAPTER 26  The Structure and Function of Allergens

Epithelial Permeability

1h

3h

116

d ate

l

Tre

ntro

kDa

Co

Tre

kDa

ate

l

d

A ntro

Allergens and Tight Junctions.  Concentrations of HDM proteases mimicking daily exposures cause cleavage of TJs in human airway epithelial cells (Fig. 26.3) by attacking their extracellular domains,53,54 which, in turn, initiates intracellular proteolytic processing of the TJ plaque protein ZO-1. The consequence of TJ cleavage by protease allergens is a nonspecific increase in epithelial permeability permissive for any allergen, which, additionally, increases the probability of allergen detection. The potential importance of Der p 1 protease activity is indicated by the observation that when TJ cleavage is blocked, transepithelial delivery of allergen does not occur,53 and when the proteolytic activity is inhibited, intranasally administered HDM allergens no longer evoke allergic sensitization. This suggests that HDM allergens with serine protease activity, which also have the ability to cleave TJs, have a subsidiary role in driving allergic sensitization compared with the group 1 allergens. In the skin, HDM proteases cause the epidermis to become leaky and impede its restitution while at the same time promoting cytokine and chemokine production like the airway response. Similar effects have also been described for Pen ch 13, a major serine protease allergen of P. chrysogenum, indicating a general property of inhaled, proteolytically active allergens. Epithelial cohesion, specifically TJ integrity, depends on a delicate balance between synthesis of junctional components and their breakdown. This is temporarily and reversibly perturbed by protease allergens, but orchestrated reinstatement of TJs occurs on allergen removal.53 These actions of protease allergens thus differ from the epithelial damage that characterizes the lung pathology of severe asthma. The localized deposition of inhaled allergens delivers them in high concentration at the point of impact, resulting in effects that are spatially restricted, unlike the diffuse effects of chronic inflammation that, in severe uncontrolled asthma, may cause epithelial cell exfoliation. However, processes activated by protease allergens promote the loss of polarity and downregulation of intercellular junctions that is associated with epithelial-mesenchymal transition (EMT) and central to airway remodeling in asthma. Adherens junctions (zonulae adherentes, ZAs), which encircle epithelial cells forming homotypic interactions with neighboring cells without occluding paracellular channels, are also targets for HDM proteases. In the airways, ZAs consist of E-cadherin, a single transmembrane domain Ca2+-binding protein that forms complexes with α−, β− and γ-catenins through its cytoplasmic domain. In this way ZAs are linked to the cytoskeleton and thus, indirectly, to other membrane-associated proteins such as TJs. Despite the existence of theoretical cleavage sites within it, proteolysis of E-cadherin by HDM protease allergens is slower than

Control

Co

Tight Junction Proteins.  Further events are necessary to achieve effective antigen presentation to networks of DCs or LCs. The airways, as a simple epithelium with well-characterized TJs, are the better studied in this regard, whereas parallel events in skin are less defined because the role of TJs in regulating the permeability of skin was itself unclear until the beginning of the 21st century. In the process of sensitization, allergens must circumvent the epithelial barrier, either by crossing it themselves and/or promoting the ability of APCs to sample from the external environment. Paracellular pathways offer a potential route for this, particularly as the dendrites of APCs intercalate into paracellular spaces. In both simple (airway, gut) and stratified (skin) epithelia, access to these paracellular routes is regulated by interepithelial tight junctions (TJs). TJs comprise transmembrane proteins (occludin, the claudin family, and junctional adhesion molecules) and others that are localized cytoplasmically in contact with the cytoskeleton. The transmembrane proteins are adhesive and regulate paracellular permeability, whereas the cytoplasmic proteins transduce intercellular signaling.

220 74 197 65 52

83 48.9

43

220 192 164

115

34

33.1

89

B

Occludin

ZO-1

Fig. 26.3  (A) The effect of house dust mite serine protease allergens on epithelial tight junctions (TJs) (green fluorescence) and desmosomes (red fluorescence). Cell images were acquired by two-photon molecular excitation microscopy after fluorescence 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 at 1 hour after exposure to the allergens, small discontinuities become evident as the TJ rings break down. This is accompanied by an increase in epithelial permeability (not illustrated). At 3 hours after allergen exposure, loss of TJs in these cells is total. (B) Representative immunoblots revealing the proteolytic processing of a transmembrane TJ protein, occludin, and a plaque protein, ZO-1, after exposure of bronchial epithelial cells to house-dust mite serine protease allergens. In the case of occludin, the proteolytic processing is initiated by direct cleavage of residues in the extracellular domains, whereas cleavage of ZO-1 arises by activation of intracellular processing pathways after perturbation by the protease allergens. Regarding events in both (A) and (B) similar effects are produced by the cysteine protease allergen Der p 1.

424

SECTION B  Aerobiology and Allergens

the TJs, indicating that ZAs may be sterically inaccessible to the HDM allergens until TJs are cleaved. However, as described later, additional mechanisms for ZA disruption can also be invoked by group 1 HDM allergens and contribute to the initiation of EMT. Further mechanisms for transient disruption of airway epithelial permeability are known. Cockroach and fungal allergens do this by inducing vascular endothelial growth factor (VEGF) and tumor necrosis factor α (TNF-α)55, although whether the action is related to allergen or nonallergen adjuvant components is unclear. Analogously, pollen extracts elicit a protease-dependent disruption of epithelial TJ integrity, which may reflect the presence of cysteine proteases in weed (e.g., Amb a 11) and grass pollens (e.g., Cyn d CP). In addition, a proteolytically active Aspergillus oryzae protein has been shown to desquamate epithelium, resulting in epithelial permeability, extracellular matrix degradation, and airway hyperreactivity because of direct protease action on airway smooth muscle cells residing below the epithelial layer. Finally, the mite serine protease allergen Der f 3 has been shown to generate bradykinin via activation of the plasma kallikrein-kininogen pathway resulting in increased vascular permeability and cleavage of the cysteine protease inhibitor domains released because of kininogen degradation.

Cell Signaling and Tissue Remodeling

Allergens, Cytokines, Chemokines, and Alarmin Release.  Airway epithelial cells, keratinocytes, dendritic cells, and mast cells offer an extensive palette of innate immune sensors including TLRs, nucleotidebinding oligomerization-like domain (NOD)–like receptors, retinoic acid inducible gene (RIG) I–like receptors, CTLRs, protease activated receptors (PARs), and purinoceptors. Ligation of these, or activation of transduction mechanisms that converge with signaling from these receptors, is a burgeoning interest yielding new insights into allergy. HDM protease allergens stimulate the production of various cytokines in airway epithelial cells as well as initiating the IgE-independent release of histamine and IL-4 in mast cells.56 Both Der p 1 and Der p 9 upregulate cytokine gene transcription and cytokine production (IL-6, IL-8, and granulocyte macrophage–colony stimulating factor [GM-CSF]) in bronchial epithelial cells, and extracts of Aspergillus fumigatus or purified fungal allergens behave similarly, suggesting similar effector pathways are activated by allergens from diverse sources. In addition to cytokines and chemokines, epithelial cells exposed to protease allergens release alarmins or danger-associated molecular patterns (e.g., uric acid, ATP, HMGB1, and S100 proteins), all of which drive TH2 immunity.

Cytokines and Innate Lymphoid Cells.  Type 2 innate lymphoid cells (ILC2) play an instrumental role in the development of allergic sensitization through their ability to secrete cytokines such as IL-5 and IL-13 in response to cytokines (TSLP, IL-25, IL-33, IL-2) released by epithelial cells.57-61 Along with alarmins, these play an essential role in driving allergic inflammation involving ILC2, APC, and T cells. Thymic stromal lymphopoietin (TSLP), which has a Th2 polarizing effect on DCs and increases expression of the costimulatory molecule OX40L, is upregulated in the asthmatic airways62-64 and is important in ILC2 survival. TSLP is also released from basophils after stimulation with the cysteine protease papain, supporting a role for all in antigen presentation. Papain is both an occupational allergen and a surrogate for the group 1 HDM allergens. It increases the concentration of IL-25 recoverable by bronchoalveolar lavage in sensitized mice and upregulates mRNA for both IL-25 and TSLP in mouse airway epithelial cells in a protease-dependent, MAP kinase-transduced manner. A similar effect on IL-25 and TSLP mRNA has been demonstrated for Der p 1. Although the proteolytic event triggered by papain or Der p 1 is unknown, it is now known that Der p 1 leads to TLR4 signaling.65

IL-33 acts indispensably and in parallel with TSLP in driving the early events of innate immunity. Both mediators link atopic dermatitis (in which sensitization to inhalant allergens is common) with airway events, prompting speculation that they drive the “allergic march.” Like IL-25, it is a potent stimulator of ILC2, is associated with Th2 cytokine production, mimics disease phenotype, and is found in elevated amounts in asthma.66-69 In IL-33-deficient mice the reaction to HDM challenge is markedly attenuated, suggesting that its role in the complex profile of responses to HDM allergens is central to allergy. Although the mechanistic linkage between the allergens and IL-33 is unclear, silencing of DUOX-1 (but not DUOX-2) inhibits HDM-dependent IL-33 release in human airway epithelial cells, indicating a role for reactive oxidant species (ROS) in its execution.

Protease-Activated Receptors.  The role of proteases in some of the mentioned studies has led to the proposition that cleavage of proteaseactivated receptors (PARs), especially PAR-2, is an important molecular recognition system for protease allergens. However, the potential role of PAR-2 in asthma is complex. In animal models, PAR-2 is associated with an inhibition of inflammatory responses, consistent with the receptor mediating cytoprotective events, whereas other evidence suggests a proinflammatory role related to the release of IL-6, IL-8, CCL-11, and GM-CSF. Studies in PAR-2 deficient mice suggest that the receptor is unnecessary for sensitization and challenge responses and does not readily explain the efficacy of HDM cysteine protease allergens in generating cytokine release. Notably, for group 1 HDM allergens, data both implicate and exclude PAR-2 cleavage as the primary recognition event, and PAR-2 deficiency produces only slight attenuation of responses to papain used as a surrogate for cysteine protease allergens such as Der p 1. As a prothrombinase, Der p 1 facilitates the canonical activation of PAR-1 and PAR-4 by forming a ternary complex of thrombin with receptor heterodimers.70,71 This eventuates in the generation of ROS and reactive nitrogen species (RNS) through a process of receptor transactivation involving epidermal growth factor receptor (EGFR), the operation of myosin motors, and the opening of pannexons, which facilitate the release of ATP without necrosis. Autocrine and paracrine signaling mediated by ATP stimulation of P2X7 and P2Y2 purinoceptors activates ADAM 10 and leads to the stimulation of TLR4 by endogenous ligands, which provide a late checkpoint in the generation of ROS and RNS.70 Although subordinate to PAR-1 and PAR-4, activation of PAR-2 also contributed to ROS generation and its consequences.71 Intriguingly, the pathway activated by Der p 1 converges with signaling activated by detection of viral RNA (by ligation of TLR3 and TLR7) such that all these routes lead to myosin motor–dependent gating of pannexons for ATP release and TLR4 activation.65 This innate signaling mechanism is notable in several respects. First, some reactive oxidant production occurs in mitochondria via the two electron-dependent reduction of oxygen to superoxide anion by the electron transport chain.71 Mitochondria-derived oxidants have wellestablished roles in regulating the transcription of inflammatory cytokines, especially those operated by NF-κB. Second, the pathway from group 1 HDM allergen to reactive molecule regulators of gene transcription involves mechanisms strongly linked to allergy (e.g., ADAM 10, which inter alia regulates IgE synthesis) or which are implicated in asthma pathophysiology (e.g., EGFR and ADAM 10). Third, asthma is associated with deficits in antioxidant defenses, at least some of which are genetically defined, and which may partially explain the susceptibility of individuals to allergens. Allergens and Toll-Like Receptor.  Studies with other allergens have identified further protease-dependent mechanisms. Extracts of

CHAPTER 26  The Structure and Function of Allergens Aspergillus oryzae or A. niger elicit allergic sensitization in mice and the expression of a disease phenotype.72,73 In one study, the development of disease characteristics was dependent on TLR4 ligation by fibrinogen cleavage products and the recruitment of type 2 innate lymphoid cells, whereas TLR4 was dispensable for IL-4 and IgE production.72 This finding is interesting in the context of innate lymphoid cells driving airway hyperreactivity without adaptive immunity. Separate work has suggested that Aspergillus protease activity leads to Th2 responses by forming fibrinogen cleavage products that ligate mast cell–expressed TLR4, causing IL-13 (but not IL-4) release and inducing programmed cell death 1 ligand 2+ (PD-L2+) dendritic cells known to favor allergy development and which correlated with the numbers of ensuing IL-4+ and IL-13+ T cells.73 In contrast to Aspergillus, mechanistic studies in mice show that expression of TLR4 by airway epithelial cells is indispensable for the development of sensitization to HDM allergens because TLR4 activation initiates the release of IL1α, GM-CSF, and IL-33, which are crucial for the transition to adaptive responses.57,58 Regardless of whether TLR4 is dispensable for IgE production in response to Aspergillus and the cell type whose expression is decisive in directing effector mechanisms, TLR4 is clearly a leading actor in the development of an allergic disease phenotype. For HDM sensitization, TLR4 activation has been thought to be related to endotoxin present in HDM extracts and group 2 HDM allergens, which show structural similarities to the TLR4 coreceptor, myeloid differentiation protein-2 (MD-2).74,75 Indeed, in MD-2–deficient mice (but not those with TLR4 deficiency), Der p 2 reconstitutes the development of allergic sensitization. This observation, and the function of MD-2 as a lipid-binding protein, have prompted speculation that MD-2 surrogacy or lipid binding may be relevant to sensitization to allergens in which predictive bioinformatics indicates a hydrophobic binding pocket (for example, Groups 5, 7, 13, 22 from HDM and others such as Blo t 13, Bos d5, d6, Can f 2, Can f3, Pru av 1, Gly m 4, Fel d2) (Table 26.3), although the bound lipid partner may be variable because Der p 7, for example, is thought not to bind bacterial endotoxin. An additional explanation for the centrality of epithelial TLR4 in sensitization to HDM has recently emerged after the discovery that Der p 1 activation of intracellular ROS/RNS production involves TLR4 activation by endogenous ligands.65

Airway Remodeling.  Loss of epithelial cohesion, thickening of the epithelial basement membrane, and hyperplasia of mucus cells and smooth muscle are well-known components of chronic asthma in which TGF-β-driven EMT plays a decisive role. HDM allergen extracts and group 1 HDM allergens activate latent TGF-β and promote EMT characterized by a reduction of E-cadherin present at cell membranes that is dependent on PAR-2 and EGFR.76,77 Downregulation of E-cadherin is associated with an upregulation of CCL17 (thymus and activationregulated chemokine [TARC]) and TSLP expression, which itself is dependent on EGFR.78 With the discovery that group 1 HDM allergens are prothrombinases (thereby cleaving PAR-4, implicated in EMT) and that they consequently trigger EGFR signaling and activate ADAM10, a key sheddase of E-cadherin,65 a reasonable inference is that the protease nature of group 1 HDM allergens makes a significant contribution to EMT.

ALLERGENS AND THE EPITHELIAL-DENDRITIC CELL AXIS Once epithelial layers are breached, or the airway lumen probed by DC dendrites, allergen interactions with DCs span the core function of antigen recognition, processing and presentation to T cells, and other effects such as regulation of their recruitment and phenotypic plasticity,

425

which affect allergenicity. For example, Der p 1 promotes the selective recruitment of DCs by releasing chemokines such as CCL-20 from epithelial cells by a mechanism that may involve TLR4.57,79 CCL-20 signals via the receptor CCR6, which is expressed on Langerhans cell–like precursors of myeloid DCs that are known to accumulate at mucosal sites during inflammation. CCL2-CCL2R signaling is also relevant to allergy development because CCR2+Ly6chi monocytes are precursors of inflammatory CD11b+ DCs.57,80 The CCL-20-CCR6 axis activated by Der p 1 highlights additional differences between protease allergens and the effete bystander allergens whose DC-recruiting effects are independent of CCR-6. Potential interactions of allergens with DCs have also been considered and proteolytic effects of Der p 1 identified, notably a downregulation of indoleamine 2,3-dioxygenase in HDM-sensitive individuals. Predictively, impairment of this immune checkpoint enzyme associates with a loss tolerance at the level of DC signaling.81 Computational analysis of cell surface proteins expressed on DC identified the C-type lectin receptor dendritic cell–specific intercellular adhesion molecule 3–grabbing nonintegrin (DC-SIGN) (Table 26.21) and its homolog DC-SIGNR as potential substrates of Der p 1. Other CTLRs on DCs have been implicated in the effects of allergen extracts from HDM and fungal sources (Alternaria and Aspergillus species). Notably, among these are dectin-1 and dectin-2 (CLEC7A and CLEC6A, respectively), which transduce the release of ROS and a range of proallergic cytokines, chemokines, and cysteinyl leukotrienes through mechanisms that may be TLR2dependent or independent. TSLP, whose production as noted earlier is enhanced by protease allergens, has pronounced effects on DCs, and its presence correlates with the CCL-17- and CCL-22-dependent recruitment of Th2 cells to airways. DCs and T cells interact through the ligation of costimulatory molecules and, in the context of driving allergic sensitization, the interaction between OX40L (CD134L) and OX40 (CD134) is of interest. TSLP stimulates DC expression of HLA-DR and CD134L,62,82 key elements in Th2 responses, although its unique centrality is questionable given that: (1) TSLP upregulation is also a feature of chronic obstructive pulmonary disease, and: (2) Th2 immunity against parasites survives its deficiency.83

CONCLUSION As summarized in this chapter, many aeroallergens, ingested allergens, and injected allergens of more or less well-characterized structure and function are recognized. Not unexpectedly, allergens fall into previously delineated protein groups. Thanks to efforts directed toward the identification, purification, and characterization of clinically important allergens as well as identifying potential adjuvants, it has become possible to appreciate why some of these substances lead to disease, particularly via modulation of the epithelium and DCs. In addition, it is clear that interactions between allergens and adjuvants also are likely to be important in defining the outcome of sensitization. The ability of at least some proteolytically active allergens to override tolerance and unmask the allergenicity of comparatively innocuous proteins suggests that there may now be a simple and instructive way of categorizing allergens that transcends their traditional classification based on a particular biologic origin. Through such an understanding, the prospects for improved treatment and even prevention of allergy may be possible.

Acknowledgments The authors’ work referred to in this chapter was originally supported by Australia’s National Health and Medical Research Council (NHMRC), Asthma Foundation WA, Asthma UK, The Medical Research Council (UK), The Wellcome Trust, and The Royal Society.

426

SECTION B  Aerobiology and Allergens

REFERENCES 1. Padavattan S, Flicker S, Schirmer T, et al. High-affinity IgE recognition of a conformational epitope of the major respiratory allergen Phl p 2 as revealed by X-ray crystallography. J Immunol 2009;182:2141–51. 2. Chruszcz M, Pomes A, Glesner J, et al. Molecular determinants for antibody binding on group 1 house dust mite allergens. J Biol Chem 2012;287:7388–98. 3. Malandain H. IgE-reactive carbohydrate epitopes–classification, cross-reactivity, and clinical impact. Eur Ann Allergy Clin Immunol 2005;37:122–8. 4. Platts-Mills TA, Schuyler AJ, Hoyt AE, et al. Delayed anaphylaxis involving IgE to galactose-alpha-1,3-galactose. Curr Allergy Asthma Rep 2015;15:12. 5. Chua KY, Stewart GA, Thomas WR, et al. Sequence analysis of cDNA coding for a major house dust mite allergen, Der p 1. Homology with cysteine proteases. J Exp Med 1988;167:175–82. 6. Radauer C. Navigating through the jungle of allergens: features and applications of allergen databases. Int Arch Allergy Immunol 2017;173:1–11. 7. Radauer C, Bublin M, Wagner S, et al. Allergens are distributed into few protein families and possess a restricted number of biochemical functions. J Allergy Clin Immunol 2008;121:847–52.e7. 8. Vidal-Quist JC, Ortego F, Rombauts S. Dietary shifts have consequences for the repertoire of allergens produced by the European house dust mite. Med Vet Entomol 2017;31:272–80. 9. Valenta R, Duchene M, Ebner C, et al. Profilins constitute a novel family of functional plant pan-allergens. J Exp Med 1992;175:377–85. 10. Valenta R, Lidholm J, Niederberger V, et al. The recombinant allergen-based concept of component-resolved diagnostics and immunotherapy (CRD and CRIT). Clin Exp Allergy 1999;29:896–904. 11. Wickman M, Lupinek C, Andersson N, et al. Detection of IgE reactivity to a handful of allergen molecules in early childhood predicts respiratory allergy in adolescence. EBioMedicine 2017;26:91–9. 12. Habenicht HA, Burge HA, Muilenberg ML, et al. Allergen carriage by atmospheric aerosol. II. Ragweed-pollen determinants in submicronic atmospheric fractions. J Allergy Clin Immunol 1984;74:64–7. 13. Grote M, Valenta R, Reichelt R. Abortive pollen germination: a mechanism of allergen release in birch, alder, and hazel revealed by immunogold electron microscopy. J Allergy Clin Immunol 2003;111:1017–23. 14. Garcia-Mozo H. Poaceae pollen as the leading aeroallergen worldwide: a review. Allergy 2017;72:1849–58. 15. Valenta R, Hayek B, Seiberler S, et al. Calcium-binding allergens: from plants to man. Int Arch Allergy Immunol 1998;117:160–6. 16. Behrendt H, Tomczok J, Sliwa-Tomczok W, et al. Timothy grass (Phleum pratense L.) pollen as allergen carriers and initiators of an allergic response. Int Arch Allergy Immunol 1999;118:414–18. 17. Gadermaier G, Hauser M, Ferreira F. Allergens of weed pollen: an overview on recombinant and natural molecules. Methods 2014;66:55–66. 18. Breiteneder H, Ebner C. Molecular and biochemical classification of plant-derived food allergens. J Allergy Clin Immunol 2000;106:27–36. 19. Finkina EI, Melnikova DN, Bogdanov IV, et al. Lipid transfer proteins as components of the plant innate immune system: structure, functions, and applications. Acta Naturae 2016;8:47–61. 20. Asam C, Hofer H, Wolf M, et al. Tree pollen allergens-an update from a molecular perspective. Allergy 2015;70:1201–11. 21. Twaroch TE, Curin M, Valenta R, et al. Mold allergens in respiratory allergy: from structure to therapy. Allergy Asthma Immunol Res 2015;7:205–20. 22. Green BJ, Sercombe JK, Tovey ER. Fungal fragments and undocumented conidia function as new aeroallergen sources. J Allergy Clin Immunol 2005;115:1043–8. 23. Wagner GE, Gutfreund S, Fauland K, et al. Backbone resonance assignment of Alt a 1, a unique beta-barrel protein and the major allergen of Alternaria alternata. Biomol NMR Assign 2014;8:229–31. 24. Shen HD, Tam MF, Tang RB, et al. Aspergillus and Penicillium allergens: focus on proteases. Curr Allergy Asthma Rep 2007;7:351–6.

25. Zahradnik E, Raulf M. Respiratory allergens from furred mammals: environmental and occupational exposure. Vet Sci 2017;4:38. 26. Pomes A, Mueller GA, Randall TA, et al. New insights into cockroach allergens. Curr Allergy Asthma Rep 2017;17:25. 27. Mueller GA, Randall TA, Glesner J, et al. Serological, genomic and structural analyses of the major mite allergen Der p 23. Clin Exp Allergy 2016;46:365–76. 28. Raulf M, Quirce S, Vandenplas O. Addressing molecular diagnosis of occupational allergies. Curr Allergy Asthma Rep 2018;18:6. 29. Matsuo H, Yokooji T, Taogoshi T. Common food allergens and their IgE-binding epitopes. Allergol Int 2015;64:332–43. 30. Cabanillas B, Jappe U, Novak N. Allergy to peanut, soybean, and other legumes: recent advances in allergen characterization, stability to processing and IgE cross-reactivity. Mol Nutr Food Res 2018;62:(1). doi: 10.1002/mnfr.201700446. 31. Bartuzi Z, Cocco RR, Muraro A, et al. Contribution of molecular allergen analysis in diagnosis of milk allergy. Curr Allergy Asthma Rep 2017;17:46. 32. Kuehn A, Swoboda I, Arumugam K, et al. Fish allergens at a glance: variable allergenicity of parvalbumins, the major fish allergens. Front Immunol 2014;5:179. 33. Zhu L, She T, Zhang Y, et al. Identification and characterization of ovary development-related protein EJO1 (Eri s 2) from the ovary of Eriocheir sinensis as a new food allergen. Mol Nutr Food Res 2016;60:2275–87. 34. de Blay F, Pauli G, Bessot JC. Cross-reactions between respiratory and food allergens. Allergy Proc 1991;12:313–17. 35. Blank S, Bilo MB, Ollert M. Component-resolved diagnostics to direct in venom immunotherapy: important steps towards precision medicine. Clin Exp Allergy 2018;48(4):354–64. 36. Jakob T, Müller U, Helbling A, et al. Component resolved diagnostics for hymenoptera venom allergy. Curr Opin Allergy Clin Immunol 2017;17:363–72. 37. Hoffman DR. Ant venoms. Curr Opin Allergy Clin Immunol 2010;10:342–6. 38. Wanandy T, Gueven N, Davies NW, et al. Pilosulins: a review of the structure and mode of action of venom peptides from an Australian ant Myrmecia pilosula. Toxicon 2015;98:54–61. 39. Cantillo JF, Puerta L, Puchalska P. Allergenome characterization of the mosquito Aedes aegypti. Allergy 2017;72:1499–509. 40. Cantillo JF, Puerta L, Lafosse-Marin S, et al. Allergens involved in the cross-reactivity of Aedes aegypti with other arthropods. Ann Allergy Asthma Immunol 2017;118:710–18. 41. Tyagi N, Farnell EJ, Fitzsimmons CM, et al. Comparisons of allergenic and metazoan parasite proteins: allergy the price of immunity. PLoS Comput Biol 2015;11:e1004546. 42. Kennedy MW. The polyprotein allergens of nematodes (NPAs) - structure at last, but still mysterious. Exp Parasitol 2011;129:81–4. 43. Acevedo N, Sanchez J, Erler A, et al. IgE cross-reactivity between Ascaris and domestic mite allergens: the role of tropomyosin and the nematode polyprotein ABA-1. Allergy 2009;64:1635–43. 44. Nissen D, Pedersen LJ, Skov PS, et al. IgE-binding components of staphylococcal enterotoxins in patients with atopic dermatitis. Ann Allergy Asthma Immunol 1997;79:403–8. 45. Nagasaki T, Matsumoto H, Oguma T, et al. Sensitization to Staphylococcus aureus enterotoxins in smokers with asthma. Ann Allergy Asthma Immunol 2017;119:408–414.e2. 46. Voorhorst R. The human dander atopy. I. The prototype of auto-atopy. Ann Allergy 1977;39:205–12. 47. Valenta R, Seiberler S, Natter S, et al. Autoallergy: a pathogenetic factor in atopic dermatitis? J Allergy Clin Immunol 2000;105:432–7. 48. Weidinger S, Mayerhofer A, Raemsch R, et al. Prostate-specific antigen as allergen in human seminal plasma allergy. J Allergy Clin Immunol 2006;117:213–15. 49. Vroling AB, Jonker MJ, Breit TM. Comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway. Allergy 2008;63:461–7. 50. Vroling AB, Jonker MJ, Luiten S, et al. Primary nasal epithelium exposed to house dust mite extract shows activated expression in allergic individuals. Am J Respir Cell Mol Biol 2008;38:293–9.

CHAPTER 26  The Structure and Function of Allergens 51. Fattouh R, Pouladi MA, Alvarez D, et al. House dust mite facilitates ovalbumin-specific allergic sensitization and airway inflammation. Am J Respir Crit Care Med 2005;172:314–21. 52. Kheradmand F, Kiss A, Xu J, et al. A protease-activated pathway underlying Th cell type 2 activation and allergic lung disease. J Immunol 2002;169:5904–11. 53. Wan H, Winton HL, Soeller C, et al. Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest 1999;104:123–33. 54. 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 2001;31:279–94. 55. Gandhi VD, Vliagoftis H. Airway epithelium interactions with aeroallergens: role of secreted cytokines and chemokines in innate immunity. Front Immunol 2015;6:147. 56. 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 1996;26:2972–80. 57. Hammad H, Chieppa M, Perros F, et al. House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells. Nat Med 2009;15:410–16. 58. Willart MA, Deswarte K, Pouliot P, et al. Interleukin-1alpha controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33. J Exp Med 2012;209:1505–17. 59. Kool M, Willart MA, van Nimwegen M, et al. An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma. Immunity 2011;34:527–40. 60. Klein Wolterink RG, Kleinjan A, et al. Pulmonary innate lymphoid cells are major producers of IL-5 and IL-13 in murine models of allergic asthma. Eur J Immunol 2012;42:1106–16. 61. Chu DK, Llop-Guevara A, Walker TD, et al. IL-33, but not thymic stromal lymphopoietin or IL-25, is central to mite and peanut allergic sensitization. J Allergy Clin Immunol 2013;131:187–200.e1-8. 62. Ito T, Wang YH, Duramad O, et al. TSLP-activated dendritic cells induce an inflammatory T helper type 2 cell response through OX40 ligand. J Exp Med 2005;202:1213–23. 63. Ying S, O’Connor B, Ratoff J, et al. Thymic stromal lymphopoietin expression is increased in asthmatic airways and correlates with expression of Th2-attracting chemokines and disease severity. J Immunol 2005;174:8183–90. 64. Harada M, Hirota T, Jodo AI, et al. Thymic stromal lymphopoietin gene promoter polymorphisms are associated with susceptibility to bronchial asthma. Am J Respir Cell Mol Biol 2011;44:787–93. 65. Zhang J, Chen J, Mangat S, et al. Pathways of airway oxidant formation by house dust mite allergens and viral RNA converge through myosin motors, pannexons and Toll-like receptor 4. Immun Inflamm Dis 2018;6:276–96. 66. Angkasekwinai P, Park H, Wang YH, et al. Interleukin 25 promotes the initiation of proallergic type 2 responses. J Exp Med 2007;204:1509–17.

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67. Milovanovic M, Volarevic V, Radosavljevic G, et al. IL-33/ST2 axis in inflammation and immunopathology. Immunol Res 2012;52:89–99. 68. Prefontaine D, Nadigel J, Chouiali F, et al. Increased IL-33 expression by epithelial cells in bronchial asthma. J Allergy Clin Immunol 2010;125:752–4. 69. Christianson CA, Goplen NP, Zafar I, et al. Persistence of asthma requires multiple feedback circuits involving type 2 innate lymphoid cells and IL-33. J Allergy Clin Immunol 2015;136:59–68.e14. 70. Chen J, Zhang J, Tachie-Menson T, et al. Allergen-dependent oxidant formation requires purinoceptor activation of ADAM 10 and prothrombin. J Allergy Clin Immunol 2017;139:2023–6.e9. 71. Zhang J, Chen J, Allen-Philbey K, et al. Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1. J Allergy Clin Immunol 2016;138:1224–7. 72. Millien VO, Lu W, Shaw J, et al. Cleavage of fibrinogen by proteinases elicits allergic responses through Toll-like receptor 4. Science 2013;341:792–6. 73. Cho M, Lee JE, Lim H, et al. Fibrinogen-cleavage products and TLR4 promote the generation of programmed cell death 1 ligand 2(PD-L2)+ dendritic cells in allergic asthma. J Allergy Clin Immunol 2018;142(2):530–41.e6. 74. Trompette A, Divanovic S, Visintin A, et al. Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein. Nature 2009;457:585–8. 75. Ichikawa S, Takai T, Yashiki T, et al. Lipopolysaccharide binding of the mite allergen Der f 2. Genes Cells 2009;14:1055–65. 76. Heijink IH, van Oosterhout A, Kapus A. Epidermal growth factor receptor signalling contributes to house dust mite-induced epithelial barrier dysfunction. Eur Respir J 2010;36:1016–26. 77. Heijink IH, Postma DS, Noordhoek JA, et al. House dust mite-promoted epithelial-to-mesenchymal transition in human bronchial epithelium. Am J Respir Cell Mol Biol 2010;42:69–79. 78. Heijink IH, Kies PM, Kauffman HF, et al. Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity. J Immunol 2007;178:7678–85. 79. Pichavant M, Charbonnier AS, Taront S, et al. Asthmatic bronchial epithelium activated by the proteolytic allergen Der p 1 increases selective dendritic cell recruitment. J Allergy Clin Immunol 2005;115:771–8. 80. Robays LJ, Maes T, Lebecque S, et al. Chemokine receptor CCR2 but not CCR5 or CCR6 mediates the increase in pulmonary dendritic cells during allergic airway inflammation. J Immunol 2007;178:5305–11. 81. Maneechotesuwan K, Wamanuttajinda V, Kasetsinsombat K, et al. Der p 1 suppresses indoleamine 2, 3-dioxygenase in dendritic cells from house dust mite-sensitive patients with asthma. J Allergy Clin Immunol 2009;123:239–48. 82. Wang YH, Ito T, Wang YH, et al. Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-activated dendritic cells. Immunity 2006;24:827–38. 83. Massacand JC, Stettler RC, Meier R, et al. Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function. Proc Natl Acad Sci USA 2009;106:13968–73.

CHAPTER 26  The Structure and Function of Allergens

427.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following pairs of statements concerning arthropod allergens is correct? a. Der p 2 allergen – a cysteine protease structurally similar to papain. b. Cockroach tropomyosin – a marker allergen for cockroach allergy. c. Bla g 2 – a pseudoprotease. d. Arginine kinases – a group of allergenic enzymes restricted to Lepidoptera species. 2. Which of the following statements about pollen allergens is incorrect? a. The group 1 allergens, such as Phl p 1, represent useful marker allergens in diagnosing grass pollen allergy. b. Pectate lyase allergens are found in gymnosperm tree pollen species as well as in Asteraceae species. c. The majority of ragweed allergens are associated with subpollen particles. d. The group 1 allergens from trees belonging to the Fagales order possess polygalacturonase activity. 3. Which of the following statements about allergens from domesticated furry animal pets is correct? a. The dog allergen, Can f 5, is a prostatic kallikrein and shows sequence identity with human PSA. b. Lipocalin allergens such as Fel d 1 are minor allergens. c. IgE from furry animal-allergic patients is directed primarily against conformational epitopes on the surface of the IgA and IgM allergens. d. Serum albumin allergens have molecular weights in the region of about 30 kDa.

4. Which of the following statements about ingested allergens is incorrect? a. The beta-parvalbumins are important allergens in cartilaginous fish species muscle tissue. b. The casein proteins in bovine milk represent a significant group of major allergens. c. Tropomyosins present in crustacean and mollusk species are associated with arthropod-shellfish cross-reactivity syndromes. d. Because of their ubiquity in most allergen sources, profilins are referred to as pan-allergens. 5. Which of the following statements concerning allergens with protease activity is correct? a. Group 1 from Dermatophagoides farinae has cysteine protease activity. b. Group 2 from Alternaria alternata has metalloprotease activity. c. Group 11 from Aspergillus fumigatus has serine peptidase activity. d. Group 2 from Blatella germanica has aspartic protease activity.

27  Aerobiology of Outdoor Allergens Janet M. Davies, Richard W. Weber

CONTENTS Introduction (Aeroallergen Sources), 428 General Principles of Allergen Aerobiology, 428 Characterized Allergens, 431 Aeroallergen Sampling, 432

SUMMARY OF IMPORTANT CONCEPTS • Outdoor aeroallergens have a variety of sources to include plants, fungi, and animals, and concentrations are affected by variables such as floristic zones, weather and season. • Methods of monitoring aeroallergens vary and have separate advantages and disadvantages. • Although aeroallergen burdens have distinct, seasonality, geographic location, warming trends and other aspects of climate change have modified timing and duration of seasons as well as intensity of exposure. • The clinical impact of exposure is affected by the allergen similarity, or cross-reactivity, between related sources.

INTRODUCTION (AEROALLERGEN SOURCES) Aerobiology is the science of airborne particles of biologic origin. A small portion of that discipline is of concern to allergists: airborne allergens. Aeroallergens are dispersed through the air within particles of varying size and arise from differing sources (Table 27.1).1 The originating source may be easily seen, such as plants, vermin, detritus, or farm or pet animals; at the limits of visual detection like dust mites; or microscopic, such as bacteria or protozoa. The airborne particle may be a cell; intact or fragmented pollen grains or their cytoplasmic components; fungal spore or hyphal fragment; protein adhering to epidermal scales or dust particles; or protein dissolved in water droplets. Outdoor sources are likely to be of plant origin, whereas animals are more common indoor allergen sources, but there are exceptions. Fungal spores may be troublesome both inside and outdoors. Once entrained into airstreams, aeroallergens may be deposited on conjunctival membranes, inducing allergic conjunctivitis; aeroallergens may be deposited on nasal membranes, causing allergic rhinitis; or fragments smaller than 5 micron can be inhaled into the lungs, inducing allergic asthma; certain perennial allergens have been incriminated in atopic dermatitis as well.

GENERAL PRINCIPLES OF ALLERGEN AEROBIOLOGY Pollen Vascular plants propagate through extension—via trunk or root shoots, rhizomes, or stolons—or by seed. Transport of the male gamete, the

428

Representative Pollens, 434 Representative Fungi, 443 Meteorologic Variables, 446 Impact of Climate Change on Aeroallergens, 447

pollen, to the female gamete, the ovary, accomplishes sexual reproduction. Pollen dispersal mechanisms are with the wind (anemophily), or through a vector such as an insect (entomophily). Insect-pollinated plants are uncommon inducers of hay fever, but there are exceptions. Some plants using both mechanisms, being primarily insect-pollinated, produce sufficient pollen that becomes airborne (amphiphilic).

Fungi Fungi comprise one of seven kingdoms of living organisms and are more closely related to the animal kingdom than to the plant kingdom. Fungi fulfill an ecologic role of decomposers and recyclers, whereas plants are considered as producers, and animals as consumers. Fungi are eukaryotic organisms with chromosomes within membrane-bound nuclei, dividing through mitosis. These organisms have chitin-containing cell walls, a polysaccharide found also in insect exoskeletons. Fungi may be unicellular; syncytial with many nuclei not divided into separate cells; or multicellular with nuclei separated by septa. Life cycles are frequently very complex, with multiple life stages and either sexual or asexual reproduction.2

Animal Although animals are primarily sources of indoor allergens, some may be significant outdoor allergens as well. Heavy hatches of caddis flies or mayflies or miller moth infestations have been reported to induce allergic symptoms.3,4 Occupational exposures to tussock moths in Douglas fir may bother lumberjacks, and sewer flies may bother municipal sanitation workers.5,6 Horse dander allergen can be sampled outdoors downstream of stables.7

Submicronic Allergenic Particles That ragweed hay fever symptoms may persist days after intact airborne pollen is no longer detectable spurred studies that demonstrated aero­ allergens in submicronic particles, presumably from fragmented pollen grains.8,9 Airborne birch allergenic activity has also been demonstrated on particles smaller than 2.4 microns.10 Starch granules are prominent in the cytoplasm of certain pollens such as the grasses (Poaceae) and docks (Rumex, Polygonaceae).11 Such grass starch granules have heavy concentrations of major allergen groups 1, 5, and 13.12–14 It is proposed that humidity in the cloud base or raindrops disrupt pollen grains by osmotic shock, releasing large amounts of respirable allergen-laden

CHAPTER 27  Aerobiology of Outdoor Allergens

429

TABLE 27.1  Common Aeroallergen

BOX 27.1  Characteristics of Wind-

Allergen Source

Particle Type

Bacteria

Cells, fragments, metabolites

Thermophilic actinomycetes

Spores, metabolites

Algae

Cells, fragments, metabolites

Protozoa

Metabolites

• Incomplete flowers—spatially separate male (staminate) and female (pistillate) • Male flowers (stamens) exposed to the wind • Petals and sepals inconspicuous or absent • Absent vector attractants such as color, aroma, or nectar • Pollen grains small, dry, with reduced surface ornamentation

Fungi

Spores, hyphal fragments

Ferns & mosses

Spores

Grasses, weeds, & trees

Pollens, cytoplasmic particles

Arthropods

Feces, saliva, body parts

Birds

Feces, epidermal debris

Mammals

Dander, saliva, urine

Sources*

Pollinated (Anemophilous) Plants

*Modified from Burge HA. Monitoring for airborne allergens. Ann Allergy 1992;69:9–18.

particles.12 Taylor and colleagues demonstrated experimentally that a moisture-drying cycle of grass pollen will result in starch granules emanating through the aperture or as a result of fragmentation.15 Ruptured grass pollen has been observed to coincide with thunderstorm asthma episodes and is suspected to be a contributing causative factor.16 (See also Chapter 58.) Fungal spores are greatly variable in shape and size, ranging from 2 to over 200 µm. Individual conidiospores of Penicillium and Aspergillus are spheroidal and usually 4 to 5 µm to slightly larger, and thus, easily respirable. Alternaria spores, on the other hand, may range from 12 to 150 µm, and Cladosporium spores are also variable in size, from 4 to 20 µm.17 Therefore some fungal spores are easily respirable into the lungs, whereas others are not. However, about a quarter of airborne hyphal fragments can be demonstrated to have allergenic activity on immunostaining, a number of which are of respirable size.18

Fig. 27.1  Dangling catkin of pollen-producing male flowers of hophornbeam, Ostrya virginiana.

Characteristics of Wind-Pollinated Plants By comparison to extant relatives, the most primitive vascular plants in the fossil record appeared to be wind-pollinated. Although wind pollination appears to be a simpler process than vector-facilitated pollination, it is extravagant in requiring a large amount of pollen to be produced to ensure successful reproduction. Erdtman studied numerous trees and grasses and showed that wind-pollinated plants produce extraordinary amounts of pollen.19 Each catkin may have more than 200 individual tiny flowers. He reported that a single birch catkin produced about 6 million pollen grains, and an alder catkin 4.5 million. An English oak catkin released 1.25 million grains. Erdtman then tabulated the number of catkins per tree and calculated the amount of pollen produced. A birch tree released more than 5.5 billion grains over a single year, alder 7.2 billion, and an oak less at 0.6 billion grains. Spruce also produced about 5.5 billion grains in a year. Cereal rye (Secale cereale) contained 4.25 million pollen grains per inflorescence.19 Insect pollination followed as a more efficient technique. However, certain anemophilous plants such as grasses give evidence of losing entomophilous characteristics and returning to wind pollination as a later evolutionary ploy.20 Anemophilous characteristics are summarized in Box 27.1. Such plants have incomplete flowers—that is, male and female functions are found on separate structures. The pollen-producing flowers are exposed to the wind. On taller plants or trees this is frequently on dangling catkins, having hundreds of small individual flowers

Fig. 27.2  Inflorescence of Kentucky bluegrass, Poa pratensis.

(Fig. 27.1). On weeds or grasses the inflorescences are thrust up into the air on the higher portions of the plant (Fig. 27.2). Female flowers may be lower, often at axils of leaves, or at stem junctions. Petals and sepals, rather than being showy, are insignificant or absent, and other attractants such as color, fragrance, or nectar are absent. The pollen grains themselves tend to be small and dry with little surface resin and with reduced ornamentation to minimize turbulence in air. In 1930, August Thommen set out five necessary principles for a plant to be an important inducer of pollinosis (Box 27.2).21 Referred to as Thommen’s Postulates, they continue to be generally correct, although with some caveats. The “excitant of hay fever” appears to be a protein or glycoprotein that is easily eluted on contact with water, or

430

SECTION B  Aerobiology and Allergens

BOX 27.2  Thommen’s Postulates on

Pollen Causation of Hay Fever

Arctic Ocean

• The pollen must contain an excitant of hay fever • The pollen must be anemophilous, or wind-borne • The pollen must be produced in sufficiently large quantities • The pollen must be sufficiently buoyant to be carried considerable distances • The plant producing the pollen must be widely and abundantly distributed

2

1

1

Data from Thommen AA. Which plants cause hayfever? In: Coca AF, Walzer M, Thommen AA. Asthma and Hay Fever in Theory and Practice. Springfield, IL: Charles C. Thomas Publisher; 1931: p. 546–54.

coated on respirable cytoplasmic particles. Although most pollinosisinducing plants are wind pollinated, in the proper setting entomophilous plants can release sufficient airborne pollen to cause sensitization. A single point source could lead to individual sensitization. Although most pollen grains settle within meters of the source, pollens can be transported for hundreds of miles.2,19

Floristic Zones The distribution of individual plant species is dependent on many factors. Primary are conditions that constitute climate: average temperature ranges, prevalent humidity, and average rainfall. Selective advantage to different plants may be derived by soil conditions such as density, mineral content, and pH.22 Certain plants have adapted to diverse circumstances and are therefore found broadly distributed; others are niche specific, perhaps adapting to extremes of temperature or water availability. The range of native indigenous species may be determined by niche selectivity. However, the extent to which an introduced plant has spread will be determined by its adaptability, aggressiveness, and duration of time from introduction. One of the major tumbleweeds of the American Great Plains, Russian thistle (Salsola kali), was introduced as a contaminant of flaxseed in Bon Homme County, South Dakota, in 1886. After acclimation, it quickly spread over the entire Great Plains, western states, and Canada.23 Burning bush or summer cypress (Kochia scoparia) was introduced as an ornamental and quickly escaped cultivation and is now found throughout North America.24 Similarly, American endemic short, giant, and western ragweed (Ambrosia artemisiifolia, A. trifida, and A. psilostachya, respectively) have all been introduced into Europe and are now found throughout southern and central Europe.25 Sensitization to ragweed among Europeans has greatly increased.26 Which plants may be found in specific locales may be induced from knowledge of floristic zones. These zones or regions were originally defined by their endemic flora.27 However, the manner in which physical factors impact on the boundaries of these zones has become obvious. One of the most important is the annual mean minimum temperature. The United States Department of Agriculture (USDA) has defined “hardiness zones” based on the average annual minimum temperature: there are twelve zones in the North American continent beginning with zone 0 at less than −60° F, and progressing by 10° increments to zone 11 at more than 40° F.28 These isotherms define the northern limits of species. Comparison of the 1990 and 2015 maps shows warming by a 1–2 zone northern encroachment over much of the United States.29 The USDA map does not take into account factors such as rainfall or maximum temperature. These in turn are determined by the interplay of six additional factors: latitude, elevation, Pacific or Atlantic Ocean influence, continental air mass influence, mountains or hills, and local terrain.22 Latitude implies that the farther from the equator, either northward or southward, the longer and colder the winters. The impact

1

6

2

2 5

7

Pacific Ocean

9

3

6 8 10

Atlantic Ocean

3

5

4

11 12

14

13

N W

E

15

S

Fig. 27.3  Floristic zones of North and Central America, modified from Takhtajan.26 See also Table 27.2.

of elevation is that for each rise of 1000 feet, there is a 3° F drop in temperature, which in North America equates to a northward shift of 600 miles. The ocean (or lake) effect imparts cooler summers and milder winters. Because of the prevailing winds being from the west, the Pacific Ocean effect reaches farther inland than the Atlantic Ocean effect. In the central region, the continental air mass influence dictates hotter summers, colder winters, and more evenly spaced rainfall. Mountains and hills are barriers for precipitation, and the windward side will be wetter, whereas the leeward side (east) will be drier. Local terrain will have an impact in that cold air, like water, flows downhill and will collect in low points like creek beds, gullies, and ravines. North America can be divided into five zones illustrated in Fig. 27.3 and Table 27.2.27 Borders are estimates, because there are frequently wide transition areas between zones and biogeographical distributions of plant are altering due to climate change. Allergenic plants listed in Table 27.2 are common representative plants and not all-inclusive. The Arctic Tundra is treeless, and vegetation is limited to grasses and short shrubby growth. The vast Canadian Boreal Forest, similar to its European counterpart in Scandinavia, has a preponderance of conifers and northern deciduous trees such as alder, aspen, and birch. Weed and grasses are less important as aeroallergens. The Appalachian Eastern Agricultural zone is characterized by marked diversity in trees and weeds, and a large variety of “northern pasture grasses” (subfamily Pooideae). The deciduous forests have been replaced in part by urbanization and farming activity. Additionally, certain native trees such as elm and chestnut have been decimated in their original ranges by differing fungal blights. The Atlantic/Gulf Coastal Plain also shows great diversity among trees with the addition of live oaks and pines, and a preponderance of more southern grasses such as Bermuda, Panicum or Johnson grasses. The Great Plains Prairie comprises a great wedge beginning in Texas and expending northward across the middle of the continent through Manitoba to Alberta. Eastern reaches are described as original tall-grass prairie, with the western higher elevation abutting

CHAPTER 27  Aerobiology of Outdoor Allergens

431

TABLE 27.2  Common Representative Allergenic Plants of North American Floristic Zones Floristic Zone

Grasses

Weeds

Trees

Northern Region 1. Arctic Tundra

Phleum, Poa, Agrostis Phleum, Poa, Agrostis, Dactylis, Festuca

Artemisia, Rumex

Betula, Alnus, Populus, Salix, Pinus

Phleum, Poa, Dactylis, Lolium, Sorghum

Ambrosia, Artemisia, Chenopodium, Amaranthus, Rumex,

Quercus, Acer, Ulmus, Betula, Pinus, Juniperus, Populus

4. Atlantic/Gulf Coastal Plain

Cynodon, Sorghum, Phleum, Dactylis, Paspalum

Chenopodium, Amaranthus, Kochia, Ambrosia, Plantago, Rumex, Artemisia

Quercus, Populus, Acer, Ulmus, Pinus, Myrica, Liquidambar

5. Great Plains Prairie

Festuca, Poa, Dactylis, Lolium, Bromus

Salsola, Kochia, Amaranthus, Chenopodium, Xanthium, Ambrosia

Quercus, Populus, Ulmus, Fraxinus, Acer, Carya

Festuca, Phleum, Sorghum, Cynodon, Lolium

Artemisia, Amaranthus, Atriplex, Ambrosia, Kochia, Salsola

Pinus, Juniperus, Pseudotsuga, Populus, Alnus, Quercus

2. Canadian Boreal Forest Eastern/Midwest Region 3. Appalachian Eastern Agricultural

Western Region 6. Rocky Mountain Cordillera 7. Vancouverian Pacific Maritime

Phleum, Poa, Lolium

Amaranthus, Artemisia, Chenopodium, Rumex

Betula, Alnus, Ulmus,

8. Great Basin

Cynodon, Sorghum, Poa, Dactylis

Artemisia, Amaranthus, Atriplex, Salsola

Populus, Juniperus, Ulmus

9. Californian Lowlands

Cynodon, Festuca, Poa, Bromus

Amaranthus, Chenopodium,

Cupressus, Pinus, Juniperus, Quercus, Olea

10. Sonoran Desert

Sorghum, Cynodon

Ambrosia, Larrea

Prosopis, Cercidium, Acacia

11. Chihuahuan Desert

Sporobolus, Muhlenbergia

Atriplex

Prosopis, Cercidium, Acacia

Southern Region 12. Tamaulipan

Chloris, Bouteloua

Atriplex, Artemisia, Salsola

Celtis, Taxodium, Prosopis,

13. Sierra Madre Highlands

Festuca,

Senecio

Quercus, Pinus, Pseudotsuga, Abies

14. West Indian Tropical

Cynodon, Paspalum, Sorghum

Amaranthus, Ambrosia, Chenopodium

Casuarina, Myrica, Taxodium, Quercus, Acacia

15. Central American Tropical

Paspalum, Cynodon, Sorghum

Amaranthus, Chenopodium

Acacia, Mimosa, Musa

See also Fig. 27.3. Modified from Takhtajan AL. Floristic Regions of the World. Berkeley, CA: University of California Press; 1986.

on the Rocky Mountains being short-grass prairie. The Rocky Mountain Cordillera demonstrates diversity of flora related to extreme changes in elevation and local terrain. Although aeroallergenic plants are found in the tropical zones, generally pollens appear to be lesser inducers of symptoms compared with mold spores and perennial allergens like dust mites.30,31 Vegetation within a city, the “urban forest,” may defy limitations of the floristic zones in which those cities are found. Urban planning of city parks and selection of street trees may incorporate nonendemic species. Survival of more southerly trees and shrubs may be assured by the presence of the heat bubble encompassing most cities. Marked alteration of the aeroallergen profile over time has been documented in some cities such as Tucson, Arizona.32 Moreover, airborne allergens within cities may arise from local urban landscapes as well as rural sources transported to populated regions by wind.33 The impact of climate change on the distribution of plant aeroallergen sources is incompletely understood but changes in temperature, rainfall, and carbon dioxide affect the phenology of flowering, biogeographical range, biomass of subtropical and temperate grass species, and the content of allergen.34,35 Additionally, the impact of urbanization further modifies distribution of allergenic plant sources, with, for example, the creation of niches for ragweed establishment, and modification of allergenicity by gaseous and diesel pollutants in complex ways.36–38

CHARACTERIZED ALLERGENS Nomenclature Numerous allergens from plant, fungal, and animal sources have now been fully or partially characterized. A list of those that have been sequenced is maintained and updated online by the International Union of Immunological Societies (IUIS).39 Nomenclature for newly recognized allergens is now assigned with the first three letters of the genus followed by the first one or two letters of the species, and a number. For example, the major allergen of short ragweed (Ambrosia artemesiifolia) is Amb a 1, formerly known as Antigen E. A lower number may signify importance or order of discovery. Occasionally allergens are renamed to conform to the numbers of biochemically related allergens from other sources. Isoallergens are homologous allergens that share at least 67% identity in primary amino acid sequence. Isoallergens may also differ in posttranslational modifications (for example glycosylation), with or without observable differences in molecular weight, charge of an allergen, and, importantly, allergen function or IgE binding capacity. Isoallergens may have additional minor variants and are closely related (more than 90%). Isoallergens and variants are defined by suffixes, with the first two digits after the decimal point indicating the isoform and the next two digits indicating the variant (e.g., the isoallergens of timothy grass group 5, Phl p 5.0101 and Phl p 5.0201, whereas Phl p 5.0102 is a variant of the former).

432

SECTION B  Aerobiology and Allergens

Cross-Reactivity of Pollens and Fungi Cross-reactivity is the ability of an antigen to bind with an antibody that was raised to a different antigen. It may arise by one of two mechanisms: shared epitopes on multivalent antigens or conformational similarity of epitopes. In the former case, antibodies should bind with the same affinity; in the latter case, antibodies would bind with lesser affinity. Sequence amino acid homology implies complete cross-reactivity, but this is not always the case, because differences in tertiary folding may result in different epitopes. For example, despite high amino acid homology of the NH2-terminal between Lol p 1 and Cyn d 1, crossreactivity of IgG monoclonal antibodies and human IgE between perennial ryegrass and Bermuda grass is weak.40 In the context of allergy, immunologic cross-reactivity with allergens may occur with conformational surface epitopes for serum IgE binding, as well as at a T cell level with short linear peptide epitopes in the context of a trimolecular interaction between the epitope, major histocompatibility antigen presentation molecules, and T cell receptor. Why should cross-reactivity of pollens and fungi be of concern? It is because such relationships have impact both on diagnosis and therapy. For example, skin test positivity or serum IgE reactivity to a nonendemic plant may be explained either by prior exposure in a mobile population or cross-reactivity with endemic plants. The decision whether to include such a nonendemic extract in an allergen immunotherapy formulation depends on several factors, one of which is whether related plants provide adequate coverage. At a time of constricting availability of particular pollen and fungal extracts, this issue becomes crucial. Initially, because pollen cross-allergenicity data were limited, plant systematics had been used to infer the likelihood of cross-reactivity. The validity of this process depends on two premises: first, that more closely related plants will share greater similarities and antigens; second, that the accepted botanical classification reflects phylogeny, that two plants in the same genus evolved from a common ancestor, two in the same family from a more distant ancestor, and so on.41 Two plants in the same genus would be expected to have the greatest number of shared allergens, those in the same family fewer, and distantly related plants would be expected to show little cross-reactivity. Research generally supports the use of this approach, the exceptions being panallergens such as the profilins, calmodulins, or pathogenesis-related protein families (PR), which are both pollen and food allergens, and some of which may also be found in animal tissue. The explosion of gene sequencing technology and the subsequent cloning of numerous allergens via yeast and bacterial vectors (see www.allergen.org) has revolutionized the study of allergenic protein function and cross-reactivity. Cross-reactivity with groups of related allergenic plants will be commented on in the sections on Representative Pollens and Representative Fungi.

TABLE 27.3  Types of Aeroallergen

Samplers Type

Example

Comments

Durham Petrie dish

No longer adequate for research Smaller particles underrepresented Particles/surface area (p/cm2) Colonies/plate

Gravimetric

Volumetric Impaction Intermittent rotary

Particles/volume air (p/m3) Rotorod

Overloads with continuous sampling; collects 1 in every 9 minutes Poor capture efficiency particles 250-500

>500

Fluticasone propionate (HFA)

100-250

>250-500

>500

Mometasone furoate

110-220

>220-440

>440

Triamcinolone acetonide

400-1000

>1000-2000

>2000

Children 6-11 Years Beclomethasone dipropionate (CFC)a

100-200

>200-400

>400

50-100

>100-200

>200

Budesonide (DPI)

100-200

>200-400

>400

Budesonide (nebules)

250-500

>500-1000

>1000

Beclomethasone dipropionate (HFA)

Ciclesonide

80

>80-160

>160

Fluticasone furoate (DPI)

n.a.

n.a.

n.a.

Fluticasone propionate (DPI)

100-200

>200-400

>400

Fluticasone propionate (HFA)

100-200

>200-500

>500

110

≥220-800-1200

>1200

Mometasone furoate Triamcinolone acetonide

CFC, Chlorofluorocarbon propellant; DPI, dry powder inhaler; HFA, hydrofluoroalkane propellant; n.a., not applicable. a Beclomethasone dipropionate CFC is included for comparison with older literature. ©2017 Global Initiative for Asthma, reprinted with permission.

A detailed review of these various classes of compounds can be found elsewhere in this textbook; however, a few remarks specific to the medication class in relation to the pediatric population will be reviewed.

Inhaled Corticosteroids The increased appreciation of the role of inflammation in mild to moderate asthma has elevated the use of ICSs to first-line controller therapy in all age groups1 based on the results of several pediatric studies.19-23 This class of medication is noted to reduce airway hyperresponsiveness, inhibit airway inflammation, block the late-phase reaction to allergen, and reduce asthma symptom burden and risk of exacerbations.1,2,24 However, based on the results of prevention trials, ICSs do not appear to alter the underlying severity or progression of disease.20,25,26 The effectiveness of ICSs must also be weighed against the concerns about systemic toxicity of ICSs, particularly in relationship to growth. However, the risk-benefit ratio is complicated, resulting in part from difficulties in distinguishing statistically significant research findings from clinically relevant adverse effects.27 Collectively, numerous studies have established the relative safety of ICS therapy in children.27 Nonetheless, the potential of ICSs to decrease growth rate exists.27,28 These effects may be influenced by the dose and potency of the specific ICS; the type of delivery device used; the age, gender, and weight of the child; and the individual susceptibility to steroid-induced growth suppression. Furthermore, the small risk of side effects must be balanced with the ability of ICSs to improve both impairment and risk outcome parameters when used on a long-term basis.19 The choice of which ICS to be use depends on a number of factors, including potency, systemic absorption, taste, delivery system, and cost, among others. There are multiple ICS formulations and devices available, including dry powder inhaler (DPI) and hydrofluoroalkane propellent (HFA). In addition, budesonide is the only FDA-approved nebulized ICS in the United States and is effective in treating asthma in children as young as 1 year of age.27 Systemic bioavailability of ICS results from a combination of oral (swallowed fraction) and lung components. For some ICSs (e.g., budesonide), a higher clearance rate and shorter plasma half-life have been shown in children compared with adults, suggesting an increase in the ratio between local and systemic side effects.27 Relative binding affinities and lipophilicity can also affect the potency of various ICSs and thus affects the estimated daily dosages. In part related to side effects and adherence rates that are often less than 50% for daily ICS treatment, alternate ICS dosing strategies have also been studied. A step-up short-term or “yellow zone” approach in the preschool age group, in high-risk children 12 to 53 months of age with severe episodic disease, found no difference in the rate of exacerbations or symptoms between daily low-dose budesonide and intermittent high-dose budesonide during respiratory tract illnesses over the course of 1 year10 (Table 50.1). Of note, the children in the intermittent highdose ICS group received 30% of the total ICS exposure compared with the daily low-dose group. A rescue ICS (step-up intermittent) approach, using ICS whenever rescue beta-agonist is required, has also been studied in children 5 to 18 years with promising results16 (Table 50.1). In this study, children who received beta-agonists only had higher rates of exacerbations than children that received beclomethasone whenever they used albuterol or received daily low-dose beclomethasone. Notably, there was not a significant difference in exacerbations between children treated with daily vs. rescue ICSs, and the cumulative ICS dose was significantly lower in the rescue ICS group. Moreover, effects on growth velocity were not seen with the rescue ICS approach but were seen when ICSs were used at least on a daily basis.16 However, ICS/SABA combination inhalers are not currently available in the United States, making use of this strategy a challenge from a practical perspective.

CHAPTER 50  Management of Asthma in Infants and Children GINA endorses increasing ICS doses at the early signs of loss of asthma control in children treated with daily ICS. However, a recent study of 5- to 11-year-old children receiving daily low-dose ICSs found that quintupling the dose of ICS for 7 days at onset of loss of asthma control (i.e., “in the yellow zone”) did not reduce severe asthma exacerbations treated with systemic corticosteroids and was associated with possible small effects on linear growth.29 Combined with the rescue ICS findings in children, it appears that although daily ICSs are effective in improving asthma control in children, temporary increases from the daily ICS dose do not provide additional benefit.

Long-Acting Bronchodilators Long-acting beta agonists.  Salmeterol and formoterol are two long-acting beta-agonists (LABA) that have been evaluated in children27; currently, salmeterol has been approved for children aged 12 years and older delivered by metered dose inhaler (MDI) and 4 years and older delivered by DPI, whereas formoterol is approved for children 6 years and older. When administered in a single dose, salmeterol has a delayed (10 to 15 minute) onset of action compared with albuterol (5 minutes), but the duration of significant bronchodilation is much longer (12 to 18 hours vs. 3 to 6 hours).27 Formoterol has a similar onset of action to albuterol in addition to providing prolonged bronchodilation.30 Several metaanalyses of trials in patients older than 12 years reported a greater benefit in decreasing asthma symptoms and exacerbations and improvement in lung function with the addition of a LABA compared with increasing the inhaled corticosteroid dose alone.31,32 However, the bronchodilating effect of long-acting beta-agonists may diminish with time.33 Unlike regular use of SABAs, daily use of LABAs has not produced an increase in bronchial hyperresponsiveness.1 It should be noted that there is no evidence of antiinflammatory effects from inhaled long-acting β2-agonists. Thus use of long-acting β2-agonists as monotherapy is contraindicated for long-term control of persistent asthma.1 Discontinuation of ICSs after starting a LABA results in an increase in asthma exacerbations.34 In 2005, the FDA placed a Black Box Warning label on products in the United States containing either salmeterol or formoterol in response to reports of an increased risk of severe asthma exacerbations associated with LABA use.1 However, several large recent studies provided reassurance as to the safety of LABA therapy in combination with ICS,35,36 including children as young as 4 years of age,36 and the FDA has removed this warning from products containing LABAs.37,38 In patients aged 12 years and older, ICS/LABA combination therapy may permit reductions (but not elimination) of inhaled corticosteroid doses without a significant worsening of asthma control and may be more efficacious than further increasing the dose of ICS.27,34 Some investigators have suggested that the significant consistent beneficial effect noted in adult patients after the introduction of combination ICS and LABA therapy needs to be better defined in children.27 The EPR-3 and GINA stress that LABA should not be used as monotherapy but should be used for adjunctive therapy in patients older than 5 years who have asthma that is not controlled on a low to moderate dose of ICSs.1,2 The BADGER study comparing low-dose ICS plus LABA to low-dose ICS plus LTRA or medium-dose ICSs demonstrated that low-dose ICS plus LABA provided the greatest chance for a superior treatment response as assessed by a composite outcome of asthma impairment and risk.39 Long-acting muscarinic antagonists.  The long-acting muscarinic antagonist (LAMA) medication tiotropium has also been studied in children and was recently approved in the United States for use in children aged 6 years and older. Studies in adults have demonstrated a modest reduction in asthma exacerbations (21%). Recently published studies in children were not powered to assess an exacerbation endpoint but demonstrated improvements in lung function.40,41 The relative

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benefits of add-on therapy with tiotropium compared with LABA, LTRA, or increased doses of ICSs have not been assessed in children.

Leukotriene Modifiers.  Leukotriene modifiers target a specific component of the asthmatic inflammatory cascade. A role for leukotrienes in the pathogenesis of asthma has been suggested by their biologic activities on bronchoconstriction, mucus secretion, and inflammatory cell infiltration into the airway.27 Leukotriene modifiers include either inhibitors of leukotriene production (5-lipoxygenase inhibitors) or leukotriene receptor antagonists (LTRAs), which bind to cell surface receptors. Only montelukast is approved for use in children in the United States. Montelukast modestly improved lung function and response to allergen and exercise challenges when used as monotherapy in school-age children.27 Similarly, montelukast provided improvement in symptoms and exacerbations compared with placebo in children younger than 5 years.42,43 However, when overall efficacy of LTRA was compared with low-dose monotherapy with ICSs in children with persistent asthma, most outcome measures (symptoms, exacerbations, and lung function) significantly favored ICS.22 Furthermore, it has been demonstrated that children who have higher levels of eosinophilic/ allergic airway inflammation (nitric oxide, immunoglobulin E [IgE] levels, total eosinophil levels) or low pulmonary function (measured by forced expiratory volume in 1 second [FEV1]/forced vital capacity [FVC] or FEV1) are more likely to demonstrate benefit with ICS compared with LTRA. Children who do not have these characteristics appear to respond to either treatment equally.23,44,45 The use of LTRA as add-on therapy in asthma that is not controlled on low-dose inhaled steroids alone (moderate or severe asthma) has not been studied satisfactorily in children aged 5 to 11 years and not at all in children younger than 5 years.1 Three studies in adults and one study in children1 demonstrate a trend showing that LTRA improved lung function and some measures of asthma control compared with a fixed dose of ICS. A study comparing LTRA and LABA in children 6 to 17 years of age as add-on therapy with low-dose ICS demonstrated that the greatest chance of achieving the best response at step 3 care was with LABA.39 However, a substantial number of children did achieve their best response with ICS plus LTRA treatment. Because 98% of children enrolled in this study responded best to one of these three step 3 treatment options (medium-dose ICS, low dose ICS plus LABA, or low-dose ICS plus LTRA), if a clinician prescribes one of those treatments and it does not achieve acceptable asthma control, one of the other two should be considered before the clinician moves to step 4 care.39 Long-term safety issues with the use of leukotriene modifiers in children remain to be fully addressed, but most adverse events reported in clinical trials have been mild. However, in a recent real-world practice study, a small but significant proportion of young children started on montelukast experienced neuropsychiatric side effects leading to medication discontinuation.46 Presumably, the convenience of once-daily oral dosing for montelukast has been an attraction for many primary care physicians who treat childhood asthma. Based on the studies to date, GINA and EPR-3 recommend that LTRA be used as an alternative, not preferred, treatment option for mild persistent asthma, and as an alternative, not preferred, adjunctive treatment with ICS in moderate or severe asthma.1,2

Theophylline.  Theophylline, a methylxanthine, has been shown to be effective as monotherapy for the attenuation of persistent asthma in children related to its effects as a bronchodilator, antiallergic, and antiinflammatory compound.47 Although theophylline has oral steroid-sparing effects in children with moderate to severe persistent asthma,48 other data demonstrate that theophylline has less clinical efficacy than ICS to control persistent asthma.1 As an adjunctive therapy to ICSs, theophylline

838

SECTION E  Respiratory Tract

produces a small improvement in lung function similar to doubling the dose of ICSs.1 Thus GINA and EPR-3 recommend that sustained-release theophylline be used as an alternative, not preferred, adjunctive therapy with ICSs.1,2 When theophylline is prescribed, careful attention should be paid to appropriate initial and follow-up dosing strategies and the monitoring of serum theophylline levels (target level of 5 to 15 µg/ml). It may be necessary to decrease daily dosing during illness or with the use of concomitant medications such as macrolide antibiotics.

Biologic Agents.  Biologic agents bind specific molecular targets or antibody receptors (e.g., IgE, IL-5, IL-4R humanized monoclonal antibodies [hMab]) that inhibit specific cell signaling pathways relevant to asthma pathogenesis. These agents are more comprehensively reviewed in other chapters. The most extensively studied immunomodifier in children, omalizumab, is a recombinant mouse hMab directed against human IgE. Studies of omalizumab in children have demonstrated efficacy from 5 to 18 years of age.13,49,50 Used as add-on therapy to ICS or ICS/LABA combination, omalizumab leads to improvement in symptoms but has the greatest benefit in the prevention of exacerbations.13,50 Omalizumab is FDA-approved for use in children aged 6 years and above. GINA suggests that omalizumab be considered for adjunctive therapy in persons older than 6 years with severe asthma (step 5, Fig. 50.1) who have allergies and are not controlled on medium to high doses of ICSs and LABAs.2 Both mepolizumab and benralizumab, which target the IL-5 pathway in patients with eosinophilic asthma, are approved in adolescents aged 12 years and older in the United States. These anti-IL5 agents have been demonstrated to reduce the risk of asthma exacerbations in adolescents. Additional biologic therapies are currently being evaluated in children. Immunotherapy.  Specific subcutaneous allergen immunotherapy (SCIT) has been shown to potentially modify existing allergic sensitization and secondarily reduce allergic asthma in regard to specific exposures.51 A metaanalysis of 75 pediatric and adult studies supports the effectiveness of immunotherapy in asthma with reductions in asthma burden and medication use and improvement in bronchial hyperreactivity.52 However, a large placebo-controlled clinical trial evaluating the efficacy of multiallergen immunotherapy in children did not demonstrate a significant effect on a variety of asthma outcome measures.27 In contrast, a recent large study in adults of sublingual immunotherapy with dust mite demonstrated a reduction in multiple asthma outcomes, including exacerbations.53 GINA and EPR-3 have recommended allergen immunotherapy in patients with stable asthma who are sensitized to a particular allergen if there is clear association between symptoms and allergen exposure.1,2

Environmental Control A number of studies have documented the association of the risk of developing childhood asthma and aeroallergen sensitization.54,55 The level of house dust mite27 exposure in older children correlates with the presence of both wheezing and airway hyperresponsiveness. Greater exposure to cockroach allergen in children with asthma is associated with skin test sensitization,27 particularly in children of African-American race and low socioeconomic status.56 Pet dander exposure can occur in environments without the presence of an animal27 and may trigger worsening asthma in sensitized children.27 Furthermore, exposure to the specific allergen to which both adults and children are sensitized can lead to allergic inflammation and hyperresponsiveness in the airways.27 Reduction of house dust mite exposure in the indoor environment decreases asthma symptoms and bronchial hyperresponsiveness in children.57 Because exposure to antigens such as dust mite, pet dander, and cockroaches can occur in public buildings27 including schools,

environmental allergen avoidance measures both within and outside the home may influence overall disease activity.58 Thus GINA and EPR-3 recommend that children with persistent asthma be evaluated for allergen-related symptoms by history for seasonal fluctuations in control with substantiation by skin testing or specific IgE antibodies to the indoor and/or outdoor allergens to which the patient is exposed.1,2 It is important to educate families on a multifaceted allergen control strategy for the home and school settings to reduce cockroach, dust mite, pet, rodent, or mold allergen exposure in a sensitized child with asthma.1,2,58 Although numerous studies have shown that passive smoke exposure adversely influences asthma incidence, airway hyperresponsiveness, symptoms, exacerbations, and overall lung function over time,27 parents and caregivers often continue to smoke. Smoking cessation resources and education on avoidance of smoke exposure in the home and daycare should be addressed regularly.1,2 As noted previously, viral infections can be a major contributor to wheezing episodes in infants and children and can be a major trigger for asthma exacerbations once the disease process has become established.27 Exposure to daycare facilities is associated with an increasing incidence of lower respiratory illnesses and recurrent wheezing.27 Likely, these associations are related to the rapid and frequent transmission of viruses among the children within these facilities. However, it should be noted that early daycare exposure in the first 6 months of life has also been associated with a reduced risk of asthma in children;59 thus avoidance of daycare may be more important in children past infancy with an established pattern of recurrent wheeze or asthma. In sum, control of factors from both the outdoor and indoor environment are important in the overall treatment of children with established asthma.

Psychosocial Factors Several observational studies have identified an association between stress and depression and poorly controlled asthma. Stress is associated with an increased prevalence of asthma60 and an increased risk of exacerbations,61 especially in children with very negative life events and brittle asthma.27 Emotions can influence objective measurements of airway function, interpretation of clinical symptoms, and the ability to react appropriately to them.27 Conversely, asthma as a chronic disease may influence various psychosocial adaptations to a child’s home and school environment. Children with asthma have been reported to have significantly more total anxiety disorders, lower self-esteem, greater functional impairment, past school problems, past psychiatric illnesses, and intrafamilial stress.27 Other psychosocial factors are also associated with poor asthma outcomes, such as conflict between family and the medical staff, inappropriate asthma self-care, depressive symptoms, behavioral problems, and disregard of perceived asthma symptoms.62 The influence of asthma on the family and the converse are also noteworthy. Some investigators have demonstrated that controlled asthma can be related to the correct use of medication, which is significantly influenced by a more rigid, structured, and interdependent family environment.27 Conversely, when the benefits of a structured family are disrupted by negative, stressful events, asthmatic children can be at increased risk of asthma exacerbations. In addition to personal factors, parental mental status is a strong predictor of asthma morbidity, hospitalization, and poor adherence to therapy.27 GINA and EPR-3 recommend patients with poorly controlled asthma should have evaluation of the potential role of stress or depression of the child or caretaker in asthma management and be provided with additional education regarding these issues.1,2

Asthma Education Several benefits of asthma education have been documented. The education of children with asthma on skills of self-assessment, use of

CHAPTER 50  Management of Asthma in Infants and Children medications, and actions to prevent or control exacerbations is associated with reduction in urgent care visits and hospitalizations, reduction in school absences, and improvement in health status.1,2,58 Many of these studies were performed in a wide range of points of care (e.g., clinics, hospitals, schools, and doctor’s offices) using a variety of educators. A metaanalysis showed that the addition of a written asthma action plan to asthma self-management education significantly decreased hospitalizations and ED visits for asthma in adults.63 Similar positive findings have been reported in children.64 Asthma action plans can also be successfully implemented in the electronic medical record (Fig. 50.2). Key topics covered in various educational programs include: (1) understanding of the basic facts of asthma; (2) roles of medications and importance of appropriate medication use; (3) skills (device use, spacer use, how to assess asthma control, symptom monitoring, PEF monitoring, recognition, and appropriate response to symptoms); (4) importance of reducing environmental triggers of asthma; and (5) when and how to adjust treatment using a written asthma action plan and responding to changes in asthma control.1,2,58 As discussed, comprehensive and effective asthma education is a dynamic process that requires time and dedication by health care professionals. The following four characteristics summarize the most cogent points regarding asthma education programs: 1. Reach agreement on goals in partnership with the physician. Families must learn that asthma is a chronic disease rather than an intermittent problem. Caretakers, families, and patients must agree on treatment goals. Goals should include living a normal lifestyle with little or no symptoms of asthma. 2. Rehearsal. Parents and patients should be able to react appropriately to hypothetical scenarios that may mimic events surrounding exacerbations specific to the patient and his or her environment. 3. Repetition. Review of the treatment goals and the specifics of asthma education are critical to an effective program. 4. Reinforcement. As progress is made, positive reinforcement from the asthma care team is important. Families and patients derive further reinforcement as the symptoms of asthma are better controlled.47,65 GINA and EPR-3 recommend that asthma self-management education be incorporated into routine care of children who have asthma in a variety of points of care (clinic, pharmacy, emergency department, and hospital settings).1,2 In addition, partnerships among the patient, the family, the clinician, and school personnel should be created to establish a “circle of support” (see next section) to promote effective asthma management both at home and within the school setting.58

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School-Based Asthma Management Programs Morbidity from childhood asthma adversely affects school performance, with one in two children reporting school absences related to asthma each year.1,118 These asthma-related absences influence academic achievement, leading to decreased levels of reading proficiency and increased risk of learning disabilities.66 Improving health and school-related outcomes for children with asthma will require the use of school-based partnerships that focus on integrated care coordination among families, clinicians, and school nurses. In this regard, clinicians who care for children with asthma have an obligation to coordinate asthma care with the schools. Aside from routine clinical care of asthma, providers must educate the family and child about the need for an asthma treatment plan in school and support the school nurse meeting the needs of the student requiring schoolbased asthma care. Developmentally appropriate asthma management, effective communication, and partnership with the schools are essential for quality asthma care. Recently a group of stakeholders from a variety of different disciplines and organizations developed the School-based Asthma Management PROgram (SAMPRO).58 SAMPRO consists of four components (Table 50.4) essential to creating an effective partnership between schools and providers centered around childhood asthma care. These four items include the development and implementation of the following: 1. A Circle of Support that facilitates communication among the child, the family, clinicians, school nurses, and the community. 2. Asthma Management Plans that include both the Asthma Emergency Treatment Plan (AEP) and an Asthma Action Plan (AAP). The AEP details an emergency management plan for all students with asthma, including stock albuterol and a way to deliver the medication. An Asthma Action Plan for home and school includes medical authorization for self-carry and administration of asthma medications, along with parental release of information. 3. A comprehensive education plan for all school personnel. 4. A plan for assessment of the school environment and remediation of school-based asthma triggers. These four components are described in detail in this paper. In addition to a complete description and recommendations regarding these components, SAMPRO also provides an asthma education toolbox that contains many reference tools and educational videos to complement, supplement, and enhance the utility of using SAMPRO for providing comprehensive school-based care for children with asthma. The

TABLE 50.4  Components of a School-Based Asthma Management Program (SAMPRO) Components

Description

1. Development and implementation of a Circle of Support that facilitates communication among clinicians, school nurses, families, and the community.

A communication network integrating the child, their family, the health care provider, and the school nurse into the asthma care team.

A “generic” asthma emergency treatment plan to address children 2. Implementation of Asthma Management Plans (AMP). The AMP includes experiencing an asthma exacerbation, when to use rescue the Asthma Emergency Treatment Plan (AEP) for emergency management medication, how much rescue medication to administer, and when of asthma symptoms including stock albuterol where state law allows and a way to to call 911. An AAP includes individualized instructions for the deliver the medication. Each individual student should have an Asthma Action student’s asthma management plan and instructions on when to Plan (AAP) for home and school with medical authorization for self-carry and step up asthma care. administration of asthma medications, along with parental release of information. 3. A comprehensive asthma educational plan for all school personnel.

A plan for school personnel regarding what is asthma, how to recognize and respond to an asthma exacerbation.

4. Assessment and remediation of school-based asthma triggers.

A plan to assess the school environment and respond to asthma triggers.

From Lemanske RF Jr., Kakumanu S, Shanovich K, et al. Creation and implementation of SAMPRO: a school-based asthma management program. J Allergy Clin Immunol 2016;138(3):711–23.

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SECTION E  Respiratory Tract

Fig. 50.2  Asthma action plan. (From Lemanske RF Jr., Kakumanu S, Shanovich K, et al. Creation and implementation of SAMPRO: a school-based asthma management program. J Allergy Clin Immunol 2016;138(3):711–23.)

CHAPTER 50  Management of Asthma in Infants and Children entire SAMPRO programs can be accessed on line (http://www.aaaai.org/ SAMPRO).

MANAGING EXACERBATIONS Asthma exacerbations are acute or subacute periods of worsening asthma symptoms: cough, wheezing, dyspnea, and chest tightness. During exacerbations, decreases in expiratory flow measured by lung function or peak expiratory flow can also be documented. Management of acute asthma can be considered in three settings: the home (mild exacerbation), the office or emergency department, and the hospital (more severe exacerbation) with appropriate types and doses of medications.1,2 Although asthma that is well controlled will decrease the frequency of exacerbations, exacerbations can still occur, particularly with viral infections. In the Childhood Asthma Management Program study of schoolaged children with mild to moderate asthma, approximately 40% of the children in the ICS-treated group experienced exacerbations (first course of prednisone) compared with approximately 75% in those treated with placebo.19

Home Management The best strategy for management of exacerbations is early treatment. Home management of an asthma exacerbation avoids delays in therapy, prevents exacerbations from becoming severe, and enhances the child’s and family’s sense of control over asthma. The essentials of early treatment are education of the child and their family regarding following a written asthma action plan, recognition of early signs of an exacerbation, appropriate intensification of therapy, removal of precipitating environmental factors or events, and prompt communication with the provider to discuss significant deterioration in symptoms or poor response to therapy. Written asthma action plans typically have a “green,” “yellow,” and “red” zone intervention strategy based on changes in symptoms and/or PEF values (Fig. 50.2). In addition to PEF values in older children or in children too young or otherwise incapable of performing objective measurements of pulmonary function, parents and caretakers should be taught the signs and symptoms to be evaluated (color changes, respiratory rate, location and extent of retractions, duration of inspiratory and expiratory phases, presence or absence of cough or wheezing). Initial therapeutic intervention is usually directed toward assessing the extent and duration of reversibility following bronchodilator administration with inhaled SABA.27 Inhaled SABA can be delivered by metered dose inhaler (MDI) with a spacer device, dry powdered formulations, or by handheld nebulizers. It should be noted that young children younger than 5 years require the use of a mask with nebulizer treatments or with an MDI and valved spacer system for effective delivery of medication into the airways.1,2 The efficacy of treatment by inhalation will be dependent on the technique of the patient with some patients and their families preferring nebulizer treatments. However, both highdose MDI and nebulized SABA delivery have been shown to be equally efficacious in the emergency room and hospital settings.27 If the acute episode is severe and unresponsive to bronchodilator therapy, the family and/or patient should be instructed to contact a health care provider by phone or in person. Home therapy may involve the introduction of OCSs for a short course, which may reduce the duration of the exacerbation and prevent hospitalization.67 However, while still the standard of care, the efficacy of oral corticosteroids in preschool children has recently been called into question by some,68,69 but not all investigators.70 In older children and adults, oral corticosteroids have been shown to be more effective than high-dose ICS.71 Patients with a history of severe exacerbations should have beta-agonists and appropriate equipment (spacer, nebulizer) for treating exacerbations

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available in their home.1,2 The increased SABA use during an exacerbation should continue until the asthma symptoms and PEFs return to the patient’s baseline.

Office or Emergency Department Management If early intervention at home fails to improve the worsening symptoms or PEFs, the family should be instructed to take the child to the treating health care provider’s office, urgent care, or hospital emergency department for further management. The initial assessment should include a brief history, physical examination focused on the work of breathing, PEF or spirometry, and oxygen saturation (Fig. 50.3). Blood gases are not routinely indicated except if a patient has a severe exacerbation that is poorly responsive to initial bronchodilator therapy. In this scenario, an arterial blood gas can be helpful in predicting the potential for subsequent respiratory failure (increased pCO2) and possible intubation. Routine chest radiographs are not necessary unless potential complications (pneumothorax, pneumomediastinum, pneumonia, atelectasis, or aspiration) are suspected based on history or the initial physical examination.27 In a mild to moderate exacerbation (FEV1 or PEF above 50% of baseline), initial therapy includes oxygen to keep oxygen saturations above 90% and up to three doses in the first hour of inhaled SABA by either nebulizer or MDI with spacer, and possibly OCSs if no immediate response to bronchodilators occurs (Fig. 50.3). In severe exacerbations (FEV1 or PEF below 50% of baseline), therapy would include prompt administration of oxygen, high-dose inhaled SABA plus ipratropium every 20 minutes or continuously for the first hour, and oral corticosteroids.1 Severe exacerbations are potentially life threatening, and therefore transfer to an ED is required in most cases for close observation for deterioration, repeated assessments, and frequent treatment.1 Impending or actual respiratory arrest should result in intubation and mechanical ventilation with 100% oxygen, continuous inhaled SABA, ipratropium, and intravenous corticosteroids and admission to the intensive care unit (Fig. 50.3). Adjunctive bronchodilation with the subsequent or simultaneous administration of ipratropium bromide by inhalation remains controversial. Some authors have reported improved symptoms particularly when used in the first 24 hours of the exacerbation, whereas others have found no additional beneficial effects47; these conclusions are supported by recent trials and reviews.1,72 Most, but not all, studies evaluating the use of intravenous theophylline in the treatment of acute exacerbations have been unable to demonstrate any additional benefit over aggressive intervention with beta-agonists; thus its use in hospitalized asthmatic children also remains controversial.1

Hospital Management An incomplete response in symptoms or lung function (FEV1 or PEF of 40% to 69%) despite aggressive treatment should warrant hospitalization for continued inhaled SABA, systemic corticosteroids, oxygen (if needed), and close monitoring. Those with a poor response to therapy (FEV1 or PEF below 40%, pCO2 above 42 mm Hg, severe respiratory distress, drowsiness, or confusion) should be admitted to the hospital intensive care unit for oxygen, inhaled SABA, intravenous corticosteroids, and possible intubation and mechanical ventilation. Special attention should be given to children with risk factors for fatal asthma as outlined previously. It can be difficult to assess the severity of an asthma exacerbation in young children and infants because they cannot perform lung function measures or give detailed histories; therefore a combination of symptoms and physical examination findings is used.1,2 The anatomic and physiologic features of infants predispose them to airway narrowing. Frequent pulse oximetry and lower threshold for arterial blood gas monitoring should be used, because infants are more prone to becoming hypoxic and entering respiratory failure than adults given

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SECTION E  Respiratory Tract

PRIMARY CARE

Patient presents with acute or sub-acute asthma exacerbation

Is it asthma? ASSESS the PATIENT

Risk factors for asthma-related death? Severity of exacerbation?

MILD or MODERATE

SEVERE

Talks in phrases, prefers sitting to lying, not agitated Respiratory rate increased Accessory muscles not used Pulse rate 100–120 bpm O2 saturation (on air) 90–95% PEF 50% predicted or best

Talks in words, sits hunched forwards, agitated Respiratory rate >30/min Accessory muscles in use Pulse rate > 120 bpm O2 saturation (on air) 60−80% of personal best or predicted

ARRANGE at DISCHARGE Reliever: continue as needed Controller: start or step up Check inhaler technique, adherence

Oxygen saturation >94% room air

Prednisolone: continue, usually for 5−7 days (3−5 days for children)

Resources at home adequate

Follow up: within 2−7 days

FOLLOW UP Reliever: reduce to as-needed Controller: continue higher dose for short term (1−2 weeks) or long term (3 months), depending on background to exacerbation Risk factors: check and correct modifiable risk factors that may have contributed to exacerbation, including inhaler technique and adherence Action plan: Is it understood? Was it used appropriately? Does it need modification? O2: oxygen; PEF: peak expiratory flow; SABA: short-acting beta2-agonist (doses are for salbutamol). Fig. 50.3  Global Initiative for Asthma (GINA) management of exacerbations in children 6 years of age and older. (©2017 Global Initiative for Asthma, reprinted with permission.)

CHAPTER 50  Management of Asthma in Infants and Children their special ventilation/perfusion and anatomic characteristics. Hospitalization should be considered in an infant with an oxygen saturation below 92% on room air.73 In addition, both respiratory acidosis and metabolic acidosis can be observed in children during exacerbations, which are unique to children, because metabolic acidosis is not often observed in adults.27 Thus the administration of sodium bicarbonate to young patients in status asthmaticus to correct the metabolic acidemia seems rational, provided there is sufficient alveolar ventilation to eliminate the CO2 produced.47 Additionally, the development of hypokalemia as a consequence of frequent beta-agonist administration may also need to be addressed.27 Although the use of intravenous magnesium sulfate may be of benefit in children failing bronchodilator and corticosteroid therapy, doubleblind, placebo-controlled studies have both supported and refuted its efficacy.27 Heliox-driven albuterol nebulization can be considered in patients with life-threatening exacerbations; however, a metaanalysis did not find significant improvement in lung function or symptoms compared with those receiving oxygen or air.74 Intravenous montelukast, a potential bronchodilator, has been shown during exacerbations to significantly improve lung function within 10 minutes of administration, with the effect lasting 2 hours.75 However, LTRAs given orally would not provide symptom relief for at least 90 minutes.76 Lastly, the use of intravenous beta-agonists in the management of acute asthma is controversial.27 In addition to the acute management of the exacerbation, one of the most important aspects of this event should be a goal of preventing its recurrence. Asthma education is appropriate in the clinic, emergency room, and hospital settings. 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 both maintenance and intervention strategies that are all age and language appropriate.1,2 In addition, consideration should be given for placing a child on controller therapy, most likely with an ICS, and a follow-up appointment with an asthma-care provider prior to discharging the patient from the emergency department or hospital.1,2 Children requiring ICU care are at particularly high risk of future asthma morbidity and should receive close follow up with an asthma specialist.

ASTHMA PREVENTION Asthma prevention continues to be an important goal, and recent advances in our understanding of asthma phenotypes and their associated risk factors (see Chapter 49) may soon help promote effective preventive strategies. Prevention can be thought of as either primary or secondary. Primary prevention strategies are those that promote immune and airway development away from a proasthmatic response. Secondary prevention strategies target high-risk infants or children who have already developed asthma-related symptoms in an effort to reduce the severity and morbidity of the disease and prevent damage to the developing respiratory system. For the most part, secondary prevention strategies conducted to date have targeted symptomatic, high-risk infants or children to avoid treating a large number of children who are likely to outgrow their disease. The use of ICSs early in the course of asthma development in asthma prevention has been addressed in several studies. Bisgaard and colleagues studied the effects of intermittent ICS treatment during wheezing episodes in infants with a maternal history of asthma and at least one episode of wheezing.26 By 3 years of age, there was no significant difference between treatment groups in asthma burden or lung function. In the Prevention of Early Asthma in Kids (PEAK) study, 2- and 3-year-old children with a positive modified asthma predictive

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index77 (recurrent wheezing and parental history of asthma or signs of personal atopy) were randomized to receive 2 years of daily treatment with a low-dose ICS or placebo.20 During the treatment period, the ICS group had lower symptom burden and improved lung function compared with controls. However, shortly after the ICS was discontinued, there was loss of any significant difference between groups in measures of either asthma burden or lung function. Murray et al. randomized 200 infants with an atopic parent to receive either daily ICSs or placebo after their first episode of prolonged wheezing (longer than 1 month) or two medically confirmed episodes of wheeze.25 At 5 years of age, there were no differences between treatment groups in the prevalence of doctor-diagnosed asthma, use of asthma medications, lung function, or airway reactivity. Leukotriene receptor antagonists and antihistamines have also been studied as potential disease-altering therapies. Bisgaard and colleagues randomized infants and toddlers hospitalized with RSV bronchiolitis to treatment with montelukast or placebo within 7 days of the onset of symptoms for up to 20 weeks. There was no difference in respiratory symptoms between treatment groups.78 The Early Treatment of the Atopic Child (ETAC) study demonstrated that toddlers with atopic dermatitis and a positive family history of atopy79 had no significant difference in the cumulative prevalence of asthma whether they were treated with cetirizine or placebo for 18 months. Similarly, treatment of young children with recurrent ENT infections and two or fewer wheezing episodes with loratadine for 12 months provided no reduction in the number of respiratory infections or pulmonary exacerbations relative to placebo during the 12 months after stopping the medication.80 In sum, to date there has been no demonstration of sustained positive effects on asthma burden or lung function later in life after early intervention with ICS, LTRA, or antihistamines; however, different interventions and/or more accurate identification of high-risk children in infancy may enhance the effectiveness of this approach. It has been postulated with the “hygiene hypothesis” that exposure to certain infections (microbes) and vaccines might skew the immune response away from the development of atopic diseases.81 Several studies have demonstrated reduced risk of atopic disease among children exposed to large amounts of microbial products in the environment during early childhood, such as exposure to farm livestock and farm milk,47 daycare, or older siblings.59 The protective effect of pet ownership in early life is not consistent, with some investigators finding an association with decreased wheezing82 and others finding an increased risk.83 Results have also been conflicting on the effect of breast-feeding on the risk on the development of asthma and other atopic diseases, with some studies revealing a protective effect84,85 and others findings an increased risk of asthma.86,87 A number of studies have examined the primary prevention strategy of house dust mite (HDM) avoidance measures started before birth, with several studies showing an association with transient reduction in sensitization88-90 and one study demonstrating a significant reduction in the occurrence of severe wheeze in the intervention group compared with controls.91 Early life viral respiratory tract infections, including respiratory syncytial virus (RSV) and rhinovirus (RV), are associated with heightened risk for recurrent wheezing and subsequent asthma. Several trials using the anti-RSV hMab palivizumab to prevent RSV infection in infants born late-preterm have demonstrated reductions in early life recurrent wheeze but no significant effect on the risk of atopic asthma.92-95 No RV prevention strategies are currently available. Strategies using immunomodulators theorize that these agents will promote immune development away from a proasthmatic response in high-risk, young children with a positive family history and atopic manifestations. Kalliomaki and colleagues treated pregnant women with a family history of atopic disease with either oral Lactobacillus GG or

SECTION E  Respiratory Tract

placebo daily and then placed the infant on this supplement after birth.96 By 2 years of age, significantly fewer infants developed atopic dermatitis, a result subsequently replicated.97 Newborns with at least one biologic parent with a history of asthma were randomized within 4 days after birth to receive either daily Lactobacillus rhamnosus GG supplementation or placebo for 6 months.98 There was no difference in the cumulative incidence of eczema at 2 years of age, nor was there a difference in the cumulative incidence of asthma at 5 years of age. Vaccination is associated with a lower risk of asthma compared with those who are not vaccinated.99 Specific allergen immunotherapy has been shown to reduce asthma burden and reduces the frequency of development of both new sensitization100 and asthma101-103 in children. Administration of sublingual immunotherapy to grass for 3 years to children 5 to 12 years of age with grass pollen allergy, but without history of signs of asthma, followed by 2 years of follow up, did not alter the time to onset of asthma (based upon a composite endpoint including asthma symptoms, medication use, and demonstration of reversible airflow obstruction) but did significantly reduce the risk of asthma symptoms or asthma medication use.104 Lower maternal vitamin D intake during gestation has been associated with an increased risk of early life recurrent wheezing.105,106 Based upon these associations, two recent trials have examined whether prenatal vitamin D supplementation would reduce the risk of early life recurrent wheezing and asthma. In a Danish study, 623 pregnant women were randomized to receive either the standard recommended dose of vitamin D during pregnancy (400 IU/d) or an additional 2400 IU/day (total of 2800 IU/d) starting at week 24 of gestation and continuing through 1 week postpartum.107 There was a nonsignificant effect on the risk of persistent wheeze during the first 3 years of life, nor was there a difference in asthma prevalence at 3 years of age, rates of respiratory tract infections, allergic sensitization, or eczema. In a multicenter trial from the United States, 881 pregnant women at risk of having a child with asthma based upon a parental history of asthma, allergic rhinitis, or eczema, the control group was randomized to receive 400 IU/d of vitamin D3, and the supplementation group received an additional 4000 IU/d (total of 4400 IU/d) starting at 10 to 18 weeks’ gestation.108 Asthma and recurrent wheeze did not differ significantly between treatment and control groups (hazard ratio 0.8; 95% CI, 0.6-1.0; P = 0.051). Although these two trials did not indicate an asthma-protective effect of prenatal vitamin D supplementation, a combined analysis of these trials did demonstrate a statistically significant 25% reduction in the risk of recurrent wheeze or asthma during the first 3 years of life.109 Epidemiologic evidence suggests that diets deficient in antiinflammatory n-3 long-chain polyunsaturated fatty acids (LCPUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) during pregnancy may increase the risk of asthma or wheezing in children.110 Recent trials of LCPUFA supplementation during pregnancy, using fish oils, demonstrated a significant reduction in the risk of persistent wheeze or asthma at age 3 to 5 years, and a lower risk of hospital discharges for asthma by age 24 years.111,112 Several asthma prevention studies have used multiple interventions simultaneously, including both dietary and allergen avoidance strategies in high-risk infants. The studies have demonstrated a reduction in the outcomes of sensitization,113,114 prevalence of asthma,113,115 asthma symptom burden,114,115 and atopic disease,116 but not in bronchial hyperresponsiveness or lung function115 compared with controls. Overall, these multiintervention strategies in early life have demonstrated mixed results. Interestingly, and highlighting the complexities of prevention trials, an analysis of three trials suggests that success or adverse outcomes in these trials was effected by CD14 genotype117 (Fig. 50.4).

Normal lung function

100

Percent of predicted FEV1

844

Decline in lung function associated with smoking

Severe childhood asthma

80

Early origins of airflow Iimitation

0 Birth

6

25

60

Age (yr) Fig. 50.4  Origins of airflow limitation. (From Martinez FD. Earlylife origins of chronic obstructive pulmonary disease. N Engl J Med 2016;375(9):871–8.)

CONCLUSION Effective asthma management in children includes a team approach to care, incorporating a partnership among health care providers, families, and children. Asthma guidelines promote a step-wise approach to asthma therapy in children and adults, with education and reevaluation of patients to assess their individual response to therapy as critical components of care. Personalized approaches to asthma therapy and novel strategies aimed at disease prevention in early life are important goals moving forward.

REFERENCES 1. National Asthma Education and Prevention Program. Expert Panel Report 3 (EPR-3): Guidelines for the Diagnosis and Management of Asthma-Summary Report 2007. J Allergy Clin Immunol 2007;120(5 Suppl.):S94–138. 2. GINA Scientific Committee. Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention; 2017. www.ginasthma.org. 3. Kloepfer KM, Lee WM, Pappas TE, et al. Detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations. J Allergy Clin Immunol 2014;133(5):1301–7. 4. Thomas AO, Lemanske RF Jr, Jackson DJ. Infections and their role in childhood asthma inception. Pediatr Allergy Immunol 2013;25(2):122–8. 5. Gern JE. Picornavectors. Viruses that spread bacteria. Am J Respir Crit Care Med 2017;196(9):1095–6. 6. Jartti T, Gern JE. Role of viral infections in the development and exacerbation of asthma in children. J Allergy Clin Immunol 2017;140(4):895–906. 7. Calışkan M, Bochkov YA, Kreiner-Møller E, et al. Rhinovirus wheezing illness and genetic risk of childhood onset asthma. N Engl J Med 2013;368(15):1398–407. 8. Bisgaard H, Hermansen MN, Bønnelykke K, et al. Association of bacteria and viruses with wheezy episodes in young children: prospective birth cohort study. BMJ 2010;341:c4978. 9. Bacharier LB, Guilbert TW, Mauger DT, et al. Early administration of azithromycin and prevention of severe lower respiratory tract illnesses in preschool children with a history of such illnesses: a randomized clinical trial. JAMA 2015;314(19):2034–44. 10. Zeiger RS, Mauger D, Bacharier LB, et al. Daily or intermittent budesonide in preschool children with recurrent wheezing. N Engl J Med 2011;365(21):1990–2001.

CHAPTER 50  Management of Asthma in Infants and Children 11. Ducharme FM, Lemire C, Noya FJ, et al. Preemptive use of high-dose fluticasone for virus-induced wheezing in young children. N Engl J Med 2009;360(4):339–53. 12. Kew KM, Quinn M, Quon BS, et al. Increased versus stable doses of inhaled corticosteroids for exacerbations of chronic asthma in adults and children. Cochrane Database Syst Rev 2016;(6): CD007524. 13. Busse WW, Morgan WJ, Gergen PJ, et al. Randomized trial of omalizumab (anti-IgE) for asthma in inner-city children. N Engl J Med 2011;364(11):1005–15. 14. Esquivel A, Busse WW, Calatroni A, et al. Effects of Omalizumab on Rhinovirus Infections, Illnesses, and Exacerbations of Asthma. Am J Respir Crit Care Med 2017;196(8):985–92. 15. Durrani SR, Montville DJ, Pratt AS, et al. Innate immune responses to rhinovirus are reduced by the high-affinity IgE receptor in allergic asthmatic children. J Allergy Clin Immunol 2012;130(2):489–95. 16. Martinez FD, Chinchilli VM, Morgan WJ, et al. Use of beclomethasone dipropionate as rescue treatment for children with mild persistent asthma (TREXA): a randomised, double-blind, placebo-controlled trial. Lancet 2011;377(9766):650–7. 17. Calhoun WJ, Ameredes BT, King TS, et al. Comparison of physician-, biomarker-, and symptom-based strategies for adjustment of inhaled corticosteroid therapy in adults with asthma: the BASALT randomized controlled trial. JAMA 2012;308(10):987–97. 18. Papi A, Canonica GW, Maestrelli P, et al. Rescue use of beclomethasone and albuterol in a single inhaler for mild asthma. N Engl J Med 2007;356(20):2040–52. 19. The Childhood Asthma Management Program Research Group. Long-term effects of budesonide or nedocromil in children with asthma. N Engl J Med 2000;343(15):1054–63. 20. Guilbert TW, Morgan WJ, Zeiger RS, et al. Long-term inhaled corticosteroids in preschool children at high risk for asthma. N Engl J Med 2006;354(19):1985–97. 21. Teper AM, Colom AJ, Kofman CD, et al. Effects of inhaled fluticasone propionate in children less than 2 years old with recurrent wheezing. Pediatr Pulmonol 2004;37(2):111–15. 22. Sorkness CA, Lemanske RF Jr, Mauger DT, et al. Long-term comparison of 3 controller regimens for mild-moderate persistent childhood asthma: the Pediatric Asthma Controller Trial. J Allergy Clin Immunol 2007;119(1):64–72. 23. Fitzpatrick AM, Jackson DJ, Mauger DT, et al. Individualized therapy for persistent asthma in young children. J Allergy Clin Immunol 2016;138(6):1608–18.e12. 24. National Asthma Education and Prevention Program. Expert Panel Report 3: guidelines for the diagnosis and management of asthma: clinical practice guidelines. Bethesda, MD: NIH/National Heart, Lung, and Blood Institute; 2007. August(08): p. 4051. 25. Murray CS, Woodcock A, Langley SJ, et al. Secondary prevention of asthma by the use of Inhaled Fluticasone propionate in Wheezy INfants (IFWIN): double-blind, randomised, controlled study. Lancet 2006;368(9537):754–62. 26. Bisgaard H, Hermansen MN, Loland L, et al. Intermittent inhaled corticosteroids in infants with episodic wheezing. N Engl J Med 2006;354(19):1998–2005. 27. Jackson DJ, Lemanske RF Jr, Guilbert TW. Management of Asthma in Infants and Children. In: Adkinson NF Jr, Bochner BS, Burks AW, et al, editors. Middleton’s Allergy: Principles and Practice. Philadelphia, PA: Elsevier; 2014. p. 876–91. 28. Guilbert TW, Mauger DT, Allen DB, et al. Growth of preschool children at high risk for asthma 2 years after discontinuation of fluticasone. J Allergy Clin Immunol 2011;128(5):956–63.e1-7. 29. Jackson DJ, Bacharier LB, Mauger DT, et al. Quintupling inhaled glucocorticoids to prevent childhood asthma exacerbations. N Engl J Med 2018;378(10):891–901. 30. Kips JC, Pauwels RA. Long-acting inhaled beta(2)-agonist therapy in asthma. Am J Respir Crit Care Med 2001;164(6):923–32. 31. Ni CM, Greenstone IR, Ducharme FM. Addition of inhaled long-acting beta2-agonists to inhaled steroids as first line therapy for persistent

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asthma in steroid-naive adults. Cochrane Database Syst Rev 2005;(2):CD005307. 32. Masoli M, Weatherall M, Holt S, et al. Moderate dose inhaled corticosteroids plus salmeterol versus higher doses of inhaled corticosteroids in symptomatic asthma. Thorax 2005;60(9):730–4. 33. Simons FE. A comparison of beclomethasone, salmeterol, and placebo in children with asthma. Canadian Beclomethasone DipropionateSalmeterol Xinafoate Study Group. N Engl J Med 1997;337(23):1659–65. 34. 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 2001;285(20):2594–603. 35. Peters SP, Bleecker ER, Canonica GW, et al. Serious asthma events with budesonide plus formoterol vs. budesonide alone. N Engl J Med 2016;375(9):850–60. 36. Stempel DA, Szefler SJ, Pedersen S, et al. Safety of Adding Salmeterol to Fluticasone Propionate in Children with Asthma. N Engl J Med 2016;375(9):840–9. 37. Seymour SM, Lim R, Xia C, et al. Inhaled corticosteroids and LABAs - Removal of the FDA’s boxed warning. N Engl J Med 2018;378(26):2461–3. 38. Busse WW, Bateman ED, Caplan AL, et al. Combined analysis of asthma safety trials of Long-Acting beta2-Agonists. N Engl J Med 2018;378(26):2497–505. 39. Lemanske RF Jr, Mauger DT, Sorkness CA, et al. Step-up therapy for children with uncontrolled asthma receiving inhaled corticosteroids. N Engl J Med 2010;362(11):975–85. 40. Hamelmann E, Bateman ED, Vogelberg C, et al. Tiotropium add-on therapy in adolescents with moderate asthma: a 1-year randomized controlled trial. J Allergy Clin Immunol 2016;138(2):441–50.e8. 41. Szefler SJ, Murphy K, Harper T 3rd, et al. A phase III randomized controlled trial of tiotropium add-on therapy in children with severe symptomatic asthma. J Allergy Clin Immunol 2017;140(5):1277–87. 42. Bisgaard H, Zielen S, Garcia-Garcia ML, et al. Montelukast Reduces Asthma Exacerbations in 2 to 5 year old Children with Intermittent Asthma. Am J Respir Crit Care Med 2005;171(4):315–22. 43. 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 2001;108(3):E48. 44. Szefler SJ, Phillips BR, Martinez FD, et al. Characterization of within-subject responses to fluticasone and montelukast in childhood asthma. J Allergy Clin Immunol 2005;115(2):233–42. 45. Zeiger RS, Szefler SJ, Phillips BR, et al. Response profiles to fluticasone and montelukast in mild-to-moderate persistent childhood asthma. J Allergy Clin Immunol 2006;117(1):45–52. 46. Benard B, Bastien V, Vinet B, et al. Neuropsychiatric adverse drug reactions in children initiated on montelukast in real-life practice. Eur Respir J 2017;50(2). pii: 1700148. 47. Moss MH, Gern JE, Lemanske RF Jr. Asthma in Infancy and Childhood. In: Adkinson NF Jr, Yunginger JW, Busse WW, et al, editors. Middleton’s Allergy: Principles and Practice. Philadelphia, PA: Mosby; 2003. p. 1225–55. 48. Nassif EG, Weinberger M, Thompson R, et al. The value of maintenance theophylline in steroid-dependent asthma. N Engl J Med 1981;304(2): 71–5. 49. Lanier B, Bridges T, Kulus M, et al. Omalizumab for the treatment of exacerbations in children with inadequately controlled allergic (IgE-mediated) asthma. J Allergy Clin Immunol 2009;124(6):1210–16. 50. Teach SJ, Gill MA, Togias A, et al. Preseasonal treatment with either omalizumab or an inhaled corticosteroid boost to prevent fall asthma exacerbations. J Allergy Clin Immunol 2015;136(6):1476–85. 51. Joint Task Force on Practice Parameters. Allergen immunotherapy: a practice parameter. American Academy of Allergy, Asthma and Immunology. Ann Allergy Asthma Immunol 2003;90(1 Suppl. 1):1–40. 52. Abramson MJ, Puy RM, Weiner JM. Allergen immunotherapy for asthma. Cochrane Database Syst Rev 2003;(4):CD001186. 53. Virchow JC, Backer V, Kuna P, et al. Efficacy of a house dust mite sublingual allergen immunotherapy tablet in adults with allergic asthma: a randomized clinical trial. JAMA 2016;315(16):1715–25.

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SECTION E  Respiratory Tract

54. Halonen M, Stern DA, Wright AL, et al. Alternaria as a major allergen for asthma in children raised in a desert environment. Am J Respir Crit Care Med 1997;155(4):1356–61. 55. Sears MR, Burrows B, Flannery EM, et al. Atopy in childhood. I. Gender and allergen related risks for development of hay fever and asthma. Clin Exp Allergy 1993;23(11):941–8. 56. Sarpong SB, Hamilton RG, Eggleston PA, et al. Socioeconomic status and race as risk factors for cockroach allergen exposure and sensitization in children with asthma. J Allergy Clin Immunol 1996;97(6):1393–401. 57. Morgan WJ, Crain EF, Gruchalla RS, et al. Results of a home-based environmental intervention among urban children with asthma. N Engl J Med 2004;351(11):1068–80. 58. Lemanske RF Jr, Kakumanu S, Shanovich K, et al. Creation and implementation of SAMPRO: a school-based asthma management program. J Allergy Clin Immunol 2016;138(3):711–23. 59. Ball TM, Castro-Rodriguez JA, Griffith KA, et al. Siblings, day-care attendance, and the risk of asthma and wheezing during childhood. N Engl J Med 2000;343(8):538–43. 60. Wright RJ, Cohen S, Carey V, et al. Parental stress as a predictor of wheezing in infancy: a prospective birth-cohort study. Am J Respir Crit Care Med 2002;165(3):358–65. 61. Sandberg S, Paton JY, Ahola S, et al. The role of acute and chronic stress in asthma attacks in children. Lancet 2000;356(9234):982–7. 62. Strunk RC. Death due to asthma. New insights into sudden unexpected deaths, but the focus remains on prevention. Am Rev Respir Dis 1993;148(3):550–2. 63. Gibson PG, Powell H, Coughlan J, et al. Self-management education and regular practitioner review for adults with asthma. Cochrane Database Syst Rev 2003;(1):CD001117. 64. Ducharme FM, Zemek RL, Chalut D, et al. Written action plan in pediatric emergency room improves asthma prescribing, adherence, and control. Am J Respir Crit Care Med 2011;183(2):195–203. 65. Guilbert TW, Moss MH, Lemanske RF Jr. Approach to Infants and Children with Asthma. In: Busse WW, Adkinson NF, Bochner BS, editors. Middleton’s allergy principles and practice. Philadelphia, PA: Mosby; 2008. p. 1319–39. 66. Bruzzese JM, Evans D, Kattan M. School-based asthma programs. J Allergy Clin Immunol 2009;124(2):195–200. 67. Rachelefsky G. Treating exacerbations of asthma in children: the role of systemic corticosteroids. Pediatrics 2003;112(2):382–97. 68. Bush A. Practice imperfect–treatment for wheezing in preschoolers. N Engl J Med 2009;360(4):409–10. 69. Beigelman A, King TS, Mauger D, et al. Do oral corticosteroids reduce the severity of acute lower respiratory tract illnesses in preschool children with recurrent wheezing? J Allergy Clin Immunol 2013;131(6):1518–25. 70. Foster SJ, Cooper MN, Oosterhof S, et al. Oral prednisolone in preschool children with virus-associated wheeze: a prospective, randomised, double-blind, placebo-controlled trial. Lancet Respir Med 2018;6(2):97–106. 71. Schuh S, Dick PT, Stephens D, et al. High-dose inhaled fluticasone does not replace oral prednisolone in children with mild to moderate acute asthma. Pediatrics 2006;118(2):644–50. 72. Everard ML, Bara A, Kurian M, et al. Anticholinergic drugs for wheeze in children under the age of two years. Cochrane Database Syst Rev 2005;(3):CD001279. 73. Sole D, Komatsu MK, Carvalho KV, et al. Pulse oximetry in the evaluation of the severity of acute asthma and/or wheezing in children. J Asthma 1999;36(4):327–33. 74. Ho AM, Lee A, Karmakar MK, et al. Heliox vs air-oxygen mixtures for the treatment of patients with acute asthma: a systematic overview. Chest 2003;123(3):882–90. 75. Camargo CA Jr, Smithline HA, Malice MP, et al. A randomized controlled trial of intravenous montelukast in acute asthma. Am J Respir Crit Care Med 2003;167(4):528–33. 76. Dockhorn RJ, Baumgartner RA, Leff JA, et al. Comparison of the effects of intravenous and oral montelukast on airway function: a double blind,

placebo controlled, three period, crossover study in asthmatic patients. Thorax 2000;55(4):260–5. 77. Guilbert TW, Morgan WJ, Krawiec M, et al. The Prevention of Early Asthma in Kids study: design, rationale and methods for the Childhood Asthma Research and Education network. Control Clin Trials 2004;25(3):286–310. 78. Bisgaard H, Flores-Nunez A, Goh A, et al. Study of montelukast for the treatment of respiratory symptoms of post-respiratory syncytial virus bronchiolitis in children. Am J Respir Crit Care Med 2008;178(8):854–60. 79. Warner JO. 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 2001;108(6):929–37. 80. Grimfeld A, Holgate ST, Canonica GW, et al. Prophylactic management of children at risk for recurrent upper respiratory infections: the Preventia I Study. Clin Exp Allergy 2004;34(11):1665–72. 81. Strachan DP. Family size, infection and atopy: the first decade of the “hygiene hypothesis”. Thorax 2000;55(Suppl. 1):S2–10. 82. Bufford JD, Reardon CL, Li Z, et al. Effects of dog ownership in early childhood on immune development and atopic diseases. Clin Exp Allergy 2008;38(10):1635–43. 83. Apelberg BJ, Aoki Y, Jaakkola JJ. Systematic review: exposure to pets and risk of asthma and asthma-like symptoms. J Allergy Clin Immunol 2001;107(3):455–60. 84. Chulada PC, Arbes SJ Jr, Dunson D, et al. Breast-feeding and the prevalence of asthma and wheeze in children: analyses from the Third National Health and Nutrition Examination Survey, 1988-1994. J Allergy Clin Immunol 2003;111(2):328–36. 85. Oddy WH, Holt PG, Sly PD, et al. Association between breast feeding and asthma in 6 year old children: findings of a prospective birth cohort study. BMJ 1999;319(7213):815–19. 86. Wright AL, Stern DA, Halonen M. The association of allergic sensitization in mother and child in breast-fed and formula-fed infants. Adv Exp Med Biol 2001;501:249–55. 87. Sears MR, Greene JM, Willan AR, et al. Long-term relation between breastfeeding and development of atopy and asthma in children and young adults: a longitudinal study. Lancet 2002;360(9337): 901–7. 88. Tsitoura S, Nestoridou K, Botis P, et al. Randomized trial to prevent sensitization to mite allergens in toddlers and preschoolers by allergen reduction and education: one-year results. Arch Pediatr Adolesc Med 2002;156(10):1021–7. 89. van Strien RT, Koopman LP, Kerkhof M, et al. Mattress encasings and mite allergen levels in the Prevention and Incidence of Asthma and Mite Allergy study. Clin Exp Allergy 2003;33(4):490–5. 90. Corver K, Kerkhof M, Brussee JE, et al. House dust mite allergen reduction and allergy at 4 yr: follow up of the PIAMA-study. Pediatr Allergy Immunol 2006;17(5):329–36. 91. Custovic A, Simpson BM, Simpson A, et al. Effect of environmental manipulation in pregnancy and early life on respiratory symptoms and atopy during first year of life: a randomised trial. Lancet 2001;358(9277):188–93. 92. Park J, Munagala I, Xu H, et al. Interferon signature in the blood in inflammatory common variable immune deficiency. PLoS ONE 2013;8(9):e74893. 93. Simoes EA, Carbonell-Estrany X, Rieger CH, et al. The effect of respiratory syncytial virus on subsequent recurrent wheezing in atopic and nonatopic children. J Allergy Clin Immunol 2010;126(2): 256–62. 94. Mochizuki H, Kusuda S, Okada K, et al. Palivizumab prophylaxis in preterm infants and subsequent recurrent wheezing. Six-Year Follow-up study. Am J Respir Crit Care Med 2017;196(1):29–38. 95. Erratum, respiratory syncytial virus and recurrent wheeze in healthy preterm infants. N Engl J Med 2016;374(24):2406. 96. Kalliomaki M, Salminen S, Arvilommi H, et al. Probiotics in primary prevention of atopic disease: a randomised placebo-controlled trial. Lancet 2001;357(9262):1076–9.

CHAPTER 50  Management of Asthma in Infants and Children 97. Viljanen M, Savilahti E, Haahtela T, et al. Probiotics in the treatment of atopic eczema/dermatitis syndrome in infants: a double-blind placebo-controlled trial. Allergy 2005;60(4):494–500. 98. Cabana MD, McKean M, Caughey AB, et al. Early probiotic supplementation for eczema and asthma prevention: a randomized controlled trial. Pediatrics 2017;140(3). 99. Martignon G, Oryszczyn MP, Annesi-Maesano I. Does childhood immunization against infectious diseases protect from the development of atopic disease? Pediatr Allergy Immunol 2005;16(3):193–200. 100. DesRoches A, et al. Does specific immunotherapy to Dermatophagoides pteronyssinus prevents the onset of new sensitizations in monosensitized children? J Allergy Clin Immunol 1997;99(1):524. 101. Moller C, Dreborg S, Ferdousi HA, et al. Pollen immunotherapy reduces the development of asthma in children with seasonal rhinoconjunctivitis (the PAT-Study). J Allergy Clin Immunol 2002;109(2):251–6. 102. Niggemann B, Jacobsen L, Dreborg S, et al. Five-year follow-up on the PAT study: specific immunotherapy and long-term prevention of asthma in children. Allergy 2006;61(7):855–9. 103. Novembre E, Galli E, Landi F, et al. Coseasonal sublingual immunotherapy reduces the development of asthma in children with allergic rhinoconjunctivitis. J Allergy Clin Immunol 2004;114(4):851–7. 104. Valovirta E, Petersen TH, Piotrowska T, et al. Results from the 5-year SQ grass sublingual immunotherapy tablet asthma prevention (GAP) trial in children with grass pollen allergy. J Allergy Clin Immunol 2018;141(2):529–38.e13. 105. Devereux G, Litonjua AA, Turner SW, et al. Maternal vitamin D intake during pregnancy and early childhood wheezing. Am J Clin Nutr 2007;85(3):853–9. 106. Camargo CA Jr, Rifas-Shiman SL, Litonjua AA, et al. Maternal intake of vitamin D during pregnancy and risk of recurrent wheeze in children at 3 y of age. Am J Clin Nutr 2007;85(3):788–95. 107. Chawes BL, Bønnelykke K, Stokholm J, et al. Effect of vitamin D3 supplementation during pregnancy on risk of persistent wheeze in the offspring: a randomized clinical trial. JAMA 2016;315(4):353–61.

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108. Litonjua AA, Carey VJ, Laranjo N, et al. Effect of prenatal supplementation with vitamin D on asthma or recurrent wheezing in offspring by age 3 years: the VDAART randomized clinical trial. JAMA 2016;315(4):362–70. 109. Wolsk HM, Chawes BL, Litonjua AA, et al. Prenatal vitamin D supplementation reduces risk of asthma/recurrent wheeze in early childhood: a combined analysis of two randomized controlled trials. PLoS ONE 2017;12(10):e0186657. 110. Willers S, Devereux G, Craig LC, et al. Maternal food consumption during pregnancy and asthma, respiratory and atopic symptoms in 5-year-old children. Thorax 2007;62(9):773–9. 111. Bisgaard H, Stokholm J, Chawes BL, et al. Fish Oil-Derived fatty acids in pregnancy and wheeze and asthma in offspring. N Engl J Med 2016;375(26):2530–9. 112. Hansen S, Strøm M, Maslova E, et al. Fish oil supplementation during pregnancy and allergic respiratory disease in the adult offspring. J Allergy Clin Immunol 2017;139(1):104–11.e4. 113. Arshad SH, Bateman B, Matthews SM. Primary prevention of asthma and atopy during childhood by allergen avoidance in infancy: a randomised controlled study. Thorax 2003;58(6):489–93. 114. Peat JK, Mihrshahi S, Kemp AS, et al. Three-year outcomes of dietary fatty acid modification and house dust mite reduction in the Childhood Asthma Prevention Study. J Allergy Clin Immunol 2004;114(4):807–13. 115. Chan-Yeung M, Ferguson A, Watson W, et al. The Canadian Childhood Asthma Primary Prevention Study: outcomes at 7 years of age. J Allergy Clin Immunol 2005;116(1):49–55. 116. Bruno G, Giampietro PG, Businco L. Results of a multicentric study for the prevention of atopic allergy. 48 months of follow up. Minerva Pediatr 1996;48(10):413–19. 117. Kerkhof M, Daley D, Postma DS, et al. Opposite effects of allergy prevention depending on CD14 rs2569190 genotype in 3 intervention studies. J Allergy Clin Immunol 2012;129(1):256–9. 118. Centers for Disease Control and Prevention. National Surveillance of Asthma: United States, 2001–2010. Available from: https://www.cdc.gov/ nchs/data/series/sr_03/sr03_035.pdf.

CHAPTER 50  Management of Asthma in Infants and Children

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SELF-ASSESSMENT QUESTIONS 1. Which of the following strategies is effective in preventing schoolaged children receiving daily maintenance inhaled glucocorticoid therapy who are entering into their “yellow zone,” from progressing on to an asthma exacerbation requiring a course of oral prednisone? a. Doubling the dose of inhaled glucocorticoids b. Tripling the dose of inhaled glucocorticoids c. Quintupling the dose of inhaled glucocorticoids d. None of the above 2. Of the following treatments, which one is the best form of therapy in the majority of children who require step 2 care for preschool asthma? a. Inhaled glucocorticoids b. Oral leukotriene receptor antagonists c. Scheduled daily albuterol d. Theophylline

3. Which of the following should be included in school-based asthma management programs? a. Written asthma action plan b. Asthma education for the child, family members, school nurses, and other school personnel c. An evaluation of the school and home environment d. The creation of a circle of support that includes the child, the family, the clinician, and relevant school personnel e. All of the above 4. Which of the following are desired goals to achieve within the risk domain of asthma severity and asthma control? a. Prevention of recurrent exacerbations b. Minimization of adverse effects of therapy c. Prevention of loss of lung function over time d. A and C e. A, B, and C

51  Diagnosis of Asthma in Adults Louis-Philippe Boulet, Krystelle Godbout

CONTENTS Introduction, 848 Definition of Asthma, 848 Main Components of Asthma, 848 Overdiagnosis and Underdiagnosis of Asthma, 850 Airway Inflammation and Remodeling, 850 Evaluation of Asthma, 850 Assessment of Asthma Control and Future Risks of Events, 853

Assessment of Asthma Severity, 854 Evaluation of Asthma-Related Quality of Life, 854 Phenotyping and Endotyping of Asthma, 854 Assessment of Comorbidities, 854 Special Considerations in Regard to Asthma Diagnosis, 855 Differential Diagnosis: Conditions That May Mimic Asthma, 856 Conclusions and Perspectives, 856

SUMMARY OF IMPORTANT CONCEPTS

DEFINITION OF ASTHMA

• Diagnosis of asthma should be confirmed by objective measures of variable airway obstruction or the demonstration of airway hyperresponsiveness. • Pulmonary function tests and assessment of asthma control criteria should be obtained both at the time of diagnosis and at follow-up visits. • Asthma triggers and inducers (sensitizers) should be identified at the time of diagnosis and their possible contribution to asthma regularly assessed. • The allergic status and the presence of comorbid conditions that might influence asthma control should be identified. • Noninvasive measures of airway inflammation are increasingly used to better characterize the asthma phenotype. • In patients with normal airway function while on controller treatment, a step down of this medication may be required to confirm the diagnosis.

The Global Initiative for Asthma (GINA) defines asthma as “a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness, and cough that vary over time and in intensity, together with variable expiratory airflow limitation. Therefore a diagnosis of asthma requires the presence of both symptoms and demonstration of such variability in airflow limitation.”2 The definition of asthma has been a subject of debate for a long time. This seems related to the heterogeneity of the disease, which subsequently led asthma to be qualified as an umbrella term. Different phenotypes and endotypes, each with different characteristics, are indeed regrouped under the word asthma (Box 51.1). In this regard, a joint American Thoracic Society/European Respiratory Society task force previously indicated that:3 “Asthma is a heterogeneous condition. Its natural history includes acute episodic deterioration (exacerbations) against a background of chronic persistent inflammation and/or structural changes that may be associated with persistent symptoms and reduced lung function. Trigger factor exposure combines with the underlying phenotype, the degree of hyperresponsiveness and of airflow obstruction, and the severity of airway inflammation to cause wide variability in the manifestations of asthma in individual patients.” There is, nevertheless, a general consensus that asthma should be defined according to its key clinical features, although its subtypes should be carefully identified, using biomarkers whenever available, to offer the best evaluation and treatment strategies.

INTRODUCTION Asthma is a common respiratory disease; the current prevalence is more than 300 million individuals worldwide and continues to increase.1 In recent decades, progress has been made not only with treatment but in establishing the diagnosis and better assessing asthma, as well as defining its subtypes or phenotypes. However, there is evidence that overdiagnosis of asthma is still common, although this seems to be true also for underdiagnosis. This situation often occurs because of the lack of objective measurements of physiologic features of asthma, such as in documentation of variable airway obstruction or hyperresponsiveness. Furthermore, there has been an increasing interest in evaluation of inflammation, the key underlying mechanism of asthma, since the development and standardization of noninvasive measures of airway inflammatory features. This chapter describes current recommendations on the diagnosis and evaluation of bronchial asthma in adults.

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MAIN COMPONENTS OF ASTHMA Confirmation of asthma diagnosis is the first step in the management of this condition. It requires the presence of respiratory symptoms that are variable in intensity and over time, the demonstration of reversible airway obstruction, and the exclusion of other conditions (Table 51.1).

CHAPTER 51  Diagnosis of Asthma in Adults

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BOX 51.1  Phenotypes of Asthma

TABLE 51.1  Diagnosis of Asthma in Adults

Based on Airway Inflammation • Eosinophilic • Neutrophilic • Mixed • Pauci-granulocytic

Respiratory Symptoms Breathlessness, Variable in intensity and over time wheezing, cough, Triggered by viral infection, exercise, chest tightness, laughter, irritants or allergens phlegm production

Based on Clinical Features • Mild, moderate, or severe asthma • Exacerbation-prone • Treatment-resistant • Early-onset or late-onset asthma • Asthma in the elderly

Variable Airway Obstruction Spirometry FEV1/FVC 60 L/min (minimum ≥20%) after bronchodilator or a course of controller therapy Diurnal variability >8% (twice-daily PEF) or > 20% (multiple PEF daily)

Bronchoprovocation Tests Methacholine or PC20 < 4 mg/mL or PD20 1x200-dose canister/month) • Low FEV1, especially if 1.5, ACT 3 days each) in the previous year 3. Serious exacerbations: at least one hospitalization, intensive care unit stay, or mechanical ventilation in the previous year 4. Airflow limitation: after appropriate bronchodilator withheld FEV1 0.75-0.80 in adults, >0.90 in children). Positive bronchodilator (BD) reversibility test* (more likely to be positive if BD medication is withheld before test: SABA ≥4 hours, LABA ≥15 hours)

Adults: increase in FEV1 of >12% and >200 mL from baseline, 10-15 minutes after 200-400 μg albuterol or equivalent (greater confidence if increase is >15% and >400 mL). Children: increase in FEV1 of >12% predicted.

Excessive variability in twice-daily PEF over 2 weeks*

Adults: average daily diurnal PEF variability >10%** Children: average daily diurnal PEF variability >13%

Significant increase in lung function after 4 weeks of antiinflammatory treatment

Adults: increase in FEV1 by 12% and >200 mL (or PEF by >20%) from baseline after 4 weeks of treatment, outside respiratory infections.

Positive exercise challenge test*

Adults: fall in FEV1 of >10% and >200 mL from baseline. Children: fall in FEV1 of >12% predicted, or PEF 12% and >200 mL between visits, outside of respiratory infection. Children: variation in FEV1 of >12% in FEV1 or >15% in PEF between visits (may include respiratory infections).

*These tests can be repeated during symptoms or in the early morning. **Daily diurnal PEF variability is calculated from twice daily PEF as ([day’s highest minus day’s lowest]/mean of day’s highest and lowest), and averaged over one week. FEV1, Forced expiratory volume in 1 second; FVC, forced vital capacity; LABA, long-acting β2-agonists; PEF, peak expiratory flow; SABA, short-acting β2-agonists. From Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention, 2018. Available from: www.ginasthma.org

The Goal of Asthma Management is:

TABLE 52.3  Domains of Outcomes Used

in the Assessment of Asthma Severity and Disease Control

Overall Asthma Control achieving

reducing

Current control

Future risk

defined by

defined by

Symptoms

Reliever use

Instability/ worsening

Exacerbations

Activity

Lung function

Loss of lung function

Adverse effects of medication

Fig. 52.3  The goal of asthma management is to achieve overall asthma control as measured by features of current control and reducing future risks.5,157

Impairment • Symptoms • Nighttime awakenings • Use of short-acting β2-agonists for symptom control • Interference with normal activity • Lung functions Risk • Exacerbations • Progressive loss of lung function • Side effects from medications From National Heart, Lung, and Blood Institute. Expert Panel Report 3 (EPR-3): guidelines for the diagnosis and management of asthma— full report 2007. August 28, 2007. Available from: https://www.nhlbi .nih.gov/health-topics/guidelines-for-diagnosis-management-of-asthma.

CHAPTER 52  Management of Asthma in Adolescents and Adults

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TABLE 52.4  Assessment of Asthma Control in Patients 12 Years of Age and Older ASTHMA CONTROL CLASSIFICATION: FREQUENCY/NATURE OF COMPONENT

Control Category/ Component

Well Controlled

Not Well Controlled

Very Poorly Controlled

Impairment Symptoms

≤2 day/wk

>2 day/wk but not daily

Throughout the day

Nighttime awakenings

≤ 2×/mo

3-4×/mo

7×/wk

Interference with normal activity

None

Minor limitation

Extremely limited

SABA use for symptom control

≤2 day/wk

>2 day/wk

Several times a day

FEV1 or peak flow

>80% predicted or personal best

60%-80% predicted or personal best

240-480 µg

>480 µg

Ciclesonide MDI 80 µg/puff, 160 µg/puff

80-240 µg

240-320 µg

>320 µg

Budesonide DPI 90, 180, or 200 µg/inhalation

180-600 µg

>600-1200 µg

>1200 µg

88-264 µg 100-300 µg

>264-440 µg >300-500 µg

>440 µg >500 µg

Fluticasone furoate   DPI: 100 or 200 µg

100 µg

N/A

200 µg

Mometasone DPI 200 µg/inhalation

200 µg

400 µg

>400 µg

Fluticasone propionate   HFA/MDI: 44, 110, or 220 µg/puff   DPI: 50, 100, or 250 µg/inhalation

DPI, Dry powder inhaler; HFA, hydrofluoroalkane; MDI, metered-dose inhaler. From National Heart, Lung, and Blood Institute. Expert Panel Report 3 (EPR-3): Guidelines for the diagnosis and management of asthma—full report 2007. August 28, 2007. Available from: https://www.nhlbi.nih.gov/health-topics/guidelines-for-diagnosis-management-of-asthma.

in asthma exacerbations to enhance control of airway dysfunction and airflow limitation by reducing inflammation. Corticosteroids. ICSs are the primary controller medications used in asthma and improve outcomes associated with both impairment and risks. They do not, however, have any disease-modifying activities, and their effects will diminish once they are no longer used.38,39 The antiinflammatory activities of corticosteroids are broad spectrum and affect many cell types associated with inflammation, including lymphocytes, principally on T helper cell type 2 (Th2), as well as inflammatory cell migration and activation (e.g., eosinophils) (Chapter 96). ICSs have minimal long-term side effects when used in conventional doses. Multiple formulations of ICSs are available; these may be used in low, medium, or high doses, depending on the underlying need for asthma control (Table 52.6). Fluticasone furoate is approved for once per day dosing. Although similar to fluticasone propionate, the furoate derivative has enhanced affinity for the glucocorticoid receptor with a similar onset of action but of longer duration. Compared with other long-term control medications, such as leukotriene receptor antagonists (LTRAs), ICSs generally are more effective.40 Oral corticosteroids have antiinflammatory actions similar to those of ICS and generally are used in short-term bursts for relief from exacerbations or loss of control. Regular use of systemic corticosteroids is limited by associated side effects and are given primarily to patients with the most severe asthma—GINA step care 5, for which doses of ICSs alone are not sufficient to achieve control, and whose use is being reduced by intervention with biologics. Leukotriene modifiers. Leukotriene modifiers interfere with the effects of products of the leukotriene pathway, which leads to airflow obstruction and inflammation. Included in this group are the LTRAs montelukast and zafirlukast and also zileuton, which inhibits the 5-lipoxygenase pathway (Chapter 97). Leukotriene modifiers are considered as alternative choices for treatment of mild persistent asthma, but for some patients, they may be as effective as ICSs.41 Combination with ICSs gives added benefit for achieving disease control but not to the degree seen with long-acting β2-agonists (LABAs).40,42 Leukotriene modifiers may also help to alleviate symptoms of exercise-induced bronchospasm (EIB). Cromolyn sodium and nedocromil sodium. Cromolyn sodium and nedocromil sodium interfere with mast cell chloride channels to

prevent mediator release. Although these compounds are extremely safe, their use in patients older than 12 years of age is limited. LABAs. LABAs—salmeterol and formoterol—are inhaled bronchodilators that improve airflow by relieving obstruction for at least 12 hours (Chapter 93). Vilanterol is highly selective for the β2-adrenorecptor and has a 24-hour duration of activity. Indacaterol is a LABA with a 24-hour duration of bronchodilation, but its use in asthma is not currently approved by the US Food and Drug Administration (FDA). Based on data from the Salmeterol Multicenter Asthma Research Trial (SMART) as well as some other metaanalyses, the FDA had raised significant concerns for usage of LABA in asthma and therefore had mandated additional safety trials and label warnings.43 The results of three large multicenter trials demonstrated that the combination inhalers (ICS/LABA) did not pose a higher risk of serious asthma-related events compared with ICS-alone preparations.44-46 These findings have led to the removal of the box safety warning that had been placed on LABAs. These compounds, in contrast with their use in COPD, are not to be used alone in asthma for safety reasons (see later text) and typically are given in combination with an ICS (fluticasone propionate–salmeterol; budesonide–formoterol; and mometasone–formoterol; fluticasone furoate–vilanterol) (Table 52.7). Combination ICS/LABA medications are available in low, medium, and high doses based on the concentration of the ICS. The ICS dose determines its use in different levels of step care. When used in combination with ICSs, LABAs improve control of impairment and reduce exacerbations. Furthermore, equivalence for asthma control with combination therapy is achieved with lower doses of ICSs. Methylxanthines. Sustained-release theophylline has modest bronchodilator activity, and its use in asthma has become limited because of concerns over toxicity and less efficacy than for other long-term controllers (Chapter 94). Toxicity, although rare, can occur, and monitoring of serum theophylline levels is required. Long-acting anticholinergics. Tiotropium, in contrast, is a LAMA and has greater selectivity for M2 and M3 receptors with evidence for once per day dosing. Initially approved for COPD, tiotropium is an approved long-acting anticholinergic agent for use in asthma patients 6 years of age and older. Tiotropium is used in asthma in combination with ICS alone or in combination with ICA/LABAs.47-50 Other

CHAPTER 52  Management of Asthma in Adolescents and Adults

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TABLE 52.7  Usual Dosages for Combination Treatment (ICS/LABA) for Patients 12 Years of

Age or Older

Combination Agent

Formulation

Dose

Comments

Fluticasone–salmeterol (Advair®)

DPI 100 µg/50 µg, 250 µg/50 µg, or 500 µg/50 µg HFA 45 µg/21 µg, 115 µg/21 µg, or 230 µg/21 µg

1 inhalation bid; dose depends on severity of asthma

• 100/50 DPI or 45/21 HFA: for patients whose asthma is not controlled on low- to medium-dose ICS • 250/50 DPI or 115/21 HFA: for patients whose asthma is not controlled on medium- to high-dose ICS

Budesonide–formoterol (Symbicort®)

HFA, MDI 80 µg/4.5 µg, 160 µg/4.5 µg

2 inhalations bid; dose depends on severity of asthma

• 80/4.5: for patients whose asthma is not controlled on low- to medium-dose ICS • 160/4.5: for patients whose asthma is not controlled on medium- to high-dose ICS

Mometasone–formoterol (Dulera®)

HFA, MDI 50 µg/5 µg, 100 µg/5 µg, or 200 µg/5 µg,

2 inhalations bid; dose depends on the severity of asthma

• 50/5: for patients whose asthma is not controlled on low-dose ICS • 100/5: for patients whose asthma is not controlled on medium-dose ICS • 200/5: for patients whose asthma is not controlled on high-dose ICS

Fluticasone furoate– vilanterol (Breo®)*

100 µg/25 µg, 200 µg/25 µg

1 inhalation daily

• 100/25: for patients whose asthma is not controlled on low-dose ICS • 200/25: for patients whose asthma is not controlled on medium-dose ICS

*FDA-approved for ages 18 and older. DPI, Dry powder inhaler; HFA, hydrofluoroalkane; MDI, metered-dose inhaler. From National Heart, Lung, and Blood Institute. Expert Panel Report 3 (EPR-3): Guidelines for the diagnosis and management of asthma—full report 2007. August 28, 2007. Available from: https://www.nhlbi.nih.gov/health-topics/guidelines-for-diagnosis-management-of-asthma.

LAMAs include glycopyrrolate and aclidinium, but these are not currently approved for asthma. Immunomodulation/biologics. In the past decade, there has been tremendous progress with the usage and availability of biologic therapy for asthma. At present, four biologic therapies (omalizumab, mepolizumab, reslizumab, and benralizumab) are approved for use in various phenotypes of asthma (Chapter 91). One other biologic, dupilumab (anti IL-4/IL-13 monoclonal antibody), is currently FDA-approved for atopic dermatitis and asthma and has demonstrated benefit in reducing exacerbations and corticosteroid dependence in numerous asthma trials. Omalizumab (anti-IgE) is given as an injectable monoclonal antibody to bind to IgE, thus preventing this antibody from binding to its receptor (Chapter 90). At present, omalizumab is recommended for use in patients (older than 6 years) with severe allergic asthma (i.e., GINA step 5) and where underlying evidence for IgE-dependent allergic asthma exists (a positive skin test response or serologic evidence of allergen-specific IgE). Its effects in asthma are noted on symptom control, decreased need for ICS, and a reduction of asthma exacerbations. Mepolizumab, reslizumab, and benralizumab belong to a class of biologic therapy that abrogates the IL-5 pathway. All of these biologics have demonstrated benefit in mitigating asthma exacerbations, reducing systemic corticosteroid usage among moderate to severe eosinophilic asthma subjects and with variable improvements in lung function metrics. These compounds will be discussed in greater detail later. Other immunomodulators— methotrexate, cyclosporine, and intravenous immunoglobulin—have been evaluated in asthma, with inconsistent effects. Bronchial thermoplasty (BT). Bronchial thermoplasty targets the structural changes, specifically smooth muscle hypertrophy, that occur within the airway smooth muscles of asthmatic patients. BT delivers a controlled amount of thermal energy to targeted areas within the airway to possibly affect airway smooth muscle function. BT reduces asthma exacerbations, emergency department (ED) visits, and hospitalizations as well as improving quality-of-life parameters. BT was approved for use in 2010 in severe asthma patients who

remain uncontrolled despite high-dose ICS and LABA combination therapies.51 Despite its approval in 2010, BT has yet to garner widespread use owing to questions about which phenotype of asthma responds best to this therapy, and it remains an offering primarily at centers with expertise in managing severe asthma. Nonpharmacologic interventions and comorbid conditions. At any step level of care, particularly if asthma control is not achieved and maintained, attention should be directed toward patient adherence and techniques in medication use as well as other factors including environmental control (e.g., removal of pets, control of dust mites) and treatment of coexisting conditions, such as chronic rhinosinusitis, gastroesophageal reflux disease, ABPM, obesity, and psychosocial issues, should be addressed. Moreover, alternative diagnoses, such as paradoxical vocal cord motion and eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome), should be considered, especially if the patient is not responding to standard therapies.

STEP CARE APPROACH TO ASTHMA MANAGEMENT AND CONTROL Asthma guidelines for treatment recommend that specific therapy be tailored to the needs, circumstances, and responsiveness of the individual patient. Although current guidelines recommend a stepwise approach to the management of “all” asthma patients, a paradigm shift in asthma management is emerging with a transition toward personalized care such that asthma patients may be broadly phenotyped, and, eventually, endotyped by using clinical and biomarker profiles, which, in turn, would allow selection of targeted therapies to achieve better outcomes, because the chosen intervention is designed to modify patient-specific features or pathways of their disease. Although current information does not provide a full complement of biomarkers to either fully direct or achieve this goal, personalized care for patients can meet the needs of individual variations in asthma and follows the emerging information that multiple asthma phenotypes exist.8,52 In addition, at each step in the care of patients with asthma, it is important to review and identify

868

SECTION E  Respiratory Tract treatment has become based upon achieving control of impairment and future risk. A brief description of the asthma clinical parameters at each level of step care are discussed in the following sections.

Step 1  Care: As-Needed Reliever Inhaler Patients with intermittent asthma have infrequent markers of impairment (e.g., symptoms on fewer than 2 days per week; nighttime awakenings fewer than twice per month, use of SABA fewer than 2 days per week; no interference with normal activity; and normal lung function). In addition, most have few exacerbations, 0 to 1 per year, that require intervention with systemic corticosteroids. Patients with intermittent asthma are still at risk for exacerbations, which, although infrequent, can be severe.

SE ON

T

AD

A

JU

Diagnosis Symptom control & risk factors (including lung function) Inhaler technique & adherence Patient preference

S ES SS

Symptoms Exacerbations Side-effects Patient satisfaction Lung function

REVIEW R ES P

comorbid conditions that may influence the likelihood of achieving control. The provision of a continuous educational interaction to improve the patient’s understanding of their disease and the use and mechanism of their medications are important, as is assessing adherence to treatment. The step care approach to asthma (Fig. 52.5) is based on the premise that control is most effectively achieved by greater amounts of medication, particularly with agents directed at regulation of underlying inflammation, or with the use of combination therapy. This approach may not, however, be effective in all patients, especially those with more severe asthma. In previous asthma guidelines, EPR-3, step-care treatment for asthma was based upon severity: intermittent and persistent (mild, moderate, and severe). The classification of asthma by severity still exists, but step-care

ST TREATM

EN

Asthma medications Non-pharmacological strategies Treat modifiable risk factors

STEP 5 STEP 4 PREFERRED CONTROLLER CHOICE

STEP 1

STEP 2

STEP 3

Low dose ICS

Low dose ICS/LABA

Med/high ICS/LABA

Med/high dose ICS Low dose ICS + LTRA (or + theophylline)

Add tiotropium High dose ICS + LTRA (or + theophylline)

Other Consider low Leukotriene receptor antagonists (LTRA) controller dose ICS Low dose theophylline options

RELIEVER

REMEMBER TO...

As needed SABA

Refer for add-on treatment (eg, tiotropium, anti-lgE, or anti-IL5)

Add low dose ICS

As needed SABA or low dose ICS/formoterol

• Provide guided self-management education (self-monitoring + written action plan + regular review) • Treat modifiable risk factors and comorbidities (e.g., smoking, obesity, anxiety) • Advise about non-pharmacological therapies and strategies (e.g., physical activity, weight loss, avoidance of sensitizers) where appropriate

SLIT added as an option

• Consider stepping up if ... uncontolled symptoms, exacerbations or risks, but check diagnosis, inhaler technique and adherence first • Consider adding SLIT in adult HDM-sensitive patients with allergic rhinitis who have exacerbations despite ICS treatment, provided FEV1 is >70% predicted • Consider stepping down if ... symptoms controlled for 3 months + low risk for exacerbations. Ceasing ICS is not advised. Fig. 52.5  Stepwise approach to control symptoms and minimize future risk, from Global Initiative for Asthma (GINA), Global Strategy for Asthma Management and Prevention, 2018. SLIT, Sublingual immunotherapy. Available from: www.ginasthma.org.156

CHAPTER 52  Management of Asthma in Adolescents and Adults For patients with intermittent asthma, SABAs, such as albuterol, are effective in relieving their infrequent symptoms and preventing compromises in pulmonary function.5,6 Short-acting anticholinergic agents, such as ipratropium, generally are not recommended owing to a slower onset of action and less bronchodilation. Furthermore, use of ipratropium in combination with SABAs has not been found to be more effective at this level of disease severity.37 Patients with EIB benefit from pretreatment with a SABA taken shortly before exercise.5,6 Montelukast has also been shown to be helpful in attenuating symptoms of EIB in both adult and pediatric patients53 when taken at least 2 hours before exercise.54 SABAs may be used every 4 to 6 hours for 24 hours, or longer, by patients with intermittent asthma in whom a loss of asthma control follows a viral respiratory infection, for example. If regular use of SABAs for more than 24 hours is needed to achieve control, additional treatment usually is of benefit, such as a short course of oral corticosteroids, to regain control.5 Furthermore, if episodes requiring more frequent use of SABAs occur more often than every 6 weeks, consideration should be given to step-up treatment with the addition of long-term controllers. Consideration for the addition of ICSs would be the presence of risk factors like an FEV1 below 80% predicted, or personal best, or an exacerbation in the past 12 months. In addition, there is evidence that use of daily low-dose ICSs, particularly in recent onset asthma, may improve disease control, reduce risks for exacerbation, and prevent decline in lung function.55

Step 2  Care: Low-Dose Controller Medication Plus As-Needed Reliever Medication Patients considered for step 2 treatment have mild, chronic asthma characterized by symptoms on more than 2 days per week but not daily; nighttime awakenings three to four times per month; use of SABAs on more than 2 days per week but not daily; mild limitation to normal activity; and normal lung function between asthma exacerbations (FEV1 of 80% or greater and FEV1/forced vital capacity [FVC] normal for age). In addition, patients beginning step 2 level of treatment usually have a history of 0 to 1 exacerbation in the preceding year. Low-dose daily ICS (Table 52.6) is the currently preferred treatment for patients with step 2 care asthma. This recommendation has been substantiated by systematic reviews of several well-designed randomized controlled trials.38,39 For example, the Inhaled Steroid Treatment as Regular Therapy in Early Asthma (START) study enrolled 7241 asthmatic subjects 5 to 66 years of age who were recently diagnosed with asthma.56 START compared low-dose inhaled budesonide (200 to 400 µg) daily and placebo over a 3-year period of evaluation to determine whether the initiation of low-dose ICS within 2 years of the diagnosis of mild persistent asthma would prevent both severe asthma-related events (i.e., need for systemic corticosteroids) and an accelerated decline in lung function. Subjects who were treated with budesonide had fewer symptoms, a decreased risk for asthma exacerbations, and improved lung function.56 Although some loss of lung function was documented in all treatment groups, a secondary analysis found that patients who had received inhaled budesonide initially experienced less of a reduction in lung function compared with those receiving placebo and subsequently experienced an exacerbation with the need for prednisone.57 These data suggest that the use of a low dose of ICS may prevent the loss in lung function that may be associated with exacerbations in patients with recent onset and mild disease. ICSs are preferred over other medications including LTRAs such as montelukast and zafirlukast and theophylline.5,6 LTRAs relieve symptoms and improve lung function58,59 but are, as a rule, less effective than ICSs.40,60 However, LTRAs may be a preferred option in nonadherent patients61 or with concomitant EIB. LTRAs are also considered in more

869

severe asthma with existing aspirin-exacerbated respiratory disease (AERD).62a,62b To illustrate the rationale for a stratified or personalized treatment for individual subjects with persistent asthma, Malmstrom and associates41 compared montelukast and inhaled beclomethasone in 895 adult patients with chronic asthma. Both beclomethasone and montelukast produced significantly greater improvement in the FEV1 than placebo, with beclomethasone being nearly twice as effective on comparison of the mean values of improvement in the two groups. When the investigators evaluated the distribution of treatment responses to individual subjects, they found considerable variability in the degree to which patients responded to the two treatments. Some patients demonstrated a positive response to beclomethasone, whereas other patients exhibited a more complete response to the LTRA. These early observations pointed to the need, and potential advantage, of considering individual responsiveness to specific treatments. Theophylline functions primarily as a bronchodilator but has limited antiinflammatory effects5,6 and a narrow therapeutic profile. Consequently, its benefits must be balanced against concerns for toxicity (Chapter 94). Theophylline can be considered as an alternative treatment if expense is an issue or if a tablet form of medication is preferred. Owing to insufficient evidence and the possibility of rare but lifethreatening outcomes, LABAs such as salmeterol or formoterol are not recommended for stand-alone treatment at any level of step care.5,6 For many patients, adherence to medication regimens remains a significant issue (Chapter 89). Furthermore, some interest has focused on whether intermittent treatment of mild persistent asthma may be as effective and safe as continuous use of medications.63 After initiation of treatment, follow-up visits are needed to assess level of control. For patients who have evidence of not being well controlled, a “step-up” in treatment by one level of care, with a reevaluation in 2 to 6 weeks, is recommended. By contrast, patients in the very poorly controlled category are likely to need two interventions: (1) a short course of systemic corticosteroids and (2) a step-up in treatment level of one to two steps. With treatment changes and an inherent concern regarding further loss of control, EPR-3 recommends a follow-up visit in 2 weeks to help assess the effectiveness of the new treatment approach and to ensure that an improved level of control has been achieved. At present, these recommendations are largely empirical.

Step 3  Care: One or Two Controllers (ICS/LABA) Plus As-Needed Reliever Medication Patients requiring treatment at the step 3 care level experience daily symptoms, nighttime awakening more than once per week, need for daily SABA use, limitation in daily activity, and compromises in lung function with an FEV1 usually greater than 60% but less than 80%. Over the preceding year, these patients usually experience an average of two exacerbations. It is recommended that before increasing treatment to step 3 care, mitigating factors be considered as reasons for the apparent ineffective control: inhaler technique, adherence to treatment, and environmental exposures, particularly allergens, that might contribute to diminished control. EPR-3 discussed the comparative advantages to its two preferred recommendations for step 3 care: a medium dose of ICS or combination therapy with low-dose ICS and a LABA (Table 52.7). The addition of LABAs to low-dose ICSs has consistently demonstrated greater efficacy than doubling the dose of ICSs64-66; the benefit derived by the addition of a LABA to an ICS appears to relate to the addition of bronchodilation, because little evidence is available to suggest supplemental antiinflammatory action with a LABA. The clinical effects of adding a LABA to the ICS regimen include improvement in lung function, reduction in

870

SECTION E  Respiratory Tract

symptoms, decreased frequency of exacerbations, and a lessened need for SABAs. Currently available and approved combinations of ICS/ LABA include low doses of fluticasone propionate/formoterol, fluticasone propionate/salmeterol, beclomethasone/formoterol, budesonide/ formoterol and mometasone/formoterol, all of which are used twice per day. Fluticasone furoate/vilanterol is available for once-per-day dosing. In the Gaining Optimal Asthma ControL (GOAL) study, for example, Bateman and coworkers64 found that an increase in ICS dose for symptomatic asthma improved control. However, the benefit was, as a rule, greater with combination therapy, even at a lower ICS dose. In GINA, step 3 care recommends use of a low-dose ICS with a LABA as the preferred treatment. To address the question of what may be the best treatment approach at step 3 care, increasing ICS versus adding an adjunctive medication such as a LABA, Thomas and coworkers67 used data from the Canadian General Practice database. Both approaches were equivalent in achieving symptom control. When the effects of these two approaches were evaluated on number of courses of oral corticosteroids needed (1, 2, or 3 or more), reflecting frequencies of exacerbations, an increase in ICS dose was found to be more effective than adding a LABA. These observations raised the possibility that ICSs act principally to reduce inflammation and translate to fewer exacerbations. The primary rationale for use of two preferred treatments at step 3 in EPR-3, medium-dose ICS or low-dose ICS plus LABA, despite evidence suggesting superiority of combination therapy, had been safety concerns—namely, the risk of life-threatening asthma-related events, including death, with LABA use. A major impetus for this concern was the SMART study, a randomized controlled clinical trial that involved 26,355 asthma subjects who were randomized to receive salmeterol alone or albuterol, presumably along with ICS; among patients in the salmeterol treatment group, an increased incidence in life-threatening events and respiratory-related or asthma-related deaths were noted.68 In addition to other studies,69,70 a metaanalysis performed by the FDA seemed to confirm an added risk with LABA use.43 As a result, the FDA issued an advisory statement and applied a “boxed warning” label to all asthma treatment products that contained salmeterol or formoterol while advising the public and physicians that LABAs should not be used as monotherapy. To further evaluate the LABA safety profile, the FDA mandated the enrollment of large numbers of children, adolescents, and adults into double-blind randomized controlled trials to compare, particularly, the safety of the addition of LABAs to ICSs versus ICSs alone, with the primary outcome being occurrence of severe adverse events—that is, death or near-death intubations from asthma. These large multicenter trials have been completed and did not substantiate these safety concerns. In AUSTRI,44 for example, 11,679 adults or adolescents who had an asthma exacerbation in the previous year were assigned to fluticasonesalmeterol or fluticasone only for 26 weeks, with the primary safety endpoint being first serious asthma-related event (death, intubation, or hospitalization) and a primary efficacy endpoint being time to first severe asthma exacerbation. Similar safety findings were noted by Peters et al.46 when 11,693 subjects were evaluated with budesonide vs. budesonide/formoterol combination therapy. The fixed dose combination inhaler did not have significantly higher severe asthma-related events but importantly had fewer asthma exacerbations. As a consequence of these results, the FDA has removed the boxed warning for safety for LABAs. However, LABA use alone is not recommended in asthma. Although not approved by the FDA in the United States, the combination of budesonide and formoterol has been shown to prevent exacerbations when given in addition to maintenance medications (Fig. 52.5). O’Byrne and associates71 evaluated three treatment regimens: budesonide 320 µg twice daily plus SABA as needed; budesonide

80 µg–formoterol 4.5 µg twice per daily plus SABA as needed; and budesonide 80 µg–formoterol 4.5 µg twice daily plus the same treatment for acute relief, rather than a SABA. The dose of ICS for monotherapy was selected on the basis of earlier observations that a quadrupled dose of ICS was required to prevent exacerbations.72 The frequency of asthma exacerbations was similar in the high-dose budesonide and the low-dose budesonide–formoterol combination regimens with which SABAs were used for rescue treatment; however, when the low-dose budesonide–formoterol combination was used for both maintenance and relief, the frequency of exacerbations was reduced by approximately 50%. Precisely how an early intervention with an ICS/LABA reduces exacerbations is not established. This approach to exacerbation prevention is noted by GINA. Another therapeutic option at step 3 would be the addition of a LAMA, such as tiotropium, to current treatment regimens with ICS or ICS/LABA. A double-blind, triple-dummy crossover trial involving 210 subjects with poorly controlled asthma compared the addition of tiotropium to an ICS and doubling the dose of the ICS or the addition of LABA to ICS.50 Tiotropium addition was superior to doubling the ICS and equivalent to the addition of LABAs in outcomes evaluated, including peak expiratory flow (PEF), symptoms, and lung function.50 The benefit of tiotropium in asthma has been substantiated in a large clinical trial that demonstrated improved disease control with the addition of LAMA in patients with uncontrolled asthma on ICS/LABA.47 Acceptable, nonpreferred alternatives for add-on therapy include LTRAs and theophylline. In one study, the addition of LTRA to ICS was found to be equivalent to doubling the ICS dose73; however, a systematic review of studies comparing the add-on of LABAs to ICSs and of LTRAs to ICSs found superiority with the LABA-ICS combination in preventing corticosteroid-requiring exacerbations, improving lung function, and reducing symptoms and need for SABA rescue.74 The treatment recommendations in guidelines are based primarily upon double-blind, placebo-controlled trials. Price and coworkers61 suggest that pragmatic trials also may serve to complement traditional randomized studies and bring decisions on treatment selections to “real-world practice.” These investigators61 conducted studies to address key questions using a pragmatic trial design that involved an open-label approach allowing subjects and their treating physicians to cross over treatment arms, depending on a patient’s clinical response. The first phase of this study showed that ICSs and LTRAs were equivalent when used as first-line medications; a second phase, using outcomes of higher quality-of-life scores, reduced symptoms, and improved PEF, demonstrated that LABA and LTRA were equivalent as add-ons to low-dose inhaled beclomethasone. These studies represent a new approach to assessing treatment-comparative effectiveness but have certain limitations, including significant cross-over between treatment arms, the lack of a placebo group, and a possible primary diagnosis of COPD rather than asthma in some of the subjects. Nonetheless, the results point to an alternative approach to assess the effectiveness of treatments that the investigators suggest may align more fully with clinical practice than randomized, double-blind, placebo-controlled trials. Theophylline remains an acceptable alternative for add-on therapy in poorly controlled asthma, especially in patients for whom cost may be an issue, those who demonstrate poor adherence with inhaled medication regimens, or those whose disease is apparently refractory to treatment or who suffer adverse effects with other medications. One randomized controlled trial found theophylline add-on therapy to be as beneficial as doubling the dose of inhaled budesonide (400 to 800 µg/ day) in uncontrolled asthma in terms of reducing symptoms and rescue medication use and superior in improving lung function.46 Of interest, these effects were achieved at a lower median dose and a lower plasma concentration (8.7 µg/mL) than the recommended therapeutic range

CHAPTER 52  Management of Asthma in Adolescents and Adults for theophylline (10 to 20 µg/mL); adverse effects were similar in both groups.75 Similarly, studies have found that in patients on high-dose ICSs (with or without oral corticosteroids) and theophylline, withdrawal of theophylline led to a significant worsening of symptoms.75,76 “Headto-head” direct comparison studies of theophylline as an add-on to LABA and/or LTRA are lacking, however. The addition of theophylline may be of benefit in a subset of patients with uncontrolled disease.77

Step 4  Care: Two or More Controllers Plus As-Needed Reliever Medication Patients needing step 4 level of care have symptoms present throughout the day, frequent daily awakenings from sleep, need for SABA use several times per day, limitations to normal activity, and a significant compromise in lung function (with FEV1 often less than 60% of predicted). These patients have one or more exacerbations per year. For patients with asthma uncontrolled at step 3 care, GINA recommends to increase treatment to a medium to high-dose ICS in combination with a LABA, especially in those patients with a history of recurrent exacerbations requiring oral corticosteroids, emergency department (ED) visits, or hospitalizations.5,72 Often patients who are seen for the first time and have levels of asthma control that correspond to the step 4 or higher level of care will require, and benefit from, a short course of oral corticosteroids in combination with ICS plus LABA. Patients remaining symptomatic with two relievers, such as ICS plus LABA, may benefit from the addition of a LAMA like tiotropium. Tiotropium bromide had previously been approved in the EU (European Union) as add-on therapy in patients on ICS or ICS/LABA with suboptimal control of symptoms. It garnered US regulatory approval for asthma use in 2015 based on data from 12 trials involving 5000 patients with varying severities of asthma on at least an inhaled corticosteroid. Kerstjens et al.47 compared the efficacy and safety of once-per-day tiotropium, 5 µg and 10 µg, to placebo as add-on to high-dose ICS/ LABA. There was an improvement in FEV1 values, an overall reduction in the frequency of exacerbations by 21% and in the need for rescue medications as well as an improved quality of life. Subgroup analyses of the benefit from tiotropium did not identify sex, percent predicted FEV1, reversibility, smoking status, or asthma duration as characteristics more likely associated with a favorable outcome. Side effects were minimal and limited to an increased frequency of dry mouth at the 10 µg dose. A recent Cochrane database review of tiotropium as add-on therapy for asthma in adolescents and adults affirms its efficacy and safety in this population with demonstrated benefit in reducing oral corticosteroid needs, improving lung function, and reducing asthma flares.78 Experience with use of LTRA as add-on therapy in more severe disease is limited; when available, the evidence does not support a beneficial effect79; however, clinical data suggest a possible benefit with the use of zileuton in patients with severe asthma and aspirin sensitivity.50 The level of asthma control achieved with treatment in patients who require step 4 care or higher is often less than with outcomes at lower levels of care. In the GOAL study, for example, patients who ultimately received the highest doses of either fluticasone, 500 µg twice per day, or fluticasone 500 µg plus salmeterol 50 µg twice per day, continued to have symptoms and exhibit compromises in lung function.64 Less than 70% of patients who eventually received the highest dose of fluticasone (500 µg twice per day) or highest-dose fluticasone-salmeterol combination did not achieve well-controlled status as defined by GINA.7 Although the subjects on high-dose combination therapy had an improved level of control over that obtained with ICS alone, overall control was lower than that in guideline recommendations for wellcontrolled disease. Collectively, these findings indicate that patients with more severe asthma do not always achieve optimal disease control despite high-dose combination ICS/LABA therapy. An additional concern

871

is that at high doses of ICS, adrenal function may be suppressed. Moreover, despite the overall good safety profile of long-term high-dose ICS, cataracts or osteoporosis may develop in some patients. Nevertheless, the risks with high-dose ICS are far less than those associated with systemic corticosteroid use.

Step 5  Care For patients at the GINA step 5 level of care, it is recommended to advance the daily ICS dose from a medium level used at step 4 to higher doses (Table 52.6). Evidence for added effectiveness with ICS treatment beyond medium-level doses is limited, and relevant studies tend to involve smaller numbers of patients.5,72,80 In the GOAL study, high-dose combination therapy (fluticasone 500 µg plus salmeterol 50 µg, twice per day) was more effective than high-dose ICS (fluticasone 500 µg twice a day) alone for all patients studied.64 The frequency of achieving either good or well-controlled asthma was lower in patients with more severe disease. Pauwels and colleagues72 initially provided evidence to indicate an added benefit with a higher dose of ICS by comparing dosing of budesonide 100 µg twice daily vs. 400 µg twice daily along with addition of formoterol 12 µg twice daily to the two dosing schedules of budesonide. In the Formoterol and Corticosteroids Establishing Therapy (FACET) study, rates of exacerbations were reduced with the addition of formoterol to both dose levels of budesonide. Moreover, the higher dose of budesonide (a fourfold increase) alone was approximately twice as effective in reducing exacerbations as the low-dose ICS. Finally, the most effective treatment to reduce exacerbations was high-dose budesonide plus formoterol. This study was important in pointing out that a fourfold increase in ICS dose may be necessary to reduce pending exacerbations of asthma. Subsequent studies by Harrison and colleagues81 and FitzGerald and associates82 found that doubling the dose of ICS at the time of a pending exacerbation was not beneficial or, at best, was of limited benefit. In a recent pragmatic trial on 1922 adults and adolescents with asthma who had at least one exacerbation in the prior 12 months, McKeever et al. evaluated the efficacy of a self-directed plan of temporarily quadrupling the dose of inhaled corticosteroids at the first sign of loss of asthma control in blunting the incidence of severe asthma exacerbations. The study demonstrated that such an approach resulted in fewer severe asthma exacerbations, but less than what was expected. It was also shown that the “quadrupling” group had a higher rate of treatment-related adverse effects. Given the discussed results, the authors conclude that patients, physicians, and guideline committees need to consider whether the clinical benefit-to-risk ratio warrants such an approach.83

APPROACHES TO TREATMENT OF SEVERE ASTHMA Airway Inflammation and Associated Biomarkers: A Targeted Personalized Approach to Asthma Management In the past decade, tremendous progress has been made to understand the pathophysiologic mechanisms contributing to chronic inflammation in asthma and the associated clinical phenotype. The inflammatory milieu in asthma is elaborate and includes an array of cells and cytokines that contribute to inflammation in an independent but redundant fashion. Mast cells, neutrophils, eosinophils, lymphocytes (Th2 subtype), epithelial cells, and myriad cytokines—IL-4, 5, 6, 9, 13, 25, 31, 33, among others—have been recognized as key players in asthma pathogenesis and inflammation. The complex interplay between resident tissues, cytokines, recruited cells, and environmental factors, such as a

872

SECTION E  Respiratory Tract

respiratory virus, an aeroallergen, or an irritant, results in the airway inflammation and injury that sustains the symptoms of asthma and may contribute to airway remodeling, which is often not fully responsive to current treatments with ICSs and LABAs. Airway remodeling is a conglomerate of structural changes observed in large and small airways of some asthmatic patients and associated with reticular basement membrane thickening, subepithelial fibrosis, goblet cell hyperplasia, smooth muscle hypertrophy, and neovascularization. However, the mediators of inflammation and remodeling in asthma play a differential role in its pathophysiology. Th2 lymphocytes, eosinophils, and selected cytokine products and growth factors of these cell types have taken center stage in this process. IL-3, GM-CSF, and IL-5, produced by Th2 and innate lymphoid cells (ILC)-2, promote eosinophil growth, differentiation, and recruitment of these cells to the airway. IL-4 and IL-13 regulate IgE class switching in B cells. IL-33 and thymic stromal lymphopoietin (TSLP) are cytokines derived from epithelial cells and fibroblasts that enhance the maturation and activation of dendritic cells (DCs) and naïve type 2 lymphoid cells (Th0). IL-6, IL-23, and TGF-β activate dendritic cells and together can induce

regulatory T cells (Treg) and Th17 cells that secrete IL-17 to add to the inflammatory entourage already in place. The activation and polarization of Th0 cells augments production of IL-4, IL-5, IL-13 that further skew the inflammatory phenotype toward a Th2 pattern. This intricate inflammatory framework presents potential biologic targets for intervention and drug development (Fig. 52.6). Recognition of these events has led to diligent efforts by investigators and clinicians to categorize asthma patients into more distinct and informative phenotypes based on the presence of certain clinical features and associated treatment-directive biomarkers. Some of the biomarkers of interest in asthma that indicate a skewing toward a T2 phenotype include sputum and blood eosinophils, IgE, serum periostin, fractional exhaled nitric oxide (FeNO), and dipeptidyl peptidase 4 (DPP4). Elevations in these markers have been purported to represent activation of specific pathways of inflammation in asthma and possibly indicate treatment responsiveness in T2-high disease (Table 52.8). This evolving effort and resulting insights have further led to the proposed classification or stratification of asthma into “endotypes,” which effectively couples the phenotype to an identified pathway or molecular mechanism.84,85

T2-high

T2-low

Allergic eosinophilic

Non-allergic eosinophilic Pollutants, oxidative stress, microbes

Allergens Epithelium and RBM DP2/PGD2 Dendritic cell

Mast cell IgE

IL13

B cells

TSLP

TSLP

TSLP

IL4/IL13 via IL4α

TH0

TH2

TSLP

DP2/PGD2

TSLP Mast cell

IL5(R)

IL4/IL13 via IL4α

IL33 IL25

DP2/PGD2

IL13 IL13 via IL4α

Eosinophils DP2/PGD2

DP2/PGD2 ILC2

IL13 via IL4α

DP2/PGD2

IL5(R) Eosinophils

IL13

IL13

DP2/PGD2

IgE IL13 via IL4α

DP2/PGD2

Airway smooth muscle Target

Airway eosinophils

Exacerbations

Symptoms/ health status

FEV1

Target

Airway eosinophils

Exacerbations

Symptoms/ health status

FEV1

IgE

↓

↓↓

↓

↑

IgE

↔

↔

↔

↑↓

IL5(R)

↓

↓↓

↓

↑

IL5(R)

↓

↓↓

↓

↑

IL4α

?

↓↓↓

↓↓

↑↑

IL4α*

?

?

?

?

DP2

↓

?

↓↓

↑

DP2

↓

?

↓↓

↑

TSLP

?

↓↓↓

↓↓

↑↑

TSLP

?

↓↓↓

↓↓

↑↑

Fig. 52.6  Biologics and emerging small molecule therapies for severe asthma licensed or in phase 3; their targets in the immunopathology of the disease and clinical impact. ↑, improve; ↓, attenuate; ↔, no effect; ?, awaiting data or not studied; ↑↓, variable reports. *Dupilumab studies included nonatopic subjects in phases 2 and 3. Effects in nonatopic subjects were not reported in phase 2, but a phase 3 full report is awaited. IL, Interleukin; IgE, immunoglobulin E; TSLP, thymic stromal lymphopoietin; DP2/PGD2, prostaglandin D2; R, receptor; RBM, reticular basement membrane.154

CHAPTER 52  Management of Asthma in Adolescents and Adults

873

TABLE 52.8  Properties of Type 2 High Biomarkers Marker

Advantages

Limitations

Sputum eosinophils

Identifying T2-mediated airway inflammation. Correlates with disease severity, health care utilization and corticosteroid responsiveness. Predictive for therapeutic responses to corticosteroid therapy and T2-targeting therapies. Exacerbation risk in general and after corticosteroid therapy discontinuation.

Inadequate samples are common. Risky procedure for patients with severe asthma. Procedure is not feasible for children younger than 8 years of age.

Blood eosinophils

Can distinguish between eosinophilic and noneosinophilic asthma based on a cut-off value of 300 cells/mL. Correlate with disease severity and exacerbation risk. Predictive of therapeutic responses to corticosteroids as well as IL-5 targeting biologic therapies. A cut-off value of 150 eosinophils per mL can be used to gain a 72% reduction in exacerbation frequency using mepolizumab.

No consensus exists about the optimal cut-off value. A wide range of cut-off values employed in clinical trials investigating IL-5 targeting therapeutics varying from 150-400 mL. Marker offers no insight into the function or amount of eosinophils residing in the lungs under anti–IL-5 therapy. Other conditions such as infections, autoimmune disorders, allergies, and its diurnal variation can increase peripheral eosinophil numbers. The sensitivity of blood eosinophils is reduced under IL-5 targeted therapy.

FeNO

Simple and noninvasive method for determining epithelial activity and eosinophilic airway inflammation. ATS/ERS guidelines recommend 50 ppb as cut-off points for excluding or demonstrating T2-mediated airway eosinophilia, respectively. High levels of FeNO in mild to moderate asthma appear predictive of corticosteroid responsiveness. FeNO assessments can be used to monitor correct use of inhalation corticosteroids, therapy compliance, and corticosteroid resistance.

FeNO-based therapy optimization does not lead to better clinical outcomes and is therefore not recommended by asthma guidelines. Distribution of FeNO concentrations is positively skewed, with the upper limit overlapping with asthmatic populations. NO concentrations are influenced by smoking, atopy, age, height, weight, sex, comorbidities. Cannot be performed reliably in children younger than 5 years old.

Serum IgE

Determination of atopy status. Dosing of omalizumab. Free IgE levels in the serum appeared useful for monitoring response to therapy, whereby decreases in serum IgE concentrations of at least 90% result in the strongest reduction in exacerbation frequency.

Serum IgE levels cannot predict therapeutic responses to omalizumab. Low specificity for detecting sputum eosinophilia. Poor correlations with airflow obstruction and disease severity.

Serum periostin

—

Inconsistent results about the association of periostin serum concentrations and eosinophilic airway inflammation. Inconsistent results regarding the use of periostin serum levels for predicting treatment response to IL-13 targeting therapeutic lebrikizumab. The distribution of serum periostin concentrations is right skewed and lacks a universal cut-off value as well as standardization of measurement technique. Periostin serum concentrations can also be increased by conditions other than asthma, such as atopic dermatitis, allergic rhinitis, scleroderma, bone metastases, bone fractures, osteoporosis, renal insufficiency, and cardiovascular disorders.

Dipeptidyl peptidase 4 (DPP-4)

Predictive of improvements of lung function and quality of life before treatment with IL-13 targeting therapy tralokinumab. High serum levels of periostin appear better at predicting treatment response to tralokinumab than high periostin levels.

Role in asthma is unclear as both upregulation and downregulation of the enzyme have been reported. Several cell types are able to produce the enzyme and can exert both stimulatory and inhibitory effects on the immune system. No universal cut-off value has been established. Whether DPP-4 is indeed a superior marker to periostin requires further investigation.

Eosinophilic cationic protein (ECP)

Potential marker for diagnosing eosinophilic airway inflammation. Serum ECP concentrations appear to be inversely correlated with FEV1 and decrease with corticosteroid therapy. Increased concentrations of serum ECP predict a higher prevalence of asthma exacerbations in the upcoming 3 months.

Replication of the results and validation of measurement technique are essential before this marker can be used in clinical practice. Universal cut-off values and standardization of measurement technique are currently lacking.

874

SECTION E  Respiratory Tract

TABLE 52.8  Properties of Type 2 High Biomarkers—cont’d Marker Cysteinyl leukotrienes

Advantages A positive correlation was found between urinary concentrations of cysteinyl leukotrienes and sputum eosinophilia. Increased urinary concentrations of leukotriene E4 seems predictive of therapeutic response to leukotriene receptor antagonists in both pediatric and adult asthma patients.

Limitations Replication of the results and validation of measurement technique are essential before this marker can be used in clinical practice. Universal cut-off values and standardization of measurement technique are currently lacking.

FeNO, fractional exhaled nitric oxide; FEV1, forced expiratory volume in 1 second. From Richards, et al Curr Opin Allergy Clin Immunol 2018;18:96-108.153

TABLE 52.9  Properties of Type 2 Low Biomarkers Marker

Advantages

Limitations

Sputum neutrophils

Sputum neutrophilia has been associated with severe asthma and can be indicative for non–T2-mediated asthma. Inverse correlation between sputum neutrophils and FEV1 values.

Wide range of cut-off values have been applied varying between 40% and more than 60%. Role of neutrophils in asthma pathology is unclear. Corticosteroid therapy can also increase sputum neutrophil numbers. Sputum neutrophils are unable to predict therapeutic responses to macrolide antibiotic therapy.

IL-17

Holds possible diagnostic value for the identification of non–Th2mediated asthma. Concentrations in BAL fluid, sputum, serum and biopsies correlate with asthma severity. Associations between serum concentrations of IL-17 and neutrophil counts in blood and sputum have been demonstrated.

Universal cut-off values and standardization of measurement technique are currently lacking. Interleukin is not disease specific. Hence, IL-17 levels in the systemic circulation can be modified by other diseases as well.

YKL-40

Serum and sputum levels of YKL-40 are increased in severe asthma and correlate with disease severity, airway obstruction, and membrane thickness regardless of airway inflammation type. Serum concentrations of YKL-40 appear to correlate with sputum neutrophilia.

It is suggested YKL-40 is involved in asthma pathology, but its role remains to be determined. Determination of YKL-40 has not been standardized or validated. Furthermore, currently no universal cut-off values have been established.

IL-6

Possible indicator for metabolic dysfunction and tissue damage in asthma patients. Serum concentrations of IL-6 correlate with disease activity and severity.

IL-6 is not specific for asthma or other pulmonary and nonpulmonary conditions. Universal cut-off values and standardization of measurement technique are currently lacking.

BAL, Bronchoalveolar lavage; FEV1, forced expiratory volume in 1 second; T2, type 2. From Richards, et al. Curr Opin Allergy Clin Immunol 2018;18:96-108.153

This stratification approach would provide the advantage of allowing clinicians to selectively choose a biologic therapy, which would be more likely to produce a favorable response in the patient and ultimately provide a roadmap for personalized medicine. At present, asthma can be more broadly categorized into groups: Th2-high (T2 high) and Th2-low (T2 low). The T2 high group typically displays an increased eosinophil presence in the sputum, airways, and peripheral circulation while the T2 low group classically exhibits a neutrophilic or a paucigranulocytic profile in sputum and airways (Fig. 52.6 and Table 52.9). IL-6 is another cytokine that is also being actively evaluated in asthma whereby it may serve as a marker of systemic inflammation and metabolic dysfunction in obese patients, for example, who have severe asthma. Among obese subjects with asthma recruited from the Severe Asthma Research Project (SARP) cohort, it was demonstrated that the FEV1 was significantly lower in IL-6 high patients compared with IL-6 low patients along with higher rates of exacerbations noted in the IL-6 high group. A similar pattern was also observed in nonobese asthmatic patients with severe asthma. Based on this observation, IL-6 may serve as a biomarker for a new “metabolic asthma” endotype and provide a basis to consider trials that use IL-6 antagonists

(i.e., tocilizumab or sarilumab) to reduce metabolic dysfunction in subsets of severe asthma patients and determine whether this improves asthma control.33 Based on current understanding of the inflammatory perspectives in asthma, this type of classification can still present a therapeutic dilemma in patients with overlapping or redundant and competing inflammatory mechanisms contributing to asthma pathophysiology. An example of such a stratification is presented in Fig. 52.7. Based upon this expanded understanding of asthma heterogeneity and multiple inflammatory pathways, attention has been dedicated to the development of monoclonal antibodies that specifically target key mediators of inflammation that may exist in some asthma patients and contribute to a resulting pathophysiology. These targets include IgE, IL-5, IL-4, IL-13, IL-17, TSLP, and CRTH2 or DP2 receptor. Early studies of biologics in asthma had included all asthma phenotypes and severities and used traditional lung function improvement (i.e., FEV1) as the primary outcome and failed to meet that endpoint. Since then, intervention efforts have refocused the approach to targeting specific subgroups, or asthma phenotypes, as well as more relevant outcome measures, perhaps more reflecting activation of key

875

CHAPTER 52  Management of Asthma in Adolescents and Adults

Severe refractory asthma

Consider ongoing protocols on experimental compounds for noneligible/unresponsive patients to available therapeutic approaches

• High rate of exacerbations despite best standard therapy/OCS • Spirometry FEV1 300 cells/L • Serum lgE 30 to1500 IU/mL • Perennial allergens sensitization

Omalizumab (Ideal setting: allergens, severe refractory asthma)

Non-responder consider alternate T2 antibody

Responder

Yes Continue omalizumab therapy

Patients eligible for T2 biologics (mepolizumab, reslizumab, benralizumab, dupilumab)

No

Blood eosinophils >300 cells/L

Yes T2 Biologics (mepolizumab, reslizumab, benralizumab, dupilumab) (Ideal setting: severe uncontrolled eosinophilic asthma regardless of atopic or nonatopic state)

No Bronchial thermoplasty (Ideal setting: severe uncontrolled asthma with FEV1 60% unsuitable for the currently available biodrugs)

Blood eosinophils >300 cells/L Responder Yes T2 Biologics (mepolizumab, reslizumab, benralizumab, dupilumab)

No Bronchial thermoplasty/Alternate T2 antibody

Yes Continue anti-T2 monoclonal antibody or alternate T2 antibody

No Bronchial thermoplasty /AlternateT2 antibody

Fig. 52.7  Flowchart for the selection of different treatment options. OCS, Oral corticosteroids.158

inflammatory pathways, such as the prevention of exacerbations and a sparing of the need for systemic or large doses of inhaled corticosteroids. There is also interest in gauging whether biologic therapy would result in disease-modifying effects. This strategy of modifying key features that represent underlying pathophysiology of asthma has led to a renewed interest in the usage of biologics with clinically relevant outcomes being evaluated. This chapter focuses on select targets and biologic therapies that are currently approved for use as well as ones that have garnered prominent attention based on preliminary studies (Fig. 52.8). As will be discussed, targeted treatment with biologics has begun to fill important and clinically relevant unmet needs for many patients, particularly those with severe disease.

Biologics for Use in Severe Asthma (Table 52.10) Anti-IgE (Omalizumab).  Omalizumab is an injectable monoclonal antibody directed against the epsilon C3 domain of IgE and prevents an interaction with the high-affinity IgE receptor (FCεR1) on mast cells, basophils, eosinophils, Langerhans cells, and dendritic cells. It was the first approved biologic therapy for allergic asthma. Currently, omalizumab is approved for use in pediatric patients (ages 6 to 12 years with an IgE range of 30 to 1300 IU/mL) and adult patients (older than 12 years with an IgE range of 30 to 700 IU/mL) with allergic asthma. The dosing for omalizumab is based on a nomogram that incorporates the

patient’s weight and IgE level. Omalizumab is currently recommended as add-on therapy to consider for patients in EPR-3 step 5 and 6 care or GINA step 5. It has a label warning for anaphylaxis, and current guidelines recommend a 2-hour wait with the first three administrations. The most common adverse events noted in adults were arthralgias, generalized pain, fatigue, and dizziness, with nasopharyngitis being more common in children. A pooled analysis of seven early studies indicated that omalizumab, as add-on therapy, significantly reduces the rate of exacerbations and ED visits independent of patient age, sex, and baseline IgE level, with an effect that is most profound in severe persistent asthma.86 Hanania et al. evaluated the efficacy of omalizumab in uncontrolled severe asthma patients on high-dose ICS/LABA therapy with or without additional controller therapy, patient groups most akin to step 5 and 6 in EPR-3; the omalizumab-treated group had a decrease in exacerbations by 25% and also improved asthma symptom scores and asthma quality-of-life outcome metrics.87 Benefit from omalizumab was not seen in oral corticosteroid-dependent subjects. A more recent RDBPC study by Busse et al. showed that omalizumab when added to guidelinesbased therapy in a group of high-risk inner-city adolescents ages 6 to 17 years, with seasonal exacerbations of asthma in fall and spring, abolished these seasonal peaks in exacerbations, improved asthma control, and reduced the need for controller medications, including ICS.88

876

SECTION E  Respiratory Tract

Airway epithelium IL-4Rα

Dupilumab

Dendritic cells B cells

AMG 157 (Tezepelumab)

IL-13

Lebrikizumab Tralokinumab Smooth airway muscle

IgE

TSLP FcεRI Omalizumab

ILC2 cells

Bronchial thermoplasty

T2 cells

Mast cells PGD2R

SGF KIT

Eosinophils

Imatinib

IL-5 IL-5Rα

Fevipiprant Mepolizumab Reslizumab

Benralizumab

Fig. 52.8  Mechanisms of action of new and emerging treatments for severe asthma. Therapeutic targets for selected biologics and other novel treatments for patients with severe, uncontrolled asthma. FcεRI, High affinity IgE receptor; ILC2, innate lymphoid type 2; PGD2R, prostaglandin D2 receptor; SCF, stem cell factor. (Adapted with permission from Fajt ML, Wenzel SE. Development of new therapies for severe asthma. Allergy Asthma Immunol Res 2017;9:3-14.)

A pooled analysis of omalizumab in moderate to severe persistent allergic asthma receiving moderate- to high-dose ICS indicated that omalizumab significantly reduced peripheral blood eosinophil counts compared with placebo along with improved clinical outcomes; this observation corroborated early findings that omalizumab has an inhibitory effect on peripheral blood eosinophil counts.89 There remains uncertainty regarding clinical or biomarker parameters that would predict which patients are most likely to respond to omalizumab therapy. Busse et al. made the observation that in a postmarketing study omalizumab appeared to reduce protocol-defined exacerbations more significantly in subgroups with higher eosinophils (more than 300/µL) at baseline compared with those that had low eosinophil counts, thus supporting the value of an eosinophil count as a T2 inflammatory biomarker to predict efficacy of omalizumab.90 The EXTRA omalizumab study also addressed this question and found a greater reduction in exacerbations in subgroups of patients who had elevated FeNO, periostin, and blood eosinophil counts (260 or more cells/µL).91 The PROSPERO study (Prospective Observational Study to Evaluate Predictors of Clinical Effectiveness in Response to Omalizumab) has provided a greater understanding of the profile of patients, including disease and comorbidity burden, who were initiated on omalizumab therapy based on a clinical need in a “real-world” setting. The authors characterized this group as a poorly controlled, highly allergic, heterogeneous group with varying biomarker patterns and significant disease burden.92 Also of interest is a unique study that demonstrated the efficacy of omalizumab in adult nonatopic, severe, refractory asthma by improving FEV1 and downregulating FcεR1 expression on basophils and dendritic cells. Contrary to other early studies with omalizumab, lung functions improved, particularly in a nonallergic cohort.93 Omalizumab’s efficacy

in treating chronic idiopathic urticaria (a non-IgE-mediated disease), coupled with observations in nonatopic asthma, raises the interesting possibility that omalizumab may mediate its effect through additional unknown mechanisms that transcend its traditional role in downregulating IgE and FcεR1.94 Other anti-IgE biologics, particularly quilizumab and ligelizumab, have also been studied in allergic asthma but have not shown similar efficacy in preliminary studies nor translated as of yet to clinical trials.95,96

Anti-IL-5 Therapy.  Eosinophilia is a prominent feature in at least 50% of asthma patients across all levels of severity. IL-5, commonly produced by Th2 lymphocytes and mast cells, as well as ILC2, is a key cytokine responsible for eosinophil differentiation, activation, and recruitment. Consequently, IL-5 is another important target to consider in biologic therapy for asthma. Currently, three anti-IL-5 mAbs are approved for use as add-on therapy for severe eosinophilic asthma. Interest in anti-IL-5 therapy dates back to the late 1990s when it was demonstrated that such therapy significantly reduced airway hyperresponsiveness and eosinophil counts in bronchoalveolar lavage (BAL) fluid in ovalbumin-challenged mice.97 However, these findings did not readily translate to clinical success, because initial studies focused on multiple asthma phenotypes and used improvement in lung function as the primary outcome. Mepolizumab is an IgG1 kappa monoclonal antibody directed against IL-5, and the first to undergo trials to get approval for use in severe asthma patients aged 12 years or older. Haldar et al. found mepolizumab reduced exacerbations and improved Asthma Quality of Life Questionnaire (AQLQ) scores in patients with severe eosinophilic asthma along with reducing blood and sputum eosinophil counts but without leading to FEV1 improvement.98 This finding led

877

CHAPTER 52  Management of Asthma in Adolescents and Adults

TABLE 52.10  Biologics for Treatment of Severe Asthma Drug/Target

Reference

Population

Dose/Duration

Primary Outcomes

Secondary Outcomes

Mepolizumab Anti-IL-5

Chupp LRM 2017 MUSCA Phase 3b59 Bel NEJM 2014 SIRIUS Phase 358 Ortega NEJM 2014 MENSA Phase 357

Adults and children (>12 y), n = 556   ≥2 exacerbations in last year   Background therapy: high-dose inhaled corticosteroid plus additional controller(s)   Prebronchodilator FEV1 < 80% in adults ≥18 y ( 6 mo   Blood eosinophil count ≥150 cells/µL at screening or ≥300 cells/µl in the last year Adults and children (aged ≥12 y), n = 576   ≥2 exacerbations in last year   Background therapy (≥880 µg/d fluticasone propionate equivalent) for >3 mo and an additional controller   Blood eosinophil count ≥150 cells/µL at screening or ≥300 cells/µL in the last year

100 mg SC Q4W   24 wk 100 mg SC Q4W   20 wk 75 mg IV Q4W or 100 mg SC Q4W   32 wk

SGRQ: ↓ score by 7.7 vs placebo Oral corticosteroid use: ↓ ~50% Exacerbation rate: ↓ ~50%

↓ exacerbation rate 42% ↑ FEV1 120 mL ↓ exacerbation rate ~32% ↓ ACQ (~0.52) ↑ FEV1 (100 mL) ↓ ACQ (~0.43), SGRQ (~7)

Reslizumab Anti-IL-5

Corren Chest 2016 Phase 391 Bjermer Chest 2016 Phase 392 Castro LRM 2015 Phase 362a

Adults and children (12-65 y), n = 492  ACQ ≥1.5   Background therapy (≥440 µg/d fluticasone propionate equivalent) for >1 mo and an additional controller   Bronchodilator response >12% Adults and children (12-75 y), n = 315  ACQ ≥1.5   Background therapy (≥440 µg/d fluticasone propionate equivalent) for >1 mo and an additional controller   Blood eosinophil count ≥400 cells/µL Adults and children (12-75 y), n = 953 (study 1 n = 489, study 2 n = 464)   ≥1 exacerbation in last year  ACQ ≥1.5   Background therapy (≥440 µg/d fluticasone propionate equivalent) for >1 mo and an additional controller   Bronchodilator response >12%   Blood eosinophil count ≥400 cells/µL

3 mg/kg IV Q4W   16 wk 0.3 mg/kg IV Q4W  or   3 mg/kg IV Q4W   16 wk 3 mg/kg IV Q4W   52 wk

FEV1 at week 16   ↔ FEV1 FEV1 at week 16 in eosinophilHigh:   ↑ FEV1 (~140 mL) Exacerbation rate (eosinophilHigh only recruited >400 cells/µL):   ↓ exacerbation   rate ~60-80%

EosinophilHigh:   ↑ FEV1 (270 mL)   ↓ ACQ (0.49)   No benefits in eosinophilLow group ↓ ACQ (~0.3),   ↑ AQLQ (~0.3) ↑ FEV1 (100 mL)   ↓ ACQ (~0.25),   ↑ AQLQ (~0.23)

Benralizumab Anti-IL-5R

Nair NEJM 2017 ZONDA Phase 374 Bleecker Lancet 2016 SIROCCO Phase 371 Fitzgerald Lancet 2016 CALIMA Phase 370

Adults (18-75 y), n = 220   Background therapy (7.5-40 mg/d prednisolone or its equivalent for > 6 mo)   Blood eosinophils ≥150 cells/µl at screening Adults and children (12-75 y), n = 1205   ≥2 exacerbations in last year  ACQ ≥1.5   Background therapy: ICS plus LABA for ≥1 y before enrollment (high-dose ICS in adults and moderate to high in children) and another controller   Prebronchodilator FEV1 < 80% (adults), 12%   As per SIROCCO, n = 306

30 mg SC Q4W or  Q8W   28 wk 30 mg SC Q4W or  Q8W   48 wk 30 mg SC Q4W or  Q8W   56 wk

Oral corticosteroid use:   ↓ 75% Exacerbation rate in eosinophilHigh:   ↓ ~50% Exacerbation rate in eosinophilHigh:   ↓ ~50%

↓ exacerbation rate 55-70% ↑ FEV1 (110 mL)   ↓ ACQ (~0.25),   ↑ AQLQ (~0.25) ↑ FEV1 (~120 mL)   ↓ ACQ (~0.2),   ↑ AQLQ (0.2)

Continued

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SECTION E  Respiratory Tract

TABLE 52.10  Biologics for Treatment of Severe Asthma—cont’d Drug/Target

Reference

Population

Dose/Duration

Primary Outcomes

Secondary Outcomes

Dupilumab Anti-IL-4Rα

QUEST Phase 378 Wenzel Lancet 2016 Phase 2b77

Adults and children (≥12 y), n = 1902   Background therapy (≥250 µg of fluticasone BD - 2000 µg/d vs fluticasone) and another controller Adults (≥18 y)   ≥1 exacerbation in last year  ACQ ≥ 1.5   Background therapy (≥500 µg/d fluticasone proprionate equivalent + LABA) for > 1 mo   Prebronchodilator FEV1 40-80%, bronchodilator response >12%

200 mg SC Q2W or   300 mg SC Q2W   52 wk 200 mg SC Q2W or   Q4W or   300 mg Q2W or  Q4W   24 wk

Exacerbation rate:   ↓ 46%  FEV1 at week 12:   ↑ 130 mL at 300 mg dose FEV1 at week 12 in eosinophilHigh:   ↑FEV1 (~210 mL)

Rate of LOAC events/severe exacerbation events Time to LOAC event/ severe exacerbation event ↓ exacerbation rate ~60-80%   ↓ ACQ (~0.5)   ↑ AQLQ (~0.6)

Fevipiprant Anti-DP2

Gonem LRM 2016 Phase 284

Adults (≥18 y), n = 61   ≥1 exacerbation in last year or  ACQ ≥1.5   Background therapy: low- to high-dose ICS   Sputum eosinophil count ≥2% at screening

225 mg BD PO   12 wk

↓ Sputum eosinophils

↑ FEV1 (160 mL)   ↓ ACQ (0.56),   ↑ AQLQ (0.59)

Tezepelumab Anti-TSLP

Corren NEJM 2017 Phase 290

Adults (18-75 y), n = 584   ≥2 exacerbations requiring OCS or   ≥ 1 severe exacerbation that led to hospitalization in last year  ACQ ≥1.5   Background therapy (250-500 µg/d fluticasone + LABA) or high dose (>500 µg/d fluticasone) for >6 mo   Prebronchodilator FEV1 40%-80%, bronchodilator response ≥12%, ≥200 mL

70 mg SC Q4W or   210 mg SC Q4W or 280 mg SC Q2W   52 wk

Exacerbation rate:   ↓ ~60–70%

↑ FEV1   (~100-150 mL)   ↓ ACQ, ↑AQLQ at high dose

ACQ, Asthma Control Questionnaire; ACT, Asthma Control Test; ATAQ, Asthma Treatment Assessment Questionnaire; FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; LOAC, Annualized Rate of Loss of Asthma Control; SGRQ, St George’s respiratory questionnaire. From Diver et al. Clin Exp Allergy 2018;48:241-52.154

to three hallmark trials (DREAM, MENSA, and SIRIUS) as well as the recently completed MUSCA trial that demonstrated efficacy for mepolizumab in severe uncontrolled eosinophilic asthma patients on highdose ICS therapy by reducing exacerbations, demonstrating OCS-sparing effects, improving ACQ scores and health-related quality of life scores, and leading to modest improvement on lung function metrics.98-101 Currently, mepolizumab is approved for severe eosinophilic asthma and is administered subcutaneously at a fixed dose of 100 mg every 4 weeks for patients with an eosinophil cutoff of at least 150 cells/µL in the last 8 weeks or a greater than 300 cells/µL cutoff in the past 12 months. Rare hypersensitivity reactions and herpes zoster infections have been reported with mepolizumab usage. Aside from asthma, mepolizumab has also been recently approved for use in eosinophilic granulomatosis with polyangiitis (formerly Churg-Strauss syndrome) at a dose of 300 mg given every 4 weeks.102,103 Reslizumab, an IgG4 kappa monoclonal antibody also directed against IL-5, has been approved for use in severe eosinophilic asthma patients aged 18 years and older with an eosinophil cutoff of 400 cells/µL or higher and is administered intravenously at a standard dose of 3 mg/kg every 4 weeks. It carries a boxed warning for anaphylaxis with oropharyngeal pain as the main reported adverse reaction. Two large, duplicate, RDBPC trials of reslizumab in asthmatic patients aged 12 to 75 years with uncontrolled asthma on a medium- to high-dose ICS and eosinophil counts equal to or greater than 400 cells/µL, demonstrated

significant reductions in exacerbation rates compared with placebo.104 In a recent phase 3 study of asthmatic patients aged 12 to 75 years who were suboptimally controlled on a medium-dose ICS and had a blood eosinophil count greater than 400 cells/µL, reslizumab significantly improved FEV1, ACQ, and AQLQ scores, findings not consistently demonstrated in studies with mepolizumab.105 When a similar analysis was conducted across a broad range of eosinophil counts, reslizumab demonstrated improvements in lung function but primarily in those with peripheral blood eosinophil counts of 400 cells/µL or more.106 This difference in lung function improvement may be a consequence of patient selection. Patients in the reslizumab trials had higher baseline eosinophil counts compared with those in the mepolizumab trials and may be more reflective of a “true” eosinophilic phenotype of asthma. A recent trial evaluating subcutaneous reslizumab failed to show efficacy. Consequently, reslizumab remains available only by intravenous administration.107 Benralizumab is a humanized monoclonal antibody directed against the alpha subunit of the IL-5 receptor and is administered subcutaneously. Given its unique mechanism of action on the IL-5 receptor, benralizumab enhances eosinophil apoptosis via antibody-dependent cell-mediated cytotoxicity (ADCC) and effectively abrogates cells that express the IL-5-Rα. There is also a reduction in basophils. The question remains whether this effect translates to superior clinical efficacy. Two phase 3 studies (SIROCCO and CALIMA) of benralizumab on

CHAPTER 52  Management of Asthma in Adolescents and Adults Eosinophils ≥ 300 cells per µL 1.4

(1.12–1.58)

Percentage reduction relative to placebo –51% –45%

Annual asthma exacerbation rate ratio (95% CI)

1.2 1.0 p < 0.0001 (0.60–0.89)

0.8

p < 0.0001 (0.53–0.80)

0.6 0.4 0.2 0

A

Placebo (n = 267)

Benralizumab 30 mg Q4W (n = 275)

Benralizumab 30 mg Q8W (n = 267)

Eosinophils < 300 cells per µL Percentage reduction relative to placebo –17% –30%

1.4

Annual asthma exacerbation rate ratio (95% CI)

1.2

(0.96–1.52)

1.0

p = 0.047 (0.65–1.11)

p = 0.269 (0.78–1.28)

0.8 0.6 0.4 0.2 0

B

Placebo (n = 140)

Benralizumab 30 mg Q4W (n = 124)

Benralizumab 30 mg Q8W (n = 131)

Fig. 52.9  Annual asthma exacerbation rate estimates at 48 weeks according to baseline blood eosinophil concentrations. Data for patients with baseline blood eosinophils (A) ≥300 cells per µL and (B) 42 mm Hg • Physical exam: symptoms severe, drowsiness, confusion • Continue fetal assessment

Individualized Decision re: Hospitalization Discharge Home • Continue treatment with inhaled β2-agonist • Continue course of oral systemic corticosteroids • Initiate or continue inhaled corticosteroids until review at medical follow-up • Patient education • Review medicine use • Review/initiate action plan • Recommend close medical follow-up

Admit to Hospital Ward • Inhaled β2-agonist every 4 hours and as needed • Systemic (oral or intravenous) corticosteroids • Oxygen • Monitor FEV1 or PEF, O2 saturation, pulse • Continue fetal assessment until patient stabilized

Admit to Hospital Intensive Care • Inhaled β2-agonist hourly or continuously • Intravenous corticosteroids • Oxygen • Possible intubation and mechanical ventilation • Continue fetal assessment until patient stabilized

Improve

Discharge Home • Continue treatment with inhaled β2-agonist • Continue course of oral systemic corticosteroids • Initiate or continue inhaled corticosteroids until review at medical follow-up

• Patient education • Review medicine use • Review/initiate action plan • Recommend close medical follow-up

Fig. 55.1  National Asthma Education and Prevention Program (NAEPP) recommendations for management of asthma exacerbations: emergency department and hospital-based care. FEV1, Forced expiratory volume in 1 second; PEF, peak expiratory flow.

and could be considered in pregnant patients with poorly responsive exacerbations, especially with coexisting pregnancy-induced hypertension or preterm uterine contractions. For patients not helped by the initial therapy, ABG levels must be carefully observed, particularly in pregnant patients with Pao2 less than 70 mm Hg or Paco2 greater than 35 mm Hg. Criteria for admission and hospital management of the pregnant woman with acute severe asthma probably should be more lenient than criteria for the nongravid patient. When the pregnant asthmatic patient is hospitalized, both

medical and obstetric supervision are required. The use of intubation and assisted ventilation for life-threatening asthma during pregnancy is occasionally necessary; ventilator management of respiratory failure during pregnancy is described elsewhere.78,79 Intravenous cefuroxime is recommended initially for hospitalized pregnant asthmatic patients with suspected bacterial respiratory infection. Erythromycin should be included as part of initial therapy if Mycoplasma pneumoniae, Chlamydia pneumoniae, or Legionella infection is suspected.

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SECTION E  Respiratory Tract

Asthma During Labor.  Although asthma exacerbations during labor are uncommon, patients should continue their medical therapy during labor. Patients experiencing some asthma symptoms during labor usually require no medication or are adequately controlled by inhaled β-agonists. If the patient’s asthma responds poorly to β-agonist therapy, methylprednisolone should be administered intravenously. Patients receiving regular OCS or who have received frequent courses of OCS during pregnancy should receive supplemental corticosteroids for the stress of labor, delivery, and the puerperium. Adrenal insufficiency has been reported only rarely in infants of mothers receiving corticosteroids during pregnancy.7 Consequently, although such infants should be carefully observed for any evidence of adrenal hypofunction, prophylactic treatment is not warranted. Obstetric Management of Asthmatic Women.  Joint clinical management of the pregnant asthmatic patient by a team of the allergist or pulmonary specialist and the obstetrician is essential. Because asthma may be associated with IUGR and preterm birth, it is important to establish pregnancy dating accurately by first-trimester ultrasound when possible.71 The obstetric management of the well-controlled patient with mild to moderate asthma is generally the same as that for nonasthmatic women.66 However, patients with poorly controlled asthma or asthma requiring frequent oral glucocorticoids must be carefully monitored for IUGR and preeclampsia. In addition, intensive fetal monitoring is essential during acute episodes. Many medications potentially used for obstetric indications should be avoided in patients with asthma, because these agents have been shown to trigger bronchospasm. These include nonselective β-adrenergic blockers, 15-methylprostaglandin F2α, transcervical or intraamniotic prostaglandin E2, methylergonovine or ergonovine, and nonsteroidal antiinflammatory drugs (NSAIDs) in aspirin-sensitive asthmatic patients.7 Use of PGE2 gel or suppositories80 or PGE1 tablets81 for cervical ripening or labor induction has not been reported to cause clinical exacerbations in asthmatic patients. Magnesium sulfate and calcium channel blockers, which have bronchodilator properties, should also be well tolerated in pregnant asthmatic subjects. As in nonasthmatic women, regional anesthesia is preferred to general anesthesia during delivery. If general anesthesia is required, however, the medications recommended because of their bronchodilating properties are preanesthetic use of atropine or glycopyrrolate, induction of anesthesia with ketamine, and low concentrations of halogenated anesthetics.7

RHINITIS Although precise prevalence estimates vary, rhinitis is one of the most common medical conditions reported during pregnancy.82 Rhinitis does not appear to directly affect the outcome of pregnancy. However, uncontrolled rhinitis can indirectly affect pregnancy by interfering with sleep and potentially causing snoring and obstructive sleep apnea, which may be associated with gestational hypertension and IUGR.83,84 In addition, uncontrolled rhinitis may exacerbate coexisting asthma. In a recent study of 218 pregnant women with asthma,85 the presence and severity of rhinitis was associated with the level of asthma control. Nasal symptoms may begin with pregnancy. Some women experience “vasomotor rhinitis of pregnancy” (pregnancy rhinitis), a syndrome of nasal congestion and vasomotor instability lasting at least 6 weeks without evidence of allergy or infection and limited to the gestational period. Symptoms tend to be most prominent in the second half of pregnancy and usually disappear within 2 weeks after delivery.86 Pregnancy-associated hormones, such as estrogen and progesterone, have direct and indirect effects on nasal blood flow and mucous glands,

but their role in causing or aggravating rhinitis during pregnancy has not been clearly established. Ellegard87 found elevated levels of serum placental growth hormone in patients with pregnancy rhinitis versus those without pregnancy rhinitis but found no differences between groups in serum levels of estradiol, progesterone, or insulin-like growth factor. Smoking88 and increased BMI89 have been associated with an increased risk of nasal congestion during pregnancy. The incidence of pregnancy rhinitis has been variable in the primarily small series reported,90 but the prevalence was reported to be 22% in the largest series reported, which was a cross-sectional questionnaire survey study of 599 participants.91 Another important cause of gestational nasal symptoms is preexisting rhinitis. As with asthma, chronic rhinitis may improve, worsen, or remain unchanged during pregnancy. The authors have tabulated their experience with preexisting rhinitis in 348 pregnant asthmatic patients, approximately 90% of whom were atopic on skin testing.92 Nasal symptoms worsened during pregnancy in 34% of these women, improved in 15%, and remained unchanged in 45%, and the course could not be evaluated in 6% of the women. The course of gestational rhinitis appears to parallel the course of asthma during pregnancy in patients with both conditions.85,93 The most common causes of nasal symptoms necessitating treatment during pregnancy are allergic rhinitis, rhinitis medicamentosa, sinusitis, and vasomotor (or pregnancy) rhinitis.92 Allergic rhinitis is often preexisting but may occur or be recognized for the first time during pregnancy. Rhinitis medicamentosa from increased use of nasal decongestant sprays during pregnancy and sinusitis may complicate viral respiratory infections or another cause of chronic rhinitis. Vasomotor rhinitis (of pregnancy) can be defined as previously, but other causes of rhinitis must be excluded.

Therapy Appropriate therapy of rhinitis during pregnancy depends on establishing the cause of the rhinitis.94 The correct diagnostic classification can usually be established based on the history; physical examination of the eyes, ears, nose, throat, and sinuses; and nasal cytologic features (see Chapter 40). Many women can tolerate their nasal symptoms during pregnancy with little or no pharmacologic therapy. Although this is desirable, especially during the first trimester, bothersome symptoms warrant treatment. For intermittent substantial nasal obstruction, oxymetazoline drops or spray, used at the minimum dose necessary (and no more than 2 sprays in each nostril twice daily) may suffice. The patient should be clearly informed of the potential for development of rebound congestion and rhinitis medicamentosa. For more continuous nasal obstruction or for intermittent obstruction inadequately controlled by topical therapy, pseudoephedrine may be used, with attention paid to possible side effects of increased blood pressure, palpitations, tremor, and sleep disturbance. In addition, some human studies suggest increased risk for certain relatively rare congenital anomalies with first-trimester use of pseudoephedrine; therefore, if used, pseudoephedrine should be given after the first trimester if possible. For patients with allergic rhinitis, intranasal cromolyn may be considered first-line, because it is generally regarded as safe. For patients inadequately controlled by intranasal cromolyn, antihistamine therapy may be useful. Chlorpheniramine, cetirizine, and loratadine have reassuring animal studies as well as reassuring human data with a substantial number of exposures (Table 55.1). Evidence also suggests that intranasal lavage with hypertonic saline solution three times daily over the 6 weeks of pollen season reduced symptoms in pregnant women with seasonal allergic rhinitis.95 Because there are no human pregnancy data for intranasal antihistamines, they would not generally be recommended for

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy use during pregnancy except if uniquely effective for an individual patient. A generally more effective alternative for the treatment of eosinophilic (allergic or nonallergic) rhinitis is intranasal corticosteroid (INCS) therapy. Based on a recent study of the gestational use of INCS96 and the data for ICS, budesonide, fluticasone, or mometasone could be recommended as the INCS of choice during pregnancy.97 OCS may rarely be considered for patients with severe eosinophilic rhinitis not responding to antihistamine-decongestants and INCS, ideally after the first trimester. Most patients with nasal polyps require INCS during pregnancy. For patients with severe nasal polyps inadequately controlled by both INCS and treatment of secondary or complicating infection, OCS therapy is recommended. Several nonpharmacologic approaches may be useful for patients with substantial vasomotor rhinitis during pregnancy.90 Exercise may also be useful, because exercise leads to physiologic nasal vasoconstriction. Raising the head of the bed to an angle of 30 to 45 degrees can help nocturnal nasal congestion. Nasal saline irrigation may be helpful for pregnancy rhinitis90 and allergic rhinitis.98 Finally, an external nasal dilator has been effective for patients with pregnancy-related nocturnal nasal congestion.99

SINUSITIS In one study, the reported 1.5% incidence of antral lavage–proven sinusitis during pregnancy was estimated to be six times greater than in the general population.100 However, in a more recent large retrospective database study,101 women were less likely to receive antibiotics for upper respiratory infection in general or sinusitis specifically during pregnancy compared with the year before or after pregnancy. Maternal asthma, rhinitis, and sinusitis appeared to be risk factors for needing antibiotics during pregnancy.101 Physicians need to establish the diagnosis of bacterial sinusitis during pregnancy as accurately as possible. In establishing the diagnosis of sinusitis during pregnancy, more reliance is put on symptoms, because x-ray exposure is usually avoided. Symptoms of 7 or more days’ duration after an upper respiratory infection suggest the diagnosis. The symptoms most specifically associated with sinusitis are purulent secretions, cough, and pain localized to the upper teeth. Although these symptoms can be specific, they are not very sensitive. Periorbital, temporal, occipital, or frontal head pain also has a very low correlation to bacterial sinusitis. Sinus radiographs or limited screening computed tomography (CT) scan with appropriate shielding may be used during pregnancy when indicated, particularly when a clinically diagnosed sinus infection is not responding to antibiotic therapy or when a probable clinical diagnosis cannot be made without radiologic confirmation.

Treatment Patients with chronic sinusitis should continue saline lavage and INCS during pregnancy.102 Expansion of maternal extravascular volume, increased maternal glomerular filtration rate, and rapid passage of some antibiotics across the placenta may all contribute to lower serum levels of certain antibiotics during pregnancy. Thus higher doses of amoxicillin, erythromycin, and cephalosporins than normally prescribed may be needed during pregnancy.103 The most common sinus pathogens are Haemophilus influenzae and Streptococcus pneumoniae. Amoxicillin, amoxicillin–clavulanic acid, or a second- or third-generation cephalosporin is considered first-line treatment in the management of sinusitis in pregnant women who are not allergic to penicillin. Macrolides, particularly azithromycin, are recommended in the true penicillin-allergic patient.28 In penicillin-allergic patients, a

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course of trimethoprim-sulfamethoxazole also may be considered after the first trimester and before 32 weeks’ gestation.103 Tetracyclines and fluoroquinolones are not recommended for use during pregnancy.103 Three weeks of therapy may be appropriate for acute sinusitis during pregnancy to prevent development of recurrent sinusitis, especially in patients with chronic sinusitis. Systemic corticosteroids may be necessary for severe disease102 to reduce inflammation and promote drainage. If improvement does not ensue, sinus imaging should be obtained, and specific sinus irrigation may be necessary as both a diagnostic and a therapeutic modality.

ANAPHYLAXIS A statewide determination in Texas of the frequency of anaphylaxis based on hospital discharge diagnosis noted 19 cases of anaphylaxis in pregnant women, with a prevalence of 2.7 (95% CI 1.7-4.2) cases per 100,000 deliveries.104 Antibiotics were the most common cause, with six cases from cephalosporin, five from penicillin, and two from other antibiotics. The other causes were oxytocic agents (n = 2), and one each for antiemetic, antihypertensive, contrast, and antirheumatic agents. The fetus appears to be relatively protected from anaphylaxis, perhaps because the placenta does not transmit specific IgE antibodies.105 However, maternal hypoxia or hypotension associated with anaphylaxis may be catastrophic, not only to the mother but also to her fetus. Maternal anaphylaxis has been associated with fetal distress manifested by repetitive late decelerations in the fetal heart rate and multicystic fetal or infantile encephalomalacia.106 Maternal anaphylaxis and the associated fetal distress may resolve totally with prompt and aggressive medical management, without maternal or fetal compromise. Alternatively, fetal or neonatal death may occur despite maternal survival, presumably because of diminished uteroplacental perfusion as a result of the rapidity and severity of the episode or inadequate treatment.106 A review of anaphylaxis during pregnancy reported that most cases occur after the twentieth week of pregnancy, with etiologies in descending order of frequency: antibiotics (typically β-lactams), latex (which may be an added risk during pregnancy),107 succinylcholine, Laminaria, and insect stings.108 Deaths are rare in pregnancy but reported.109 A recent report of death occurred because of administration intravenously of iron sucrose for severe refractory iron-deficiency anemia.110 Lethal anaphylaxis during pregnancy was also reported to be caused by a compound amino acid solution used for nutritional support.111 Maternal anaphylaxis has been reported as a rare cause of neonatal death.105 As noted previously, Laminaria, a member of the kelp family used as a stick in obstetrics for cervical dilatation in labor induction, has been associated with anaphylaxis.112 Buccal misoprostol used for labor induction also can cause anaphylaxis.113 In addition, intracervical dinoprostone gel for preinduction114 and anti-D immunoglobulin used to prevent Rh isoimmunization115 may also cause anaphylaxis. Food-induced anaphylaxis to the common implicated foods occurs during pregnancy. Recently an unusual food culprit was associated with anaphylaxis in four pregnant women because of galacto-oligosaccharides used as a prebiotic.116 Any agent that can cause anaphylaxis in the nonpregnant state could lead to anaphylaxis in the susceptible or sensitized pregnant patient.117 Even breastfeeding has been associated with anaphylaxis in two postpartum women. The reactions occurred within the first to third day postpartum and resolved within 1 to 2 days. Changing levels of progesterone and triggering by NSAIDs were suggested as potential causes.117 Hyperfibrinolysis appears associated with anaphylaxis during pregnancy.118 Attention should be directed to the potential for anaphylaxis during labor secondary to antibiotic prophylaxis used against group B streptococci, particularly penicillin, given the beneficial effect of such

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SECTION E  Respiratory Tract

TABLE 55.6  Etiology of Anaphylaxis

During Pregnancy

First 3 trimesters, before labor and delivery Foods Stinging insect venoms Medicationsa Biologic agents, including allergen immunotherapy Natural rubber latex Otherb Labor and delivery Antibioticsc Natural rubber latex Neuromuscular blockers Oxytocin Local anesthetics Transfusion of blood or blood products a

Most commonly, antibiotics and nonsteroidal antiinflammatory medications; other medications, such as intravenous iron and intravenous vitamins B1, B6, and B12, which are used for the treatment of hyperemesis gravidarum in some countries. b Occupational allergens, including stinging insect venoms and natural rubber latex, aeroallergens (rare), radiocontrast media, and exercise. Idiopathic anaphylaxis also occurs. c Most commonly penicillin and other β-lactam antibiotics injected intrapartum for prevention of neonatal group B streptococcus infection or prevention of maternal infection after cesarean delivery. From Simon FER and Schatz M. Anaphylaxis during pregnancy. J Allergy Clinical Immunol 2012;130:597-606.

prophylaxis and the increased implementation of this recommended prophylaxis.117 Anaphylaxis was also reported during labor in a penicillinallergic parturient who received IV cefazolin chemoprophylaxis for perinatal group B streptococcal disease.119 Skin testing with penicillin major and minor determinants has been reported to be useful in identifying pregnant patients with prior histories of adverse reactions to penicillin who could safely receive penicillin during delivery for group B streptococcal prophylaxis.120 Table 55.6 provides a summary of the etiologies of anaphylaxis during the various stages of pregnancy.117

Differential Diagnosis Laryngeal obstruction, a frequent and potentially life-threatening symptom of anaphylaxis, must be differentiated from the laryngeal edema of preeclampsia and that caused by laryngopathia gravidarum and hereditary angioedema (HAE). Signs in preeclampsia, including hypertension, peripheral edema, and urinary abnormalities, as well as the prior diagnosis of preeclampsia, should prevent confusion. In laryngopathia gravidarum, laryngeal symptoms are usually less sudden, and the acute form occurs just before parturition.121 In HAE, laryngeal stridor is slower in onset, abdominal pain may be prominent, hypotension and urticaria are absent, and a family history of HAE can typically be obtained (see later). Hypotension may be a prominent feature of severe anaphylaxis and must be differentiated from other causes of hypotension during pregnancy. The coexistence of pruritus, urticaria, angioedema, or wheezing supports a diagnosis of anaphylaxis but may be absent in some cases. An elevated serum tryptase confirms the existence of mast cell activation and thus supports a diagnosis of anaphylaxis. One important alternative cause of acute cardiovascular collapse during pregnancy is amniotic fluid embolism (anaphylactoid syndrome of pregnancy). This has been estimated to occur in 1 in 15,200 and 1

in 53,800 deliveries in North America and Europe, respectively, with a case-fatality rate between 13% to 30% and 9% to 44%, respectively, and accounting for 5% to 15% of all maternal deaths in developed countries.122 Risk factors include advanced maternal age, placental abnormalities, surgical delivery, eclampsia, polyhydramnios, cervical laceration, and uterine rupture.122 Suggested criteria for amniotic fluid embolism include: 1) one or more of the clinical findings of hypotension, cardiac arrest, respiratory distress, or disseminated intravascular coagulation during pregnancy, and 2) absence of other medical explanations for the clinical course. A proposed mechanism of this syndrome is a non-IgE mediated immunologic reaction to fetal antigens leading to mast cell degranulation and release of histamine and tryptase.117 Alternatively, complement activation rather than mast cell degranulation may be a mechanism.123 Elevated tryptase levels as a marker of mast cell degranulation occur during amniotic fluid embolism–induced anaphylactoid syndrome.124 Differentiating characteristics, such as the presence of bronchospasm and absence of either large blood loss or coagulopathy, would suggest anaphylaxis rather than amniotic fluid embolism. Sialyl-Tn antigen has been proposed as a marker for severe amniotic fluid embolism but appears to be more predictive for lifethreatening cases or fatalities than for milder episodes.125

Prevention All venom-sensitive pregnant women with a history of Hymenoptera sting anaphylaxis should be reinstructed in insect avoidance measures and receive a prescription for an epinephrine autoinjector. Although only preliminary data are available supporting the safety of venom immunotherapy during pregnancy,6 benefit/risk considerations indicate that pregnant women receiving maintenance venom immunotherapy before pregnancy should continue such treatment during pregnancy. Pregnant women with histories suggestive of Hymenoptera sting anaphylaxis who have not been previously skin-tested should receive avoidance instructions and an emergency kit, but the authors recommend deferring skin testing until postpartum. If identification of venom-specific IgE is necessary, an in vitro assay may be obtained. Also, benefit/risk considerations do not appear to favor beginning venom immunotherapy during pregnancy in most women. Venom immunotherapy during pregnancy was felt contraindicated to start by 71% of members of the American Academy of Allergy Asthma and Immunology; however, 51% were comfortable with continuing the treatment.126 Only 3% of 520 members of the European Academy of Allergy and Clinical Immunology reported starting any type of allergen immunotherapy during pregnancy.127 A recent report described a successful systematic approach to protect against exercise-induced anaphylaxis during labor in an at-risk patient.128 Prepregnancy aspirin desensitization to allow preconceptional antiaggregation in a woman with aspirin sensitivity and inherited thrombophilia and recurrent miscarriage led to an uncomplicated birth.129 Pregnant women with systemic mastocytosis, being at increased risk for anaphylaxis, need to adhere to preventive medications, as noted previously in the urticaria/angioedema section.

Treatment With a few modifications, management of anaphylaxis during pregnancy is the same as described in Chapter 75. However, the literature on the treatment of anaphylaxis during pregnancy is replete with evidence of delay in appropriate treatment. The maternal-fetal morbidity and mortality caused by anaphylaxis would likely be reduced by more prompt and appropriate therapy. Recent obstetric reviews on the treatment of anaphylaxis during labor and cesarean section emphasized the importance of immediate recognition, action, and appropriate treatment.130,131

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy

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Of the routine antianaphylaxis medications, epinephrine and diphenhydramine have been implicated in some studies as causing increased fetal malformations (Tables 55.1 and 55.2). However, the tentative nature of these data and the lack of equally effective substitutes suggest that these medications should be used during pregnancy for this life-threatening emergency. In addition, evidence supportive of the safety of epinephrine for anaphylaxis during pregnancy was shown in its successful continuous use intravenously for 3.5 hours in treating refractory hypotension in a woman with anaphylaxis during labor.132 Because of the altered circulatory and respiratory physiologic characteristics existing during pregnancy, adequate intravascular volume repletion and oxygenation are particularly important in the management of anaphylaxis during pregnancy to prevent both maternal and fetal complications. In treating hypotension during pregnancy, a minimum maternal systolic blood pressure of 90 mm Hg should be maintained to ensure adequate placental perfusion. Impending or existing hypotension must be promptly reversed with IV fluids, occasionally up to 5 to 10 L. The pregnant hypotensive patient should be placed on her left side to prevent added positional hypotension from compression of the inferior vena cava by the gravid uterus.106 As previously noted, IV epinephrine may be required and indicated to treat refractory anaphylaxis, despite its potential to cause decreased uteroplacental blood flow.106 Glucocorticoids should be administered early to patients with severe anaphylaxis (see Chapter 75). Laryngeal spasm or edema unresponsive to medical management may require intubation and in, rare cases, tracheostomy.

induced by trauma, infection, or intubation. Diagnosis is made by screening for a low C4 level and then confirming with a test for functional C1 inhibitor. HAE flares during pregnancy in about half of women.143 HAE episodes in the third trimester are more common if the fetus is affected by HAE.143,144 The trauma of vaginal delivery alone rarely induces a severe attack.140 Regional anesthesia is preferred to general anesthesia with intubation in surgical deliveries. Attacks are more common in the postpartum period.145 Management of HAE before and during pregnancy relies on C1 inhibitor concentrates, although frequent treatment appears to increase the number of attacks.146 Danazol has the potential for fetal damage, including female masculinization and pseudohermaphroditism as well as spontaneous abortion, and thus attenuated androgens are considered contraindicated in pregnancy.147 Danazol may still be used for HAE symptom prevention in nonpregnant women but should be stopped at least 8 weeks before planned conception. Pregnancy termination in patients with HAE has been managed with danazol. No evidence indicates that epinephrine is useful for the treatment of acute attacks of HAE. There are no published data on use of the recombinant kallikrein inhibitor ecallantide, or the synthetic bradykinin B2 receptor antagonist icatibant, in the setting of pregnancy.139 A rare estrogen-dependent HAE-like syndrome called type III HAE exists, with symptoms occurring only with exogenous estrogen use or pregnancy.148 It has no effective prophylactic therapy and must be managed with supportive treatment only.

URTICARIA AND ANGIOEDEMA

ATOPIC DERMATITIS AND OTHER DERMATOSES

Acute urticaria with or without angioedema can occur during pregnancy from any of the causes defined in the nonpregnant state (see Chapter 35). The first therapeutic goal is the identification and avoidance of triggering factors, if possible, though most cases remain idiopathic and resolve within 6 weeks.133 When symptomatic therapy is required, loratadine or cetirizine is recommended for mild cases. For cases uncontrolled with the nonsedating antihistamines, chlorpheniramine is recommended, and hydroxyzine if this is not effective. Systemic corticosteroids are not recommended for the treatment of acute urticaria because the risks outweigh the benefits.134 Chronic urticaria, lasting more than 6 weeks, is typically not exacerbated by pregnancy. Antihistamines remain the mainstay of treatment. The use of systemic corticosteroids is not recommended for chronic urticaria. Omalizumab may also be used in the setting of pregnancy.135

Atopic dermatitis (AD) is the most common dermatosis during pregnancy, accounting for about one-third to one-half of cases149 and occurring in 49.7% of 505 retrospectively studied pregnant women with dermatosis.150 Between 60% and 80% of these cases appear to have onset during pregnancy.150 In one report cesarean section was associated with increased frequency of AD in Korean adolescents.151 The course is variable; one study reported that AD worsened in 52%, improved in 24%, and remained unchanged in 24% of 88 full-term pregnancies of 50 women.152 Most women who reported worsening experienced exacerbations of AD by 20 weeks gestation. Up to 10% worsen postpartum.149 In another study, 14 of 23 (61.0%) of women with AD reported deterioration during pregnancy.153 No data exist relative to the effect of AD on fertility or miscarriage rates.154 Prompt treatment of secondary eczema herpeticum with acyclovir or similar antivirals is important to prevent potential complications associated with herpes simplex infections during pregnancy.155 Advice on management of AD before planned conception should include avoiding potent topical corticosteroids and discontinuing methotrexate for both mother and father for at least 3 months before pregnancy and PUVA (psoralen + ultraviolet light).149 Treatment of AD during pregnancy should emphasize avoidance of triggering factors and reliance on topical treatment such as emollients to reduce dryness and pruritus, modulate inflammation, and treat secondary infections (see Chapter 33).154 Treatment of pruritus associated with AD may require oral antihistamines. Oral antihistamines should be avoided, especially in the first trimester, unless definitely indicated, then used at the lowest effective dose, starting with chlorpheniramine, loratadine, or cetirizine. Hydroxyzine is often used to treat itching in nonpregnant patients with AD, and two small cohort studies failed to show an association between hydroxyzine exposure during the first trimester of pregnancy and birth defects, whereas another one showed a slightly increased risk (Table 55.1). Cautious use of oral antihistamines during the last trimester of pregnancy is advised to avoid withdrawal symptoms and retrolental fibroplasia of the newborn, particularly in the premature.154

Mastocytosis Women with mastocytosis typically deliver healthy infants.136 A rare infant will develop cutaneous mastocytosis several years later. Onequarter of women with mastocytosis may have an exacerbation of their chronic symptoms during pregnancy, and one-third may show improvement.137 Treatment during pregnancy is symptom-driven, using primarily antihistamines and systemic corticosteroids.138

Hereditary Angioedema Hereditary angioedema is a rare disorder resulting from a functional C1 inhibitor deficiency (see Chapter 36).139 Several genetic defects can lead to the same clinical syndrome. Sex hormones worsen symptoms in HAE. The effect seems to be mediated primarily by estrogens; thus, combined oral contraception is contraindicated in women with HAE.140 Women with higher progesterone levels also have more attacks, and those with higher sex hormone–binding globulin have a lower attack frequency.141 HAE may affect women during pregnancy who are unaware they have HAE.142 Clinical symptoms include painful bowel swelling that can mimic an acute abdomen and life-threatening laryngeal edema

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Topical corticosteroids should be selected based on potency considerations, given the potential for systemic absorption and increased skin surface area of pregnancy. Severe IUGR occurred in the infant of a mother who applied 40 mg/day of topical triamcinolone cream from 12 to 29 weeks’ gestation to treat her AD.156 Topical corticosteroid treatment should be initiated, when clinically indicated, with preparations such as hydrocortisone (0.5% to 2.5%) that have the lowest potential for adrenal suppression, reserving more potent preparations for more recalcitrant areas in select patients.157 An evidence-based review concluded that “the best evidence suggests that mild/moderate topical corticosteroids are preferred to potent/very potent ones in pregnancy, because of the associated risk of fetal growth restriction with the latter.”158 Maternal exposure to topical corticosteroids is not associated with orofacial cleft, preterm delivery, or fetal death.159 OCS use after the first trimester may be considered cautiously in patients with severe dermatitis who have failed to respond to topical and other therapies (e.g., appropriately treated infections). With no adequate and well-controlled studies of topical tacrolimus or picrolimus in pregnant women, their safety in treating AD in pregnancy cannot be assessed at this time. Given the potential for increased preterm birth with systemic use of tacrolimus, topical calcineurin inhibitor use if required in severe uncontrolled AD should be restricted to localized areas only.154 Systemic immunosuppressants, such as cyclosporine, may be associated with prematurity and low birth weight, but apparently not birth defects,160 and could be considered as a last resort for severe recalcitrant AD during pregnancy.154 Methotrexate, mycophenolate mofetil, and psoralen and UVA light (PUVA) should be avoided because of reported mutagenesis and teratogenesis.154 The biologic dupilumab, which binds to the alpha subunit of the interleukin-4 receptor to modulate both the IL-4 and IL-13 pathways, is effective in moderate to severe AD (see Chapter 33). However, although animal studies are reassuring, dupilumab use in human pregnancy has not been studied. Controlling secondary bacterial and viral infections in atopic dermatitis is paramount. It is recommended to culture skin lesions suspected of infection. Staphylococcus aureus is the major culprit for infection during pregnancy, as it is during the nonpregnant state. Given their safety during pregnancy, penicillins and cephalosporins are recommended for infections with nonresistant organisms. Erythromycins may be used for those with penicillin allergy or for methicillin-resistant organisms.154 Eczema herpeticum occurs during pregnancy155 and should be considered when vesicles, scalloped erosions, or crusts appear.154 Use of acyclovir is recommended for this complication during pregnancy.154

Other Dermatoses of Pregnancy About 1.5% of pregnant women will develop a new pruritic condition associated with pregnancy.161 Pruritic urticarial papules and plaques of pregnancy (PUPPP) is the most common specific dermatosis of pregnancy and is characterized by urticarial papules and extreme itching. PUPPP typically starts abruptly on the lower abdomen within striae during the last month of pregnancy. In a minority of patients, PUPPP starts postpartum. It occurs in about 1 in 160 pregnancies but is much more common in twin/multiple pregnancies and seems to be associated with increased weight gain.162 PUPPP generally lasts about 1 month and is treated symptomatically with antihistamines and topical corticosteroids. Discussion of other specific dermatoses of pregnancy are beyond the scope of this chapter, are best treated by dermatologists, and are reviewed elsewhere.163–166

DRUG HYPERSENSITIVITY The management of drug hypersensitivity during pregnancy is very much like the management of drug hypersensitivity in any patient.

Most reported drug allergies in the electronic health record (EHR) are not drug hypersensitivities and are not reproducible upon rechallenge.167 Multiple (more than two) nonrelated drug allergies in a single patient does not increase the probability of any of them being immunologically mediated.168 Mild reactions do not always progress to more severe reactions.169 Many cases of anaphylaxis have no history of any previous hypersensitivity.170 True anaphylaxis and severe cutaneous adverse reactions (SCARs) are extremely rare and frequently overdiagnosed.142,171 Reactions occur more commonly with parenteral compared with oral drug administrations.172 Reactions occur with all drug exposures at predictable rates.173 There is no such thing as a risk-free drug exposure. Reactions to drugs will still occur at predictable rates after all negative tests and challenges.174 When evaluating pregnant women with drug allergies it is important to try to determine the potential mechanism(s) of the historical reaction, whether the drug is really needed, whether an alternative is safer than reexposure, and whether avoidance is more dangerous than testing and/or rechallenge. Whenever possible, medication avoidance is preferred during pregnancy. However, if a life-saving medication is needed during pregnancy that has been associated with a hypersensitivity reaction, many pregnant patients can still be safely tested for tolerance, and, if confirmed to be acutely hypersensitive, treated via desensitization. All drug hypersensitivity that can be managed by desensitization involves mast cell activation. Most clinically significant T cell–mediated hypersensitivity requires complete avoidance, as desensitization is not possible for T cell–mediated reactions. Needless avoidance of essential medications, such as penicillin in the setting of group B streptococcus (GBS) colonization or a syphilis infection, can cause increased morbidity. Antibiotic, anticoagulant, insulin/other hormone, latex, local anesthetic, and other protein hypersensitivity merit special comment in the settings of conception, pregnancy management, parturition, and assisted-reproduction technology. Ohel and coworkers from Israel in 2010 reported on a cohort of 186,443 deliveries, and noted that 8647 (4.6%) of the mothers reported at least one drug allergy.175 As expected, having any drug allergy was associated with advanced maternal age and age-related comorbidities including recurrent abortions, fertility treatments, hypertension, and diabetes. In their multivariable analysis they noted that any drug allergy, and specifically penicillin allergy, was associated with increased risks of IUGR and preterm deliveries. Desai and coworkers in 2017 reported on a cohort of pregnant women cared for by Kaiser Permanente in Southern California, representing about 1% of the U.S. population over a 6-year period.176 They noted about 70 total reported drug allergies for every 100 pregnant women, and that about 9% carried a specific penicillin allergy label. Penicillins and cephalosporins are first-line antibiotics during pregnancy.177,178 All pregnant women with an unconfirmed penicillin allergy should consider undergoing penicillin allergy testing, particularly if GBS positive or anticipating a cesarean section. Cesarean section– associated surgical infections are more common if beta-lactam antibiotics are avoided. Penicillin remains the drug of choice for group B streptococcal therapy. Penicillin skin testing has also been shown to be safe in pregnant women with a history of penicillin allergy and positive group B streptococci cultures.179 Cephalosporins can be safely used in pregnant women with a low risk history of penicillin allergy in whom cephalosporins are indicated for group B streptococcal treatment.180,181 Reactions to heparins include IgE-mediated reactions, cell-mediated DTH, and heparin-induced thrombocytopenia. Most reactions to unfractionated heparin during pregnancy may be managed by using any of several low-molecular-weight heparins (LMWHs), including ardeparin, certoparin, dalteparin, enoxaparin, nadroparin, and reviparin.182 Risk factors for reactions to LMWH include previous use of LMWHs and inherited protein C deficiency. Fondaparinux has been used in pregnant

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy women with reactions to multiple LMWHs, although danaparoid is still considered the drug of choice for LMWH intolerance in pregnancy.183 Hirudin derivatives are not recommended. Human insulin allergy may occur extremely rarely in the setting of gestational diabetes and has been managed with continuous subcutaneous infusion.184 Although rare, anaphylactoid reactions to oxytocin have been reported.185 Latex allergy is still a concern but has been less of a problem over the past 2 decades with the avoidance of materials containing significant amounts of water-soluble natural rubber latex protein in the health care environment.186 If suggested by a history of urticaria or respiratory symptoms with exposure to latex-containing materials, IgE-mediated latex allergy should be confirmed by measuring anti–latex-specific IgE. The treatment is latex protein avoidance during labor and delivery. IgE-mediated reactions to local anesthetics are extremely rare, although reactions to methylparabens have been documented.187 Provocative dose testing is appropriate in pregnant patients with a history of adverse reactions in whom local anesthetic use is anticipated during pregnancy or delivery. Cases of seminal fluid protein allergy have been reported, some managed by desensitization.188 Cases resulting in infertility have been managed using intrauterine insemination with washed sperm.189

SUMMARY Pregnancy may modify the clinical manifestations of asthma and allergy, and asthma and anaphylaxis can affect the outcome of pregnancy. Asthma typically improves or worsens during pregnancy, and an increased risk of perinatal complications has been associated with more poorly controlled asthma during pregnancy. Anaphylaxis, drug reactions, and rhinitis may occur in pregnant women from the same causes as in nonpregnant patients, but these conditions may also be associated with unique pregnancy circumstances. Management of asthma, rhinitis, sinusitis, anaphylaxis, urticaria, angioedema, atopic dermatitis, and drug allergy in pregnant women is similar to such management in nonpregnant patients, although certain specific medications are preferred based on human gestational safety information.

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36. Murphy VE, Clifton VL, Gibson PG. Asthma exacerbations during pregnancy: incidence and association with adverse pregnancy outcomes. Thorax 2006;61:169–76. 37. Blais L, Forget A. Asthma exacerbations during the first trimester of pregnancy and the risk of congenital malformations among asthmatic women. J Allergy Clin Immunol 2008;121:1379–84. 38. Rejno G, Lundholm C, Gong T, et al. Asthma during pregnancy in a population-based study—Pregnancy complications and adverse perinatal outcomes. PLoS ONE 2014;9:e104755. 39. Firoozi F, Lemiere C, Ducharme FM, et al. Effect of maternal moderate to severe asthma on perinatal outcomes. Resp Med 2010;104:1278–87. 40. Namazy JA, Murphy VE, Powell H, et al. Effects of asthma severity, exacerbations and oral corticosteroids on perinatal outcomes. Eur Resp J 2013;41:1082–90. 41. Blais L, Kettani F-Z, Forget A, et al. Asthma exacerbations during the first trimester of pregnancy and congenital malformations: revisiting the association in a large representative cohort. Thorax 2015;70:647–52. 42. Holland SM, Thomson KD. Acute severe asthma presenting in late pregnancy. Int J Obstet Anesth 2006;15:75–8. 43. Bodker B, Hvidman L, Weber T, et al. Maternal deaths in Denmark 2002-2006. Acta Obstet Gynecol 2009;88:556–62. 44. Juniper EF, Newhouse MT. Effect of pregnancy on asthma: a systematic review and meta-analysis. In: Schatz M, Zeiger RS, Claman HN, editors. Asthma and immunologic diseases in pregnancy and early infancy. New York: Marcel Dekker; 1998. p. 401–27. 45. Gluck JC, Gluck PA. The effect of pregnancy on the course of asthma. Immunol Allergy Clin North Am 2006;26:63. 46. Schatz M, Dombrowski MP, Wise R, et al. Asthma morbidity during pregnancy can be predicted by severity classification. J Allergy Clin Immunol 2003;112:283–8. 47. Murphy VE, Gibson P, Talbot PI, et al. Severe asthma exacerbations during pregnancy. Obstet Gynecol 2005;106:1046–54. 48. Murphy V, Powell H, Wark PAB, et al. A prospective study of respiratory viral infection in pregnant women with and without asthma. Chest 2013;144:420–7. 49. Murphy VE, Clifton VL, Gibson PG. The effect of cigarette smoking on asthma control during exacerbations in pregnant women. Thorax 2010;65:739–44. 50. Murphy VE, Jensen ME, Powell HP, et al. Influence of maternal body mass index and macrophage activation on asthma exacerbations in pregnancy. J Allergy Clin Immunol Pract 2017;5(4):981–7.e1. 51. Ali Z, Nilas L, Ulrik CS. Excessive gestational weight gain in first trimester is a risk factor for exacerbation of asthma during pregnancy: a prospective study of 1283 pregnancies. J Allergy Clin Immunol 2018;141(2):761–7. 52. Powell H, McCaffery K, Murphy VE, et al. Psychosocial variables are related to future exacerbation risk and perinatal outcomes in pregnant women with asthma. J Asthma 2013;50:383–9. 53. Cydulka R, Emerman CL, Schreiber D, et al. Acute asthma among pregnant women presenting to the emergency department. Am J Respir Crit Care Med 1999;160:887–92. 54. McCallister JW, Benninger CG, Frey HA, et al. Pregnancy-related treatment disparities of acute asthma exacerbations in the emergency department. Resp Med 2011;105:1434–40. 55. Cossette B, Beauchesne M-F, Forget A, et al. Systemic corticosteroids for the treatment of asthma exacerbations during and outside of pregnancy in an acute-care setting. Res Med 2014;108:1260–7. 56. Dombrowski MP, Schatz M, Wise R, et al. Asthma during pregnancy. Obstet Gynecol 2004;103:5–12. 57. Bobrowski RA. Pulmonary physiology in pregnancy. Clin Obstet Gynecol 2010;53:285–300. 58. Bidad K, Heidarnazhad H, Pourpak Z, et al. Frequency of asthma as the cause of dyspnea in pregnancy. Int J Gynecol Obstet 2010;111:140–3. 59. Pfister R, Frank KF, Rosenkranz S, et al. Severe dyspnea during late pregnancy in a woman with a history of asthma. Clin Res Cardiol 2011;100:1119–21. 60. Murphy VE, Gibson PG, Talbot PI, et al. Asthma self-management skills and the use of asthma education during pregnancy. Eur Resp J 2006;26:435–41.

61. Sawicki E, Stewart K, Wong S, et al. Medication for chronic health conditions by pregnant women attending an Australian maternity hospital. Aust NZ J Obstet Gynecol 2011;51:333–8. 62. Veras de Araujo G, Leite DFB, Rizzo JA, et al. Asthma in pregnancy: association between the Asthma Control Test and the Global Initiative for Asthma classification and comparisons with spirometry. Eur J Obstet Gynecol Reprod Biol 2016;203:25–9. 63. Palmsten K, Schatz M, Chen PH, et al. Validation of the Pregnancy Asthma Control Test. J Allergy Clin Immunol Pract 2016;4:310–15. 64. Powell H, Murphy VE, Taylor DR, et al. Management of asthma in pregnancy guided by measurement of fraction of exhaled nitric oxide: a double-blind, randomized controlled trial. Lancet 2011;378:983–90. 65. Nittner-Maeszalska M, Liebhart J, Pawlowicz R, et al. Fractioned exhaled nitric oxide (FeNO) is not a sufficiently reliable test for monitoring asthma during pregnancy. Nitric Oxide 2013;33:56–63. 66. Schatz M, Dombrowski MP. Asthma in pregnancy. N Engl J Med 2009;360:1862–9. 67. Cossette B, Beauchesne M-F, Forget A, et al. Relative perinatal safety of salmeterol vs formoterol and fluticasone vs budesonide use during pregnancy. Ann Allergy Asthma Immunol 2014;112:459–64. 68. Charlton RA, Snowball JM, Nightingale AL, et al. Safety of fluticasone proprionate prescribed for asthma during pregnancy: a UK population-based cohort study. J Allergy Clin Immunol Pract 2015;3:772–9. 69. Cossette B, Forget A, Beauchesne M-F, et al. Impact of maternal use of asthma-controller therapy on perinatal outcomes. Thorax 2013;68:724–30. 70. Eltonsy S, Forget A, Beauchesne M-F, et al. Risk of congenital malformations for asthmatic pregnant women using a long-acting β2-agonist and inhaled corticosteroid combination versus higher-dose inhaled cort8icosteroid monotherapy. J Allergy Clin Immunol 2015;135:123–30. 71. National Asthma Education and Prevention Program Working Group on Managing Asthma during Pregnancy. Recommendations for pharmacologic treatment: update 2004. NIH Pub No 05-3279. US Department of Health and Human Services, National Institutes of Health, National Heart, Lung and Blood Institute; 2005. 72. Tan H, Sarwate C, Singer J, et al. Impact of controller medications on clinical, economic, and patient-reported outcomes. Mayo Clin Proc 2009;84:575–84. 73. Price D, Musgrave SD, Shepstone L, et al. Leukotriene anatagonists as first-line or add-on asthma controller therapy. N Engl J Med 2011;364:1695–707. 74. Cavero-Carbonell C, Vinkel-Hansen A, Rabanque-Hernández MJ, et al. Fetal exposure to montelukast and congenital anomalies: a population-based study in Denmark. Birth Defects Res 2017;109: 452–9. 75. Body C, Christie JA. Gastrointestinal diseases in pregnancy. Gastoetnerol Clin N Am 2016;45:267–83. 76. Pasternak B, Huiid A. Use of protein-pump inhibitors in early pregnancy and the risk of birth defects. N Engl J Med 2010;363: 2114–23. 77. National Asthma Education and Prevention Program Expert Panel Report 3. Guidelines for the diagnosis and management of asthma: summary report 2007. J Allergy Clin Immunol 2007;120:S93–138. 78. Schwaiberger D, Karcz M, Menk M, et al. Respiratory failure and mechanical ventilation in the pregnant patient. Crit Care Clin 2016;32:85–95. 79. Lapinsky SE. Management of acute respiratory failure during pregnancy. Semin Resp Crit Care Med 2017;38:201–7. 80. Towers CV, Briggs GG, Rojas JA. The use of prostaglandin E2 in pregnant patients with asthma. Am J Obstet Gynecol 2004;190:1777–80. 81. Thompson MR, Towers CV, Howard BC, et al. The use of prostaglandin E1 in peripartum patients with asthma. Am J Obstet Gynecol 2015;212(392):e1–3. 82. Law A, McCoy M, Lynen R, et al. The additional cost burden of preexisting medical conditions during pregnancy and childbirth. J Womens Health 2015;24:924–32.

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy 83. Ayrim A, Keskin EA, Ozol D, et al. Influence of self-reported snoring and witnessed sleep apnea on gestational hypertension and fetal outcome in pregnancy. Arch Gynecol Obstet 2011;283:195–9. 84. Franklin KA, Holmgren PA, Jonsson F, et al. Snoring, pregnancy-induced hypertension, and growth retardation of the fetus. Chest 2000;117:137–41. 85. Powell H, Murphy VE, Hensley MJ, et al. Rhinitis in pregnant women with asthma is associated with poorer asthma control and quality of life. J Asthma 2015;52:1023–30. 86. Ellegard E. Clinical and pathogenetic characteristics of pregnancy rhinitis. Clin Rev Allergy Immunol 2004;26:149–59. 87. Ellegard E, Oscarsson J, Bougoussa M, et al. Serum level of placental growth hormone is raised in pregnancy rhinitis. Arch Otolaryngol Head Neck Surg 1998;124:439–43. 88. Ellegard E, Karlson G. IgE-mediated reactions and hyperreactivity in pregnancy rhinitis. Arch Otolaryngol Head Neck Surg 1999;125:1121–5. 89. Ulkumen B, Ulkumen BA, Pala HG, et al. Pregnancy rhinitis in Turkish women: do gestational week, BMI and parity affect nasal congestion. Pak J Med Sci 2016;32:950–4. 90. Orban N, Maughan E, Bleach N. Pregnancy-induced rhinitis. Rhinology 2013;51:111–19. 91. Ellegard E, Hellgren M, Toren K, et al. The incidence of pregnancy rhinitis. Gyncol Obstet Invest 2000;49:98–101. 92. Schatz M, Zeiger RS. Diagnosis and management of rhinitis during pregnancy. Allergy Proc 1988;9:545–54. 93. Kircher S, Schatz M, Long L. Variables affecting asthma course during pregnancy. Ann Allergy Asthma Immunol 2002;89:463–6. 94. Incaudo GA, Takach P. The diagnosis and treatment of allergic rhinitis during pregnancy and lactation. Immunol Allergy Clin North Am 2006;26:137–54. 95. Garavello W, Somigliana E, Acaia B. Nasal lavage in pregnant women with seasonal allergic rhinitis: a randomized study. Int Arch Allergy Immunol 2010;151:137–41. 96. Berard A, Sheehy O, Kurzinger M-L, et al. Intranasal triamcinolone use during pregnancy and the risk of adverse pregnancy outcomes. J Allergy Clin Immunol 2016;138:97–104. 97. Namazy JA, Schatz M. The safety of intranasal steroids during pregnancy: a good start. J Allergy Clin Immunol 2016;138:105–6. 98. Garavello W, Somigliana E, Acaia B, et al. Nasal lavage in pregnant women with seasonal allergic rhinitis: a randomized study. Int Arch Allergy Immunol 2010;151:137–41. 99. Turnbull GL, Rundell OH, Rayburn WF, et al. Managing pregnancy-related nocturnal nasal congestion. The external nasal dilator. J Reprod Med 1996;41:897–902. 100. Sorri M, Hartikainen-Sorri A-L, Karja J. Rhinitis during pregnancy. Rhinology 1980;18:83–6. 101. Namazy JA, Schatz M, Yang S-J, et al. Antibiotics for respiratory infections during pregnancy: prevalence and risk factors. J Allergy Clin Immunol Pract 2016;4:1256–7. 102. Lai D, Jategaonkar AA, Borish L, et al. Management of rhinosinusitis during pregnancy: systematic review and expert panel recommendations. Rhinology 2016;54:99–104. 103. Bookstaver PB, Bland CM, Griffin B, et al. A review of antibiotic use during pregnancy. Pharmacotherapy 2015;35:1052–62. 104. Mulla ZD, Ebrahim MS, Gonzalez JL. Anaphylaxis in the obstetric patient: analysis of a statewide hospital discharge database. Ann Allergy Asthma Immunol 2010;104:55–9. 105. Berenguer A, Couto A, Brites V, et al. Anaphylaxis in pregnancy: a rare cause of neonatal mortality. BMJ Case Rep 2013;2013. 106. Chaudhuri K, Gonzales J, Jesurun CA, et al. Anaphylactic shock in pregnancy: a case study and review of the literature. Int J Obstet Anesth 2008;17(4):350–7. 107. Draisci G, Zanfini BA, Nucera E, et al. Latex sensitization: a special risk for the obstetric population? Anesthesiology 2011;114(3):565–9. 108. Chaudhuri K, Gonzales J, Jesurun CA, et al. Anaphylactic shock in pregnancy: a case study and review of the literature. Int J Obstet Anesth 2008;17:350–7.

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109. Simon MR, Mulla ZD. A population-based epidemiologic analysis of deaths from anaphylaxis in Florida. Allergy 2008;63:1077–83. 110. Mishra A, Dave N, Viradiya K. Fatal anaphylactic reaction to iron sucrose in pregnancy. Indian J Pharmacol 2013;45(1):93–4. 111. Meng WJ, Li Y, Zhou ZG. Anaphylactic shock and lethal anaphylaxis caused by compound amino acid solution, a nutritional treatment widely used in China. Amino Acids 2012;42(6):2501–5. 112. Sierra T, Figueroa MM, Chen KT, et al. Hypersensitivity to laminaria: a case report and review of literature. Contraception 2015;91(4):353–5. 113. Schoen C, Campbell S, Maratas A, et al. Anaphylaxis to buccal misoprostol for labor induction. Obstet Gynecol 2014;124(2 Pt 2 Suppl. 1):466–8. 114. Vaidya M, Ghike S, Jain S. Anaphylactoid reaction after use of intracervical dinoprostone gel. J Obstet Gynaecol Res 2014;40(3):833–5. 115. Rutkowski K, Nasser SM. Management of hypersensitivity reactions to anti-D immunoglobulin preparations. Allergy 2014;69(11):1560–3. 116. Soh JY, Chiang WC, Huang CH, et al. An unusual cause of food-induced anaphylaxis in mothers. World Allergy Organ J 2017;10(1):3. 117. Simons FER, Schatz M. Anaphylaxis during pregnancy. J Allergy Clin Immunol 2013;130:597–606. 118. Truong HT, Browning RM. Anaphylaxis-induced hyperfibrinolysis in pregnancy. Int J Obstet Anesth 2015;24(2):180–4. 119. Jao M-S, Cheng P-J, Shaw S-W, et al. Anaphylaxis to cefazolin during labor secondary to prophylaxis for group B streptococcus. J Reprod Med 2006;51:655–8. 120. Macy E. Penicillin skin testing in pregnant women with a history of penicillin allergy and group B streptococcus colonization. Ann Allergy Asthma Immunol 2006;97:164–8. 121. Bhatia PL, Singh MS, Jha BK. Laryngopathia gravidarum. Ear Nose Throat J 1981;60(9):408–12. 122. Conde-Agudelo A, Romero R. Amniotic fluid embolism: an evidence-based review. Am J Obstet Gynecol 2009;201:445–53. 123. Tamura N, Farhana M, Oda T, et al. Amniotic fluid embolism: pathophysiology from the perspective of pathology. J Obstet Gynaecol Res 2017;43(4):627–32. 124. Busardo FP, Frati P, Zaami S, et al. Amniotic fluid embolism pathophysiology suggests the new diagnostic armamentarium: beta-tryptase and complement fractions C3-C4 are the indispensable working tools. Int J Mol Sci 2015;16(3):6557–70. 125. Harboe T, Benson MD, Oi H, et al. Cardiopulmonary distress during obstetrical anaesthesia: attempts to diagnose amniotic fluid embolism in a case series of suspected allergic anaphylaxis. Acta Anaesthesiol Scand 2006;50:324–30. 126. Calabria CW, Hauswirth DW, Rank M, et al. American Academy of Asthma, Allergy & Immunology membership experience with venom immunotherapy in chronic medical conditions and pregnancy, and in young children. Allergy Asthma Proc 2017;38(2):121–9. 127. Rodriguez Del RP, Pitsios C, Tsoumani M, et al. Physicians’ experience and opinion on contraindications to allergen immunotherapy: the CONSIT survey. Ann Allergy Asthma Immunol 2017;118(5):621–8. 128. Hindmarsh D, Mahadasu S, Meneni D, et al. Managing labour with a history of Exercise Induced Anaphylaxis. Eur J Obstet Gynecol Reprod Biol 2016;198:167–8. 129. Santos N, Gaspar A, Livramento S, et al. Aspirin desensitization in a woman with inherited thrombophilia and recurrent miscarriage. Eur Ann Allergy Clin Immunol 2012;44(6):256–7. 130. Adriaensens I, Vercauteren M, Soetens F, et al. Allergic reactions during labour analgesia and caesarean section anaesthesia. Int J Obstet Anesth 2013;22(3):231–42. 131. Hepner DL, Castells M, Mouton-Faivre C, et al. Anaphylaxis in the clinical setting of obstetric anesthesia: a literature review. Anesth Analg 2013;117(6):1357–67. 132. Gei AF, Pacheco LD, Vanhook JW, et al. The use of a continuous infusion of epinephrine for anaphylactic shock during labor. Obstet Gynecol 2003;102:1332–5. 133. Lawlor F. Urticaria and angioedema in pregnancy and lactation. Immunol Allergy Clin North Amer 2014;34:149–56.

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134. Barniol C, Dehours E, Mallet J, et al. Levocetirizine and prednisone are not superior to levocetirizine alone for the treatment of acute urticaria: a randomized double-blind clinical trial. Ann Emerg Med 2018;71(1): 125–31.e1. 135. Gonzalez-Estrada A, Geraci SA. Allergy medications during pregnancy. Am J Med Sci 2016;352:326–31. 136. Ciach K, Niedoszytko M, Abacjew-Chmylko A, et al. Pregnancy and delivery in patients with mastocytosis treated at the Polish Center of the European Competence Netwok on Mastocytosis (ECNM). PLoS ONE 2016;11:e0146924. 137. Matito A, Alvarez-Twose I, Morgado JM, et al. Clinical impact of pregnancy in mastocytosis: a study of the Spanish network on mastocytosis (EMA) in 45 cases. Int Arch Allergy Immunol 2011;156:104–11. 138. Lei D, Akin C, Kovalszki A. Management of mastocytosis in pregnancy: a review. J Allergy Clin Immunol Pract 2017;5:1217–23. 139. Caballero T, Farkas H, Bouillet L, et al. International consensus and practical guidelines on the gynecologic and obstetric management of female patients with hereditary angioedema caused by C1 inhibitor deficiency. J Allergy Clin Immunol 2012;129:308–20. 140. Gompels MM, Lock RJ, Abinun M, et al. C1 inhibitor deficiency: consensus document. Clin Exp Immunol 2005;139:379–94. 141. Visy B, Fust G, Varga L, et al. Sex hormones in hereditary angioneurotic oedema. Clin Endocrinol 2004;60:508–15. 142. Salamon L, Morovic-Vergles J. Initial presentation of hereditary angioedema as abdominal pain and ascities in puerperium: case report. Acta Dermatovenerol Croat 2010;18:261–3. 143. Chinniah N, Katelaris CH. Hereditary angioedema and pregnancy. Aus NZ J Obstet Gynecol 2009;49:2–5. 144. Czaller I, Visy B, Csuka D, et al. The natural history of hereditary angioedema and the impact of treatment with human C1-inhibitor concentrate during pregnancy: a long-term survey. Eur J Obstet Gynecol Reprod Biol 2010;152:44–9. 145. Gonzalez-Quevedo T, Larco JI, Marcos C, et al. Management of pregnancy and delivery in patients with hereditary angioedema due to C1 inhibitor deficiency. J Investig Allergol Clin Immunol 2016;26:161–7. 146. Bork K, Hardt J. Hereditary angioedema: increased number of attacks after frequent treatments with C1 inhibitor concentrate. Am J Med 2009;122:780–3. 147. Banerji A, Riedl M. Managing the female patient with hereditary angioedema. Womens Health 2016;12:351–61. 148. Soltanifar D, Afzal S, Harrison S, et al. Caesarean delivery in a parturient with type III hereditary angioedema. Int J Obstet Anesth 2014;23:398–9. 149. Weatherhead S, Robson SC, Reynolds NJ. Eczema in pregnancy. BMJ 2007;335(7611):152–4. 150. Ambros-Rudolph CM, Mullegger RR, Vaughan-Jones SA, et al. The specific dermatoses of pregnancy revisited and reclassified: results of a retrospective two-center study on 505 pregnant patients. J Am Acad Dermatol 2006;54:395–404. 151. Yu M, Han K, Kim DH, et al. Atopic dermatitis is associated with Caesarean sections in Korean adolescents, but asthma is not. Acta Paediatr 2015;104(12):1253–8. 152. Kemmett D, Tidman MJ. The influence of the menstrual cycle and pregnancy on atopic dermatitis. Br J Dermatol 1991;125:59–61. 153. Cho S, Kim HJ, Oh SH, et al. The influence of pregnancy and menstruation on the deterioration of atopic dermatitis symptoms. Ann Dermatol 2010;22(2):180–5. 154. Babalola O, Strober BE. Treatment of atopic dermatitis in pregnancy. Dermatol Ther 2013;26(4):293–301. 155. Gurvits GE, Nord JA. Eczema herpeticum in pregnancy. Dermatol Reports 2011;3(2):e32. 156. Katz VL, Thorp JM Jr, Bowes WA Jr. Severe symmetric intrauterine growth retardation associated with the topical use of triamcinolone. Am J Obstet Gynecol 1990;162(2):396–7. 157. Boiko S, Zeiger RS. Diagnosis and treatment of atopic dermatitis, urticaria, and angioedema during pregnancy. In: Schatz M, editor. Immunology and allergy clinics of North America 20[4]. Philadelphia: WB Saunders; 2000. p. 839–55.

158. Chi CC, Kirtschig G, Aberer W, et al. Evidence-based (S3) guideline on topical corticosteroids in pregnancy. Br J Dermatol 2011;165:943–52. 159. Chi CC, Wang SH, Wojnarowska F, et al. Safety of topical corticosteroids in pregnancy. Cochrane Database Syst Rev 2015;(10):CD007346. 160. Bar OB, Hackman R, Einarson T, et al. Pregnancy outcome after cyclosporine therapy during pregnancy: a meta-analysis. Transplantation 2001;71(8):1051–5. 161. Kroumpouzos G, Cohen LM. Specific dermatoses of pregnancy: an evidence-based systematic review. Am J Obstet Gynecol 2003;188:1083–92. 162. Rudolph CM, Al-Fares S, Vaughan-Jones SA, et al. Polymorphic eruption of pregnancy: clinicopathology and potential trigger factors in 181 patients. Br J Dermatol 2006;154:54–60. 163. Danesh M, Pomeranz MK, McMeniman E, et al. Dermatoses of pregnancy: nomenclature, misnomers, and myths. Clin Dermatol 2016;34(3):314–19. 164. Beard MP, Millington GW. Recent developments in the specific dermatoses of pregnancy. Clin Exp Dermatol 2012;37(1):1–4. 165. Bechtel MA, Plotner A. Dermatoses of pregnancy. Clin Obstet Gynecol 2015;58(1):104–11. 166. Lambert J. Itch in pregnancy management. Curr Probl Dermatol 2016;50:164–72. 167. Macy E, Romano A, Khan D. Practical management of antibiotic hypersensitivity in 2017. J Allergy Clin Immunol Pract 2017;5:577–86. 168. Macy E, Ho NJ. Multiple drug intolerance syndrome: prevalence, clinical characteristics, and management. Ann Allergy Asthma Immunol 2012;108:88–93. 169. Ardern-Jones MR, Friedmann PS. Skin manifestations of drug allergy. Br J Clin Pharmacol 2011;71:672–83. 170. Gonzalez-Estrada A, Silvers SK, Klein A, et al. Epidemiology of anaphylaxis at a tertiary care center: a report of 730 cases. Ann Allergy Asthma Immunol 2017;118:80–5. 171. Davis RL, Gallagher MA, Asgari MM, et al. Identification of Stevens-Johnson syndrome and toxic epidermal necrolysis in electronic health records databases. Pharmacoepidemiol Drug Saf 2015;24:684–91. 172. Macy E, Contreras R. Adverse reactions associated with oral and parenteral use of cephalosporins: a retrospective population-based analysis. J Allergy Clin Immunol 2015;135:745–52. 173. Macy E, Poon K-YT. Self-reported antibiotic allergy incidence and prevalence; age and sex effects. Am J Med 2009;122:788.e1–7. 174. Macy E, Ho NJ. Adverse reaction associated with therapeutic antibiotic use after penicillin skin testing. Perm J 2011;15:31–7. 175. Ohel I, Levy A, Zweig A, et al. Pregnancy complications and outcome in women with history of allergy to medicinal agents. Am J Reprod Immunol 2010;64:152–8. 176. Desai SH, Kaplan MS, Chen Q, et al. Morbidity in pregnant women associated with unverified penicillin allergies, antibiotic use, and group B Streptococcus infections. Perm J 2017;21:16–80. 177. Mylonas I. Antibiotic chemotherapy during pregnancy and lactation period: aspects for consideration. Arch Gynecol Obstet 2011;283:7–18. 178. Muanda FT, Sheely O, Berard A. Use of antibiotics during pregnancy and the risk of major congenital malformations: a population based cohort study. Br J Clin Pharm 2017;83(11):2557–71. 179. Macy E. Penicillin skin testing in pregnant women with a history of penicillin allergy and group B streptococcus colonization. Ann Allergy Asthma Immunol 2006;97:164–8. 180. Policy Statement. Recommendations for the prevention of perinatal group B streptococcus (GBS) disease. Pediatrics 2011;128:611016. 181. Philipson EH, Lang DM, Gordon SJ, et al. Management of group B Streptococcus in pregnant women with penicillin allergy. J Reprod Med 2007;52:480–4. 182. Schindewolf M, Gobst C, Kroll H, et al. High incidence of heparin-induced allergic delayed-type hypersensitivity reaction in pregnancy. J Allergy Clin Immunol 2013;132:131–9. 183. Lindhoff-Last E, Kreutzenbeck HJ, Magnani HN. Treatment of 51 pregnancies with danaparoid because of heparin intolerance. Thromb Haemost 2005;93:63–9.

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy 184. Durand-Gonzalez KN, Guillausseau N, Anciaux ML, et al. Allergy to insulin in a woman with gestational diabetes mellitus: transient efficiency of continuous subcutaneous insulin lispro infusion. Diabetes Metab 2003;29:432–4. 185. Lin MC, Hsieh TK, Liu CA, et al. Anaphylactoid shock induced by oxytocin administration – a case report. Acta Anaesthesiol Taiwan 2007;45:233–6. 186. Draisci G, Zanfini BA, Nucera E, et al. Latex sensitization. A special risk for the obstetric population? Anest 2011;114:565–9.

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187. Macy E, Schatz M, Zeiger RS. Immediate hypersensitivity to methylparaben causing false-positive results of local anesthetic skin testing or provocative dose testing. Perm J 2002;6:17–21. 188. Berstein JA. Human seminal plasma hypersensitivity: an underrecognized woman’s health issue. Postgrad Med 2011;123:120–5. 189. Frapsauce C, Berthaut I, de Larouziere V, et al. Successful pregnancy by insemination of spermatozoa in a woman with human seminal plasma allergy: should in vitro fertilization be considered first? Fertil Steril 2010;94:753.e1–3.

CHAPTER 55  Asthma and Allergic Diseases During Pregnancy

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SELF-ASSESSMENT QUESTIONS 1. Which of the following is true regarding asthma during pregnancy? a. Asthma usually improves during pregnancy, even in patients with severe asthma. b. Asthma is associated with increased pregnancy complications, but no relationship between asthma control and the occurrence of pregnancy complications has been demonstrated. c. Based on the existing data, budesonide or fluticasone would be recommended if inhaled corticosteroids are begun during pregnancy. d. Montelukast is favored over long-acting beta agonists as add-on therapy during pregnancy. e. Although animal studies are reassuring, no human data exist for the use of asthma biologics during pregnancy. 2. Which of the following is true regarding rhinitis and sinusitis during pregnancy? a. Preexisting rhinitis may improve or worsen during pregnancy and may be concordant with asthma course. b. Pregnancy rhinitis is usually most prominent in the first half of pregnancy and is less common in smokers. c. Chlorpheniramine is recommended as the antihistamine of choice during pregnancy due to the lack of reassuring human data for second generation antihistamines. d. No data exist to inform the choice of specific intranasal corticosteroids for use during pregnancy. e. Amoxicillin-clavulanate but not cefuroxime or azithromycin is recommended for the treatment of sinusitis during pregnancy. 3. Which of the following is true regarding anaphylaxis during pregnancy? a. Continuation of inhalant immunotherapy is not recommended during pregnancy due to the risk of anaphylaxis. b. The most common causes of anaphylaxis during pregnancy are antibiotics. c. Because of increased risks of congenital malformations and decreased uteroplacental blood flow with epinephrine, ephedrine is the initial treatment of choice for gestational anaphylaxis.

d. In treating hypotension during pregnancy, a minimum maternal systolic blood pressure of 70 mm Hg should be maintained to ensure adequate placental perfusion, and the patient should be placed on her right side. e. Anaphylactoid syndrome of pregnancy (amniotic fluid embolism) may mimic anaphylaxis during pregnancy, but an elevated serum tryptase excludes this diagnosis. 4. Which of the following is true regarding skin conditions during pregnancy? a. Mastocytosis usually worsens during pregnancy. b. Vaginal delivery frequently triggers attacks, so pregnant women with hereditary angioedema should usually have a cesarean section delivery. c. Danazol or C1 esterase inhibitor concentrates are appropriate for management of hereditary angioedema during pregnancy. d. Atopic dermatitis is the most common dermatosis during pregnancy but usually improves during pregnancy. e. High doses of potent topical steroids should be avoided during pregnancy. 5. Which of the following is true regarding drug hypersensitivity during pregnancy? a. Only about 1% of pregnant women report a history of drug allergy. b. Cephalosporins should not be used in pregnant women with a low risk history of penicillin allergy. c. A history of penicillin allergy has not been associated with complications of pregnancy. d. Most reactions to unfractionated heparin during pregnancy can be managed by using any of several low-molecular-weight heparins. e. Penicillin skin testing is contraindicated during pregnancy.

56  Occupational Allergy and Asthma Catherine Lemière, Olivier Vandenplas

CONTENTS Introduction and Definitions, 940 Epidemiologic Aspects, 941 Pathogenesis and Etiology, 941 Clinical Features, 945 Patient Evaluation, Diagnosis, and Differential Diagnosis, 945 Outcomes and Treatment, 949

SUMMARY OF IMPORTANT CONCEPTS • Occupational allergic respiratory diseases represent a significant public health concern because of their high prevalence, long-term respiratory health consequences, socioeconomic consequences for the affected workers, and society. • Occupational asthma (OA) and rhinitis (OR) should be evaluated in all workers who develop or experience worsening of asthma and/or rhinitis symptoms in relation to their work environment. • The diagnosis of OA and OR should be established with the highest level of accuracy by performing a comprehensive investigation to avoid unwarranted socioeconomic consequences. • Complete avoidance of the causal agent remains the recommended treatment, because available information indicates that reduction of exposure is a less beneficial management option. • Incomplete avoidance of exposure to the causal agent is associated with substantial long-term respiratory health morbidity, because it is associated with a low rate of recovery, especially when the diagnosis is delayed. • Primary prevention should be directed toward reducing exposure to levels that prevent the onset of asthma in all workers, regardless of their individual susceptibility.

INTRODUCTION AND DEFINITIONS Occupational Asthma During the last decade, there has been a growing recognition that workrelated asthma (WRA) is a major public health concern because of its high prevalence and societal burden. Work-related asthma is a broad term indicating that asthma is worsened by the workplace.1 Work-related asthma encompasses occupational asthma (OA), which is asthma caused by a specific agent at the workplace, and work-exacerbated asthma (WEA), which corresponds to asthma exacerbated by nonspecific stimuli at the workplace, but not caused by it2 (Fig. 56.1). Several definitions of OA have been proposed. A widely cited definition emphasized the

940

Prevention, 950 Socioeconomic Impact and Medicolegal Aspects, 950 Specific Agents Causing Occupational Asthma and Rhinitis, 950 Work-Related Anaphylaxis, 952 Summary, 952

causal relationship between asthma and the workplace: “OA is a disease characterised by airway inflammation, variable airflow limitation, and airway hyperresponsiveness due to causes and conditions attributable to a particular occupational environment and not to stimuli encountered outside the workplace.”3 Another definition published by the American College of Chest Physicians defined the nature of the agents that can cause OA4: “Occupational asthma refers to de novo asthma or the recurrence of previously quiescent asthma (i.e., asthma as a child or in the distant past that has been in remission) induced by either sensitization to a specific substance (e.g., an inhaled protein [high-molecular-weight (HMW) protein of molecular mass >10 kDa] or a chemical at work [low-molecular-weight (LMW) agent]), which is termed sensitizerinduced OA, or by exposure to an inhaled irritant at work, which is termed irritant-induced OA”.4 Sensitizer-induced OA has also been defined as “asthma with a latency period,” suggesting the presence of an underlying immunologic mechanism responsible for a latency period occurring between the beginning of the occupational exposure and the onset of asthma symptoms.3 In contrast, irritant-induced asthma (IIA), also called OA without a latency period, encompasses a wide spectrum of clinical presentations. The most typical form is represented by reactive airways dysfunction syndrome (RADS),5 which refers to a type of OA without latency and immunologic sensitization, occurring after a single massive irritant exposure, causing severe airway injury resulting in persistent airway inflammation and nonspecific bronchial hyperresponsiveness (NSHB). In many cases, the onset of asthma is not sudden and follows repeated low-dose exposure to one or more bronchial irritants. Various names have been proposed for this phenotype of irritant-induced asthma: low-dose RADS, not so sudden irritant-induced asthma, and low intensity chronic exposure dysfunction syndrome (LICEDS). Because this textbook focuses on allergic diseases, this chapter only covers “sensitizer-induced OA.”

Occupational Rhinitis Occupational rhinitis (OR) has been defined as an “inflammatory disease of the nose, which is characterized by intermittent or persistent symptoms

CHAPTER 56  Occupational Allergy and Asthma

941

TABLE 56.1  Principal Agents Causing Immunologic Occupational Asthma Agent

Workers/Occupations at Risk

High Molecular Weight Agents Cereals (flour) Wheat, rye, barley, buckwheat

Millers, bakers, pastry makers

Latex

Gloves

Health care workers, laboratory technicians

Animals

Mice, rats, cows, seafood

Laboratory workers, farmers, seafood processors

Enzymes

α-Amylase, maxatase, alcalase, papain, bromelain, pancreatin

Baking products manufacture, bakers, detergent production, pharmaceutical industry, food industry

Low Molecular Weight Agents Isocyanates Toluene diisocyanate (TDI), methylene diphenyl-diisocyanate (MDI), hexamethylene diisocyanate (HDI)

Polyurethane production, plastic industry, molding, spray painters, insulation installers

Metals

Chromium, nickel, cobalt, platinum

Metal refinery, metal alloy production, electroplaters, welders

Biocides

Aldehydes, quaternary ammonium compounds

Health care workers, cleaners

Persulfate salts

Hair bleach

Hairdressers

Acid anhydrides

Phthalic, trimellitic, maleic, tetrachlorophthalic acids

Epoxy resin workers

Reactive dyes

Reactive black 5, pyrazolone derivatives, vinyl sulfones, carmine

Textile workers, food industry workers

Acrylates

Cyanoacrylates, methacrylates, di- and triacrylates

Manufacture of adhesives, dental and orthopedic materials, sculptured fingernails, printing inks, paints and coatings

Wood dusts

Red cedar, iroko, obeche, oak

Sawmill workers, carpenters, cabinet and furniture makers

(i.e., nasal congestion, sneezing, rhinorrhea, itching), and/or variable nasal airflow limitation and/or hypersecretion due to causes and conditions attributable to a particular work environment and not to stimuli encountered outside the workplace”6 (Fig. 56.1). As for OA, the broad spectrum of rhinitis syndromes related to the work environment should be classified into the following categories: (1) allergic OR characterized by immunologically mediated hypersensitivity directed toward a specific occupational agent; (2) nonallergic OR caused by the work environment through irritant, nonimmunologic mechanisms, best typified by the acute onset of rhinitis after single or multiple exposures to very high concentrations of irritant substances, which is referred to as the reactive upper airways dysfunction syndrome; and (3) work-exacerbated rhinitis, a poorly defined condition in which a preexisting or concurrent (allergic or nonallergic) rhinitis is worsened by a wide variety of conditions at work, including irritant agents, physical factors, emotions, secondhand smoke, and strong odors.

EPIDEMIOLOGIC ASPECTS Estimates of the frequency of OA can be derived from various sources: (1) cross-sectional and longitudinal studies in high-risk workplaces; (2) population-based studies; (3) compulsory occupational disease registries and medicolegal statistics; and (4) surveillance programs based on voluntary notification by physicians. A pooled analysis of all studies published up to 2007 indicated that 17.6% of all cases of adult-onset asthma are attributable to workplace exposures.7 Cross-sectional surveys of workforces exposed to sensitizing agents reported highly variable prevalence rates of OA, but these estimates are largely affected by the criteria used to identify the disease and selection biases, mainly, the “healthy worker effect.” This selection bias refers to the fact that for people who are employed, morbidity and mortality rates are lower than in the general population. The chief reason for this advantage is that healthy persons are likely to gain employment and to remain employed, whereas those with health problems often are excluded from employment. Cohort studies reported incidence rates of 2.7 to 3.5 cases of OA per 100 person-years among workers exposed to

laboratory animals,8 4.1 per 100 person-years among those exposed to wheat flour,9 and 1.8 per 100 person-years among dental health apprentices exposed to latex gloves.10 In the same cohorts, the incidence of OR was much higher than OA in workers exposed to laboratory animals (7.3 to 12.1 per 100 person-years) and flour (11.8 to 13.1 per 100 person-years). Incidence rates estimated from notification schemes and compensation statistics ranged from 17 to 174 new cases per million active workers per year (Table 56.1). These data suggest that the disease is underestimated in most countries. Differences from one country to another could also result, at least in part, from heterogeneity in diagnostic criteria and data collection schemes. Although there is a lack of recent data reporting the current incidence of OA, a recent review reports a decline of the incidence of OA based upon physicians’ reporting or accepted compensated claims.11 Population surveys allow for minimizing the survivor bias but are limited by the lack of confirmation of OA through objective tests. Information on the prevalence and incidence of OR in the general population is largely lacking, although surveys of workforces exposed to sensitizing agents indicate that OR is two to four times more common than OA.6 Convincing evidence shows that OR is associated with a five- to sevenfold increased risk for the development of asthma and OA. The proportion of subjects with OR in whom OA will subsequently develop remains uncertain.

PATHOGENESIS AND ETIOLOGY Pathophysiology The pathophysiology of sensitizer-induced OA and OR involves in most cases an immunoglobulin E (IgE)–dependent mechanism, especially for HMW agents. However, in several cases of OA induced by LMW agents, the production of specific IgE antibodies or the upregulation of IgE receptors has not been identified.12

Immunological, Immunoglobulin E–Mediated.  The pathophysiology of OA induced by IgE-dependent agents is similar to that of allergic

942

SECTION E  Respiratory Tract

asthma unrelated to work. The process of sensitization begins by the uptake of an antigen by immature dendritic cells in the subepithelial space.13 After the antigen uptake, the dendritic cells migrate to regional lymph nodes, undergo maturation, and express antigenic peptide on its surface in conjunction with major histocompatibility type 2 molecules. The dendritic cells eventually interact with CD4+ T cells expressing high-affinity receptors for the antigen-MHC II complex.14 Subsequently, the T cells expand and exit the lymph node. Th2 cells will eventually re-settle in the airways; CD4+ T cells may react to antigen presentation locally on future exposures,15 synthesizing interleukins (IL) 4, 5, and 13. Th2 cells are key for the isotype switching of B cells and IgE production. Although HMW agents (proteinaceous products, such as animal proteins and flour) act as complete antigens and induce the production of specific IgE antibodies, some LMW occupational agents, including platinum salts and trimellitic anhydride as well as other acid anhydrides, can also induce specific IgE antibodies, probably by acting as haptens and binding with proteins to form functional antigens.

Immunological, Non-IgE Mediated.  Many LMW chemicals, including isocyanates and plicatic acid (the agent causing Western red cedar asthma), cause OA but do not consistently induce specific IgE antibodies. Specific IgE antibodies were found in only 20% of subjects with Western red cedar–associated asthma, and these antibodies may merely reflect prior red cedar exposure, because in vitro they do not passively sensitize human lung fragments.16 Isocyanate-induced asthma is associated with both specific IgE and IgG17 antibodies, although their pathogenic significance is unclear. However, specific IgE to toluene diisocyanates has been detected in a minority of symptomatic workers. The absence of specific IgE antibodies to LMW compounds may also be related to a technical inability to identify those antibodies or to the existence of another immunologic mechanism such as cell-mediated reactions. Although the predominant immune response to chemical respiratory allergens may be of the Th2 type, other cells may play important support or regulatory roles. CD4+ as well as CD8+ T cells and different cytokines such as IL-1, IL-4, IL-5, IL-6, and IL-15 have been found in biopsies, bronchoalveolar lavage (BAL), and the sputum of patients with isocyanate-induced asthma.12 There is evidence of a mixed Th1/Th2 cytokine production in subjects with red cedar–induced asthma.18 Furthermore, specific inhalation challenge (SIC) induced a mixed Th2/ Th1 response in which CD8+ T cells were the main producers of IFNgamma in workers with positive SIC.19 There is also evidence that isocyanates can stimulate human innate immune responses by upregulating immune pattern-recognition receptors on monocytes and increasing the chemokines that regulate monocyte/

macrophage trafficking (macrophage migration inhibitory factor [MIF], monocyte chemoattractant protein 1 [MCP-1]).20

Histopathology Histopathologic changes in the airways of subjects with OA do not differ from those in persons with non–work-related asthma: epithelial desquamation and ciliary abnormalities, smooth muscle hyperplasia, subepithelial fibrosis, and increased numbers of eosinophils and lymphocytes in the airways. In addition, such changes have not been found to differ with the various sensitizers that induce OA. Removal from exposure for a period ranging from 5 to 21 months in subjects with diisocyanate-induced asthma was associated with a reduction in subepithelial fibrosis, but persistence of inflammatory cell infiltration of the airways was noted.21 Elevated levels of CD4+ as well as CD8+ T cells and cytokines such as IL-1β, IL-4, IL-5, IL-6, IL-15, and tumor necrosis factor (TNF)-α were found in biopsy specimens, bronchoalveolar lavage fluid, and sputum samples from patients with isocyanate-induced asthma.22 Isocyanates also have been shown to induce a predominant activation of neutrophils, along with increased levels of myeloperoxidase and IL-8.

Agents Causing Occupational Asthma and Rhinitis.  The workplace agents known to cause immunologically mediated OA and OR usually are categorized into high-molecular-weight (molecular mass >10 kDa) and low-molecular-weight agents. HMW agents are (glyco) proteins of vegetable and animal origin, whereas LMW agents include reactive chemicals, transition metals, and wood dusts. The agents and occupations most commonly implicated are listed in Table 56.2. The main differences between HMW and LMW agents are summarized in Table 56.3. The principal etiologic agents are specifically addressed later in the chapter. A very large number of substances (more than 400) used in the workplace have been documented as causing immunologic OA and OR. However, data derived from voluntary notification programs and compensation statistics of OA in various countries show that only a few agents—specifically, flour, isocyanates, persulfate salts, quaternary ammonium compounds, animals, wood dusts, metals, and enzymes— account for the vast majority of reported cases. Nevertheless, the distribution of causative agents may vary across geographic areas, depending on the pattern of industrial activities. Several lists of the occupational agents that are the most frequently encountered in different countries have been published.23–25 Workers in occupations with the highest incidence rates of OA are bakers and pastry makers, other food processors, spray painters,

TABLE 56.2  Main Differences Between High and Low Molecular Weight Agents Causing

Occupational Asthma Feature

High Molecular Weight Agents

Low Molecular Weight Agents

Nature

(Glyco)proteins derived from plants and animals

Highly reactive chemicals, metals, and wood dusts

Immunologic mechanisms

IgE-mediated

Uncertain; specific IgE for some agents (e.g., platinum salts, reactive dyes, acid anhydrides)

Type of airway inflammation

Eosinophils

Eosinophils and sometimes neutrophils

Type of asthmatic reactions

Immediate and dual

Often isolated late and atypical

Common (∼90%) Rare, but protein contact dermatitis may occur (e.g., flour, seafood) Frequent with some agents (e.g., latex)

Less common (∼50%) May occur (e.g., epoxy resins, acrylates, metals)

Associated disorders  Rhinoconjunctivitis   Contact dermatitis   Urticaria and anaphylaxis

Rare

CHAPTER 56  Occupational Allergy and Asthma

943

TABLE 56.3  Summary of Potential Risk Factors for Development of Occupational Asthma (OA) Risk Factor

Evidence

Environmental Risk Factors High level of exposure Strong

Agents/Settingsa

Reporting Studies

HMW agents: Wheat flour,1,2 α-amylase,3 laboratory animals,4,5 detergent enzymes,6 snow crab allergens7 LMW agents: Platinum salts,8 acid anhydrides9,10

1. Houba R, et al. Am J Respir Crit Care Med 1998;158:1499-503 2. Jacobs JH, et al. Allergy 2008;63:1597-604 3. Houba R, et al. Am J Respir Crit Care Med 1996;154:130-6 4. Hollander A, et al. Am J Respir Crit Care Med 1997;155:562-7 5. Cullinan P, et al.8 6. Cullinan P, et al. Lancet 2000;356:1899-900 7. Gautrin D, et al. Occup Environ Med 2009;67:17-23. 8. Merget R, et al. J Allergy Clin Immunol 2000;105:364-70 9. Barker RD, et al. Occup Environ Med 1998;55:684-91 10. Welinder H, et al. Allergy 2001;56:506-11 11. Meredith SK, et al. Occup Environ Med 2000;57:830-6 12. Pronk A. Am J Respir Crit Care Med 2007;176:1090-7

Moderate

Diisocyanates11,12

Skin exposure

Weak

Diisocyanates13

13. Bello D, et al. Environ Health Perspect 2007;115:328-35

Cigarette smoking

Moderate

IgE sensitization: Laboratory animals,4 seafood,7,14,15 psyllium16 green coffee,16,17 enzymes,18 acid anhydrides,9,19 platinum,8,20 reactive dyes21

Weak

Clinical OA: Laboratory animals,22,23 enzymes18

14. McSharry C, et al. Clin Exp Immunol 1994;97:499-504 15. Douglas JD, et al. Lancet 1995;346:737-40 16. Zetterstrom O, et al. BMJ 1981;283:1215-7 17. Romano C, et al. Clin Exp Allergy 1995;25:643-50 18. Johnsen CR, et al. Occup Environ Med 1997;54:671-5 19. Venables KM. BMJ 1985;290:201-4 20. Calverley AE. Occup Environ Med 1995;52:661-6 21. Park HS, et al. Clin Exp Allergy 1991;21:357-62 22. Venables K, et al. Br J Ind Med 1988;45:667-71 23. Fuortes LJ, et al. Am J Ind Med 1997;32:665-9

Individual Risk Factors Atopy Strong

HMW agents: Flour,24 laboratory animals,5,25 latex26; snow crab,7 detergent enzymes,27 α-amylase3

24. Hur GY, et al. Respir Med 2008;102:548-55 25. Ruoppi P, et al. Allergy 2004;59:295-301 26. Gautrin D, et al. Am J Respir Crit Care Med 2000;162:1222-8 27. Flood DF, et al. Br J Ind Med 1985;42:43-50 28. Baker DB, et al. Am J Ind Med 1990;18:653-64

Weak

LMW agents: Platinum28, acid anhydrides10

Genetic Markers HLA class II alleles

Moderate

LMW agents: Diisocyanates,29–32 red cedar,33 acid anhydrides,34,35 platinum salts,36 HMW agents: Laboratory animals,37 latex38

29. Bignon JS, et al. Am J Respir Crit Care Med 1994;149:71-5 30. Balboni A, et al. Eur Respir J 1996;9:207-10 31. Mapp CE, et al. Clin Exp Allergy 2000;30:651-6 32. C hoi JH, et al. Int Arch Allergy Immunol 2009;150:156-63. Horne C, et al. Eur Respir J 2000;15:911-4 34. YoungRP, et al. Am J Respir Crit Care Med 1995;151:219-21 35. Jones MG, et al. Clin Exp Allergy 2004;34:812-6 36. Newman Taylor, et al.23 37. Jeal H, et al. J Allergy Clin Immunol 2003;111:795-9 38. Rihs HP, et al. J Allergy Clin Immunol 2002;110:507-14

IL-4RA (I50V) II variant

Weak

Diisocyanates39

39. Bernstein DI, et al. Ann Allergy Asthma Immunol 2006;97:800-6

Antioxidant enzymes

a

40–42

Moderate

Diisocyanates

40. Piirilä P, et al. Pharmacogenetics 2001;11:437-45 41. Wikman H, et al. Pharmacogenetics 2002;12:227-33 42. Mapp CE, et al. J Allergy Clin Immunol 2002;109:867-72

Preexisting rhinitis

Weak

IgE sensitization to HMW agents43,44

43. Gautrin D, et al. Eur Respir J 2002;19:96-103 44. Gautrin D, et al. Am J Respir Crit Care Med 2008;177:871-9

Work-related rhinitis

Strong

Development of asthma in workers with OR45; development of OA in workers with OR related to laboratory animals46,47

45. Karjalainen A, et al.24 46. Gautrin D, et al.25 47. Elliott L, et al. J Allergy Clin Immunol 2005;116:127-32

Preexisting nonspecific bronchial responsiveness

Weak

Apprentice workers exposed to HMW agents44,48

48. Gautrin D, et al. Am J Respir Crit Care Med 2001;163:899-904

HMW, High molecular weight; IgE, immunoglobulin E; IL4RA, interleukin-4 receptor α chain; LMW, low molecular weight; OR, occupational rhinitis; TLR4, Toll-like receptor 4. a Data from cited studies.

944

SECTION E  Respiratory Tract

Work-related asthma and rhinitis

OA and rhinitis Caused by the work environment

Work-exacerbated asthma and rhinitis Exacerbated by the work environment

Immunologic

Nonimmunologic

Sensitizer-induced OA • IgE-mediated - HMW and some LMW agents • IgE-independent - LMW agents

• Acute onset - Single exposure: RADS, RUDS - Multiple exposures: IIA • Progressive onset - Low-dose RA - Not-so-sudden IIA - LICEDS

Unknown mechanisms Irritant chemicals, dusts, and fumes, second-hand smoke, common allergens, work site temperature, physical exertion, emotional stress, etc.

Fig. 56.1  Classification of work-related asthma and rhinitis. HMW, High molecular weight; IgE, immunoglobulin E; IIA, irritant-induced asthma; LICEDS, low-intensity chronic exposure dysfunction syndrome; LMW, low molecular weight; OA, occupational asthma; RADS, reactive airways dysfunction syndrome; RUDS, reactive upper airways dysfunction syndrome.

hairdressers, wood workers, health care workers, cleaners, farmers, laboratory technicians, and welders. In more recent years, population-based studies conducted in various countries worldwide have consistently found that cleaning activities were associated with an excess risk of asthma26 and work-related asthma symptoms.27 Industrial and domestic cleaners are exposed to a wide variety of products containing irritant chemicals (e.g., detergents, acids, alkali, solvents, chelating compounds) as well as some potentially sensitizing substances, including biocides (e.g., chloramine-T, aldehydes, quaternary ammonium derivatives), ethanolamines, enzymes, and latex gloves. Predicting the potential hazard of a given chemical is difficult. However, a quantitative structure activity relationship (QSAR) model can help clinicians to predict a chemical asthma hazard.28

Environmental Risk Factors.  OA and OR result from the complex interaction between environmental and individual factors (Fig. 56.1). A summary of data from important studies on this topic is presented in Table 56.3. Level of exposure.  The intensity of exposure to sensitizing agents is currently the most characterized and the most important environmental risk factor for the development of OA (Table 56.3). A number of studies have provided strong evidence supporting a dose-response relationship between the level of exposure to occupational agents and the development of IgE-mediated sensitization and work-related respiratory symptoms for agents acting through an IgE-mediated mechanism. Determination of dose-response relationships is a key step for establishing permissible exposure levels at the workplace. However, exposureresponse relationships may be affected by the nature of the sensitizing agent, individual susceptibility, and timing of exposure. Individual susceptibility factors may affect the exposure-response relationships. For instance, certain human leukocyte antigen (HLA) class II alleles were found to be stronger determinants of sensitization to occupational agents at low levels of exposure.29 The timing of exposure may also play a role, because the prevalence of onset of work-related asthma symptoms is consistently higher within the early period of exposure to the occupational agents, and exposure-response gradients are more

clearly documented in those workers who develop these outcomes soon after the onset of exposure.30 There is some suggestion that nonrespiratory routes of exposure might increase the risk of sensitization to occupational agents. Animal experiments have consistently demonstrated that dermal exposure to LMW occupational agents can initiate IgE-mediated respiratory sensitization with a predominant Th2-like immune response, as well as the development of eosinophilic airway inflammation and AHR to these agents. Much less is known about the potential impact of skin exposure on the initiation of OA in humans, because the effects of dermal contact cannot be easily quantified or differentiated from those of inhalation exposure, because both occur simultaneously. Smoking and exposure to other pollutants.  A number of studies indicate that exposure to cigarette smoke can increase the risk of IgEmediated sensitization to some HMW and LMW agents (Table 56.3), but the evidence supporting an association between smoking and the development of clinical OA is still very weak. Growing evidence indicates that environmental pollutants, such as ozone, nitrogen dioxide, tobacco smoke, diesel exhaust particles, and endotoxin can act as adjuvants in allergic responses to common inhalant allergens. Only limited information, however, is available on the potential interactions between other pollutants and sensitizing agents at the workplace. Occupational exposure to vapors, gas, dust, and fumes (VGDF) seems to increase the risk of asthma and rhinitis. Exposure to chemicals increases the risk of developing rhinitis [OR, 95% CI: 1.29, (1.10 to 1.52)], asthma [(1.42, (1.15 to 1.77)], and both asthma and rhinitis [1.60, (1.31 to 1.96)].31

Individual Risk Factors Atopy.  Atopy has consistently been demonstrated as an important host risk factor for the development of IgE-mediated sensitization, OA, and OR related to HMW agents (Table 56.3), whereas this association remains controversial for some LMW agents (e.g., platinum salts, anhydride) and absent for most other LMW agents. Preexposure sensitization to common allergens that are structurally related to workplace allergens, such as pets in the case of laboratory animal workers, could be a stronger predictor of OA than atopy.

CHAPTER 56  Occupational Allergy and Asthma

Genetic susceptibility.  A number of studies documented that certain HLA class II molecules (i.e., HLA-DR, HLA-DQ, and HLA-DP alleles) involved in the presentation of processed antigens to T lymphocytes are associated with either susceptibility to or protection against OA because of various and LMW and HMW occupational agents (Table 56.3). Some evidence suggests that genes associated with Th2 cell differentiation may play a role in the development of OA caused by isocyanates. Genes involved in the protection against oxidative stress, such as glutathione S-transferase (GSTP1 and GSTM1) and N-acetyltransferase (NAT), have been associated with an increased risk of isocyanate-induced OA or a protective effect (Table 56.3). Recent genetic studies conducted in subjects with isocyanate-induced OA compared with control populations identified single nucleotide polymorphisms (SNP) mapping to genes associated with antigen processing and presentation and production of immunomodulatory cytokines (TNF-α and TGF-B1).32,33 Overall, the information currently available indicates that genetic testing is limited for both diagnostic and preventive purposes. In addition, there is convincing evidence that environmental factors can interact with genetic determinants to affect disease susceptibility.

Rhinitis Epidemiologic evidence confirms that OR is associated with an increased risk for the subsequent development of asthma34 and OA.35 However, the proportion of subjects with OR who will develop OA remains uncertain. Among apprentices in animal health technology, the predictive value of work-related nasal symptoms on the subsequent development of probable OA was only 11.4% over a follow-up period of 30 to 42 months (Table 56.3). Prospective cohort studies of apprentices also have shown that rhinitis present before work exposure is an independent risk factor for IgE sensitization to HMW allergens.

Nonspecific Bronchial Hyperresponsiveness Prospective cohort studies have shown that the presence of nonspecific bronchial hyperresponsiveness (NSBH) and a physician-based diagnosis of asthma before entering exposure to HMW occupational agents are associated with an increased risk of subsequent IgE sensitization and OA.

CLINICAL FEATURES As in non–work-related asthma, the clinical features of OA include signs and symptoms of variable cough, wheezing, dyspnea associated with reversible airflow limitation, NSBH, and airway inflammation. Some clinical features are more specifically related to OA. Typically, the affected worker initially complains of cough, wheeze, and dyspnea either as soon as the work shift exposure starts or at the end of the work shift or even in the evening, after working hours, with remission during weekends and holidays. As the disease progresses, symptoms tend to occur earlier during the day and fail to remit during days off and long holidays. With further exposure, asthma symptoms may persist and become permanent despite complete withdrawal from exposure. In these instances, even prolonged removal from exposure will result in only partial reversal of the asthmatic condition. It is therefore very important to establish the diagnosis of OA early and to remove the patient from exposure. Rhinitis is associated with respiratory symptoms in a majority of cases of OA and often precedes the occurrence of respiratory symptoms, especially with exposure to HMW agents.36 Identification of direct or even indirect exposure to a known respiratory sensitizer in the workplace also is an important aspect of the history, but such exposure often is not readily apparent to the worker or the clinician. Eosinophilic bronchitis is a variant of asthma that represents 12% of the causes of chronic cough.37 It consists of cough or asthma-like symptoms related to an underlying eosinophilic inflammation without

945

airflow obstruction or NSBH. This condition is responsive to inhaled corticosteroids. Eosinophilic bronchitis can be caused by sensitization to occupational agents and has been labeled occupational eosinophilic bronchitis. The diagnostic criteria consist of the following: isolated chronic cough (lasting more than 3 weeks) that worsens at work; sputum eosinophilia with counts of 2.5% or greater in either spontaneous or induced samples; increases in sputum eosinophilia related to exposure to the offending agent; spirometric parameters within normal limits and not significantly affected by exposure to the offending agent; absence of NSBH to methacholine (provocative concentration of methacholine inducing a 20% fall in forced expiratory volume in 1 second [FEV1]; [PC20] greater than 16 mg/mL) both at work and away from work; and other causes of chronic cough are ruled out.38 Occupational eosinophilic bronchitis has been causally related to a number of occupational agents, including latex, wheat flour, α-amylase, egg lysozyme, isocyanates, acrylates, formaldehyde, chloramine-T, epoxy resin hardener, stainless steel welding fumes, and mushroom spores. Exposure to specific occupational agents such as textile, grain dust, or aluminum can induce conditions that are considered as variants of occupational asthma (byssinosis, potroom asthma). Exposure to those agents can lead to partially reversible airflow obstruction with chronic airflow limitation.3

PATIENT EVALUATION, DIAGNOSIS, AND DIFFERENTIAL DIAGNOSIS The diagnosis of OA is a difficult one to establish. Confirmation by objective testing is necessary to ensure accuracy in identifying this clinical entity.39,40 Although a thorough clinical and occupational history must be carefully recorded, the diagnosis of OA cannot be made solely on the basis of a compatible history, which has a low positive predictive value (Table 56.4).41 Very few items included in a clinical questionnaire are satisfactory predictors of the presence of OA; wheezing and rhinoconjunctivitis symptoms are two such predictors, but only in subjects exposed to HMW agents.36 Making an accurate diagnosis of OA is crucial because of the significant social and financial consequences associated with this diagnosis. The investigation of OA is summarized in Fig 56.3. The validity of the different diagnostic tests and their practical limitations and advantages are summarized in Tables 56.4 and 56.5.

History.  OA should be suspected in every adult patient with new-onset asthma. A good occupational history, not only of the current job and exposure but also of past jobs and exposures, is essential. However, even when the history is collected thoroughly, the clinicians are likely to overlook some occupational exposures.42 A scheme for addressing relevant points has been published and includes employment history (current and past jobs), symptoms (nature, temporal relationship to work, improvement while away from work), and potential risk factors.43 In many cases, the patient may not be aware of the exact chemical exposures at work; material safety data sheets (MSDSs) can be requested from the workplace and may be of help in clarifying the presence of a workplace sensitizer. If the content of the causative agent is less than 1%, it may not be listed in the MSDS. In such instances, the manufacturer must be contacted. It often is helpful to ask the worker to sketch or to provide pictures of the work site and the work process itself, and to indicate the locations where he or she works during the work shift. In addition to identifying potential high-risk agents, the exposure history should include the duration of exposure and the frequency and concentrations of exposure. The substances to which the worker is potentially exposed at work can be checked against a comprehensive list of agents recognized as causing OA, and the specific job for that worker checked

946

SECTION E  Respiratory Tract

TABLE 56.4  Validity of Objective

Diagnostic Test

Sensitivity (%)

Specificity (%)

8469–93

4826–72

High-molecular-weight agents (skin-prick tests)a

8170–88

6042–75

Low-molecular-weight agents (specific IgE antibodies)a

3123–41

8985–92

Serial measurements of PEFa

6443–80

7766–85

Serial measurements of PEF and nonspecific bronchial responsivenessb

84-92

61-67

Single assessment of sputum eosinophilsc   ≥1%   ≥3%

50 22

67 91

Serial assessments of sputum eosinophils at and away from workd:  Increase >1%  Increase >2%  Increase >6.4%

6545–81 52 33–71 2613–46

7657–88 8061–91 9275–98

Serial assessmentd

5024–76

7551–90

85–95

Test Single assessment of nonspecific bronchial responsivenessa Immunologic tests:

Baseline PC20 histamine ≤16 mg/mL or FeNO ≥25 ppb Baseline PC20 histamine ≤16 mg/mL or sputum eosinophils ≥1%e

91 9486–98

2921–39 178–9

Increase in sputum eos ≥3% or 2-fold decrease or greater in PC20 after exposure to the offending agentf

84

74

Sensitivity and specificity of diagnostic tests are expressed as percent, with 95% confidence interval in parentheses when available. PEF, Peak expiratory flow.aModified from Beach J, Russell K, Blitz S, et al. A systematic review of the diagnosis of occupational asthma. Chest 2007;131:569-78. b Modified from Perrin B, Lagier F, L’Archevêque J, et al. Occupational asthma: validity of monitoring of peak expiratory flow rates and non-allergic bronchial responsiveness as compared to specific inhalation challenge. Eur Respir J 1992;5:40-8; and Côté J, Kennedy S, Chan-Yeung M. Sensitivity and specificity of PC20 and peak expiratory flow rate in cedar asthma. J Allergy Clin Immunol 1990;85:592-8. c Modified from Malo JL, Cardinal S, Ghezzo H, et al. Association of bronchial reactivity to occupational agents with methacholine reactivity, sputum cells and immunoglobulin E-mediated reactivity. Clin Exp Allergy 2011;41:497-504. d Difference between the percentage of eosinophils at work and away from work. Modified from Girard F, Chaboillez S, Cartier A, et al. An effective strategy for diagnosing occupational asthma: use of induced sputum. Am J Respir Crit Care Med 2004;170:845-50. e Adapted from Beretta C, Rifflart C, Evrard G, et al. Assessment of eosinophilic airway inflammation as a contribution to the diagnosis of occupational asthma. Allergy 2018;73(1):206–13. f Adapted from Racine G, Castano R, Cartier A, et al. Diagnostic accuracy of inflammatory markers for diagnosing occupational asthma. J Allergy Clin Immunol Pract 2017;5(5):1371–7.

against the list of at-risk occupations. These lists are available from various sources (websites and published tables; see later under Specific Agents Causing Occupational Asthma and Rhinitis). If available, the occupational health record and the industrial hygiene record from the company should be reviewed.

Several algorithms have been proposed for diagnosing OA.39,40 Fig. 56.2 shows the algorithm to be used in evaluating a patient suspected of having OA. A methacholine or histamine challenge test to assess the degree of NSBH should be carried out. A negative reaction to a histamine or methacholine challenge does not exclude OA if such testing is performed when the patient is off work and free of symptoms. However, if the challenge test is performed when the patient is working and symptomatic, the negative predictive value of the methacholine challenge reaches 98%, excluding the diagnosis of OA with a high level of confidence.44 A highly positive immunologic sensitization along with the presence of airway hyperresponsiveness makes the diagnosis of OA very likely in patients with a history suggestive of OA.40 The relationship between work and asthma, if any, should then be evaluated using serial measurements of peak expiratory flow (PEF) and/or assessments of NSBH at work and off work and/or SICs in the laboratory or at the workplace.

Serial Peak Expiratory Flow Monitoring.  Serial measurement of PEF with the subject at work and away from work has been found to be useful in obtaining objective information for the confirmation of OA. As with other types of asthma, compliance with PEF monitoring has been shown to be poor, especially when patients were asked to record values four times per day. The optimal duration of recording of PEF has not been established, but monitoring should include a minimum period of 2 weeks at work and exposed to the suspected causative agent and a similar period away from work, unless significant changes are recorded earlier at work. If the patient is using inhaled corticosteroids, it is important to keep the same dose throughout the period of monitoring. Inhaled bronchodilators should be used only when necessary, and the patient’s self-dosing history should be recorded. PEF should be performed before use of bronchodilators. No uniformly accepted criteria for the interpretation of PEF recordings have been established. Attempts have been made to develop such criteria, but the sensitivity and specificity of the diagnosis based on these objective criteria were no better than for the “eyeballing” method of experienced physicians.45 A computer-assisted diagnostic aid, OASYS (observation and appraisal system), has been developed to distinguish occupational from nonoccupational causes of airflow obstruction. One version, OASYS-2, is based on discriminate analysis and has achieved a sensitivity of 75% and a specificity of at least 94%. Compared with SIC, reported sensitivity of OASYS ranges between 35% and 73%, whereas specificity is between 65% and 100%. Accordingly, PEF monitoring examined with OASYS-2 analysis is better used to confirm than to exclude OA.

Serial Measurement of Nonspecific Bronchial Responsiveness.  A normal level of nonspecific bronchial responsiveness after a period at work at which time the worker experiences the usual symptoms almost excludes OA and asthma.44 Although theoretically a decrease in nonspecific bronchial responsiveness should occur after a period away from work, this has not been found to be a reliable means of confirming OA, probably because assessments are repeated after too short an interval of being away from work (usually 2 weeks).

Specific Inhalation Challenge Tests.  SIC tests are considered to be the reference test in investigation of OA. The methods of specific challenge testing have been described in detail elsewhere.46 Laboratory-specific challenge tests are time-consuming and require specialized facilities that are available in only a few clinical centers. Specific challenge tests are useful in the following circumstances: (1) when the diagnosis of OA remains in doubt after serial monitoring of PEF or airway responsiveness; (2) when a patient clearly has OA but it is necessary for management personnel to confirm or identify the causative agent at work; (3) when a

CHAPTER 56  Occupational Allergy and Asthma

947

TABLE 56.5  Advantages and Limitations of the Diagnostic Tests Used in the Investigation of

Occupational Asthma Diagnostic Tests

Advantages and Limitations

Assessment of NSBH

• Simple, low cost. • Allows confirmation of the diagnosis of asthma. • Low specificity for diagnosis of asthma OA. The lack of AHR does not allow discarding the diagnosis of OA in subjects who have been removed from the workplace.

Immunologic tests

• Easy to perform, low cost. • Commercial extracts are available (skin prick tests or specific IgE for HMW agents). • Lack of standardization for the majority of occupational allergens except for latex. • Measure of specific IgE available for some LMW agents (anhydrides, acids, isocyanates, aldehydes) but low sensitivity. • Identify the sensitization but not the disease itself.

PEF monitoring

• Low cost. • Requires the workers’ collaboration. • Low adherence (95%)†

Negative, but NOT exposed at work

Positive Assessment of immunological sensitization (SPT and/or sIgE)

* Negative or weakly positive

Not available

Highly positive (PPV >90%)

*

Second Step Specific inhalation challenge in the laboratory

Serial measurements of PEF/FEV1 and/or NSBH and/or sputum eosinophils at work and off work Especially useful when: • The subject is exposed to multiple asthmagens at work • No agent known as causing OA has been identified at work • Facility for SIC is not easily available • The conditions of exposure at work cannot be reproduced in the lab

Negative¥

Positive

Especially useful when: • SIC can be performed efficiently and safely • The patient is no longer exposed at work • The highest level of diagnostic confidence is required • There is need to identify a particular agent • PEF records are inconclusive

Equivocal

Positive

Negative#

Occupational asthma

No occupational asthma

Fig. 56.3  Algorithm for the investigation of occupational asthma. FEV1, Forced expiratory volume in one-second; IgE, immunoglobulin E; NPV, negative predictive value; NSBH, nonspecific bronchial hyperresponsiveness; OA, occupational asthma; PEF, peak expiratory flow; PPV, positive predictive value; sIgE, specific immunoglobulin E; SPT, skin prick test. (Adapted from Vandenplas et al. Clin and Exp Allergy. 2016;47:6–18.40) *High NPV and PPV are applicable only to selected populations of subjects with a high pretest probability of OA (i.e., tertiary centers). †Consider assessment of sputum eosinophil counts to identify occupational eosinophilic bronchitis or further investigation if the clinical history is highly suggestive of OA. ¥Consider specific inhalation challenge in the laboratory if the clinical history is highly suggestive of OA. #Consider workplace inhalation challenge or serial PEF monitoring if the clinical history is highly suggestive of OA.

new agent is suspected of causing OA; and (4) when the patient cannot be returned to the incriminated workplace. Although SIC testing is still considered the gold standard for the diagnosis of OA, the potential for false-positive and false-negative responses is well recognized. A false-negative response may occur if the wrong agent is used (e.g., different types of diisocyanates), or if the exposure conditions are not comparable to those in the workplace. Even if the subject has been away from work for a long time, complete desensitization is uncommon.47 SICs can be performed in the laboratory or at the workplace. SIC testing at the workplace consists of serial spirometric measurements performed by a respiratory technologist while the worker is doing his or her usual tasks. It can be useful to perform an SIC at the workplace when the SIC result in the laboratory is negative. SICs at the workplace have been shown to be positive in 22% of the subjects with a highly suggestive history and negative SIC reaction in the laboratory.48 However, they do not allow for the identification of the causative agent. The risk of falsely negative SIC results

Immunologic Testing.  Immunologic tests in the diagnosis of OA are limited by the lack of standardized commercially available reagents for skin and in vitro tests. In a systematic review54 assessing the sensitivity and specificity of skin prick testing in patients exposed to HMW agents compared with SIC, the pooled sensitivity was 80.6% (95% CI, 69.8% to 88.1%), whereas the pooled specificity was 59.6% (95% CI, 41.7% to 75.3%). In patients exposed to LMW agents, the pooled sensitivity was 72.9% (95% CI, 59.7% to 83.0%), whereas the pooled specificity was 86.2% (95% CI, 77.4% to 91.9%). The same review assessed the sensitivity and specificity of serum-specific IgE compared with SIC. Sensitivity was higher for the HMW agents studied (73.3% (95% CI, 63.9% to 81.0%), whereas the specificity was 79.0% (95% CI, 50.5% to 93.3%). In subjects exposed to LMW agents, the pooled sensitivity was 31.2% (95% CI, 22.9% to 40.8%) and the pooled specificity was 88.9% (95% CI, 84.7% to 92.1%). A high level of sensitization to flour, latex, or obeche wood has been shown to have a high positive predictive value for OA in selected populations.55–57 Most in vitro tests to assess specific sensitization to occupational chemicals remain research tools at present. Noninvasive Measures of Airway Inflammation Sputum cell counts.  Similar to the airway inflammation found after common allergen inhalation challenges, an increase in sputum eosinophils has been observed after SICs to a number of HMW and LMW agents.58 A neutrophilic airway inflammation has been reported after exposure to isocyanates. The factors that influence the type of airway inflammation induced by the exposure to occupational agents are unclear. The concentration of and duration of exposure to the offending agents may play a role. Using sputum differential cell counts during the investigation of OA can improve its diagnosis by bringing an additional objective measure to this investigation. Although the reliability of the FEV1 and PC20 can be diminished by inadequate spirometric maneuvers, the presence or absence of airway inflammation cannot be affected by improper maneuvers during sputum collection. The best timing for the collection of induced sputum in respect to the exposure to occupational agents is likely to be 7 to 24 hours after exposure. An increase in sputum eosinophils has been shown to occur 7 hours after exposure to occupational agents and to persist 24 hours after exposure.59 In subjects with OA, the increase in sputum eosinophils precedes the occurrence of functional changes occurring after exposure to the occupational agent responsible for OA.60 Vandenplas and associates61 showed that an increase in sputum eosinophil count greater than 3% after the first day of exposure during SIC was the most accurate parameter for predicting the development of an asthmatic response on subsequent exposures, with a sensitivity of 67% and a specificity of 97%. Therefore a notable increase in the sputum eosinophil count in the absence of a fall in FEV1 should incite pursuing the investigation. The lack of increase in sputum eosinophil counts after exposure to occupational agents should not rule out the diagnosis of OA. Indeed, some subjects can experience a 20% fall in FEV1 without showing sputum eosinophilia,62 whereas others can experience a 20% fall in FEV1 accompanied by an important increase in airway inflammation without NSBH.63

CHAPTER 56  Occupational Allergy and Asthma Interfering factors that can modify the sputum cell response such as corticosteroid treatment should be considered in the interpretation. Exhaled nitric oxide.  Studies that assessed the usefulness of exhaled nitric oxide (eNO), measured as the fraction of exhaled gas (FeNO), in the investigation of OA have provided inconsistent results.59,64–67 Several issues warrant consideration in interpreting these conflicting results. One is the insufficient duration of patient monitoring after the acute exposure; the level of exhaled NO tends to increase only 24 to 48 hours after exposure.68 Second, glucocorticosteroids inhibit the induction of NO synthase, and FeNO falls after treatment with oral or inhaled corticosteroids in subjects with asthma. FeNO response might have been blunted in studies that included patients on corticosteroid treatment at the time of the test. Third, an increase in NO production in the presence of bronchoconstriction might have been underestimated. Finally, the pathophysiology of the asthmatic reaction (IgE mediated vs. non-IgE mediated) induced by occupational agents may also affect the production of FeNO. An increase in FeNO may be found more consistently in subjects with OA caused by HMW agents known to induce IgE-mediated asthmatic reactions than in OA caused by LMW agents.69 In conclusion, although the measurement of FeNO has some advantages over the analysis of induced sputum in OA, the interpretation of increased FeNO is more difficult than sputum differential cell counts, owing to its lack of specificity as well as the potential confounding factors that may influence the results. Further studies are needed to clarify the role and the interpretation of changes in FeNO in the investigation of OA. Combination of tests.  A systematic review of studies on the diagnosis of OA assessed the sensitivity and specificity of the combination of various diagnostic tools. For OA related to HMW agents, the nonspecific bronchial responsiveness test, skin-prick test (SPT), and serumspecific IgE had sensitivities greater than 73% compared with SIC, the reference test.30 Specificity was highest at 79.0% (95% CI, 50.5% to 93.3%) for specific IgE versus SIC. High specificity was demonstrated for a positive result on nonspecific bronchial responsiveness tests and SPTs alone (82.5%; 95% CI, 54.0% to 95.0%) or in combination with specific IgE (74.3%; 95% CI, 45.0% to 91.0%) versus SIC. The highest sensitivity for the diagnosis of OA related to LMW agents occurred between combined nonspecific bronchial responsiveness tests and SPTs versus SIC (100%; 95% CI, 74.1% to 100%). Compared with SIC, specific IgE and SPT had similar specificities (88.9%; 95% CI, 84.7% to 92.1%; and 86.2%; 95% CI, 77.4% to 91.9%, respectively). However this systematic review did not include recent studies. In a series of 240 subjects investigated for possible OA through SIC, 8 of 17 subjects without baseline NSBH despite a subsequent positive SIC showed a sputum eosinophil count of 2% or more, a FeNO level of 25 ppb or more, or both outcomes. The combination of a baseline measure of airway hyperresponsiveness (PC20 histamine of 16 mg/mL or less) or airway inflammation (FeNO of 25 ppb or more or sputum eosinophils of 1% or more) showed a high sensitivity (91% and 94%, respectively) for the diagnosis of OA.70 These findings indicate that adding the assessment of FeNO level and sputum eosinophils to NSBH improves the identification of subjects who may have OA and requires further objective testing before excluding the possibility of OA. A twofold or greater decrease in PC20 or a 3% or greater increase in sputum eosinophil counts after exposure to the offending agent achieved a sensitivity of 84% and a specificity of 74% with a negative predictive value of 91% for the diagnosis of OA in subjects referred for possible OA in a tertiary center.71

Differential Diagnosis The most challenging aspect of the differential diagnosis for OA is certainly the diagnosis of work-exacerbated asthma (WEA).2 In both

949

conditions, the affected worker complains of a worsening of asthma symptoms when at work. Along with those symptoms, impairment of respiratory function, as evidenced by increased airflow limitation, increased NSBH, and increased variability of serial PEF measurements, can be identified during periods at work, in comparison with periods away from work. PEF variability has been compared between workers with OA and those with WEA. Workers with OA exhibited greater PEF variability during periods at work than workers with WEA. However, when the PEF plots were analyzed visually by clinician specialists in the field of WRA, they were unable to differentiate OA from WEA.72 The reference test to differentiate OA from WEA remains the SIC. Whereas the exposure to a specific occupational asthmagen induces a 15% to 20% fall in FEV1 in workers with OA, no sustained fall in FEV1 is observed in subjects with WEA. The occurrence of an eosinophilic inflammatory process on exposure to an occupational agent also favors the diagnosis of OA.61 A substantial proportion of subjects evaluated for work-related respiratory symptoms fail to demonstrate any objective evidence of asthma.73 Therefore the first diagnostic step is to confirm the presence of asthma and to exclude conditions with asthma-like manifestations, such as vocal cord dysfunction, hyperventilation, and sick building syndrome.4

OUTCOMES AND TREATMENT Systematic reviews of existing data on the outcome of immunologic OA indicate that complete and definitive avoidance of exposure to the causative agent remains the optimal treatment for immunologic OA.74,75 Indeed, workers with immunologic OA who continue to be exposed to the causative agent are at high risk for deterioration of asthma symptoms, airway obstruction, and NSBH.74,75 Reduction of exposure to the agent causing OA has been acknowledged as an alternative option to complete avoidance when elimination of exposure or accommodation of affected workers to unexposed jobs is not possible. The limited available evidence, however, indicates that this option is less beneficial than cessation of exposure, because it is associated with a lower likelihood of asthma improvement and a higher risk of worsening.76 This management approach should therefore be restricted to selected patients, and careful medical monitoring is required to ensure early identification of asthma worsening. Immunotherapy has been tested only in workers with allergy to and/ or OA caused by HMW agents for which an IgE-dependent reaction has been demonstrated. Immunotherapy has been tested mainly in health care workers allergic to natural rubber latex (NRL). Some evidence indicates that immunotherapy can reduce cutaneous and respiratory symptoms in health care workers allergic to latex. Of note, however, this treatment can induce systemic reactions in a large number of treated subjects. Small or uncontrolled studies have reported decreases in allergic and respiratory symptoms after immunotherapy in persons with baker’s asthma or sea squirt, laboratory animal, or wood allergy and asthma.77 Whether immunotherapy can alter the course of OA in the long term remains to be determined. Further studies need to be conducted before immunotherapy can be recommended for the treatment of OA related to HMW agents. A few case reports provided some suggestion that treatment with the anti-IgE omalizumab could improve asthma control in subjects with flour-induced OA who remain exposed to the causal work environment. Further prospective investigations are required in subjects who choose to continue exposure.78 Clinicians should be aware that OA is not always reversible after cessation of exposure to the sensitizing agent. Asthma symptoms and NSBH persist in approximately 70% of the patients with OA several years after their removal from the offending environment.79 Besides

950

SECTION E  Respiratory Tract

environmental interventions, the pharmacologic treatment of OA should follow the clinical practice guidelines for asthma. Systematic treatment with high-dose inhaled corticosteroids in addition to exposure cessation provides only a slight additional benefit.75 Follow-up studies consistently show that a better outcome of asthma after cessation of causal exposure is associated with a shorter symptomatic period before removal and with less severe disease at the time of diagnosis. These findings emphasize the need for early diagnosis and intervention.79 The management of OR not only should aim at reducing nasal symptoms and their impact on quality of life but may also offer the opportunity to prevent the subsequent development of OA, in keeping with the fact that OR is regarded as an early marker of OA. Complete cessation of exposure should theoretically be recommended to workers with OR. Only a few quantitative estimates of the risk of OA in workers with OR have been derived, however; accordingly, a reduction of exposure should be considered a reasonable option when complete avoidance would have important adverse socioeconomic consequences.

PREVENTION Primary prevention aims at blocking, or at least limiting, the development of immunologic sensitization and OA by excluding susceptible workers from at-risk jobs and minimizing exposure to potentially sensitizing substances.4 Primary preventive strategies for OA and OR should focus on the control of workplace exposures, because strong evidence supports a dose-response relationship between the level of exposure to sensitizing agents and the occurrence of OA (Table 56.4). Identifying susceptible persons at the time of preemployment examination to exclude them from employment or from high-risk jobs is inefficient and unduly discriminatory.4 The currently identified markers of individual susceptibility (Table 56.4) offer only a low positive predictive value for the development of OA, especially when these markers, such as atopy, are highly prevalent in the general population. Secondary prevention of immunologic OA and OR involves detection of the disease process at an early (preferably preclinical) stage, to prevent the development of overt OA and to modify the disease process through appropriate interventions to eliminate exposure. The rationale underlying secondary prevention is the consistent finding that the outcome of OA is better with an early diagnosis and milder disease at the time of removal from exposure.79 Increasing awareness of the disease among workers and health care professionals is a key step to enhance the recognition of OA, because the condition still remains underdiagnosed and inappropriately investigated. Recent evidence suggests that appropriately designed surveillance programs are effective in identifying OA in subjects with less severe asthma and a more favorable outcome.80 A few observational studies and historical data indicate that prevention is effective in reducing the incidence of OA and OR caused by natural rubber latex in health care workers,81 enzymes in the detergent industry,82 flour,83 laboratory animals,84 and isocyanates.80 However, available data do not distinguish among the relative effects of the diverse components of prevention strategies, because such strategies usually are implemented as multicomponent programs targeting education, control of exposure, and medical surveillance.

SOCIOECONOMIC IMPACT AND MEDICOLEGAL ASPECTS In the United States, it has been estimated that the total cost of OA was $1.6 billion in 1996, including 76% in direct costs (health care expenditures), when assuming that 15% of adult asthma is attributable to workplace exposures.85 However, OA is likely to generate higher indirect costs than

non-OA, because the former condition most often requires job changes to either avoid or reduce exposure to the causative agent or agents. Followup studies of workers with OA have consistently documented that the condition is associated with a high rate of prolonged unemployment, ranging from 18% to 69%, with a reduction in work-derived income in 44% to 74% of affected workers.86 Complete avoidance of exposure to the sensitizing agent, employment in smaller-sized companies, a lower level of education, older age, and lack of effective job retraining programs are associated with a worse socioeconomic outcome.64 Data derived from Quebec’s Public Health Insurance Plan87 have shown that OA is associated with higher rates of physician visits, admission to an emergency department, and hospitalizations compared with asthma unrelated to work. Although medical resource utilization decreases after removal from exposure to the incriminated workplace, an excess rate of visits to physicians and emergency departments has been documented for such workers compared with other asthmatics. The socioeconomic impact of OR has rarely been investigated but is likely to be substantial.88 Because bronchial hyperresponsiveness to occupational agents almost never completely disappears, workers with OA should be considered to be permanently and completely disabled for jobs involving exposure to the sensitizing agent that caused their OA. They should be thoroughly informed about the possibilities for compensation, and established cases should be reported to the appropriate public health authorities, in accordance with national regulations. Evaluation of physiologic impairment should take into account the characteristic features of asthma and should be based on the level of airway obstruction, the degree of NSBH, the medication regimen required for controlling asthma, and the effects of asthma on quality of life.

SPECIFIC AGENTS CAUSING OCCUPATIONAL ASTHMA AND RHINITIS This section presents an overview of agents that are well known to cause OA, as well as agents that are increasingly reported to do so. Also highlighted are new findings in relation to those agents.

High Molecular Weight Agents

Baking Products.  Bakers and pastry makers are exposed to a variety of potentially sensitizing agents, including (1) flour from cereals (wheat, rye, and barley) and other sources (i.e., soy, lupine, and buckwheat); (2) enzymes used to improve the baking process (i.e., fungal amylase, cellulase, and xylosidase); (3) egg proteins; and (4) other ingredients (e.g., sunflower seeds, soy lecithin). Occasional cases of OA in bakers have been related to insects, storage mites, and molds.89 Baking products are the leading cause of OA in most Western countries.90 Estimates of the mean annual incidence of OA among bakery workers derived from notification programs and compensation-based statistics range from 0.8 to 2.4 cases per 1000 exposed workers. In a cohort study of bakery apprentices, the cumulative incidence rates were 12.4% for IgE-mediated sensitization to bakery allergens, 8.4% for OR, and 6.1% for OA, the latter two conditions being documented by positive immunological tests and specific nasal or bronchial provocation tests. The validity of skin-prick tests (SPTs) with commercial extracts of wheat and rye for diagnosing flour-induced OA has been recently compared with that of the determination of specific IgE antibodies using either ImmunoCAP or ELISA methods and of SICs with flour.91 Large variations were found among commercial cereal extracts in protein concentrations and composition, leading to wide differences in the sensitivity and specificity of SPT results. The sensitivity of specific IgE measurement was generally higher than that of SPT. The study investigators found that high titers of specific IgE and marked skin reactivity to flour extracts were good predictors of a positive inhalation challenge.

CHAPTER 56  Occupational Allergy and Asthma Progress has been made in the characterization of bakery allergens, particularly cereal-derived allergens. Salt-soluble proteins seem to be mainly involved in baker’s asthma, and prolamins (extractable in alcoholwater mixtures) in wheat-dependent exercise-induced anaphylaxis, whereas both protein fractions bind IgE from food-allergic patients.92

Latex.  Natural rubber latex (NRL), a milky fluid collected by tapping the tropical rubber tree Hevea braziliensis (botanical family Euphorbiaceae), is used for the production of a wide variety of medical and nonmedical materials. During the late 1980s, it became apparent that trace amounts of proteins that persist in NRL materials could cause a wide spectrum of immediate IgE-mediated hypersensitivity reactions ranging from mild urticaria to extensive angioedema and life-threatening anaphylaxis after cutaneous, mucosal, or visceral exposure. In the early 1990s, NRL proteins were identified as potential airborne allergens causing rhinitis and asthma through their binding to glove powder particles.93 Fifteen NRL allergens have been characterized at the molecular level.93 The use of recombinant or highly purified natural proteins revealed that health care workers are sensitized predominantly to Hev b5 and Hev b6.01/6.02, whereas Hev b1 and Hev b3 are the major allergens for patients with spina bifida, indicating that the pattern of IgE reactivity is dependent on the route of sensitization.94 NRL allergy reached an epidemic pattern during the late 1980s and early 1990s owing to the increased use of latex gloves and condoms for the prevention of viral infections and the change in NRL processing that resulted from the increasing demand for gloves. A metaanalysis of surveys published before 2004 showed that the prevalence of NRL allergy among health care workers was 3.9% (95% CI, 3.5% to 4.4%)—significantly higher than in the general population (1.4%; 95% CI, 0.4% to 2.3%).95 A high proportion of health care workers with NRL allergy develop OR and OA. NRL-induced OA and OR also have been documented in various nonmedical environments: those of glove manufacturing workers, hairdressers, food processors, laboratory and pharmaceutical industry workers, and greenhouse workers. With today’s deeper understanding of the public health issue posed by NRL allergy, efforts have been made to quantify and reduce the protein allergen content in gloves, and the American Society for Testing and Materials has recommended that the amount of elutable protein be less than 200 µg/m2 (approximately 50 µg/g of glove) for medical gloves (ASTM D3577 and D3578). Preventive policies have been implemented in many health care facilities. Substitution of powdered NRL gloves by gloves with a low content of protein and powder resulted in a significant reduction in NRL sensitization and OA. Of importance, several studies show that health care workers with NRL allergy and OA have been able to continue their work in health care settings in which low-allergen, low-protein, and usually powder-free gloves have been used.

Low Molecular Weight Agents

Diisocyanates.  Polyisocyanates are the most common causes of OA in many parts of the world. Diisocyanates are the forerunners of all polyisocyanates, and 2.4- and 2.6-toluene diisocyanates (TDIs) are the most important ones used commercially. TDI was the first diisocyanate suspected of causing OA.96 Diisocyanates are used in the automobile industry in the production of foam rubber cushions and for finish coating. Methylene diphenyl diisocyanate (MDI) is used in foundries in the making of all mold and core processes and for insulation, whereas hexamethylene diisocyanate (HDI) is used extensively in spray paints. A recent surveillance study reported the industries in which workers were at risk to develop WRA related to exposure to isocyanates in four American states, California, Massachusetts, Michigan, and New Jersey,

951

from 1993 to 2008. The industries with the most isocyanate-induced WRA cases were reported as transportation equipment manufacturing, rubber and miscellaneous plastics manufacturing, and chemicals and allied products manufacturing.97 Diisocyanates are characterized by the presence of an –N=C=O group. Exposure to diisocyanates can induce, in addition to OA, various respiratory conditions that include RADS and hypersensitivity pneumonitis. OA will develop in approximately 5% to 10% of workers exposed to diisocyanates. The threshold limit value has been in many jurisdictions set at 5 parts per billion (ppb). Atopy and smoking are not important risk factors in TDI-induced asthma. Although the clinical features of diisocyanate-induced asthma are those of an allergic disease, the immunologic mechanisms underlying the disease are still not known despite considerable work. Specific IgE antibodies to TDI are found in only a small proportion of patients with allergic disease documented by specific challenge tests.98 The level of increase in specific IgE has been found to be specific for the presence of OA. An increase in the level of specific IgE has been found to be a marker for the presence of OA. The level of specific IgE is higher in patients who have a better prognosis after removal from exposure, as shown by Piirila and coworkers,99 who investigated 245 subjects with OA caused by various types of diisocyanates 3 to 20 years after removal from exposure. By examining bronchial biopsy specimens from seven workers after positive diisocyanate challenges that showed the absence of Cε and IL-4 mRNA-positive cells, contrary to what is found after challenges in which the mechanism is IgE-mediated, Jones and associates100 concluded that diisocyanates do not exert their effect through an IgE-mediated mechanism, at least in workers in whom specific IgE levels are not elevated. For some, increased specific IgG is more specific for the disease than increased specific IgE.17 Having specific IgG may be associated more directly with exposure than with the disease. Levels of specific IgG are specific for the type of diisocyanate concerned (e.g., MDI) and are higher when assessed against the polymer of diisocyanates than the monomer. Bernstein and associates showed that diisocyanate antigen– stimulated monocyte chemoattractant protein 1 (MCP-1) synthesis has greater test efficiency than do IgE- and IgG-specific antibodies for identification of diisocyanate asthma.101 Levels of specific antibodies and of MCP-1 decrease after cessation of exposure. Biologic monitoring of diisocyanates by urine assay for diamine has been described. Evidence that cell-mediated hypersensitivity may be responsible for airway inflammation can be derived from animal models of TDI-induced respiratory allergy and from immunohistochemical analyses of bronchial biopsy specimens from patients with TDI-induced asthma showing an increase in activated T cells and in studies of T cell clones from such specimens demonstrating predominance of CD8+ cells producing IL-5 and IFN. HDI can conjugate to proteins to form adducts.102 Tools to investigate possible OA from diisocyanates are similar to the ones generally used (as described earlier in the discussion on patient evaluation and diagnosis). Most patients with diisocyanate-induced asthma failed to recover even years after removal from exposure, although OA related to HDI seems to carry a better prognosis than OA related to TDI and MDI.99 Tremendous efforts have been made to lower and monitor the threshold of exposures to isocyanates in the occupational environment and to improve protective measures and equipment for workers, such as respirators. Efficient surveillance programs have been implemented.80 Although isocyanates remain one of the most frequent LMW agents causing OA, the number of OA cases caused by isocyanates has decreased markedly since the late 1980s. In Ontario, a substantial decrease has occurred in the number of cases of OA related to isocyanates from that reported in the late 1990s,103 probably as a consequence of implementation of surveillance programs. There is some evidence supporting a

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SECTION E  Respiratory Tract

decline in the incidence of isocyanate-induced OA in industrialized countries.90,103,104

Wood Dust.  Exposure to wood dusts has been shown to give rise to OA, as well as to hypersensitivity pneumonitis, chronic bronchitis, mucous membrane irritation, and organic dust toxic syndrome.105 Of these, asthma is the most serious and common health problem among exposed workers. Cumulative exposure to wood dust seems to affect airway function, showing an inverse correlation between the level of cumulative exposure and FEV1 and FEV1/forced vital capacity (FVC).106 OA induced by Western red cedar is the one most extensively studied and is common in the Pacific Northwest region. It is caused by inhalation of plicatic acid, present predominantly in Western red cedar and, in smaller amounts, in Eastern white cedar.107 Plicatic acid, with a molecular weight of 440, has been shown to induce specific bronchial reactions on bronchial challenge testing in exposed workers with a history compatible with OA. Isolated late phase and biphasic reactions are the most common type of bronchial reactions induced, whereas isolated immediate reaction is uncommon. As in TDI-induced asthma, plicatic acid probably conjugates with a body protein to form a complete antigen. Still, specific IgE antibodies to plicatic acid–human serum albumin conjugate were found in only approximately 20% of patients with documented disease by specific challenge tests. As in TDI-induced asthma, studies have shown that T cells may participate directly in inducing airway inflammation in Western red cedar asthma. The prevalence of Western red cedar asthma is between 5% and 10%, depending on the degree of exposure: the higher the exposure, the higher the prevalence. The permissible concentration of Western red cedar dust was reduced from 10 mg/m3 to 2 mg/m3 in 1990 in British Columbia. Even at 2 mg/m3, cases of Western red cedar asthma were found. The presence of specific IgE antibodies has only been convincingly documented in subjects with OA induced by obeche wood dust.108

Hairdressing Products.  Studies from several countries have shown that hairdressers constitute a category of workers at high risk for development of OA and rhinitis. In Sweden, the reported incidence of asthma during subjects’ active years as hairdressers was 3.9 per 1000.109 In the French national surveillance program on OA, hairdresser was the fourth most frequent occupation reported in workers with the disorder, accounting for 6.8% of all cases of OA from 1996 to 1999.110 The agents most frequently responsible for OA and rhinitis in hairdressers are bleaching products, followed by persulfate. Hair dyes and latex also can be responsible for OA in this setting. OR is associated with 57.7% of the cases of OA in Italy.111

Quaternary Ammonium Compounds and Cleaning Agents.  Epidemiologic studies provided consistent evidence that exposure to cleaning and disinfecting products is associated with high rates of asthma-like symptoms in professional cleaners.112 In the past 2 decades, cleaningrelated exposures have been identified as one of the main causes of physician-diagnosed OA, accounting for 5% to 15% of reported cases.113–115 Cleaning materials may contain a wide variety of ingredients, some of which are respiratory irritants, such as chlorine-releasing agents, ammonia, and solvents, whereas others are potential airway sensitizers, notably quaternary ammonium compounds. SIC-based studies suggested that a substantial proportion of cleaners who experience asthma symptoms related to cleaning materials actually suffer from sensitizer-induced OA, predominantly caused by the quaternary ammonium compounds that are currently widely used as biocides in cleaning products116,117 Although the underlying pathophysiologic mechanism remains largely uncertain, a substantial proportion of asthmatic reactions induced by

quaternary ammonium compounds were associated with a significant increase in post-challenge NSBH and/or an increase in sputum eosinophils, supporting a specific hypersensitivity mechanism.

WORK-RELATED ANAPHYLAXIS Although a majority of work-related allergic symptoms are respiratory and cutaneous, occupational agents also can induce anaphylactic reactions. A recent consensus statement on occupational anaphylaxis published by the European Academy of Allergy and Clinical Immunology (EAACI) has defined occupational anaphylaxis as “anaphylaxis arising out of triggers and conditions attributable to a particular work environment.” Food proteins, medications, insect bites/stings, mammal and snakes bites, natural rubber latex, and a variety of chemicals have been reported to elicit occupational anaphylaxis.118 The acute and long-term management of this condition has been well described in a recent review.118 Aside from the initial resuscitation measures consisting mainly in the administration of adrenaline, oxygen, systemic corticosteroids, and fluid support, the long-term management of this condition relies on the identification of the triggering occupational allergen as well as the removal from exposure to this allergen at the workplace, but also in the domestic environment if applicable (e.g., latex).119

SUMMARY During the past decade it has become apparent that OA and OR are highly prevalent and underdiagnosed diseases that impose a large medical and socioeconomic burden on affected workers and society in general. Because a late diagnosis of OA is associated with a poor outcome, and because prevention programs have been shown to decrease the incidence of new cases of OA, efforts should be made to increase the awareness of OA and OR in primary care practice. Evaluating the cost-effectiveness of preventive measures and compensation systems should become a priority for assisting policy makers in elaborating rational strategies. Current understanding of the pathophysiology of OA is limited, especially regarding OA induced by LMW agents. More research is needed to identify biologic markers, allowing an easier and more accurate way of diagnosing OA and OR, as well as developing effective surveillance programs for use in high-risk work forces. Although additional tools such as noninvasive measures of airway inflammation have been made available for the investigation of OA in the past decade, its diagnosis remains difficult and requires a comprehensive investigation to avoid any misdiagnosis. Access to centers that offer a comprehensive investigation is limited. Furthermore, no current standardized approach to the investigation of OA has yet been established. Development and standardization of diagnostic procedures and consensus diagnostic algorithms for OA and OR would be helpful in formulating a consistent approach to the management of OA and OR worldwide. Determining the cost-effectiveness of different management options requires further investigations using the outcomes that have been validated for the evaluation of asthma and rhinitis, such as the level of disease control, disease-specific quality of life, and measurements of airway inflammation.

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CHAPTER 56  Occupational Allergy and Asthma

955.e1

SELF-ASSESSMENT QUESTIONS 1. A baker reporting a history of childhood asthma decided to quit his workplace because of a reoccurrence of asthma symptoms. This baker was investigated 2 months after he left his job. Which factors can exclude the diagnosis of occupational asthma? a. Childhood asthma b. Absence of airway hyperresponsiveness c. Normal spirometry d. Absence of eosinophilic inflammation e. None of the above 2. What is the most effective intervention in the management of sensitizer-induced occupational asthma? a. Complete removal from exposure b. Reduction of exposure c. Treatment with anti-IgE therapy d. Immunotherapy targeting the occupational agent. 3. Which agent has not been reported to be associated with cases of sensitizer-induced occupational asthma? a. Flour b. Cleaning agents c. Enzymes d. Solvents

4. Which test is sufficient to make a diagnosis of occupational asthma with a high level of certainty? a. Spirometry with assessment of reversibility b. Methacholine inhalation challenge test c. Skin prick-test specific to the suspected agent d. Sputum cell counts e. None of the above 5. Which are the most frequently responsible occupational agents (one or more correct answer)? a. Coffee b. Flour c. Cyanoacrylates d. Diisocyanates e. None of the above

57  Pathology of Asthma Margaret M. Kelly

CONTENTS Introduction, 956 Classifying Asthma, 956 Mild or Moderate Asthma, 957

SUMMARY OF IMPORTANT CONCEPTS • Asthma is a heterogeneous condition with considerable variability in inflammatory and structural cells in the airways throughout the bronchial tree; distinctive changes are also seen in the alveolar parenchyma. • Airway inflammation in mild or moderate asthma is eosinophilic in only approximately 50%. • Inflammation in mild, moderate, or severe asthma and exacerbations of asthma may be eosinophilic, neutrophilic, a mixture of both, or paucigranulocytic. • Airway inflammation correlates moderately well with airway hyperresponsiveness, depending on the technique used for assessment. • Airway structural changes referred to as remodeling include epithelial metaplasia, hyperplasia and loss of integrity, subepithelial fibrosis, increased airway smooth muscle mass, and neoangiogenesis. • Airway remodeling is related to persistent airflow limitation and airflow obstruction and correlates with the severity of disease. • Airway remodeling has been detected in children as young as 1 to 3 years. • Understanding the spatial localization and temporal sequence of processes such as inflammation and remodeling in asthma is important for the design of appropriate animal models, permitting translation of findings to therapeutic interventions in patients. • The current phenotype/endotype concept of asthma has a strong focus on clinical and inflammatory characteristics, and it is important to incorporate the associated pathologic structural changes in these groups.

Severe or Fatal Asthma, 963 Conclusion, 965

(http://ginasthma.org/2016-gina-report-global-strategy-for-asthmamanagement-and-prevention/). Similarly, the pathologic changes in asthma exhibit heterogeneity and are not specific. Nevertheless, in most patients with clinically diagnosed asthma, characteristic abnormalities are usually observed in the airways, which are the focus of this chapter. The pathogenesis of asthma is discussed elsewhere in this textbook. Pathologic changes occur throughout the conducting airways, and not only in the large airways. It is now recognized that the small airways, arbitrarily defined as having an internal diameter of less than 2 mm and corresponding to generations 8 to 23 down to the terminal bronchioles, are a major site of inflammation in asthma.1,2 Because of their extensive branching, the total volume and surface area of the small airways are much greater than those of the large airways and therefore comprise only approximately 10% of airway resistance in health. Disease and obstruction in the small airways is therefore difficult to detect, because up to 75% of the small airways can be diseased before being detectable by routine pulmonary function tests.3,4 It is estimated that the small airways account for 50% to 90% of the obstruction seen in asthma.5 There is also evidence of involvement of the lung parenchyma.6–8 The inflammatory changes are accompanied by structural changes in the airway walls and are collectively referred to as remodeling.

CLASSIFYING ASTHMA INTRODUCTION The term asthma encompasses a set of clinical syndromes rather than a single disease entity. The diagnosis of asthma depends primarily on clinical features, and there is no specific diagnostic test. According to the Global Initiative for Asthma (GINA): “Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation”

956

Classically, asthma has been divided into mild, moderate, and severe forms. Attempts have been made to classify asthma on the basis of clinical features and to correlate these characteristics with pathologic findings and mechanisms of disease. Two broad categories of the asthma syndrome are based on the presence or absence of atopy, evaluated by skin testing and IgE levels. Approximately 50% of patients have features of sensitization to allergens (allergic or extrinsic asthma), whereas the nonatopic patients (nonallergic or intrinsic) tend to be older at onset and in general tend to be more severe.9–11 Patients with allergic asthma usually have elevated levels of circulating immunoglobulin E (IgE), although there is substantial overlap with those levels seen in nonallergic asthma. The recognition of heterogeneity within these groups

CHAPTER 57  Pathology of Asthma has produced further categorization based on age, gender, observable characteristics including triggering factors, patterns of inflammation, severity, complications, and responses to treatment.11 These asthma phenotypes are difficult to define precisely and tend to overlap and are not necessarily directly related to the underlying pathogenic process. Therefore the concept of the “endotype,” whereby a specific biologic pathway is identified that explains the observable properties of a phenotype, may be more useful to develop therapeutic approaches targeting the causative pathomechanism.12,13 Because our understanding of mechanisms of disease in asthma remains incomplete, classification based on endotypes is still evolving, and we will refer to the allergic/nonallergic classification for purposes of convenience, recognizing its limitations. Early autopsy studies yielded information about the pathology of asthma in those who died of asthma (“fatal asthma”), and who died with, but not because of, their asthma (“nonfatal asthma”). Technical advances in the late 1970s and early 1980s, including the introduction of flexible fiberoptic bronchoscopes, made it possible to routinely perform bronchoalveolar lavage (BAL), bronchial biopsy, and segmental allergen challenge.14 In parallel, advances in immunohistochemistry and in situ hybridization have facilitated identification and phenotyping of inflammatory cells. Conclusions based on tissue sampling with bronchial biopsies need to take into consideration the fact that airway smooth muscle abundance varies along the length of the airway and with age.15 In addition, stereologic quantitation methods have only recently been applied to the study of asthma, and studies using less stringent methods may suffer from bias.16,17 The next two sections compare and contrast the pathologic changes of mild or moderate asthma with those of severe or fatal asthma.

MILD OR MODERATE ASTHMA Inflammatory Changes Asthma is a chronic inflammatory disease of the whole bronchial tree, the composition of which is heterogenous and can change rapidly. Although studies have found a poor correlation between cellular profiles in sputum (Fig. 57.1) and airway wall biopsies,18–21 sputum cell counts are more useful clinically.20,22–24 The term eosinophilic asthma is defined by the presence of a significant number of sputum or bronchial wall eosinophils (Fig. 57.1B-C). In noneosinophilic asthma, the dominant inflammatory cell type in sputum may comprise neutrophils (neutrophilic asthma) (Fig. 57.1D) or very few inflammatory cells (paucigranulocytic asthma).25 A mixed eosinophilic and neutrophilic group is also observed. Biopsies of eosinophilic asthma have reported an average of approximately 20 eosinophils/mm2 26,27 up to an average of 98 eosinophils/ mm2.28

Allergic Asthma Significant airway inflammation is present even in mild asthma.29 Bronchial biopsies typically demonstrate increased eosinophils (Fig. 57.2), T lymphocytes, IgE-producing B cells, plasma cells (Fig. 57.3), and mast cells (Fig. 57.4).30 The most well-defined asthma endotype is the T helper type 2 (Th2)-high endotype, which is correlated with allergic asthma and usually responsive to inhaled corticosteroid treatment.30–33 Th2 lymphocytes secrete interleukin-4 (IL-4), IL-5, and IL-13, but it is now recognized that these type 2 cytokines are also secreted by invariant T cells, natural killer (NK) cells, and group 2 innate lymphoid cells (ILC2s).34 Other cell types, such as regulatory T cells and T helper cell

Eos Eos Mac

Neut

Neut

Eos

A

B

CLC

Eos

Eos

Gr

C

957

D

Fig. 57.1  Light photomicrographs of Congo Red–stained cytocentrifuge preparations of induced sputum from patients with asthma. (A) Characteristic morphology of eosinophils (Eos), neutrophils (Neut), and macrophages (Mac). Neutrophils and macrophages are a normal constituent of sputum from healthy subjects, but eosinophils are not. (B and C) The majority of cells consist of eosinophils with numerous free granules (Gr) providing evidence of marked degranulation. A needle-shaped Charcot-Leyden crystal (CLC) is seen in (C). This slide is consistent with eosinophilic asthma (although not specific). (D) Majority of cells consist of neutrophils, and consistent with conditions such as neutrophilic asthma or infection. Congo Red stains eosinophil cytoplasm, granules, and Charcot-Leyden crystals strongly orange. Bar = 20 µm (A & C), 100 µm (B), 50 µm (D).

958

SECTION E  Respiratory Tract

Ep

Eos

Gr

∗ ∗ A

Nu

∗

B

∗ Nu

Gr

∗

Gr

∗ Gr

∗

∗

Gr Gr

Gr

D C

∗

Gr

Fig. 57.2  (A) Light micrograph of an endobronchial biopsy from a patient with asthma, H&E stain. The bronchial epithelium (Ep) has undergone squamous metaplasia and is infiltrated by a few lymphocytes and eosinophils (Eos), whereas the underlying lamina propria shows a marked eosinophilic and mononuclear infiltrate with numerous extracellular eosinophilic eosinophil granules present. (B–D) Transmission electron micrograph of eosinophils and granules in the bronchial wall. (B) A high power image of an eosinophil shows an intact nucleus (Nu) and plasma membrane with the cytoplasm packed with specific (secretory) granules (Gr) with their characteristic ultrastructural appearance. The granules consist of a variably electron-dense crystalline core (*) surrounded by an electron-lucent matrix. Some granules, however, have disassembled cores and reduced electron density typical of piecemeal degranulation, as described in the text. (C) An eosinophil undergoing cytolysis; the nucleus is disintegrating, the plasma membrane has dissolved, and the expelled granules (Gr) are mostly lying freely in the extracellular matrix. (D) A higher magnification showing clusters of free-lying eosinophil granules. Bar = 100 µm (A), 1 µm (B & C), 500 nm (D).

subtypes such as Th9 and Th17, also have important roles in asthma,35 illustrating that the immunology of asthma is complex and is still not fully understood. The presence of eosinophils in the bronchial mucosa has been regarded as characteristic of asthma. Airway secretions and bronchial mucosa characteristically show numerous eosinophils in various stages of degranulation. In the tissues, eosinophils undergo two main forms of degranulation. In piecemeal degranulation, changes occur in the granule matrix and core structures with the cell remaining intact (Fig. 57.2B).36 Granule contents travel in vesicles that fuse with the plasma membrane and release. By contrast, cytolysis is characterized by dissolution of the cell membranes, extrusion of DNA nets, and release of intact granules (Fig. 57.2C). These granules lie singly and as clusters of cell-free extracellular granules (Fig. 57.2D). The granules retain functional receptors and can continue to secrete their contents into the tissues. Charcot-Leyden crystals form in situations of high eosinophil turnover (Fig. 57.1C, and 57.5B). These are needle-like structures composed of the eosinophil lysophospholipase, lysolecithin acylhydrolase. Like the free granules, these structures have the potential to secrete biologically active substances and cause ongoing damage in the tissues.37

However, approximately 50% of patients with mild to moderate asthma are persistently noneosinophilic,38 and these patients are more likely to be atopic (allergic) than nonatopic. In contrast, a study in children found that the presence of eosinophilic or noneosinophilic asthma was independent of atopy.39 Neutrophils appear to be related to smoking in mild to moderate asthma (Fig. 57.3).40,41 Mast cells are broadly categorized into tryptase-positive, chymasenegative (MCT) cells and tryptase-positive, chymase-positive (MCTC) cells. MCTC cells express high levels of carboxypeptidases, whereas MCT cells do not.42 MCT cells usually predominate in the lung, with MCTC cells being present in smooth muscle, submucosal glands, blood vessels, and pleura.43 In a study using stereologic quantitative methods, mast cells in the bronchial epithelium of Th2-high mild to moderate asthma were increased compared with controls and had an unusual phenotype in that they expressed tryptase and carboxypeptidase 3, but not chymase.44 Mast cells45 also accumulate in the smooth muscle cell layer of larger airways and are identified by their characteristic granule ultrastructure (Fig. 57.4).46 Airway inflammation extends peripherally to involve the smaller conducting airways where the inflammation is transmural but external

CHAPTER 57  Pathology of Asthma

959

Ep Ep

Neuts Neuts Plasma cells

A Co Pericyte Neut

D

Nu

RER

B

Endothelium

Nu

µm Nu

Co

Gr

RER

Co

Gr

Plasma cells

Nu

Gr RER

C

E

Fig. 57.3  (A) Light micrograph of an endobronchial biopsy from a patient with asthma, with an immunohistochemical stain for neutrophil elastase (dark brown). Clusters of neutrophils (Neuts) are present in the lamina propria, and a few infiltrate the bronchial epithelium (Ep). (B–C) Transmission electron micrograph of neutrophils in the bronchial wall. (B) A neutrophil is seen exiting a blood vessel and entering the tissue, adjacent to a collagen (Co) bundle. The characteristic segmented nucleus (Nu) and variously shaped granules (including football and dumbbell shapes) identify the cell as a neutrophil. (C) Neutrophil granules in the extracellular matrix from lysed neutrophils. (D) Light micrograph of an endobronchial biopsy with an immunohistochemical stain for CD138 (dark brown), which stains plasma cells and also the basal epithelial layer. (E) Transmission electron micrograph of three plasma cells with eccentric nuclei with heterochromatin and extensive rough endoplasmic reticulum (RER). Bar = 50 µm (A, D), 1 µm (B), 500 nm (C) and 2 µm (E).

to the airway smooth muscle.1,47 T and B lymphocytes may form isolated aggregates, similar to bronchus-associated lymphoid tissue (BALT), but these aggregates are not in a subepithelial location and lack the characteristic structural features of BALT (Fig. 57.5E).48

Nonallergic Asthma Data are conflicting as to whether nonallergic asthmatics exhibit eosinophilic inflammation.27,49 This may be because many studies have relatively small numbers of subjects, but is also likely related to the variability of eosinophil (and neutrophil) counts over time.38,50 In a study based on repeated sputum cell counts, the majority of subjects with persistent noneosinophilic inflammation were allergic.38 Unlike Th2-high asthma, noneosinophilic asthmatics respond poorly to controller medications.30 As previously noted, eosinophilic airway inflammation in children is independent of their atopic status.39 There is also conflicting data as to the incidence of neutrophilic inflammation in nonallergic asthma.27,49,51 The classification of inflammatory type is made more difficult by evidence that corticosteroid therapy prolongs survival of neutrophils.52,53

Remodeling Airway remodeling is broadly defined as any change in composition, distribution, thickness, mass or volume, and/or number of structural components observed in the airway wall of patients relative to the airway wall of normal healthy individuals and is characteristic of all forms of asthma.54–56 Airway remodeling is present in other chronic lung diseases such as chronic obstructive pulmonary disease (COPD), cystic fibrosis, and chronic hypersensitivity pneumonitis, which, like asthma, involve the large and small airways but are distinctly different diseases.57–59 Approximately 20% of patients with obstructive airway disease (asthma or COPD) and 2% of the general population have persistent airflow limitation with a combination of clinical features usually associated separately with asthma or COPD.60 These patients have worse outcomes than with either disease alone.61 The pathologic features of this heterogenous group, referred to as asthmaCOPD overlap syndrome (ACOS),60 have not been well characterized. Structural cells altered in asthma include airway epithelium, interstitium, airway smooth muscle cells, nerve tissue, and bronchial

960

SECTION E  Respiratory Tract

Ep

Mc

Ep

Mc

Mc

SM Mc SM

Mc

A

B

Gr

Gr SM

SM

∗

SM Gr

Gr ∗ Co

C

SM

D

E

Fig. 57.4  (A and B) Light micrograph of an endobronchial biopsy from a patient with asthma, with an immunohistochemical stain for tryptase (dark brown). Mast cells (Mc) are present in the bronchial epithelium (Ep), lamina propria, and smooth muscle (SM). (C–E) Transmission electron micrograph of mast cells in the bronchial wall. (C) An intact mast cell lying adjacent to collagen bundles (Co). The characteristic granules (Gr) (lamellar shapes, scrolls) and lipid bodies (*) identify the cell. (D) An intact mast cell is seen lying within smooth muscle bundles. (E) Extracellular mast cell granules are scattered within smooth muscle bundles. Bar = 100 µm (A), 50 µm (B), 500 nm (C) and 1 µm (D, E).

vasculature, and the key features are summarized in Fig. 57.5.62,63 Remodeling is considered to represent adaptive changes in the quantity and/ or quality of structural cells and extracellular matrix in response to various stress stimuli.64 Airway remodeling may occur very early in the course of the disease, as subepithelial fibrosis has been detected in infancy.65–67 Most studies of remodeling have focused on the large airways, because these are most easily amenable to biopsy and to assessment by conventional pulmonary function tests. However the small airways are abnormal even in mild5,68,69 and well-controlled asthma.70 In general, the descriptions of remodeling in this chapter refer to the large airways unless otherwise specified. There are characteristic changes in the airway epithelium with increased numbers (hyperplasia) of mucin-secreting goblet cells in the large airways, whereas the resident non–mucus secreting epithelial cells in the small airways undergo metaplasia (transformation of a differentiated cell type to another) to goblet cells (Fig. 57.7A).62,63 The type of mucin secreted in Th2-high asthma is altered, with increased MUC5AC protein relative to MUC5B.71 The mucus can be markedly viscous, forming mucus “plugs” with bronchial impaction, and is commonly found in the lungs of patients dying from status asthmaticus. The mucus plugs in these cases consist of mucin, fibrin, and mixed inflammatory cells, including eosinophils. They may also contain Charcot-Leyden crystals and Curschmann’s spirals, which represent mucus originating from distal small airways (Fig. 57.8D). A rare entity, known as plastic bronchitis, can be seen in asthmatics, with characteristic casts of the bronchial tree formed from inspissated mucus.72 A cast expectorated

by a patient with asthma is seen in Fig. 57.8A. On histology, the cast consists of mucin admixed with fibrin, macrophages, lymphocytes, and eosinophils, with Creola bodies, islands of unattached ciliated bronchial epithelial cells (Fig. 57.8B-C).72 If marked eosinophilia is present with lamellated mucin, asthma may be complicated by allergic bronchopulmonary aspergillosis (ABPA), a hypersensitivity to airway saprophytic fungal hyphae, usually Aspergillus fumigatus.72 Although a study in mild and moderate asthma showed no evidence of increased epithelial cell proliferation,73 other studies have conflicting findings with increased epithelial proliferation in allergic mild asthmatics after bronchoconstriction74 and increased proliferation and apoptosis in severe asthmatics.75 In both allergic and nonallergic asthma, there is evidence of increased epithelial fragility and abnormal shedding of epithelial cells (Fig. 57.5A).65,76–78 Artefactual epithelial loss is common after bronchial biopsy, which is traumatic,79 and some studies have failed to show significant differences in epithelial cell loss between controls and asthmatics.80,81 Postmortem autolysis affects epithelial adhesions, and this may be why there is contradictory data from autopsy studies with regard to comparisons between those dying with asthma and controls.82 Nevertheless, there is convincing evidence of disruption of epithelial adherence in asthma,83–86 and markers of injury, such as increased expression of epidermal growth factor receptor (EGFR), have been demonstrated in adults87 and children88 with asthma. Dysregulation of epithelium in asthma patients has recently been extensively reviewed.89 A prominent eosinophilic hyaline layer beneath the epithelium is highly characteristic of asthma, although it is also seen in cystic fibrosis

CHAPTER 57  Pathology of Asthma

961

Eosinophilic airway secretions

Eosinophil and mononuclear infiltrate Ep desquamation

CLC

CLC

C

B

CLC

Cartilage

Increased luminal secretions

MG

G Myofibroblast hyperplasia and fibrosis

D

SM

MG

Angiogenesis

Loss of tethering to parenchyma

Goblet cell metaplasia

A

MG

Cartilage

Increased smooth muscle

SM

SM

Subepithelial fibrosis

F

MG

Lymphoid aggregates

Increased mucous glands

E

Fig. 57.5  (A) Light micrograph of a bronchial wall from an asthmatic patient who died of a nonpulmonary related injury, showing key remodeling features. Large bundles of smooth muscle (SM) and lobules of submucous glands (MG) are seen in the airway wall. (B) Endobronchial biopsy from another patient showing marked eosinophilic and mononuclear inflammatory infiltration in the lamina propria. (C) Secretions full of eosinophils, many degranulated with numerous Charcot-Leyden crystals (CLC). The proliferation of small vessels in the lamina propria is seen under high power in (D). The epithelium shows areas of desquamation. (E) Goblet cell metaplasia and a lymphoid aggregate external to the smooth muscle layer. A high power micrograph of smooth muscle and mucous glands from another airway in the lung is seen in (F). (G) Immunohistochemical stain for α-smooth muscle actin (α-SMA), with myofibroblasts staining positively (brown). Bar = 500 µm (A), 100 µm (B, C), 50 µm (D, G) and 250 µm (E, F). All stains H&E except for G.

and COPD (Fig. 57.6 and Fig. 57.7B).90–93 When this area, referred to as subepithelial fibrosis, is viewed under the electron microscope, it can be seen to consist of three layers. From superficial to deep, these are the electron-clear lamina lucida, the electron-dense lamina densa, and a fibrillary layer, the reticular basement membrane (RBM), also referred to as the lamina reticularis. The lamina lucida and densa together comprise the “true” basement membrane (or basal lamina), which is approximately 80 nm thick (below the resolution of the light microscope) and is not appreciably changed in asthma. The RBM has a normal thickness of approximately 4 µm, depending on the bronchial generation and on tissue processing and measurement technique.94 The RBM is

markedly thickened in Th2-high asthma30 but is reported as only slightly or not thickened in nonallergic asthma, independent of severity.26,27,51 Thickening of the RBM has been described in infants,95–100 and the RBM is already maximally thickened in children aged 6 to 16 years with severe asthma.101 However, studies in childhood asthma showed comparable thickening of the RBM in eosinophilic and noneosinophilic asthma65 and in allergic and nonallergic children with mild asthma.98 There is increased deposition of types I, III, and V collagens in the thickened RBM,94,102 but the collagen fibres are thinner and less banded than the usual interstitial collagens seen in fibrosing interstitial lung diseases.103 The accumulation of matrix proteins, including laminins,104

962

SECTION E  Respiratory Tract

A

C

B

D

Fig. 57.6  (A) Scanning electron micrograph of the airway mucosa from a nonasthmatic person shows the epithelium attached to a reticular basement membrane (RBM) of normal thickness, beneath which there is interstitial collagen. (B) Scanning electron micrograph from a patient with a 25-year history of asthma who died of nonrespiratory causes demonstrates a thickened RBM and damaged epithelium. (C) Transmission electron micrograph of the airway mucosa from a nonasthmatic subject shows basal (B), ciliated (C), and mucous (M) epithelial cells with their true basement membrane (arrow) and underlying bronchial vessels (V) (bar = 10 µm). (D) Transmission electron micrograph shows a thickened RBM (R) in the bronchial biopsy from an allergic asthmatic patient. After allergen challenge, inflammatory cells appear to be in the process of migrating through the thickened layer (arrows) (bar = 10 µm).

tenascin,105 periostin,106 and the proteoglycans biglycan and decorin,107 are responsible for the hyaline appearance. Whether subepithelial fibrosis has a direct functional impact is unclear, but the thickness of the RBM typically correlates well with overall thickening of the airway wall.108 The extent of subepithelial fibrosis has been correlated with increased numbers of subepithelial myofibroblasts,109 which are considered to be part of the respiratory tract epithelial-mesenchymal trophic unit110 that is activated by allergen challenge and the release of Th2 cytokines, leading to the production of growth factors that promote remodeling.61–63 Myofibroblasts are large, elongated cells with numerous synthetic organelles including rough endoplasmic reticulum, similar to fibroblasts; mitochondria; exocytotic vesicles; and a continuous basal lamina, similar to smooth muscle cells, as seen in the electron microscope (Fig. 57.5G and Fig. 57.7C-D).111 They have an enhanced ability to secrete large amounts of collagen and other extracellular matrix proteins and contain cytoplasmic microfilament bundles (“stress fibers”), which provide tensile

strength.112 Most myofibroblasts express α-smooth muscle actin (SMA), which differentiates them from fibroblasts, but is also expressed by smooth muscle cells, pericytes, and myoepithelial cells. Because there is no unique marker for myofibroblasts, the ultrastructural appearance is the gold standard for their identification, although their morphology on light microscopy combined with α-SMA staining is a reliable means of identification.113 Myofibroblasts play an important role in tissue repair but are central to the pathogenesis of fibrosis.114 In the normal lung, myofibroblasts are present in the parenchyma, specifically in the alveolar ducts, at the tips of the alveolar septae, and less commonly within the interstitium of the alveolar walls.115,116 Differing terminologies have been used to describe these cells in the airway, such as “α-SMA positive fibroblasts,” “activated (myo)fibroblasts,”117 and ‘ ‘nonorganized airway contractile elements’ ’ (NOACE).118 Compared with controls, myofibroblasts are increased from 17-fold in moderate to 47-fold in severe asthma and increase approximately 18-fold 24 hours after allergen

CHAPTER 57  Pathology of Asthma

963

Ep

Ep RBM

A

B Ep

∗

MF

MF RER SM

∗

SM

C MG

MG MF

D

2 microns Co

MG

E

100 microns

Fig. 57.7  (A) Light micrograph of bronchial mucosa stained with PAS-Alcian blue to highlight goblet cells in the epithelium (Ep), which stain dark blue. (B) Light micrograph of bronchial mucosa with prominent pink hyaline subepithelial fibrosis or reticular basement membrane (RBM). Weigerts Biebrich Scarlet stain highlights the bright red eosinophil granules present in the lamina propria. (C and D) Transmission electron micrograph of a myofibroblast (MF) in the lamina propria. A large area of dilated rough endoplasmic reticulum (RER) is present in (C) with bundles of microfilaments at the periphery of the cell (*). (D) Myofibroblast with large crenated nucleus, adjacent to bundles of collagen (Co). (E) Light micrograph with α-smooth muscle actin (α-SMA)–positive myofibroblasts beneath the epithelium and large bundles of α-SMA positive smooth muscle (SM) and lobules of submucous glands (MG). Bar = 100 µm (A, E), 50 µm (B), 2 µm (C, D).

challenge,113,119 persisting up to 7 days.117 Thus the lamina propria (the zone between the epithelium and smooth muscle) has increased inflammatory cells, extracellular matrix, myofibroblasts, and blood vessels in asthma (Figs. 57.5 and 57.7). Abnormal deposition of extracellular matrix has also been described in the submucosal and adventitial areas of the large and small airways120–124 and within the alveolar walls124,125 in asthmatic patients. An important feature of asthmatic airway remodeling is an increase in the thickness of airway smooth muscle (Fig. 57.5F, and Fig. 57.7E),15,126,127 and the muscle bundles have been found to lie closer to the epithelium compared with controls.128,129 There is still uncertainty whether there is an increase in airway smooth muscle cell volume (hypertrophy) or cell number (hyperplasia), or both.130 Using unbiased, design-based stereology,16 a biopsy study in adult patients with mild to moderate asthma demonstrated hyperplasia of airway smooth muscle, whereas no significant hypertrophy was found.131 Another study that also used stereology but was conducted on autopsy cases found that hyperplasia was only present in fatal asthma and not nonfatal asthma, which roughly correspond to severe and mild to moderate asthma respectively.2 Hypertrophy was present in the large airways in both groups compared with nonasthmatic controls.2 Several studies confirm that increased deposition of altered types of extracellular matrix in airway smooth muscle contributes to its thickness.2,107,132–134 Preserved orientation of airway smooth muscle has been demonstrated in whole lungs from patients with asthma.135

Airway smooth muscle hypertrophy and hyperplasia in the large airways has been described in children and adolescents with moderate to severe asthma.97,136,137 In addition, increased airway smooth muscle, vascularity, and mucus gland area are described in preschool children.138 However, in a postmortem study of 12 fatal cases of childhood and adolescent asthma, airway smooth muscle area was increased only in a third of large airways of fatal asthma cases and not in the nonfatal asthma cases.100 A significant source of increased mucus in the airways of asthmatics is the mucus-secreting glands in the bronchial submucosa that are increased in number and size, even in moderate asthma (Fig. 57.5F and Fig. 57.7E).82,139 Airway mucosal vascularity is increased in mild to moderate asthma in the large and small conducting airways (Fig. 57.5D).140–142 The density of small vessels (less than 25 µm in diameter) exhibits the most marked increase, consistent with ongoing angiogenesis,143 and the increased permeability of newly formed vessels may contribute to the airway wall edema often described in asthma.142 There is also evidence of remodeling of the bronchial arteries in asthma.144

SEVERE OR FATAL ASTHMA Severe asthma is a heterogenous condition that is defined by the European Respiratory Society/American Thoracic Society guidelines as asthma that requires high-dose inhaled corticosteroids with an additional

964

SECTION E  Respiratory Tract

controller medication and/or systemic corticosteroids to maintain control, or that remains uncontrolled despite therapy.145 This definition therefore includes those patients with an increased risk of frequent severe exacerbations, adverse reactions to medications, nocturnal asthma, and chronic morbidity. Although patients with severe asthma comprise only 5% to 10% of the total asthma population, they account for a large proportion of asthma morbidity and health care expenditures. Cases of fatal asthma obtained at autopsy are likely to represent severe asthma, although there is not always a reliable clinical history to confirm this. Many of the changes of mild or moderate asthma occur in exaggerated form in severe asthma, although there are some distinctive pathologic features.

Inflammatory Changes Classifying patients with severe asthma into inflammatory subtypes is difficult, reflecting the complexity of the immunobiology, treatment with corticosteroids, and the presence or absence of infection. Airway inflammation may be eosinophilic, neutrophilic, a mixture of these cells, or pauciinflammatory.10,146 In fatal asthma, the airways are usually infiltrated with large numbers of degranulated eosinophils and increased degranulated mast cells.147 A recent study found increased numbers of MCTCs in the large airways of patients with uncontrolled asthma on treatment with corticosteroids compared with patients with controlled asthma.125 There is evidence that MCTC cells are associated with remodeling and collagen in the lung.148,149 However, increased sputum neutrophils, “neutrophilic” asthma or “Th2-low” asthma, with or without eosinophils, are more often seen in severe phenotypes compared with the milder forms of the disease (Fig. 57.3A).146,150,151 Neutrophilic inflammation may be related to secretion of IL-17 by Th17 cells,152,153 or by group 3 innate lymphoid cells.154 In some cases, neutrophilic inflammation is likely a consequence of corticosteroid therapy, which selectively inhibits neutrophil apoptosis, promoting their accumulation in tissues.52 A recent study found that so called Th2/Th17-low asthma was composed of neutrophilic or pauci­ inflammatory asthma in approximately equal proportions.155 In severe asthma, there is also selective accumulation of MCTCs in the airway epithelium and submucosa.156 Studies using transbronchial biopsies or surgical specimens have revealed that inflammation in severe asthma extends to the small airways and parenchyma.6–8,47,157 This outer wall area of the small airways is infiltrated by eosinophils, T lymphocytes, mast cells, and some neutrophils, and there are increased numbers of MCTCs in the alveolar walls.125,158 Recently, an entity termed asthmatic granulomatosis has been coined, in which a subgroup of subjects with severe asthma demonstrate infiltration of small airways and alveolar septae by eosinophils and mononuclear cells and interstitial nonnecrotizing granulomas.8,159 Interstitial lung disease, aspiration pneumonia, and hypersensitivity pneumonitis were excluded clinically and radiologically. These patients have apparently responded well to immunosuppressive therapy in addition to corticosteroids, such as azathioprine. Patients with severe asthma but without asthmatic granulomatosis showed more small airway injury, RBM thickening, and neutrophilic infiltrates. Marked inflammation is also observed around small pulmonary arteries.158,160,161

Exacerbations Although most patients respond well to standard asthma management regimens, some develop acute exacerbations of their disease. Much of the serious morbidity of asthma and most of the use of health care resources are related to exacerbations.162 Most exacerbations are triggered by respiratory viral infections. Rhinoviruses, most often the subgroup human rhinovirus species C (HRV-C), are particularly associated with asthma exacerbations.

There are limited numbers of studies on the pathology of exacerbations, because invasive tests are difficult in distressed patients. The numbers of sputum eosinophils in exacerbations have been variably reported, from markedly increased to moderate or low.163 In a study of severe exacerbation requiring hospitalization, increased recruitment of eosinophils and neutrophils was seen in the airway wall.164 However, some studies have shown relatively few eosinophils in the submucosa even in those with high sputum eosinophil counts.20,165 A biopsy study in near-fatal asthma showed increased eosinophils, lymphocytes, and variable numbers of neutrophils and plasma cells.166 Neutrophilic airway inflammation is prominent in fatal asthma resulting from a sudden exacerbation rather than slow-onset fatal asthma in which eosinophilic inflammation dominates.167–169 Rapid-onset death from asthma also exhibits less mucus plugging169 and greater evidence of mast cell degranulation.170

Remodeling Characteristic changes seen in the large airways of fatal asthma include marked congestion and edema, florid inflammatory cell infiltration, and widespread shedding of the epithelium, often with only the basal layer remaining.169 Cilia in the remaining epithelial cells are abnormal,171 and squamous metaplasia is often present (Fig. 57.2A).166 The airway lumina are frequently plugged by inspissated mucus (Fig. 57.8D), which may be primarily responsible for airflow obstruction in these cases. Patients with severe asthma with chronic airflow obstruction who are steroid resistant have an increased RBM thickness compared with those who are steroid responsive.172 Epithelial hypertrophy and proliferation contribute to exaggerated airway remodeling in severe asthma,75 and patients with uncontrolled asthma have increased numbers of myofibroblasts in the large airways.124 Increased airway smooth muscle in fatal asthma is related to both smooth muscle hyperplasia and hypertrophy and to increased fibroblast accumulation, elastic fibers and fibronectin within the airway smooth muscle bundle itself.2,128,132,134 Increased thickness of airway smooth muscle is associated with airway remodeling and eosinophilia in subjects with severe asthma but not with neutrophilia.173 In fatal asthma, there is also a substantial increase in the mass of mucus-secreting submucosal glands compared with nonfatal asthma.174 Small airways also show changes of remodeling in severe and fatal asthma, with significant fibrosis mainly in the outer wall area of the airways.123 The structural alterations at the peribronchiolar level likely contribute to functional abnormalities in severe asthma, because there is also evidence of abnormal alveolar attachments in these airways.122 The magnitude of small airway abnormalities shows correlation with the severity of the disease.175 In an autopsy study of fatal and nonfatal asthma using stereologic methods, smooth muscle hyperplasia was present in small airways in fatal asthma but not in nonfatal asthma.2 In severe asthma, myofibroblasts are significantly increased in the parenchyma (alveolar ducts, alveolar septal tips, and alveolar walls) compared with normal controls and mild to moderate asthma.124,176 Collagen VI, an unusual type of collagen implicated in fibrosis,177,178 is increased in the alveolar parenchyma in patients with uncontrolled asthma compared with those with good control.125 In summary the morphologic features that distinguish severe asthma from mild disease in the large airways are increased submucosal fibroblast/myofibroblast accumulation and collagen deposition, smooth muscle hypertrophy, decreased distance of smooth muscle from the epithelium, and increased area of submucosal glands.124,128,129 In addition, smooth muscle hyperplasia in the small airways2 and the changes described in the lung parenchyma appear to be unique to severe asthma.

CHAPTER 57  Pathology of Asthma

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A B Ep

Eos

Mac

CLC

C

D

Fig. 57.8  (A) Bronchial cast expectorated by a patient with severe asthma. (B and C), Light micrograph of the histology of the cast, with a Creola body in (B) and mixed inflammation, including eosinophils (Eos) and macrophages (Mac), in (C). (D) Inspissated mucus in the airway with numerous Charcot-Leyden crystals (CLC). Bar = 5 cm (A), 50 µm (B), 100 µm (C, D). B-D, H&E stain.

CONCLUSION As the pathogenesis of this complex syndrome continues to be unraveled and alternative approaches for assessment of patients are developed, it is important for clinicians and investigators to remain conscious of the need to correlate structure and function and to understand the spatial localization of processes such as inflammation and remodeling.179 An understanding of the pathologic changes in human asthma is important for the design of appropriate animal models and the interpretation of experiments that attempt to answer basic questions about pathogenesis.180 Although animal models have yielded useful information about the processes and mechanisms of allergic inflammation, we suggest that the inability to translate these findings to therapeutic interventions for patients results from an incomplete understanding of the pathology of human asthma. Many of the pathologic changes seen in asthma can be correlated with clinical findings. The type of inflammation present in the sputum may predict response to treatment, and the degree of remodeling can be correlated with pulmonary function.33,129,181–183 In this era of precision medicine, the pathologic changes assume an even more important role as phenotypes and endotypes are being defined and attempts to target them with specific therapies are being developed.184,185 Equally important is the recognition that noninvasive biomarkers need to be correlated with the inflammatory and structural changes in the lungs of patients with asthma.186 There remain gaps in our knowledge about the pathology of asthma, particularly in the different clinical phenotypes in children,100,187 lateonset asthma, and in acute exacerbations. We do not know the origin of the enigmatic myofibroblast and how long it survives in the airway before undergoing apoptosis.119,188 There is still controversy regarding hypertrophy versus hyperplasia of airway smooth muscle, and the recent description of asthmatic granulomatosis needs further investigation in more subjects. The microbiome of the lungs and airways in asthma is

only starting to be elucidated.189 Our knowledge of the effect of various treatments on the pathologic changes in asthma is limited.190 Noninvasive and nontissue sampling techniques to investigate remodeling in the lung show promise but are beyond the scope of this chapter.191,192 However, careful validation of changes seen in these noninvasive tests with pathologic changes needs to be performed.183

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129. Pepe C, Foley S, Shannon J, et al. Differences in airway remodeling between subjects with severe and moderate asthma. J Allergy Clin Immunol 2005;116(3):544–9. 130. Ebina M, Takahashi T, Chiba T, et al. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993;148(3):720–6. 131. Woodruff PG, Dolganov GM, Ferrando RE, et al. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169(9):1001–6. 132. Bai TR, Cooper J, Koelmeyer T, et al. The effect of age and duration of disease on airway structure in fatal asthma. Am J Respir Crit Care Med 2000;162(2 Pt 1):663–9. 133. Yick CY, Ferreira DS, Annoni R, et al. Extracellular matrix in airway smooth muscle is associated with dynamics of airway function in asthma. Allergy 2012;67(4):552–9. 134. Araujo BB, Dolhnikoff M, Silva LF, et al. Extracellular matrix components and regulators in the airway smooth muscle in asthma. EurRespir J. 2008;32(1):61–9. 135. Ijpma G, Panariti A, Lauzon AM, et al. Directional preference of airway smooth muscle mass increase in human asthmatic airways. Am J Physiol Lung Cell Mol Physiol 2017;312(6):L845–54. 136. Regamey N, Ochs M, Hilliard TN, et al. Increased airway smooth muscle mass in children with asthma, cystic fibrosis, and non-cystic fibrosis bronchiectasis. Am J Respir Crit Care Med 2008;177(8):837–43. 137. Bossley CJ, Fleming L, Gupta A, et al. Pediatric severe asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines. J Allergy Clin Immunol 2012;129(4):974–82.e13. 138. Lezmi G, Gosset P, Deschildre A, et al. Airway remodeling in preschool children with severe recurrent wheeze. Am J Respir Crit Care Med 2015;192(2):164–71. 139. Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969;24:176–9. 140. Hashimoto M, Tanaka H, Abe S. Quantitative analysis of bronchial wall vascularity in the medium and small airways of patients with asthma and COPD. Chest 2005;127(3):965–72. 141. Siddiqui S, Sutcliffe A, Shikotra A, et al. Vascular remodeling is a feature of asthma and nonasthmatic eosinophilic bronchitis. J Allergy Clin Immunol 2007;120(4):813–19. 142. Harkness LM, Ashton AW, Burgess JK. Asthma is not only an airway disease, but also a vascular disease. Pharmacol Ther 2015;148:17–33. 143. Feltis BN, Wignarajah D, Zheng L, et al. Increased vascular endothelial growth factor and receptors: relationship to angiogenesis in asthma. Am J Respir Crit Care Med 2006;173(11):1201–7. 144. Green FH, Butt JC, James AL, et al. Abnormalities of the bronchial arteries in asthma. Chest 2006;130(4):1025–33.

Pathology of Severe or Fatal Asthma 145. Chung KF, Wenzel SE, Brozek JL, et al. International ERS/ATS guidelines on definition, evaluation and treatment of severe asthma. Eur Respir J 2014;43(2):343–73. 146. Ray A, Raundhal M, Oriss TB, et al. Current concepts of severe asthma. J Clin Invest 2016;126(7):2394–403. 147. Chen FH, Samson KT, Miura K, et al. Airway remodeling: a comparison between fatal and nonfatal asthma. J Asthma 2004;41(6):631–8. 148. Hirata K, Sugama Y, Ikura Y, et al. Enhanced mast cell chymase expression in human idiopathic interstitial pneumonia. Int J Mol Med 2007;19(4):565–70. 149. Kosanovic D, Dahal BK, Wygrecka M, et al. Mast cell chymase: an indispensable instrument in the pathological symphony of idiopathic pulmonary fibrosis? Histol Histopathol 2013;28(6):691–9. 150. Moore WC, Hastie AT, Li X, et al. Sputum neutrophil counts are associated with more severe asthma phenotypes using cluster analysis. J Allergy Clin Immunol 2014;133(6):1557–63.e5. 151. Bhakta NR, Woodruff PG. Human asthma phenotypes: from the clinic, to cytokines, and back again. Immunol Rev 2011;242(1): 220–32.

CHAPTER 57  Pathology of Asthma 152. Lajoie S, Lewkowich IP, Suzuki Y, et al. Complement-mediated regulation of the IL-17A axis is a central genetic determinant of the severity of experimental allergic asthma. Nat Immunol 2010;11(10):928–35. 153. Doe C, Bafadhel M, Siddiqui S, et al. Expression of the T helper 17-associated cytokines IL-17A and IL-17F in asthma and COPD. Chest 2010;138(5):1140–7. 154. Lambrecht BN, Hammad H. The immunology of asthma. Nat Immunol 2015;16(1):45–56. 155. Liu W, Liu S, Verma M, et al. Mechanism of T(H)2/T(H)17-predominant and neutrophilic T(H)2/T(H)17-low subtypes of asthma. J Allergy Clin Immunol 2017;139(5):1548–58.e4. 156. Balzar S, Fajt ML, Comhair SA, et al. Mast cell phenotype, location, and activation in severe asthma. Data from the Severe Asthma Research Program. Am J Respir Crit Care Med 2011;183(3):299–309. 157. Balzar S, Wenzel SE, Chu HW. Transbronchial biopsy as a tool to evaluate small airways in asthma. Eur Respir J 2002;20(2):254–9. 158. Andersson CK, Bergqvist A, Mori M, et al. Mast cell-associated alveolar inflammation in patients with atopic uncontrolled asthma. J Allergy Clin Immunol 2011;127(4):905–12.e1–7. 159. Wenzel SE, Vitari CA, Shende M, et al. Asthmatic granulomatosis: a novel disease with asthmatic and granulomatous features. Am J Respir Crit Care Med 2012;186(6):501–7. 160. Saetta M, di Stefano A, Rosina C, et al. Quantitative structural analysis of peripheral airways and arteries in sudden fatal asthma. Am Rev Respir Dis 1991;143:138–43. 161. Shiang C, Mauad T, Senhorini A, et al. Pulmonary periarterial inflammation in fatal asthma. Clin Exp Allergy 2009;39(10):1499–507. 162. Castillo JR, Peters SP, Busse WW. Asthma exacerbations: pathogenesis, prevention, and treatment. J Allergy Clin Immunol Pract 2017;5(4):918–27. 163. Jayaram L, Pizzichini MM, Cook RJ, et al. Determining asthma treatment by monitoring sputum cell counts: effect on exacerbations. Eur Respir J 2006;27(3):483–94. 164. Qiu Y, Zhu J, Bandi V, et al. Bronchial mucosal inflammation and upregulation of CXC chemoattractants and receptors in severe exacerbations of asthma. Thorax 2007;62(6):475–82. 165. Selivanova PA, Kulikov ES, Kozina OV, et al. Morphological and molecular characteristics of “difficult” asthma. J Asthma 2010;47(3):269–75. 166. Barbers RG, Papanikolaou IC, Koss MN, et al. Near fatal asthma: clinical and airway biopsy characteristics. Pulm Med 2012;2012:829608. 167. Sur S, Crotty TB, Kephart GM, et al. Sudden-onset fatal asthma. A distinct entity with few eosinophils and relatively more neutrophils in the airway submucosa? Am Rev Respir Dis 1993;148:713–19. 168. Carroll N, Carello S, Cooke C, et al. Airway structure and inflammatory cells in fatal attacks of asthma. Eur Respir J 1996;9(4):709–15. 169. James AL, Elliot JG, Abramson MJ, et al. Time to death, airway wall inflammation and remodelling in fatal asthma. Eur Respir J 2005;26(3):429–34. 170. Elliot JG, Abramson MJ, Drummer OH, et al. Time to death and mast cell degranulation in fatal asthma. Respirology 2009;14(6):808–13. 171. Thomas B, Rutman A, Hirst RA, et al. Ciliary dysfunction and ultrastructural abnormalities are features of severe asthma. J Allergy Clin Immunol 2010;126(4):722–9.e2. 172. Bourdin A, Kleis S, Chakra M, et al. Limited short-term steroid responsiveness is associated with thickening of bronchial basement membrane in severe asthma. Chest 2012;141(6):1504–11.

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173. Tsurikisawa N, Oshikata C, Tsuburai T, et al. Bronchial hyperresponsiveness to histamine correlates with airway remodelling in adults with asthma. Respir Med 2010;104(9):1271–7. 174. Green FH, Williams DJ, James A, et al. Increased myoepithelial cells of bronchial submucosal glands in fatal asthma. Thorax 2010;65(1):32–8. 175. van der Wiel E, ten Hacken NH, Postma DS, et al. Small-airways dysfunction associates with respiratory symptoms and clinical features of asthma: a systematic review. J Allergy Clin Immunol 2013;131(3):646–57. 176. Boser SR, Mauad T, Araujo-Paulino BB, et al. Myofibroblasts are increased in the lung parenchyma in asthma. PLoS ONE 2017;12(8):e0182378. 177. Specks U, Nerlich A, Colby TV, et al. Increased expression of type VI collagen in lung fibrosis. Am J Respir Crit Care Med 1995;151(6):1956–64.

Conclusion 178. Karsdal MA, Nielsen SH, Leeming DJ, et al. The good and the bad collagens of fibrosis - Their role in signaling and organ function. Adv Drug Deliv Rev 2017;121:43–56. 179. Hassan M, Jo T, Risse PA, et al. Airway smooth muscle remodeling is a dynamic process in severe long-standing asthma. J Allergy Clin Immunol 2010;125(5):1037–45.e3. 180. Holmes AM, Solari R, Holgate ST. Animal models of asthma: value, limitations and opportunities for alternative approaches. Drug Discov Today 2011;16(15–16):659–70. 181. Green RH, Brightling CE, McKenna S, et al. Asthma exacerbations and sputum eosinophil counts: a randomised controlled trial. Lancet 2002;360(9347):1715–21. 182. Berair R, Hartley R, Mistry V, et al. Associations in asthma between quantitative computed tomography and bronchial biopsy-derived airway remodelling. Eur Respir J 2017;49(5). 183. Aysola RS, Hoffman EA, Gierada D, et al. Airway remodeling measured by multidetector CT is increased in severe asthma and correlates with pathology. Chest 2008;134(6):1183–91. 184. Chung KF. Precision medicine in asthma: linking phenotypes to targeted treatments. Curr Opin Pulm Med 2018;24(1):4–10. 185. Muraro A, Lemanske RF Jr, Hellings PW, et al. Precision medicine in patients with allergic diseases: airway diseases and atopic dermatitis-PRACTALL document of the European Academy of Allergy and Clinical Immunology and the American Academy of Allergy, Asthma & Immunology. J Allergy Clin Immunol 2016;137(5):1347–58. 186. Peters MC, Nguyen ML, Dunican EM. Biomarkers of airway type-2 inflammation and integrating complex phenotypes to endotypes in asthma. Curr Allergy Asthma Rep 2016;16(10):71. 187. Mokhallati N, Guilbert TW. Moving towards precision care for childhood asthma. Curr Opin Pediatr 2016;28(3):331–8. 188. Schmidt M, Sun G, Stacey MA, et al. Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 2003;171(1):380–9. 189. Sokolowska M, Frei R, Lunjani N, et al. Microbiome and asthma. Asthma Res Pract 2018;4:1. 190. Berair R, Brightling CE. Asthma therapy and its effect on airway remodelling. Drugs 2014;74(12):1345–69. 191. Richards JC, Lynch D, Koelsch T, et al. Imaging of asthma. Immunol Allergy Clin North Am 2016;36(3):529–45. 192. d’Hooghe JNS, Goorsenberg AWM, de Bruin DM, et al. Optical coherence tomography for identification and quantification of human airway wall layers. PLoS ONE 2017;12(10):e0184145.

CHAPTER 57  Pathology of Asthma

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SELF-ASSESSMENT QUESTIONS 1. Changes described in the airway epithelium in asthma do NOT include which of the following? a. Squamous metaplasia b. Desquamation c. Hyperplasia d. Dysplasia e. Goblet cell metaplasia 2. With regard to induced sputum in asthma, which of the following is true? a. There is a good correlation between inflammatory cell counts in sputum and the bronchial wall. b. Cell counts are clinically useful. c. The percentage of eosinophils is highly repeatable in a patient who has not changed their therapy. d. The presence of neutrophils is always abnormal. e. Charcot-Leyden crystals are diagnostic of asthma. 3. With regard to remodeling in asthma, which one of these statements is true? a. The changes are confined to the large airways. b. Airway inflammation is present before remodeling occurs. c. Thickening of the basement membrane is a part of remodeling. d. Smooth muscle thickening is partially related to increased extracellular matrix within the muscle bundle.

4. With regard to myofibroblasts which one of the following is a true statement? a. Myofibroblasts can be reliably identified on hematoxylin and eosin (H&E) stain based on their shape and position in the airway. b. Ultrastructure is the only way to reliably identify myofibroblasts. c. Myofibroblasts can be reliably identified on light microscopy using a combination of their morphology, position in the tissue, and positive α-smooth muscle actin stain. d. Immunohistochemistry for smooth muscle myosin distinguishes smooth muscle from myofibroblasts. e. Increased myofibroblasts are decreased within the alveolar walls in patients with severe asthma.

58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis, and Epidemic Thunderstorm Asthma Mark Hew, Jo A. Douglass, Tunn Ren Tay, Robyn E. O’Hehir

CONTENTS Introduction, 970 Allergic Bronchopulmonary Aspergillosis, 971 Hypersensitivity Pneumonitis, 977

SUMMARY OF IMPORTANT CONCEPTS • Patients with well-treated but uncontrolled asthma should be screened for allergic bronchopulmonary aspergillosis (ABPA). • Diagnostic criteria for ABPA include asthma, increased total IgE levels, increased specific IgE to Aspergillus, positive skin testing to Aspergillus fumigatus, and central bronchiectasis (may be absent in ABPA serologic). • First-line therapy for ABPA is systemic corticosteroids; the antifungal itraconazole or voriconazole may be considered as a corticosteroid-sparing agent. Preliminary data suggest anti-IgE therapy with omalizumab may reduce exacerbations. • Patients with cystic fibrosis have a higher risk for developing ABPA; worsening of baseline symptoms, increased or colored sputum, onset of fever, weight loss, or decline in lung function should raise suspicion of ABPA. • Hypersensitivity pneumonitis (HP) results from an exaggerated immunologic response to inhaled antigens, causing manifestations ranging from acute infection-like symptoms to subacute and chronic interstitial disease. When exposure to the antigen persists, permanent lung damage may ensue. • First-line therapy for HP is withdrawal of contact with the offending antigen, with or without oral corticosteroids, depending on the severity of presentation. • Epidemic thunderstorm asthma usually occurs because of synergistic effects between weather conditions, airborne allergen loads drawn to ground level by outflows, and individual susceptibility. Grass pollens and fungal spores are the most common reported allergens. • Individual susceptibility is conferred by a “trifecta” of prior sensitization, clinical allergy, and airborne exposure to the offending allergen. Even sensitized individuals without prior asthma symptoms may be vulnerable to epidemic thunderstorm asthma. • Risk mitigation for thunderstorm asthma centers around optimizing asthma management in those with known asthma; treating existing allergic rhinitis in the case of grass pollen triggers (including bronchodilator access as required); forecasting systems to predict events; early warning systems to alert the populace; and flexible emergency services able to respond rapidly to surges in demand.

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Epidemic Thunderstorm Asthma, 981 Summary, 983

INTRODUCTION Allergic bronchopulmonary aspergillosis (ABPA) is a progressive disease resulting from a hypersensitivity response to the presence of the fungus Aspergillus fumigatus in the airways and usually affecting subjects with asthma and cystic fibrosis (CF). Susceptibility to development of ABPA is mediated by genetically determined inflammatory responses strongly linked to atopy. Local inflammation includes an imbalance of helper T cell type 2 (Th2) inflammatory response over Th1 response, with Th2 CD4+ T cell activation; interleukin-4 (IL-4), IL-5, and IL-13 release; IgE synthesis; and inflammatory cell recruitment with a marked eosinophilic response, exaggerated mucus production, and airway hyper­ reactivity. Early symptoms are often confounded by symptoms present in the underlying disease, so ABPA is often overlooked initially. The chronic inflammatory process ultimately leads to permanent lung damage and bronchiectasis. Hypersensitivity pneumonitis (HP) is an inflammatory lung disorder caused by an exaggerated immune response to inhaled antigens to which the subject has previously been sensitized. These antigens, mostly organic, include fungal spores, avian proteins, and mycobacteria; however, substances such as isocyanate and zinc can also cause HP. Clinical features include fever, dyspnea, interstitial infiltrates, and a restrictive ventilatory pattern on lung function testing. Some patients progress to develop lung fibrosis, which can often be mistaken for idiopathic pulmonary fibrosis. Both ABPA and HP can be overlooked or misdiagnosed until irreversible lung damage occurs. Broad awareness and early clinical suspicion are extremely important to allow early diagnosis and treatment, including removal of the culprit allergen source in the case of HP. Thunderstorm asthma describes an asthma exacerbation triggered under thunderstorm conditions. Epidemic thunderstorm asthma occurs when a thunderstorm triggers a number of asthma exacerbations that exceeds that expected for the time period. There is no consensus on the precise number or exact proportion of excess cases required to define a thunderstorm asthma epidemic. What is clear is that such freak events can strike unpredictably and with devastating swiftness. Thousands of

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis patients may exacerbate within the space of a few hours, overwhelming ambulance and health services. Bronchospasm may progress within minutes to a life-threatening stage, and multiple deaths have now been reported in a single epidemic. This chapter covers the etiology and pathogenesis, pathology, clinical presentation, diagnosis, and management of ABPA, HP, and epidemic thunderstorm asthma.

ALLERGIC BRONCHOPULMONARY ASPERGILLOSIS Historical Perspective Allergic bronchopulmonary aspergillosis is a disease of airway colonization and infection by the fungus Aspergillus fumigatus, accompanied by a vigorous immune response that, left unchecked, results in chronic pulmonary damage. Asthma and “aspergillosis” were first associated by Renon in 1897, and the first report of ABPA was published in 1952 by Hinson and colleagues,1 who described three patients with recurrent episodes of “wheezy bronchitis,” serum eosinophilia, sputum production, fever, and infiltrates on chest x-ray films. Although first described in asthma, ABPA was subsequently recognized to complicate cystic fibrosis (CF), and less frequently in other lung diseases such as chronic obstructive pulmonary disease (COPD). Aspergillus fumigatus (A. fumigatus) is the most common fungus causing ABPA, but other Aspergillus species have been implicated. Other fungi such as Candida species (spp.) have been noted to cause similar lung disease, whereby the term allergic bronchopulmonary mycosis (ABPM) is used.

Epidemiology The prevalence of ABPA in asthmatic patients varies depending on the severity of asthma, presence of immediate hypersensitivity to Aspergillus spp. and method of detection, and diagnostic criteria. Therefore the precise prevalence of ABPA in patients with asthma and in CF patients is unknown. Initial reports indicate that among those with asthma sensitized to Aspergillus spp. antigens, 28% met sufficient criteria to make a diagnosis of ABPA.2 Later, Agarwal and associates3 estimated overall prevalence of ABPA in asthmatic populations at 12.9% (95% confidence interval [CI] 7.9 to 18.9).3 The estimated pooled prevalence of Aspergillus spp. sensitization in CF patients was 39.1% (95% CI 33.3-45.1), whereas ABPA was found in 8.9% (95% CI 7.4-10.7) of CF patients.4 Colonization with Aspergillus spp. has been associated with decreased lung function5 in cross-sectional studies, but longitudinal studies have not identified accelerated lung function decline in CF patients colonized by Aspergillus spp. Usually manifesting between the third and fourth decades of life, ABPA has no gender predilection. High levels of outdoor spore counts may be associated with ABPA exacerbations, but a clear relationship between level of exposure to Aspergillus spp. and occurrence of ABPA has not been established.6

Pathogenesis and Etiology Aspergillus spp. occur widely in nature, particularly in decaying vegetable matter, and have a number of innate characteristics that render them opportunistic pathogens. Aspergillus spp. grow through the production of hyphae, from which sprout conidiophores. Aspergillus spp. secrete extracellular proteolytic enzymes of the aspartic, serine, and metalloprotease classes. Fungal spores (conidia) measure between 2.5 and 3.5 mm and are inhaled into the lower airways and alveoli. Adherence of conidia to respiratory epithelial cells occurs with subsequent accrual of cellular dysfunction, the first of which is cilial disruption. As the fungal colony grows, hyphae are produced and can invade between and through epithelial cells, leading to substantial tissue disruption.7

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Inhalation of fungal spores is ubiquitous, but Aspergillus spp. colonization and infection only occur in some patients, suggesting individual susceptibility is important. The breakdown of local nonspecific immunity (e.g., mucociliary clearance mechanisms) likely renders an individual more vulnerable to the adherence of spores to the airway epithelium. Preexisting lung disease such as bronchiectasis, as occurs in CF, also provides an environment for airway colonization, possibly from preexisting airway damage. Other factors may include the biofilm present in the airways of these patients, contributing to impaired spore removal. The presence and proliferation of Aspergillus spp. in the airways lead to inflammatory cell recruitment, early and late phase inflammatory reactions, and in the allergic host a predominance of Th2 signaling over the Th1 response, with release of specific cytokines (IL-4, IL-5, and IL-13) propagating inflammation.8 These mechanisms ultimately lead to total IgE and A. fumigatus-specific IgE production, mast cell degranulation, and an exacerbated eosinophilic response, causing tissue damage (Fig. 58.1). There is evidence to suggest that the Th2 response is triggered by a complex of fungal pathogen–associated molecular patterns (PAMPs) on the Aspergillus spp. conidia, rather than a single fungal component.9 In addition, it appears that T cell responses in ABPA are triggered not by a single antigen, but by a variety of Aspergillus spp. antigens (e.g., Asp f 1, Asp f 2, Asp f 3, Asp f 4, Cr f 1, and Catalase 1). The cytokine profiles produced by T cells in response to each antigen are largely similar.10 After fungal attachment to the respiratory epithelium, specific immune responses are invoked, and several inherited properties render an individual more susceptible to the proliferation of airway Aspergillus spp. in ABPA. Such features relate to the innate immune system, cellular immune responses, antibody responses, and genetics. In the innate immune system, toll-like receptors (TLR) 2 (TLR2), TLR4, and TLR9 are considered important for immunity to Aspergillus spp. A single-nucleotide polymorphism (SNP) in TLR9, a receptor-binding nonmethylated CpG motif, was associated with an odds ratio of 2.5 for ABPA.11 Cellular responses of individuals with ABPA also differ from individuals with asthma. There are different patterns of T cell chemokine receptor expression (CCR4 and CXCR3) in ABPA and non-ABPA allergic asthmatic patients after Aspergillus spp. antigen exposure.12 Responses to fungi may also be influenced by certain serum acute-phase reactants, the pentraxins. Specific antibody responses may differ between individuals with ABPA and those with allergic asthma. Gautum and colleagues13 used proteomics to identify 16 allergens associated with Aspergillus spp. infection, which were variably reactive between those with asthma and ABPA. Another study demonstrated the relevance of the Aspergillus spp. antigen Asp f 34, showing that 94% of the ABPA and 46% of the A. fumigatus– sensitized individuals evaluated had Asp f 34–specific serum IgE.14 Genetic associations with ABPA have been found, with a higher prevalence of cystic fibrosis transmembrane conductance regulator (CFTR) mutations identified in ABPA patients than healthy controls.15 In transgenic mice models the HLA-DR2 genotype, particularly DRB1 1503, conveys enhanced susceptibility to the pulmonary eosinophilic inflammation associated with ABPA.16 Other specific associations with ABPA have been found in IL-4 receptor polymorphisms17 and other cytokine polymorphisms, including IL-13, tumor necrosis factor-α (TNF-α),18 and IL-10.19

Clinical Features The first descriptions of ABPA were of patients with severe asthma who also had radiographic findings of pulmonary consolidation or segmental lung collapse, with associated fever, malaise, and cough productive of brown sputum.1 Diagnosis of asthma is reported to occur in more than

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SECTION E  Respiratory Tract

Early phase reaction Late phase reaction Inflammatory cell influx • Mast cell degranulation • Growth factors • Cytokines • Neutrophils • Chemokines

Release of cytokines ASTHMA CYSTIC FIBROSIS • Genetic susceptibility • Increased mucus viscosity A

BRONCHIECTASIS FIBROSIS • Bronchial damage • Increased IgE • Eosinophilic inflammation • Airway remodeling

• Epithelial barrier disruption • Impaired mucociliary clearance • Innate immune system activation

Facilitated AF growth in the airways Fungus proteolytic activity Release of antigen

Lungs Propagation of inflammation

B Dendritic cells Th2 over Th1 T cell response

IL-4, IL-13, GM-CSF

Eosinophilic response

IL-5

Eosinophils

CD 23 CD 86 B cell

IgE, IgG, IgA

Fig. 58.1  Pathogenesis of allergic bronchopulmonary aspergillosis (ABPA). In patients with genetic predisposition and increased pulmonary mucus viscosity, inhalation of Aspergillus fumigatus (AF) may result in persistent colonization and deposition of hyphae, releasing antigens. (A) Antigens disrupt epithelial barrier, damage mucociliary clearance, and activate innate immune response, ultimately resulting in influx of inflammatory cells and release of cytokines, with propagation of neutrophilic inflammation and early- and late-phase inflammatory reactions. (B) Antigens cause skewing of Th2 over Th1 cell response after processing of the antigen by dendritic cells, with release of Th2 cytokines (IL-4, IL-5, and IL-13) resulting in propagation of inflammation and significant eosinophilic inflammation, causing tissue damage and production of IgE. Tissue damage and bronchiectasis result from the pulmonary influx of eosinophils and neutrophils. GM-CSF, Granulocyte macrophage colony–stimulating factor; IL, interleukin.

90% of patients with ABPA, most with asthma for over a decade.20 ABPA was subsequently described in CF patients. Patients with asthma or CF who develop ABPA typically have wheeze, cough with thick, brown sputum, or plugs of mucus with histologic evidence of eosinophilic debris and Aspergillus spp. hyphae. Hemoptysis is described but rarely severe.21 Systemic symptoms such as fever, weight loss, and fatigue are common in ABPA and should raise suspicion of the condition when seen in patients with asthma and CF. The clinical picture is often accompanied by typical radiologic findings. Not all patients develop permanent pulmonary lesions, and pulmonary parenchymal infiltrates on chest radiography may disappear with treatment. Therefore ABPA may be diagnosed in the absence of radiologic abnormalities, referred to as ABPA serologic (ABPA-S), considered a milder form of the disease. The range of lung features observed

in ABPA may vary from the presence of all clinical features with no pulmonary opacities to the presence of classic, dominantly central bronchiectasis or end-stage fibrosis with associated respiratory failure, depending on the disease stage, which has prognostic and therapeutic implications. Staging of ABPA is described in Table 58.1.

Diagnosis Widely accepted international criteria for the diagnosis of ABPA are lacking, and different criteria have been suggested (Box 58.1). Initial reports focused on diagnostic criteria of asthma, positive Aspergillus spp. immediate skin test, serum eosinophilia, and presence of immunoglobulin G (IgG) precipitins to Aspergillus spp. antigens.22 The presence of pulmonary abnormalities, especially bronchiectasis, was also necessary for diagnosis. Subsequently, ABPA was divided further according

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis

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TABLE 58.1  Clinical Staging of Allergic Bronchopulmonary Aspergillosis Clinical

Immunologic

Radiologic

Conventional Staging Stage 1 Acute

Symptomatic

IgE >1000 IU/ml

Normal or presence of pulmonary infiltrates

Stage 2

Asymptomatic

35% decline in total serum IgE by 6 weeks and stabilization of total serum IgE for 6 months with prednisolone

Significant resolution of pulmonary infiltrates (unless fibrosis present)

a

Remission

Stage 3

Exacerbation

Symptoms may not be present

Doubling of total IgE from baseline

New pulmonary infiltrates

Stage 4

Corticosteroid-dependent asthma

Severe asthma that cannot be managed without oral corticosteroids

Elevated total serum IgE, precipitins to A. fumigatus Elevated serum IgE/IgG antibodies to A. fumigatus despite continuous prednisone

Pulmonary infiltrates

Stage 5

Fibrotic

Symptomatic from severe obstructive and often restrictive lung disease

Elevated total serum IgE and IgG antibodies to A. fumigatus

Pulmonary fibrosis

ISHAM Stagingb Stage 0 Asymptomatic

GINA definition of controlled asthma

Fulfills criteria for ABPA

Pulmonary infiltrates may be present

Stage 1

Acute

Uncontrolled asthma/ constitutional symptoms

Fulfills criteria for ABPA

Stage 1a: With mucoid impaction Stage 1b: Without mucoid impaction

Stage 2

Response

Improvement in asthma control Resolution of constitutional symptoms

Total IgE decline ≥25% of baseline at 8 weeks

Significant radiologic improvement

Stage 3

Exacerbation

Deterioration of symptoms

Increase in total IgE ≥50% from baseline

Worsening radiologic changes

Stage 4

Remission

Sustained symptomatic improvement

Total IgE remains suppressed for ≥6 months without systemic corticosteroids

Radiologic improvement

Stage 5

Glucocorticoid-dependent asthma

Uncontrolled symptoms when taken off systemic corticosteroids

Elevated total IgE

Radiologic changes

Stage 6

Advanced ABPA

Type 2 respiratory failure and/or cor pulmonale

Raised total IgE

Pulmonary fibrosis

ABPA, Allergic bronchopulmonary aspergillosis; GINA, Global Initiative for Asthma; ISHAM, International Society for Human and Animal Mycology. Modified from aGreenberger PA, Patterson R111; bAgarwal R, Chakrabarti A, Shah A, et al.24

to the presence (ABPA-CB) or absence (ABPA-S) of central bronchiectasis and the latter possessing the same clinical and serologic features but without central bronchiectasis.23 The first diagnostic criteria for ABPA were proposed by Rosenberg and Patterson22 in 1977 and later refined by Schwartz and Greenberger2 in 1991 (Box 58.1). Although widely accepted and used, the criteria for ABPA have no international consensus for definition. In an attempt to address potential deficiencies in the previous criteria, the International Society of Human and Animal Mycology (ISHAM) proposed a new diagnostic criteria (Box 58.1).24 Recent descriptions of “severe asthma with fungal sensitization” (SAFS)25 and impaired lung function in those with severe asthma who have coexistent fungal sensitization26 suggest that fungal airways disease caused by Aspergillus spp. may represent a continuum, with colonization of the airway at one end and severe fibrosis at the other, mediated by airway immunologic reactions to the fungi. The main differential diagnosis for ABPA-S is SAFS, with the serum total IgE level as the discriminating test and the (arbitrary) cut-off set at 1000  IU/mL or higher for ABPA diagnosis. Pulmonary infiltrates

from bacterial or viral pneumonia are also in the differential diagnosis, although the patient will usually have infection-related symptoms and a nonrecurrent pattern. In CF patients the recognition of ABPA is complicated by the usual concomitant bronchiectasis, with variable presence of asthma. Diagnostic criteria note the lack of utility of radiologic findings of bronchiectasis and pulmonary infiltrates as indicators of ABPA in patients with CF and emphasize the importance of increased total and specific IgE in addition to IgG to Aspergillus spp. in determining the diagnosis.21 Subsequently, international criteria for the diagnosis of ABPA in CF were established27 (Box 58.1). Given the common colonization of airways with Aspergillus spp. in this group, however, as well as the elevation of serum total IgE and problems with coexistent fungal sensitization, further serologic diagnostic markers have been sought. In particular, serum thymus- and activation-regulated chemokine (TARC, CCL17) and antibodies to recombinant Aspergillus spp. antigens or to newly defined antigens show particular promise in distinguishing patients with ABPA and CF from those with CF and fungal sensitization.28 Most recently,

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BOX 58.1  Diagnostic Classifications for Allergic Bronchopulmonary Aspergillosis RosenbergPatterson Criteriaa

Schwartz and Greenberger Criteriab,c

Primary 1. Asthma 2. Serum eosinophilia 3. Immediate skin reactivity to Aspergillus 4. Precipitins to Aspergillus 5. Elevated IgE 6. Pulmonary infiltrates (transient or fixed) 7. Central bronchiectasis Secondary 1. Aspergillus fumigatus in sputum 2. Expectoration of brown plugs 3. Late skin reactivity to Aspergillus

ABPA-CB: Minimal Essential Criteria 1. Asthma 2. Immediate skin test reactivity to Aspergillus 3. Elevated Aspergillus-specific IgE and/or IgG 4. Elevated total IgE (>1000 ng/mL) 5. Proximal bronchiectasis ABPA-S: Minimal Essential Criteria 1. Asthma 2. Immediate skin test reactivity to Aspergillus 3. Elevated total IgE (1000 ng/mL) 4. Elevated Aspergillus-specific IgE and/or IgG Additional Criteria 1. Current or previous pulmonary infiltrates 2. Mucus plugs 3. Presence of Aspergillus in sputum 4. Precipitins to Aspergillus 5. Delayed skin test positive 6. Eosinophilia (>1000/µL)

ISHAM Criteriad

CF Criteriae

Predisposing Conditions Asthma, CF Obligatory Criteria (Both Should Be Present) 1. Immediate skin test reactivity to Aspergillus or elevated Aspergillusspecific IgE 2. Elevated total IgE (>1000 IU/ml) Other Criteria (at Least 2 of 3) 1. Presence of precipitating or IgG antibodies against Aspergillus in serum 2. Radiographic pulmonary opacities consistent with ABPA 3. Total eosinophil count >500 cells/µL in steroid naive patients

Classic Case 1. Acute or subacute clinical deterioration (cough, wheeze, exercise intolerance, exerciseinduced asthma, decline in pulmonary function, increased sputum) not attributable to another etiology. 2. Serum total IgE concentration of greater than 1000 IU/mL (2400 ng/mL), unless patient is receiving systemic corticosteroids (if so, retest when steroid treatment is discontinued). 3. Immediate cutaneous reactivity to Aspergillus extract or detectable presence of serum IgE antibody to Aspergillus fumigatus. 4. Precipitating antibodies to A. fumigatus or serum IgG antibody to A. fumigatus by blood test. 5. New or recent abnormalities on chest radiography (infiltrates, mucus plugging) or chest computed tomography (bronchiectasis) that have not cleared with antibiotics and standard physiotherapy.

ABPA, Allergic bronchopulmonary aspergillosis; ABPA-CB, ABPA with central bronchiectasis; ABPA-S, ABPA serologic. Modified from aRosenberg M, Patterson R, Mintzer R, et al.22; bSchwartz HJ, Greenberger PA2; cGreenberger PA. Diagnosis and management of allergic bronchopulmonary aspergillosis. Allergy Proc 1994;15:335-9; dAgarwal R, Chakrabarti A, Shah A, et al.24; eStevens DA, Moss RB, Kurup VP, et al.27

the use of Aspergillus spp. serology, sputum Aspergillus spp. PCR, and sputum galactomannan (GM) in combination has been shown to differentiate ABPA from Aspergillus spp. sensitization and Aspergillus spp. bronchitis in CF.29 The basophil activation test (BAT) to Aspergillus spp. is another novel diagnostic marker to differentiate ABPA from Aspergillus spp. sensitization alone in CF.30,31

Skin Testing and Laboratory Investigations Key to a diagnosis of ABPA is evidence of Aspergillus spp. sensitization, demonstrated by immediate skin test hypersensitivity or serum Aspergillus spp.–specific IgE. Absence of sensitization to Aspergillus spp. excludes ABPA. On the other hand, fungal sensitization is common in asthma, especially severe eosinophilic asthma, so the presence of Aspergillus spp.–specific IgE alone is insufficient for the diagnosis. Prevalence of fungal sensitization has been reported to be as high as 66% in severe asthma populations, with sensitivity to Aspergillus spp. of 45%.25 Concordance between blood and skin-prick testing for specific IgE was only 54% for Aspergillus spp. antigens in this study, so both blood and skin testing should be performed to ascertain fungal sensitization. More than 80 allergens of Aspergillus spp. have been identified in humans. In asthma and in CF, reactivity to the antigens Asp f 1, 3, 4 and 6 are predominant. Patients with asthma recognize Asp f 1 and 3.

The presence of antibodies to either Asp f 4 or Asp f 6 has been associated with high sensitivity and specificity for ABPA,32 although test characteristics may be affected by ethnic differences and comorbid atopic dermatitis.33 Skin-prick testing and specific IgE blood testing is normally performed to crude allergen extract. In future, the use of recombinant antigen testing may provide greater diagnostic rigor. Historically, skin testing to Aspergillus spp. antigens was performed using 1 and 10 mg/mL of Aspergillus spp. extract. This was followed by an intradermal test using a concentration of Aspergillus spp. extract that elicited a 2- to 3-mm wheal on skin-prick testing, with the individual then observed for a delayed cutaneous response.34 This would occur 3 hours after injection and could persist for days. Such late cutaneous reactions, indicative of delayed immune responses and possibly mediated by immune complexes, were found in more than 95% of patients with ABPA. However, the delayed cutaneous intradermal test is usually not performed in routine clinical practice because of its prolonged course and its replacement by other currently available tests. Another key test finding in the diagnosis of ABPA is highly elevated serum total IgE level. Previous diagnostic criteria required a total IgE level of 1000 ng/mL (417 IU/mL), but Agarwal and coworkers3 more recently proposed a threshold of 1000 IU/mL as a criterion for ABPA diagnosis. The accuracy of various diagnostic criteria and their components is largely unknown because of the lack of a gold standard for

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis ABPA confirmation. To overcome this problem, one study used latent class analysis and demonstrated that serum total IgE thresholds of 1000 IU/mL as well as 1000 ng/mL had sensitivities of more than 95% for diagnosing ABPA, although the specificities for both thresholds were low at below 40%.35 The low specificity of elevated serum total IgE for diagnosing ABPA is further demonstrated in another study, which showed that only a minority of asthma patients with serum total IgE of more than 1000 IU/mL has ABPA.36 The utility of serum total IgE in ABPA diagnosis is, however, enhanced in patients without concomitant allergic rhinitis and eczema.37 The most sensitive indicator of disease progression is serial measurements of total IgE showing increasing levels of IgE. Oral corticosteroids will usually reduce blood IgE levels. A decline in serum total IgE of 35% is considered diagnostic of achieving remission of ABPA, although not all patients achieve such a reduction, especially those with lower total IgE levels of less than 2500 IU/ mL.38 Conversely, a doubling of serum total IgE is considered diagnostic of relapse of ABPA, especially in CF patients. Another test used in ABPA is detection of precipitating antibodies to Aspergillus spp. by gel diffusion, predominantly of the IgG class but occasionally IgE and IgA. Precipitating antibodies are identified in several fungal pulmonary diseases, including aspergilloma, so are not specific for ABPA. Serum may need to be concentrated fivefold to reveal positivity, and patients with treated ABPA do not typically have positive precipitating antibodies. Another nonspecific finding that may suggest ABPA is blood eosinophilia that often accompanies asthma or allergic disease with suppression by systemic corticosteroids. Aspergillus spp. hyphae in the sputum are suggestive of ABPA. However, Aspergillus spp. colonization of the airways is nonspecific for ABPA and also found in invasive fungal infections in the lung. Curschmann spirals and eosinophilic debris (e.g., Charcot-Leyden crystals) may be identified in the sputum of ABPA patients, indicating inflammatory airway response. Again, these findings are nonspecific.

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scarring, often extending to the pleura.39 Airway changes, particularly bronchiectasis involving large central airways with a predilection for the upper lobes (Fig. 58.3B), are diagnostic of ABPA. Bronchiectasis at lobar and segmental levels and involving the majority of airways is characteristic. Controversy has arisen with the finding that severe asthma can be associated with bronchiectasis on CT; however, involvement should not exceed two lobes, as typically occurs in ABPA.40 Mucoid impaction leading to airway collapse and “tree-in-bud” opacities is also described. Less frequent are cavities, emphysematous bullae, and mosaic attenuation. Severe central bronchiectasis will induce more peripheral bronchiectasis and fibrosis associated with end-stage disease. Disease progression in ABPA has been acknowledged by the development of staging systems (Table 58.2) that recognize that a diagnosis of ABPA may be made without central airways disease, and that serologic disease may regress to an inactive state with treatment. The staging criteria acknowledge that patients with ABPA-S receiving therapy may be stable but require observation to identify exacerbation and prevent

Radiologic Findings Initial reports of ABPA described “fleeting parenchymal pulmonary opacities,” which may be confused with persistent pneumonia. Pulmonary opacities commonly manifest in those with ABPA-S but without overt symptoms. When large airways are involved, radiologic changes may reveal transitory opacities, thickened airway walls and central bronchiectasis, mucus plugging, atelectasis, or more significant pulmonary collapse (Figs. 58.2 and 58.3). High-resolution computed tomography (HRCT) of the chest may reveal parenchymal lung opacification, transient in early stages but progressing to collapse or parenchymal

A

Fig. 58.2  Transitory opacities (white arrows) and lobar collapse (black arrow) in a patient with allergic bronchopulmonary aspergillosis (ABPA).

B

Fig. 58.3  (A) Central bronchiectasis in a patient with allergic bronchopulmonary aspergillosis (ABPA) (arrows). (B) Central bronchiectasis in the upper lobes (arrows).

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TABLE 58.2  Radiologic Staging of Allergic Bronchopulmonary Aspergillosis Categories

Features

Severity

ABPA-S

Serologic; all criteria except central bronchiectasis

Mild Mild to moderate airflow obstruction; Aspergillus spp. IgG precipitins positive in 50%

ABPA-CB

With central bronchiectasis

Moderate Moderate to severe airflow obstruction; high IgE; Aspergillus spp. IgG precipitins positive in 66%

ABPA-CB-ORF

With central bronchiectasis plus other radiologic features, such as pulmonary fibrosis, bleb, bullae, pneumothorax, parenchymal scarring, emphysematous change, multiple cysts, fibrocavitary lesions, aspergilloma, ground-glass appearance, collapse, mediastinal lymph node, pleural effusion, and thickening

Severe Severe airflow obstruction; tendency to have very high eosinophil counts and total IgE; Aspergillus spp. IgG precipitins positive in 100%

Modified from Kumar R. Mild, moderate, and severe forms of allergic bronchopulmonary aspergillosis: a clinical and serologic evaluation. Chest 2003;124:890-892.

clinical progression to permanent lung changes associated with central bronchiectasis (ABPA-CB). Agarwal and coworkers41 have described the finding of highattenuation mucus (HAM), airway luminal mucus at greater density than the surrounding paraspinal muscle, as characteristic of ABPA and diagnostic of its clinical course with respect to exacerbations. HAM has been correlated in 234 patients with immunologic criteria for ABPA diagnosis, suggesting greater susceptibility to exacerbation when present, as with central bronchiectasis. In advanced cases, pulmonary fibrosis may occur, with the imaging features designated as ABPA with other radiographic features (ABPA-ORF).42

Histopathologic Findings Pathologic samples are not required for the diagnosis of ABPA, but the detection of Aspergillus spp. on lung biopsy supports the diagnosis. Frequent histologic findings include inflammatory infiltration of the airways by eosinophils and lymphocytes, goblet cell hyperplasia, granulomas with distal exudative bronchiolitis, mucoid impaction, and fibrosis in end-stage disease.

Treatment The aims of treatment are to improve clinical symptoms of disease, reduce exacerbations, and prevent progression of disease to central bronchiectasis.

Corticosteroids Oral corticosteroids are the backbone of ABPA treatment, and serum total IgE is used to monitor disease activity.23 Initial recommended treatments for ABPA were at least 3 months of an oral corticosteroid such as prednisone, 0.5 mg/kg every day for 2 weeks, then alternate days for 3 months,23 followed by staging of disease. After 3 months of oral corticosteroid therapy, a repeat level of serum total IgE should be obtained to monitor disease activity, and repeated measurements of serum total IgE should be made to determine the baseline level during disease remission. Subsequent studies in larger cohorts indicated use of higher doses of corticosteroids for longer duration to prevent disease relapse. Some suggest 0.75 mg/kg/day for 6 weeks followed by 0.5 mg/ kg/day for 6 weeks, then reduction of 5 mg/day every 6 weeks, with 6 to 12 months of treatment, as determined by disease activity.43,44 To date, only one randomized controlled trial has compared the efficacy

of low versus higher dose corticosteroid regimens in ABPA.45 Exacerbation rates after 1 year of treatment and progression to corticosteroiddependent ABPA after 2 years of treatment, the primary outcomes of the study, were similar in both treatment groups. However, glucocorticoid side effects were significantly higher in the high-dose arm. Lung damage can occur even in the absence of symptoms. Thus it is important to monitor serum total IgE levels every 1 to 2 months and increase corticosteroid dosing if IgE levels double from the baseline values obtained after stability on the maintenance dose.44 Acute exacerbations should be treated with prednisone, 0.5 to 1.0 mg/kg/day for 1 to 2 weeks, followed by 0.5 mg/kg/day for 6 to 12 weeks on clinical remission, then tapering of the dose until the preexacerbation dose is reached. Alternate-day regimens may be an option for subjects who cannot be tapered off corticosteroids completely. Refinements of oral corticosteroid regimens have included monthly pulsed methylprednisolone to mitigate corticosteroid side effects,46 with 3 days of 10 to 15 mg/kg/day repeated every month. When administered with itraconazole, this regimen demonstrated effective reduction of serum total IgE and symptomatic improvement. Methylprednisolone with itraconazole has been used in patients with CF as well. High-dose intravenous corticosteroid treatments have been used in life-threatening situations involving ABPA patients and in those who have failed to respond to oral therapy with evident disease progression, particularly in CF patients.47 Monitoring for corticosteroid side effects and prevention where possible is important. This is especially pertinent for patients with established bone disease, in whom vitamin D and calcium supplementation are indicated, and further bone-sparing therapy (e.g., bisphosphonates or denosumab) should be considered, in accordance with guidelines for patients receiving long-term oral corticosteroid therapy. Case series have investigated inhaled corticosteroids for serologic ABPA. A study of budesonide and formoterol inhalation therapy in 21 patients with ABPA-S showed progressive elevation of serum IgE, indicative of disease progression, despite 6 months of therapy. All patients subsequently responded to oral corticosteroids, with reduction in total IgE levels.48 However, no patient in this trial used antifungal therapy.

Antifungal Agents A randomized trial of itraconazole in corticosteroid-dependent ABPA patients showed symptomatic improvement and decreased corticosteroid

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis

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requirement in 46% of those receiving 200 mg of itraconazole twice daily, versus 19% in the placebo group.49 Wark and colleagues50 showed reduced sputum eosinophil counts in both patients with ABPA-S and patients with ABPA-CB in remission, using daily itraconazole. Pulmonary exacerbations were also decreased in the therapy group. Both studies were only 16 weeks long, and long-term results with itraconazole therapy in ABPA have not yet been established. Nevertheless, published guidelines recommend itraconazole, 200 mg twice daily for 6 months for corticosteroid-dependent ABPA.44 Voriconazole has also been used as an alternative antifungal agent and was effective in a case series,51 although similar efficacy was not demonstrated in a controlled study of severe asthma with Aspergillus spp. sensitization.52 One of the concerns regarding the use of azoles (e.g., ketoconazole, itraconazole) in the management of ABPA is that azoles are strong inhibitors of the cytochrome P450–dependent CYP3A4 enzyme involved in the metabolism of budesonide and other corticosteroids. Also, adrenal suppression has been demonstrated by the ACTH stimulation test in patients receiving inhaled corticosteroids and itraconazole. This raises the possibility that some of the benefit of adjunctive azole therapy in ABPA may be caused by the relatively higher dose of bioavailable corticosteroid.

A landmark study of interstitial lung disease (ILD) found an overall prevalence of 30 per 100,000 population, with HP responsible for less than 2% of incident cases.60 However, regional variations in prevalence may occur, especially related to occupational and domestic exposures, such as farmer’s lung in rural areas and Japanese summer house lung (HP caused by exposure to damp wood and typical Japanese flooring, tatami). The largest prospective multicenter registry for ILD to date is from India with 1084 patients, which identified HP as the most common cause of incident ILD (47.3% of cases).61 This is in contrast to a Spanish study on ILD incidence across 23 centers nationally with 511 patients registered, where HP was only the fifth most common cause of ILD (6.6% of cases).62 Because not all exposed individuals acquire HP, there must be individual protective and promoting factors, although these have not yet been determined. Unusually, cigarette smoking appears protective for acute and subacute HP, but not for chronic HP, where it is associated with emphysematous change. The inhibitory effects of nicotine on macrophage phagocytosis likely mediate the apparent benefit. Gender differences in disease prevalence may reflect differential occupational exposure to offending antigens.63

Anti-IgE Biologics

Pathogenesis and Etiology

Given the key role of IgE in the pathology of ABPA and the effectiveness of anti-IgE treatments in asthma, anti-IgE therapy has been used in ABPA. Although the recommended dosing range of IgE for which omalizumab has proved effective is usually exceeded in patients with ABPA, a randomized, placebo-controlled cross-over trial nonetheless showed that omalizumab at 750 mg monthly significantly reduced exacerbation rates in ABPA patients within 4 months.53 Uncontrolled case series also report effective use of omalizumab as a corticosteroidsparing agent in corticosteroid-dependent CF patients.54 Others report the effective use of omalizumab with corticosteroids in life-threatening respiratory failure caused by ABPA. More controlled trials are needed before anti-IgE therapy can be recommended as a first-line treatment for ABPA.

Hypersensitivity pneumonitis results from an abnormal immune response, mainly to antigens of fungal, bacterial, avian, and vegetable origin. Most of these antigens are organic, but inorganic materials such as isocyanates and zinc serve as haptens and create antigenic complexes, when combined with human serum albumin, to trigger HP. Many of the antigens in HP are immune stimulants because of their own enzyme activity and growth patterns. Additionally, viral infection appears to occur frequently at the same time as initiation of HP, possibly increasing antigen activation. Many antigens are responsible for HP (Box 58.2). Well-known causes of HP include exposure to avian antigens and the Actinomyces spp.

Anti-IL-5 Biologics A handful of case reports suggest antiinterleukin-5 (IL-5) therapy with mepolizumab may be effective for ABPA, either in isolation or in combination with omalizumab; however, robust data are lacking.

HYPERSENSITIVITY PNEUMONITIS Historical Perspective Hypersensitivity pneumonitis (HP) was first described in the early 1900s in farmers who reported febrile episodes after exposure to moldy grains, hay, or straw (farmer’s lung). The relationship between HP and exposure to avian antigens was described with the report of pigeon breeder’s lung in 1965 and budgerigar fancier’s lung in 1978. Since then, it has been recognized that a large range of organic dusts may trigger HP in susceptible individuals. Exposure to multiple other antigen sources, including birds (bird fancier’s lung), moldy wood, humidifiers, and environments high in mold content have been recognized as causes of HP. Chemical compounds such as isocyanates55 were subsequently described as causes of HP. Zinc56 and more recently nickel57 have also been reported as causes of HP. Although the disease entity is well accepted, definitions of HP are not uniform. In 1985, Cormier and Schuyler58 proposed that HP is “an inappropriate immune response to inhaled antigens that causes shortness of breath, and a restrictive lung defect.” HP may be further characterized by “episodic bouts of fever a few hours after exposure.”59

Epidemiology

BOX 58.2  Causes of Hypersensitivity

Pneumonitis

Birds Pigeons, doves, parakeets, cockatoos, cockatiels, lovebirds, parrots, canaries, geese, ducks, turkeys, chickens, pheasants, partridges Molds Hay, grains, straw (Saccharopolyspora rectivirgula) Household molds, humidifiers Trichosporon asahii, T. mucoides, or T. cutaneum (summer-type hypersensitivity pneumonitis) Aspergillus spp. Candida spp. Cladosporium spp. Penicillium spp. Ulocladium botrytis and Phoma spp. (saxophone) Mycobacteria Mycobacterium avium complex (hot tub lung) Mycobacterium immunogenum (metalworking fluids) Chemicals Isocyanates Zinc Nickel

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found in moldy hay, straw, or grains (Saccharopolyspora rectivirgula); other antigens implicated include Mycobacterium avium causing hot tub lung disease,64 household mold, and Mycobacterium immunogenum in metalworking fluids.65 Exposure to aerosolized avian proteins, especially from feathers or bird droppings, is the most common cause of HP.66 Pigeons, doves, and parakeets are the birds involved most frequently, but other birds of the order Psittaciformes, including cockatoos, cockatiels, lovebirds, and parrots, have also been implicated. Sensitization and disease development require prolonged or heavy exposure to the antigen, often related to occupational exposure. Respirable antigen particles (smaller than 3 µm) trigger immune complex– mediated and T cell–mediated inflammation with a large influx of activated T lymphocytes in the lung tissue (Fig. 58.4). A Th1 response predominates in the early stages, whereas Th2 skewing occurs in later stages when fibrosis develops. Early inflammatory changes include IgG binding to antigen and activating complement pathways, resulting in macrophage activation and secretion of inflammatory and chemoattractant factors such as CXCL8, CCL5, and CCL3. These chemokines induce a neutrophilic airway infiltration with the secretion of enzymes such as elastase that

are destructive to surrounding lung tissue. Chemokine release from macrophages, particularly CCL18, may drive recruitment of lymphocytes into pulmonary tissues. Further evidence suggests that once in the lung, apoptosis of lymphocytes is inhibited, contributing to the large number of lymphocytes in the lungs of HP patients. However, the presence of an alveolar lymphocytosis is insufficient for diagnosis because individuals exposed to antigen may exhibit alveolar lymphocytosis without disease. Studies of lymphocyte subsets in those with acute, subacute, and chronic HP revealed that bronchoalveolar lavage (BAL) cells from patients with chronic HP had a higher median CD4+/CD8+ ratio (3.05, range 0.3 to 15) than those with subacute HP (1.3, range 0.1 to 10) or unaffected control subjects exposed to antigen (1.3, range 0.7 to 2.0). In addition, the lymphocytes in chronic HP patients displayed a Th2 cytokine pattern with increased CXCR4 surface expression and lower memory cell interferon γ (IFN-γ) secretion. This Th2 skewing in chronic HP may be associated with the fibrotic responses observed in this condition.67 Further studies of alveolar lymphocytes in HP implicated altered regulatory functions, although no differences in the numbers of regulatory T cells (Treg) have been observed. However, functional assays exploring the effects on T cell proliferation induced by CD3 and CD28 of suppressor cell

Exposure to antigen Molds Birds Chemicals Mycobacteria Viral infection Endotoxins

Genetic susceptibility

Reduced lymphocyte apoptosis

Th1 and Th2 response

Antigen-IgG immunocomplex

Large influx lymphocytes

Complement activation

Abnormal Treg function (IL-17 mediated)

Neutrophils

Th1 predominance

Macrophage

CXCL8 CCL5 CCL3

Acute HP

Th2 response skewing ↓ IFN-γ

Tissue damage fibrosis

↑ IL-4, IL-13

Subacute/chronic HP Fig. 58.4  Pathogenesis of hypersensitivity pneumonitis (HP). Chronic and repeated exposure to antigens in genetically susceptible individuals triggers an inflammatory process. A Th1 response predominates in the early stages, whereas Th2 skewing occurs in later stages, both leading to tissue damage from inflammatory propagation and cytokine release. Lung tissue lymphocytes are increased because of decreased lymphocyte apoptosis and impaired Treg function (mediated by IL-17). Early inflammatory changes include IgG binding to antigen and complement pathways, with macrophage activation and secretion of chemoattractants (CXCL8, CCL5, CCL3) that induce neutrophilic airway infiltration and tissue destruction.

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis subsets derived from HP patients showed absent regulatory capacity. In contrast, subjects exposed to antigen but not exhibiting HP had normal Treg function. Also, assays of IL-17 in serum and BAL fluid from patients showed increased IL-17 in those with HP, none in normal subjects, and an in-between value in those exposed but not with HP. This indicates that loss of the regulatory function of T cells in patients with HP may be a cause of the accumulated lymphocytic inflammatory changes characteristic of the disease. Moreover, the presence of elevated levels of the proinflammatory cytokine IL-17 may further indicate a causal mechanism.68 Studies in mouse models of HP suggest that the Th1-type cytokine responses are important early in the course of the disease, with subsequent transition to a Th2 cytokine pattern in more chronic patterns of disease leading to pulmonary fibrosis. Murine models of HP demonstrate the critical role of lung mucosal dendritic cells mediated by stem cell antigen CD34 in the pathogenesis of HP. Specifically, CD34 knockout mice did not develop HP in a model of exposure to S. rectivirgula, but adoptive transfer of wild-type or human CD34 restored susceptibility. These studies elegantly reveal the critical importance of dendritic cells and antigen presentation in the generation of HP.69 A further abnormality in HP is surfactant. Lung surfactant is a modulator of the immune response and is altered in HP, particularly with upregulation of the proinflammatory surfactant protein A.

Clinical and Radiologic Features The clinical presentation of HP can be acute, subacute, or chronic, with differing symptoms and pathology determined by the quantity and duration of exposure to the causative agent. Acute HP presents with cough, fever, sweating, myalgia, headache, and nausea that usually begins within 8 hours of exposure and may persist up to 1 month. The acute form of HP was initially described for farmer’s lung (S. rectivirgula) caused by episodic exposure to moldy hay or grain. Cough and dyspnea are indicative but not experienced by all patients, raising the differential diagnosis of a pulmonary infection. Physical examination may be normal or may reveal widespread crackles. In this phase the chest radiograph may be normal (40% of cases)70 but may also reveal a diffuse nodularity, often sparing the apices or bases. CT scanning may identify ground-glass opacities, poorly defined centrilobular micronodules, mosaic attenuation,

A

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and mediastinal lymphadenopathy. Lung function at this stage is likely to show impaired diffusion caused by ventilatory inhomogeneity, with a possible restrictive lung deficit. Complete recovery within 1 month can be expected when antigen exposure can be eliminated. Chronic exposure to antigen will lead to subacute and chronic HP, likely dependent on the level of antigen exposure (Fig. 58.5). Both these phases are manifest by shortness of breath and cough with constitutional features such as fever, malaise, and weight loss variably expressed. Importantly, many of the symptoms do overlap, and distinguishing these forms can be difficult, especially between subacute and chronic HP. In subacute HP, symptoms usually develop gradually over days or weeks, and examination findings are likely to reveal lung crackles. Lung function testing typically shows restriction with reduced gas transfer measurements in subacute disease. HRCT may identify a micronodular pattern or ground-glass attenuation, often sparing the lung periphery, which may progress to reticular or interstitial patterns. Chronic HP is often caused by low-level chronic exposure and may present with end-stage fibrotic lung disease and respiratory failure. Clinically, patients with this stage may exhibit digital clubbing and hypoxia. Chronic HP is exemplified by bird fancier’s lung, in which exposure to antigen occurs at a low intensity but high frequency. Chronic HP may still be active and progressive. Patients with the chronic form may also have exacerbations, similar to pulmonary fibrosis; this carries a poorer prognosis, with restrictive lung function manifesting as lower gas transfer measurements and lower total lung capacity. Chest x-ray films are likely to show an interstitial pattern progressing to fibrosis or honeycombing. Emphysematous changes may also be seen, especially in smokers. In chronic HP, common HRCT findings include fibrosis, reticular pattern, air trapping, honeycombing, and traction bronchiectasis, which are often difficult to distinguish from other forms of ILD. Findings of micronodules, mosaic attenuation, and sparing of the lung periphery tend to favor HP as a diagnosis over other interstitial diseases. Pleural disease is uncommon and may help distinguish HP on HRCT scan from other causes of pulmonary fibrosis.

Diagnosis

Clinical History.  A diagnosis of HP must first be considered based on history. Lacasse and coworkers71 studied patients referred with ILD,

B

Fig. 58.5  Radiologic findings in subacute (A) and chronic (B) hypersensitivity pneumonitis. (A) Interstitial fibrosis (black arrows) and emphysematous changes (red arrows) in chronic HP with superimposed subacute HP in a 77-year-old farmer. (B) Ground-glass opacities (black arrows), mosaic perfusion (white arrows), and fibrosis (red arrow) in chronic HP caused by pigeon exposure.

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TABLE 58.3  Significant Predictors of

Hypersensitivity Pneumonitis Variables Exposure to known offending antigen

Odds Ratio (95% CI) 38.8 (11.6-129.6)

Positive precipitating antibodies

5.3 (2.7-10.4)

Recurrent episodes of symptoms

3.3 (1.5-7.5)

Inspiratory crackles

4.5 (1.8-11.7)

Symptoms 4 to 8 hours after exposure

7.2 (1.8-28.6)

Weight loss

2.0 (1.0-3.9)

Modified from Lacasse Y, Selman M, Costabel U, et al. Clinical diagnosis of hypersensitivity pneumonitis. Am J Respir Crit Care Med 2003;168:952-958.

diagnosis of HP but can also be found in those exposed to antigens but who are asymptomatic, so this is not sufficient to confirm the diagnosis of HP. Alveolar lymphocytosis may also be found in sarcoidosis, collagen vascular diseases, silicosis, bronchiolitis obliterans with organizing pneumonia (cryptogenic organizing pneumonia), and drug reactions. Classically, a predominance of CD8+ T lymphocytes and a CD4+/CD8+ ratio less than 1 is described in HP,74 although a substantial proportion of patients with HP can have a CD4+/CD8+ ratio greater than 1. There is also no conclusive relationship between CD4+/CD8+ ratio and the stages or etiologies of HP.75 Novel biomarkers may aid in the diagnosis of HP. Levels of serum Krebs von den Lungen-6 (KL-6) and surfactant protein D (SP-D) have been found to be significantly higher in HP than other common ILDs.76 Another study demonstrated that IL-17 was detectable only in BAL fluid from HP patients, but not sarcoidosis and IPF.77

Lung Biopsy.  Lung biopsy should be performed for ILD of uncertain compared with those who did not have HP, and derived a symptomatic diagnostic algorithm. Six predictors were validated for HP diagnosis (Table 58.3). Exposure to a known offending antigen increased the odds ratio of a diagnosis by 39-fold. Clinical signs such as fever, crackles, clubbing, hypoxia, cyanosis, and wheezing were frequently present but did not favor a diagnosis of HP.71 More recently, Johannson and colleagues derived and validated two diagnostic models with high specificities (more than 90%) for chronic HP.72 Variables included in both models were age, a history of down feather and/or bird exposure, and specific HRCT features or radiologist’s diagnostic confidence of chronic HP. A positive score may obviate the need for surgical lung biopsy.

Specific Antibodies.  An important component of the diagnosis of HP is the demonstration of antibody against suspected causative antigens. Historically, this has been the documentation of precipitating antibodies in agar by immunodiffusion, but it now relies on the detection of IgG antibodies to putative antigens by immunoelectrophoresis or enzyme-linked immunosorbent assay (ELISA). The presence of precipitating antibodies (precipitins) was initially described as a diagnostic indicator for HP. More recent appreciation of their relevance suggests that these antibodies are markers for exposure to the antigen rather than acquisition of disease. Not all individuals with HP express precipitins, and less than 15% of those with antibodies detected to an antigen develop disease. For example, in dairy farmers with HP, 10% of those tested had developed antibodies to the causative fungus, but only 0.3% had evidence of disease.59 Key to the reliability of detection of IgG to antigens is the standardization and availability of antigen preparations that are relevant to local conditions. When carefully standardized antigens relevant to specific environmental circumstances are used, the sensitivity and specificity can be high. However, antigens for testing are not always commercially available, and even then the methods may not be standardized.

Lung Function.  Pulmonary function tests may be normal in some patients with HP but typically reveal a restrictive defect with reduced gas transfer. An obstructive defect may also be seen, especially in the chronic phenotype. Pulmonary inhalational challenge, used particularly in the occupational context, has a sensitivity and specificity of 72.7% (95% CI 62.6-80.9) and 84% (95% CI 65.3-93.6), respectively. 73 However, the antigen to be used, antigen dose, and criteria for a positive test are not standardized, so inhalation tests are not routinely used in clinical practice.

Bronchoalveolar Lavage.  For suspected HP, the BAL result is an important diagnostic tool. An alveolar lymphocytosis supports the

diagnosis. Transbronchial biopsy may provide evidence of granulomatous disease suggestive of the diagnosis, but at least six biopsies are required, and even this number may not be sufficiently accurate. Consequently, open-lung or thoracoscopic lung biopsy provides the “gold standard” for diffuse interstitial lung pathology, but the diagnostic yield ranges from 34% to 100%. Therefore a conservative approach to lung biopsy is recommended for the diagnosis of HP, with reliance on clinico­ radiologic findings in addition to BAL results to make a diagnosis before performing lung biopsy.

Differential Diagnosis The differential diagnosis of acute HP includes bacterial and viral infections, in particular cytomegalovirus pneumonitis. Differential diagnoses of chronic HP include interstitial pulmonary fibrosis and other interstitial lung diseases, such as sarcoidosis, nonspecific interstitial pneumonitis (NSIP), lymphoid interstitial pneumonitis, cryptogenic organizing pneumonia (COP), and ILD associated with collagen vascular disease. History of exposure to an offending agent, especially within 8 hours of symptom development in the acute form, and chronic exposure in the subacute and chronic forms, is important to distinguish HP from the other conditions clinically. Pathologic features are also helpful, but lung tissue is rarely obtained in HP; this is reserved for cases of unclear exposure or high suspicion of another ILD type.

Pathologic Findings In acute HP the most common pathologic features include alveolar infiltration with neutrophils and eosinophils and small-vessel vasculitis.78,79 Histopathologic samples from the lungs of patients with subacute HP reveal a triad of a lymphocytic interstitial inflammatory cell infiltrate, small nonnecrotizing granulomata, and bronchiolitis. Granulomata are poorly formed, differentiating these from the granulomata found in sarcoidosis. The lymphocytic alveolitis is accentuated at the peribronchiolar regions, also with foci of bronchiolitis obliterans and intraalveolar fibrosis.79 In contrast, chronic HP demonstrates features of significant lung architecture distortion, with centrilobular fibrosis, bridging fibrosis, and atelectatic fibrosis in the lobule. Epithelioid cell granulomata are sparse or absent, but giant cells are often seen in the interstitium. In patients with chronic HP, those with more frequent acute episodes usually present with changes of COP or NSIP, whereas those with more chronic insidious HP have changes of fibrotic NSIP or a usual interstitial pneumonia (UIP) pattern with honeycombing.79,80 Emphysematous changes are common in chronic HP patients, and thin-walled cysts may also occur.

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across the state of Victoria, many thousands were affected and treated in the community, and ten deaths have been linked to the epidemic.84

Treatment The diagnosis and pathogenesis of HP are important, because acute and subacute HP are reversible with removal of the patient from antigen exposure. Chronic changes of HP are less likely to be reversed. In some cases, when the patient’s removal from a source of antigen can mean loss of employment, steps have been taken to reduce antigen exposure, for example, ensuring that affected farmers switch to the use of dry fodder or silage, which may be partly effective but would obviously require careful observation. Occasionally, oral corticosteroids are used to suppress the lymphocytic inflammation involved in HP. These do not appear to improve prognosis in patients with the chronic form, however, so oral corticosteroids are best avoided unless acute, lifethreatening complications are evident. Azathioprine and mycophenolate mofetil have been shown in observational studies to improve gas exchange and reduce the dose of prednisolone required in patients with chronic HP.81 Lung transplantation may be considered in patients with progressive fibrosis. The posttransplant survival appears better in HP than in idiopathic pulmonary fibrosis (IPF), although recurrence of HP may occur in a minority of patients.82

EPIDEMIC THUNDERSTORM ASTHMA Historical Perspective Since the mid-1980s, thunderstorm asthma epidemics have been reported from around the globe, including the United Kingdom, Italy, North America, the Middle East, and Australia (Fig. 58.6).83 The most devastating and deadly event occurred in Melbourne, Australia, in the spring of 2016. At least 3500 individuals presented to emergency departments

Pathogenesis, Etiology, and Epidemiology Thunderstorm asthma is likely triggered by a conjunction of meteorologic, aerobiologic, and individual factors that may interact in an additive or synergistic fashion.83

Meteorologic Factors.  A number of meteorologic phenomena may occur in association with thunderstorm asthma epidemics. Wind gusts ahead of the storm front,86 a sudden drop in temperature,87 a rise in relative humidity, heavy rainfall,86 and lightning,85 have all been associated with thunderstorm epidemics. In particular, downdrafts known as thunderstorm outflows have been implicated in epidemic thunderstorm asthma in a case-control study, occurring on 33% of case days but only 3% of control days.88 These outflows are thought to draw relevant allergens to ground level, where they can be inhaled by susceptible individuals to trigger bronchospasm. Aerobiology.  Airborne allergen appears to be the predominant aerobiologic risk factor. A review of available evidence concluded that exposure to grass pollen, and some fungal species, were consistently associated with thunderstorm asthma epidemics.83 In Australia, high levels of airborne grass pollen have preceded thunderstorm asthma epidemics,89–93 with excessive counts of ruptured grass pollen detected in a number of studies.89,90 All seven major epidemics in Melbourne have occurred in November, at the peak of rye grass (Lolium perenne) pollination. It is postulated that moisture from increased ambient humidity and thunderstorm activity rupture rye

UNITED KINGDOM CANADA: Ottawa, Calgary Year of events 1993–1997 (study period ), 2000 Implicated aeroallergens Fungal spores e.g., Cladosporium, ascomycetes, grass pollen

Year of events 1983, 1984, 1989, 1994, 2002, 2005, 2013 Implicated aeroallergens Fungal spores e.g., Didymella exitialis, Sporobolomyces, Alternaria, Cladosporium IRAN ITALY

Year of event 2013

UNITED STATES OF AMERICA: Atlanta

Year of events 2004, 2010

Implicated aeroallergen Not specified

Year of events 1993–2004 (study period )

Implicated aeroallergens Olive tree pollen (Olea europaea), Parietaria

Implicated aeroallergen Not specified

SAUDI ARABIA Year of event 2002 Implicated aeroallergen Not specified

AUSTRALIA: Melbourne, Wagga Wagga, Newcastle, Canberra Year of events 1984, 1987, 1989, 1997, 1998, 2003, 2010, 2011, 2014, 2016 Implicated aeroallergen Rye grass

Fig. 58.6  Thunderstorm asthma epidemics reported worldwide. (Data from “Literature review on thunderstorm asthma and its implications for public health advice”. 2017. Available from: https://www2.health.vic.gov.au/about/ publications/researchandreports/thunderstorm-asthma-literature-review-may-2107.)

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Fig. 58.7  Scanning electron micrograph of hydrated pollen grain of Lolium perenne rupturing, releasing starch granules (ST). (Reproduced from Grote M, Vrtala S, Niederberger V, Valenta R, et al. Expulsion of allergencontaining materials from hydrated rye grass (Lolium perenne) pollen revealed by using immunogold field emission scanning and transmission electron microscopy. J Allergy Clin Immunol 2000;105:1140-1145. With permission from Elsevier.)

grass pollen grains through osmotic pressure, releasing smaller starch granules impregnated with rye grass pollen allergen.94 These 3-µm respirable granules are then inhaled into the lower airways, where they trigger bronchoconstriction (Fig. 58.7). In the United Kingdom, a number of studies found that thunderstorm events were also associated with high airborne levels of the fungi Alternaria spp., Didymellia spp., and Cladosporium spp.95,96 In at least one report, high levels of ozone have been detected before a thunderstorm asthma episode.97 Other environmental pollutants that were evaluated but not convincingly found to predispose to thunderstorm asthma include nitrogen dioxide, sulfur dioxide, fine particles with diameter of 2.5 µm or less, and fine particles with diameter of 10 µm or less.83

Individual Susceptibility Demographics.  Among reported thunderstorm asthma epidemics, young adults in their 20s and 30s tend to be the most affected individuals.88,92,98–100 Previous episodes have described a slight female predominance.83 Sensitization.  Allergen sensitization is the most common risk factor in most studies, but the specific allergen varies according to region. In Australian thunderstorm asthma epidemics, affected individuals have been almost universally sensitized to grass pollen, specifically, rye grass pollen (Lolium perenne).89,90,92 Australian data regarding sensitization to the fungus Cladosporium spp. have been conflicting.90,92 In United Kingdom epidemics, high rates of sensitization to either grass pollen99 or fungi (Cladosporium spp. and Alternaria spp.)96 have been described, whereas in Italy, affected individuals were sensitized to

either Parietaria spp.101 or olive tree pollen (Olea europaea).102 Epidemics in Saudi Arabia and Iran were not linked to putative allergens. Seasonal allergic rhinitis.  A history of seasonal allergic rhinitis is present in approximately 90% of patients affected by Australian and UK epidemics.89,90,92,95 Together with the high rates of allergen sensitization, this lends strong support to allergen inhalation as the final common pathway in triggering thunderstorm asthma. Asthma.  A history of known asthma can be obtained from 30% to 50% of patients affected by thunderstorm asthma.90,92,96 Among patients without known asthma, many are found to have asthma symptoms that were undiagnosed before the thunderstorm event.92 In one study, 4 out of 85 patients (5%) presenting to an emergency department during a thunderstorm asthma epidemic had experienced a similar attack during a previous thunderstorm asthma epidemic.92 Smoking is not commonly found among affected individuals, affecting less than 10%.92,103 A history of known asthma also appears to increase the risk of a severe asthma attack under thunderstorm conditions. In the largest thunderstorm asthma epidemic to date, the presence of known preexisting asthma predicted the need for hospital admission from the emergency department with an odds ratio of 1.9.104 In the same epidemic, all 35 patients requiring intensive care admission also had a previous asthma diagnosis.105 Ethnicity.  In the largest reported thunderstorm asthma epidemic in Melbourne, Australia, a predominance of patients from Asian (including East Asian and Southeast Asian) and Indian subcontinent ethnicities was reported among patients presenting to emergency departments.106 Further analysis of this cohort found that Asian patients born in Australia were more likely to require admission to the hospital from the emergency department, raising the possibility of an environmental-gene interaction for thunderstorm asthma susceptibility.104 This finding requires confirmation in other populations. Outdoors exposure.  The vast majority of thunderstorm-affected individuals give a history of being outdoors, or indoors with open windows.90,92 This suggests that exposure to airborne exposures are necessary to trigger thunderstorm asthma. Some patients report exposure before the actual storm. This is consistent with the hypothesis implicating thunderstorm outflows, since these usually occur ahead of the storm front.88

Diagnosis At the outset of a thunderstorm asthma epidemic, as patients begin to call for ambulances and present to emergency services with respiratory distress, there may be a significant delay before the nature of the epidemic becomes clear.107 To detect and manage an epidemic, it is essential for health systems to build integrated health information systems able to register a surge in patient numbers in real time. For individual patients, the severity of asthma exacerbations varies from mild episodes resolving with a single dose of bronchodilator, to refractory life-threatening attacks causing respiratory arrest. The majority of patients presenting to emergency departments with thunderstorm asthma can be discharged directly home. Admission rates to hospital wards from the emergency department typically run at 11% to 17%.89 Breathlessness is the most typical symptom, usually accompanied by cough and wheeze.98,103 The onset of symptoms occurs within minutes to hours of the thunderstorm or preceding wind gusts. The total exacerbation duration may be quite short, between 1 and 24 hours.100

Initial Investigations.  In the acute setting, airflow obstruction should be assessed using peak flow meter readings. Pulse oximetry is useful to detect hypoxemia. Arterial blood gas measurement is needed to diagnose respiratory muscle fatigue and incipient ventilatory failure. Blood gas analysis also detects lactatemia or hypokalemia complicating

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TABLE 58.4  Risk Factors and Management Targets for Thunderstorm Asthma Meteorology

Aerobiology

Individual Susceptiblity

Risk Factors

Thunderstorm outflow that carries the aeroallergens and concentrates them at ground level Formation of respirable fragments of biologic aeroallergens (e.g., rupture of grass pollen grain into starch granules by moisture)

High levels of ambient biologic aeroallergens (e.g., grass pollen and fungal spores)

Sensitization to aeroallergens (e.g., rye grass [Lolium perenne] pollen) Undiagnosed, untreated, or uncontrolled asthma Asian ethnicity Being outdoors during thunderstorms

Management Targets

Develop forecasting systems to predict thunderstorm asthma events (e.g., thunderstorm characteristics)

Develop pollen monitoring and notification system

Optimize treatment for patients with allergic rhinitis or allergic asthma who are sensitized to seasonal aeroallergens. Active screening for asthma in patients with seasonal allergic rhinitis. Develop an asthma action plan and increase use of inhaled corticosteroids in asthma. Advise at-risk patients to stay indoors during periods of thunderstorms and high pollen count.

Adapted from Queensland University of Technology. Literature review on thunderstorm asthma and its implications for public health advice. Available from: https://www2.health.vic.gov.au/about/publications/researchandreports/thunderstorm-asthma-literature-review-may-2107; Hew M, Sutherland M, Thien F, O’Hehir R. The Melbourne thunderstorm asthma event: can we avert another strike? Intern Med J 2017;47:485–7.

bronchodilator therapy. Under usual circumstances, routine chest radiography is not indicated unless pneumothorax or intercurrent chest infection are suspected.

Skin Testing, Laboratory Investigations, and Lung Function.  After the acute presentation, investigation for specific IgE to putative allergens is usually positive on skin prick or blood testing.92 Lung function after the event is normal in 80% of cases, even in those with a previous known diagnosis of asthma.92 Exhaled nitric oxide may remain elevated out of the season.108,109

Treatment

Acute Asthma Attack.  The treatment of acute epidemic thunderstorm asthma is similar to that for any asthma attack and is described elsewhere. However, the magnitude of presentations to a health service may require disaster contingency plans to be activated. Challenges faced by health systems include overwhelming demand for ambulances, the need to rapidly expand treatment areas in emergency departments to cope with patient influx, scaling up of health care personnel, and ensuring sufficient supplies of essential medication and equipment.107 Public Health Measures (Table 58.4).  Mitigating the impact of epidemic thunderstorm asthma presents a major public health challenge. Optimal management of epidemic thunderstorm asthma requires effective measures to identify and protect at-risk individuals; accurate forecasting of events based on meteorologic and aerobiologic conditions; systems to warn the populace of an impending event; and emergency services prepared and able to cope with an exponential surge in demand.107 At this time, there is no health system with a demonstrated capability to perform all these functions. In some jurisdictions, forecasting systems have been implemented based on a combination of meteorologic and aerobiologic data. However, the accuracy of such forecasting to predict an epidemic remains to be determined.

Identifying and Protecting At-Risk Individuals.  In the majority of circumstances, the most sensitive marker for individuals at risk of thunderstorm asthma is sensitization and symptomatic allergic rhinitis to the culprit allergen.90,92 In these cases, patients should have their

allergic rhinitis treatment optimized, although there is no controlled trial evidence confirming protection. In moderate to severe cases, allergen immunotherapy should be considered. O’Hehir and colleagues have reported that grass pollen sublingual immunotherapy for seasonal allergic rhinitis can protect from epidemic thunderstorm asthma exacerbations.109 Some guidelines recommend that hay fever sufferers in areas at high risk of epidemic thunderstorm asthma have access to a reliever during the season.110 Patients with both asthma and seasonal allergic rhinitis appear to be at greater risk.90,92 Where there is a known diagnosis of asthma, asthma management should be optimized by ensuring regular review, adequate education and self-management, an up-to-date action plan, and regular use of inhaled corticosteroids where indicated. Regular year-round use of preventers is generally recommended, except where patients clearly only experience asthma symptoms during springtime (in Australia) or summer (the United Kingdom). It also appears that many individuals present with thunderstorm asthma in the context of undiagnosed asthma.92 Addressing this scenario requires careful screening of hay fever sufferers for undiagnosed asthma in the community.

SUMMARY Better understanding of allergic bronchopulmonary aspergillosis has involved the continuum of fungal sensitization and formulation of more objective and relevant diagnostic criteria. Early diagnosis and adequate treatment are key to preventing development of permanent lung damage with bronchiectasis. Broader clinical use of new antifungal azoles with oral corticosteroids at an earlier stage in ABPA should significantly improve outcomes. Large clinical trials with these agents are needed, with studies of early diagnosis and screening in patients with asthma and cystic fibrosis using objective diagnostic criteria. These strategies and combined early therapy appear most effective in improving the prognosis and quality of life of ABPA patients. Randomized clinical trials assessing nebulized amphotericin, high-dose inhaled corticosteroids plus antifungal agents, and anti-IgE to treat ABPA are lacking. Over the last decade, new information on the pathogenesis of hypersensitivity pneumonitis has shown multiple elements determining the

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different phenotypes of HP. Concomitant viral or bacterial infection is a promoting factor for HP development, and Tregs are nonfunctional in HP, explaining the large number of lymphocytes present in the lungs of patients with active disease. Pulmonary surfactant is upregulated in HP and is also a modulator of the immune response in these patients. Future studies should explore therapies for HP targeting modulation of these inflammatory pathways. Despite these advances, the prognosis remains unchanged, and early identification of HP is critical. The acute, subacute, and chronic forms of HP may be preventable by removing the patient from antigen exposure or significantly decreasing exposure. Treatment with oral corticosteroids does not reverse chronic changes and should not be used as long-term therapy. Corticosteroids are reserved for more severe forms of acute and subacute HP and exacerbations, with removal of the antigen source. Thunderstorm asthma occurs when a combination of meteorologic, aerobiologic, and individual susceptibility factors intersect. Patients most at risk are those with hay fever and preexisting asthma. The scale, speed, and lethality of such epidemics may overwhelm health services. Risk mitigation requires preventive measures among those at risk, an effective forecasting system, the ability to warn the populace of an impending event, and emergency services able to meet exponential surges in demand.

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CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis 36. Tay TR, Bosco J, Aumann H, et al. Elevated total serum immunoglobulin E (>1000 IU/mL): implications? Intern Med J 2016;46:846–9. 37. Tay TR, Bosco J, Gillman A, et al. Coexisting atopic conditions influence the likelihood of allergic bronchopulmonary aspergillosis in asthma. Ann Allergy Asthma Immunol 2016;117:29–32.e1. 38. Agarwal R, Gupta D, Aggarwal AN, et al. Clinical significance of decline in serum IgE levels in allergic bronchopulmonary aspergillosis. Respir Med 2010;104:204–10. 39. Panchal N, Bhagat R, Pant C, et al. Allergic bronchopulmonary aspergillosis: the spectrum of computed tomography appearances. Respir Med 1997;91:213–19. 40. Menzies D, Holmes L, McCumesky G, et al. Aspergillus sensitization is associated with airflow limitation and bronchiectasis in severe asthma. Allergy 2011;66:679–85. 41. Agarwal R, Khan A, Gupta D, et al. An alternate method of classifying allergic bronchopulmonary aspergillosis based on high-attenuation mucus. PLoS ONE 2010;5:e15346. 42. Kumar R. Mild, moderate, and severe forms of allergic bronchopulmonary aspergillosis: a clinical and serologic evaluation. Chest 2003;124:890–2. 43. Agarwal R, Gupta D, Aggarwal AN, et al. Allergic bronchopulmonary aspergillosis: lessons from 126 patients attending a chest clinic in north India. Chest 2006;130:442–8. 44. Limper AH, Knox KS, Sarosi GA, et al. An official American Thoracic Society statement: treatment of fungal infections in adult pulmonary and critical care patients. Am J Respir Crit Care Med 2011;183:96–128. 45. Agarwal R, Aggarwal AN, Dhooria S, et al. A randomised trial of glucocorticoids in acute-stage allergic bronchopulmonary aspergillosis complicating asthma. Eur Respir J 2016;47:490–8. 46. Cohen-Cymberknoh M, Blau H, Shoseyov D, et al. Intravenous monthly pulse methylprednisolone treatment for ABPA in patients with cystic fibrosis. J Cyst Fibros 2009;8:253–7. 47. Thomson JM, Wesley A, Byrnes CA, et al. Pulse intravenous methylprednisolone for resistant allergic bronchopulmonary aspergillosis in cystic fibrosis. Pediatr Pulmonol 2006;41:164–70. 48. Agarwal R, Khan A, Aggarwal AN, et al. Role of inhaled corticosteroids in the management of serological allergic bronchopulmonary aspergillosis (ABPA). Intern Med 2011;50:855–60. 49. Stevens DA, Schwartz HJ, Lee JY, et al. A randomized trial of itraconazole in allergic bronchopulmonary aspergillosis. N Engl J Med 2000;342:756–62. 50. Wark PA, Hensley MJ, Saltos N, et al. Anti-inflammatory effect of itraconazole in stable allergic bronchopulmonary aspergillosis: a randomized controlled trial. J Allergy Clin Immunol 2003;111:952–7. 51. Glackin L, Leen G, Elnazir B, et al. Voriconazole in the treatment of allergic bronchopulmonary aspergillosis in cystic fibrosis. Ir Med J 2009;102:29. 52. Agbetile J, Bourne M, Fairs A, et al. Effectiveness of voriconazole in the treatment of Aspergillus fumigatus-associated asthma (EVITA3 study). J Allergy Clin Immunol 2014;134:33–9. 53. Voskamp AL, Gillman A, Symons K, et al. Clinical efficacy and immunologic effects of omalizumab in allergic bronchopulmonary aspergillosis. J Allergy Clin Immunol Pract 2015;3:192–9. 54. Zirbes JM, Milla CE. Steroid-sparing effect of omalizumab for allergic bronchopulmonary aspergillosis and cystic fibrosis. Pediatr Pulmonol 2008;43:607–10. 55. Yoshizawa Y, Ohtsuka M, Noguchi K, et al. Hypersensitivity pneumonitis induced by toluene diisocyanate: sequelae of continuous exposure. Ann Intern Med 1989;110:31–4. 56. Ameille J, Brechot JM, Brochard P, et al. Occupational hypersensitivity pneumonitis in a smelter exposed to zinc fumes. Chest 1992;101:862–3. 57. Kunimasa K, Arita M, Tachibana H, et al. Chemical pneumonitis and acute lung injury caused by inhalation of nickel fumes. Intern Med 2011;50:2035–8. 58. Cormier Y, Schuyler M. Hypersensitivity pneumonitis and organic dust toxic syndromes. New York: Marcel Dekker; 2006. 59. Cormier Y, Belanger J, Durand P. Factors influencing the development of serum precipitins to farmer’s lung antigen in Quebec dairy farmers. Thorax 1985;40:138–42.

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60. Coultas DB, Zumwalt RE, Black WC, et al. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994;150:967–72. 61. Singh S, Collins BF, Sharma BB, et al. Interstitial lung disease in India. Results of a prospective registry. Am J Respir Crit Care Med 2017;195:801–13. 62. Xaubet A, Ancochea J, Morell F, et al. Report on the incidence of interstitial lung diseases in Spain. Sarcoidosis Vasc Diffuse Lung Dis 2004;21:64–70. 63. Terho EO, Husman K, Vohlonen I. Prevalence and incidence of chronic bronchitis and farmer’s lung with respect to age, sex, atopy, and smoking. Eur J Respir Dis Suppl 1987;152:19–28. 64. Hanak V, Kalra S, Aksamit TR, et al. Hot tub lung: presenting features and clinical course of 21 patients. Respir Med 2006;100:610–15. 65. Tillie-Leblond I, Grenouillet F, Reboux G, et al. Hypersensitivity pneumonitis and metalworking fluids contaminated by mycobacteria. Eur Respir J 2011;37:640–7. 66. Chan AL, Juarez MM, Leslie KO, et al. Bird fancier’s lung: a state-of-the-art review. Clin Rev Allergy Immunol 2012;43:69–83. 67. Barrera L, Mendoza F, Zuniga J, et al. Functional diversity of T-cell subpopulations in subacute and chronic hypersensitivity pneumonitis. Am J Respir Crit Care Med 2008;177:44–55. 68. Girard M, Israel-Assayag E, Cormier Y. Impaired function of regulatory T-cells in hypersensitivity pneumonitis. Eur Respir J 2011;37: 632–9. 69. Blanchet MR, Bennett JL, Gold MJ, et al. CD34 is required for dendritic cell trafficking and pathology in murine hypersensitivity pneumonitis. Am J Respir Crit Care Med 2011;184:687–98. 70. Hodgson MJ, Parkinson DK, Karpf M. Chest X-rays in hypersensitivity pneumonitis: a metaanalysis of secular trend. Am J Ind Med 1989;16:45–53. 71. Lacasse Y, Selman M, Costabel U, et al. Clinical diagnosis of hypersensitivity pneumonitis. Am J Respir Crit Care Med 2003;168:952–8. 72. Johannson KA, Elicker BM, Vittinghoff E, et al. A diagnostic model for chronic hypersensitivity pneumonitis. Thorax 2016;71:951–4. 73. Munoz X, Morell F, Cruz MJ. The use of specific inhalation challenge in hypersensitivity pneumonitis. Curr Opin Allergy Clin Immunol 2013;13:151–8. 74. Ando M, Konishi K, Yoneda R, et al. Difference in the phenotypes of bronchoalveolar lavage lymphocytes in patients with summer-type hypersensitivity pneumonitis, farmer’s lung, ventilation pneumonitis, and bird fancier’s lung: report of a nationwide epidemiologic study in Japan. J Allergy Clin Immunol 1991;87:1002–9. 75. Caillaud DM, Vergnon JM, Madroszyk A, et al. Bronchoalveolar lavage in hypersensitivity pneumonitis: a series of 139 patients. Inflamm Allergy Drug Targets 2012;11:15–19. 76. Okamoto T, Fujii M, Furusawa H, et al. The usefulness of KL-6 and SP-D for the diagnosis and management of chronic hypersensitivity pneumonitis. Respir Med 2015;109:1576–81. 77. Bellanger AP, Gbaguidi-Haore H, Gondoin A, et al. Positive fungal quantitative PCR and Th17 cytokine detection in bronchoalveolar lavage fluids: complementary biomarkers of hypersensitivity pneumonitis? J Immunol Methods 2016;434:61–5. 78. Akashi T, Takemura T, Ando N, et al. Histopathologic analysis of sixteen autopsy cases of chronic hypersensitivity pneumonitis and comparison with idiopathic pulmonary fibrosis/usual interstitial pneumonia. Am J Clin Pathol 2009;131:405–15. 79. Takemura T, Akashi T, Ohtani Y, et al. Pathology of hypersensitivity pneumonitis. Curr Opin Pulm Med 2008;14:440–54. 80. Ohtani Y, Saiki S, Kitaichi M, et al. Chronic bird fancier’s lung: histopathological and clinical correlation. An application of the 2002 ATS/ERS consensus classification of the idiopathic interstitial pneumonias. Thorax 2005;60:665–71. 81. Morisset J, Johannson KA, Vittinghoff E, et al. Use of mycophenolate mofetil or azathioprine for the management of chronic hypersensitivity pneumonitis. Chest 2017;151:619–25. 82. Kern RM, Singer JP, Koth L, et al. Lung transplantation for hypersensitivity pneumonitis. Chest 2015;147:1558–65.

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83. Queensland University of Technology. Literature review on thunderstorm asthma and its implications for public health advice, 2017. Available from: https://www2.health.vic.gov.au/about/publications/ researchandreports/thunderstorm-asthma-literature-review-may-2017. 84. Hew M, Sutherland M, Thien F, et al. The Melbourne thunderstorm asthma event: can we avert another strike? Intern Med J 2017;47:485–7. 85. Newson R, Strachan D, Archibald E, et al. Acute asthma epidemics, weather and pollen in England, 1987-1994. Eur Respir J 1998;11:694–701. 86. Grundstein A, Sarnat SE, Klein M, et al. Thunderstorm associated asthma in Atlanta, Georgia. Thorax 2008;63:659–60. 87. Celenza A, Fothergill J, Kupek E, et al. Thunderstorm associated asthma: a detailed analysis of environmental factors. BMJ 1996;312:604–7. 88. Marks GB, Colquhoun JR, Girgis ST, et al. Thunderstorm outflows preceding epidemics of asthma during spring and summer. Thorax 2001;56:468–71. 89. Bellomo R, Gigliotti P, Treloar A, et al. Two consecutive thunderstorm associated epidemics of asthma in the city of Melbourne. The possible role of rye grass pollen. Med J Aust 1992;156:834–7. 90. Girgis ST, Marks GB, Downs SH, et al. Thunderstorm-associated asthma in an inland town in south-eastern Australia. Who is at risk? Eur Respir J 2000;16:3–8. 91. Howden ML, McDonald CF, Sutherland MF. Thunderstorm asthma–a timely reminder. Med J Aust 2011;195:512–13. 92. Lee J, Kronborg C, O’Hehir RE, et al. Who’s at risk of thunderstorm asthma? The ryegrass pollen trifecta and lessons learnt from the Melbourne thunderstorm epidemic. Respir Med 2017;132:146–8. 93. Wark PA, Simpson J, Hensley MJ, et al. Airway inflammation in thunderstorm asthma. Clin Exp Allergy 2002;32:1750–6. 94. Suphioglu C, Singh MB, Taylor P, et al. Mechanism of grass-polleninduced asthma. Lancet 1992;339:569–72. 95. Packe GE, Ayres JG. Asthma outbreak during a thunderstorm. Lancet 1985;2:199–204. 96. Pulimood TB, Corden JM, Bryden C, et al. Epidemic asthma and the role of the fungal mold alternaria alternata. J Allergy Clin Immunol 2007;120:610–17. 97. Anderson W, Prescott GJ, Packham S, et al. Asthma admissions and thunderstorms: a study of pollen, fungal spores, rainfall, and ozone. QJM 2001;94:429–33. 98. Davidson AC, Emberlin J, Cook AD, et al. A major outbreak of asthma associated with a thunderstorm: experience of accident and emergency departments and patients’ characteristics. Thames Regions Accident and Emergency Trainees Association. BMJ 1996;312:601–4.

99. Packe GE, Ayres JG. Aeroallergen skin sensitivity in patients with severe asthma during a thunderstorm. Lancet 1986;1:850–1. 100. Wardman AE, Stefani D, MacDonald JC. Thunderstorm-associated asthma or shortness of breath epidemic: a Canadian case report. Can Respir J 2002;9:267–70. 101. D’Amato G. Outdoor air pollution and allergic airway disease. Allergy and allergic diseases. Wiley-Blackwell; 2009. p. 1266–78. 102. Losappio L, Heffler E, Contento F, et al. Thunderstorm-related asthma epidemic owing to Olea europaea pollen sensitization. Allergy 2011;66:1510–11. 103. Forouzan A, Masoumi K, Haddadzadeh Shoushtari M, et al. An overview of thunderstorm-associated asthma outbreak in southwest of Iran. J Environ Public Health 2014;2014:4. 104. Hew M, Lee J, Susanto NH, et al. The 2016 Melbourne thunderstorm asthma epidemic: risk factors for severe attacks requiring hospital admission. Allergy 2019;74(1):122–30. 105. Darvall JN, Durie M, Pilcher D, et al. Intensive care implications of epidemic thunderstorm asthma. Crit Care Resusc 2018;20(4):294–303. 106. Thien F, Beggs PJ, Csutoros D, et al. The Melbourne epidemic thunderstorm asthma event 2016: an investigation of environmental triggers, effect on health services, and patient risk factors. Lancet Planet Health 2018;2(6):e255–63. 107. State of Victoria. Review of response to the thunderstorm asthma event of 21–22 November 2016 – Final Report. Inspector-General for Emergency Management, Melbourne, 2017. Available from: https://www2.health.vic .gov.au/about/publications/researchandreports/thunderstorm-asthma -igem-review-final-report-april-2017. 108. Sutherland MF, Portelli EL, Collins AL, et al. Patients with thunderstorm asthma or severe asthma in Melbourne: a comparison. Med J Aust 2017;207:434–5. 109. O’Hehir RE, Deckert K, Zubrinich CM, et al. Epidemic thunderstorm asthma protection with five-grass pollen tablet sublingual immunotherapy: a clinical trial. Am J Respir Crit Care Med 2018;198(1):126–8. 110. National Asthma Council Australia. Epidemic thunderstorm asthma. National Asthma Council Australia, Melbourne, 2017. Available from: https://www.nationalasthma.org.au/living-with-asthma/resources/ health-professionals/information-paper/thunderstorm-asthma. 111. Greenberger PA, Patterson R. Allergic bronchopulmonary aspergillosis. Model of bronchopulmonary disease with defined serologic, radiologic, pathologic and clinical findings from asthma to fatal destructive lung disease. Chest 1987;91:165S–171S.

CHAPTER 58  Allergic Bronchopulmonary Aspergillosis, Hypersensitivity Pneumonitis

986.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following is NOT part of the International Society for Human and Animal Mycology (ISHAM) criteria for diagnosis of allergic bronchopulmonary aspergillosis (ABPA)? a. Total serum IgE >1000 IU/mL b. Detection of Aspergillus spp. on lung biopsy c. Elevated Aspergillus spp.–specific IgE d. Bronchiectasis on high-resolution computed tomography (HRCT) e. Total blood eosinophil count >500 cells/µL 2. Which of the following high-resolution computed tomography (HRCT) findings has a high specificity for allergic bronchopulmonary aspergillosis (ABPA)? a. Tree-in-bud changes b. Bronchiectasis c. Fibrosis d. High-attenuation mucus e. Ground-glass nodules 3. Which of the following about hypersensitivity pneumonitis (HP) is correct? a. Bronchoalveolar lavage (BAL) neutrophilia supports the diagnosis of HP b. BAL CD4+/CD8+ ratio 1 is diagnostic for HP d. BAL lymphocytosis supports the diagnosis of HP e. BAL eosinophilia supports the diagnosis of HP

4. Which of the following about hypersensitivity pneumonitis (HP) is incorrect? a. The posttransplant survival in HP is better than in idiopathic pulmonary fibrosis (IPF). b. Granulomas may be seen in lung biopsy samples of patients with HP. c. Both obstructive and restrictive defects may be observed in HP. d. Randomized controlled studies have shown that systemic corticosteroids improve outcomes in patients with chronic HP. e. Nickel exposure can cause HP. 5. What contributes to epidemic thunderstorm asthma? a. Wind gusts ahead of a storm front, a sudden drop in temperature, and a rise in relative humidity. b. High airborne levels of aeroallergens such as rye grass pollen or fungal spores. c. Individuals sensitized to relevant aeroallergens. d. Outdoors exposure before or during the thunderstorm. e. All true.

59  Immunologic Nonasthmatic Diseases of the Lung Carol A. Langford, James H. Shelhamer, Joseph R. Fontana

CONTENTS Introduction, 987 Granulomatosis With Polyangiitis, 987 Microscopic Polyangiitis, 992

Eosinophilic Granulomatosis With Polyangiitis, 992 Sarcoidosis, 993 Antiglomerular Basement Membrane Antibody Disease, 999

SUMMARY OF IMPORTANT CONCEPTS

GRANULOMATOSIS WITH POLYANGIITIS

• Granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA) are systemic vasculitis syndromes that share clinical and pathologic features and are associated with antineutrophil cytoplasmic antibodies (ANCA). • The principal features of GPA are necrotizing granulomatous inflammation and vasculitis involving the upper and lower respiratory tract that is frequently associated with glomerulonephritis. • MPA is a form of small-vessel vasculitis that frequently involves the kidney and lung but is not associated with granulomatous inflammation. • EGPA is a distinct clinical syndrome characterized by systemic vasculitis with eosinophilia that occurs almost exclusively in individuals with asthma and allergic rhinitis. • Sarcoidosis is a systemic disease characterized by granulomatous inflammation that involves the lung on presentation in approximately 90% of the cases and that has a variable natural history. • Antiglomerular basement membrane antibody disease is characterized by the development of antiglomerular basement membrane antibodies, crescentic glomerulonephritis, and diffuse pulmonary hemorrhage.

Introduction and Historical Perspective

INTRODUCTION This chapter focuses on immunologically mediated lung diseases other than asthma. Although a variety of inflammatory and immune-mediated diseases have pulmonary involvement as part of the spectrum of disease presentation, we have chosen to address five disease processes. Three of the primary systemic vasculitis syndromes in which lung involvement is a prominent feature are discussed: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). These three diseases are forms of small-vessel systemic vasculitis that share clinical and immunopathologic features, including an association with antineutrophil cytoplasmic antibodies (ANCA), such that these have most recently been considered collectively as forms of ANCA-associated vasculitis. The other two processes discussed are sarcoidosis, which results in granulomatous inflammation of the interstitium, bronchovascular structure, and airways, and Goodpasture disease or antiglomerular basement membrane antibody disease with pulmonary hemorrhage.

GPA is characterized by granulomatous vasculitis involving the upper and lower respiratory tracts that is frequently associated with glomerulonephritis. The first case of GPA was described by Klinger in 1931 as a variant of polyarteritis nodosa.1 In 1936, Wegener recognized the syndrome as a separate clinical entity.2 Subsequently, more detailed clinical and pathologic descriptions were published and the term “Wegener’s granulomatosis” was introduced into the literature.3 In 2011, an international panel of experts in vasculitis recommended changing the name of the syndrome to granulomatosis with polyangiitis (Wegener’s, GPA) with the intention over time to remove the parenthetical term.4 Within the most recent nomenclature system for vasculitis, GPA together with MPA and EGPA have been considered to be within the category of ANCA-associated vasculitis.5

Epidemiology GPA can develop at any age, with the mean age at onset being approximately 40 years.6,7 Men and women are affected equally by GPA. The reported incidence varies from 4 to 15 per million, with a prevalence of 3 per 100,000.8–10 It is unclear whether there has been an increase in the incidence of GPA over time.10 A potential rise in the diagnosis of GPA may be related, in part, to improved recognition through the use of ANCA testing. Some studies have shown a weak association between the disease and environmental exposures such as silica, heavy metals, and farming, but these require further verification.10

Pathogenesis The immunopathogenesis of GPA is unclear, but many different aspects are actively being studied.11 Identification of genetic factors has been an important recent avenue of investigation.12 The involvement of upper airways and lungs with granulomatous inflammation suggests that GPA may be initiated by an aberrant cell-mediated immune response to an exogenous or endogenous antigen that enters through or resides in the respiratory tract. In vitro studies have demonstrated increased production of interferon-γ (IFN-γ) and tumor necrosis factor (TNF) by peripheral blood CD4+ lymphocytes obtained from patients with active GPA.13 Furthermore, monocytes from patients with GPA produce increased amounts of interleukin-12 (IL-12).13 In vitro studies have found skewed

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Th17 responses in activated peripheral blood CD4+ T cells from patients with GPA.14 These findings suggest that patients with GPA have an immunoregulatory defect that leads to excessive production of Th1/ Th17 cytokines (IL-17, TNF, and IFN-γ) in response to an exogenous trigger or autoantigen. The excessive production of TNF and IFN-γ could serve to initiate and perpetuate the granulomatous inflammatory vascular lesion that is characteristic of GPA. In addition to these abnormalities of cell-mediated immunity, a high percentage of patients with GPA develop ANCA, which has raised important clinical and pathophysiologic questions. In vasculitis, there are two major types of ANCA based on the specificity of the autoantibodies. Around 75% to 95% of patients with active GPA have detectable antibodies directed against proteinase-3 (PR3), a 29-kD serine proteinase that is present in neutrophil azurophilic granules.15 These PR3-ANCA exhibit a cytoplasmic pattern of fluorescence in an indirect immunofluorescence assay and are often referred to as cytoplasmic ANCA or cANCA. The other major type of ANCA recognizes the neutrophil enzyme myeloperoxidase (MPO) and occurs in a minority (5%-20%) of patients with otherwise typical GPA. MPO and ANCA exhibit a perinuclear pattern of fluorescence in an immunofluorescence assay and are often referred to as perinuclear ANCA or pANCA. There are a number of observations that suggest mechanisms whereby ANCA could contribute to the pathogenesis of vasculitis.16 PR3 and MPO reside in the azurophilic granules and lysosomes of resting neutrophils and monocytes, where they are inaccessible to serum antibodies. However, when myeloid cells are exposed to TNF or IL-1, PR3 and MPO translocate to the cell membrane, where they can interact with extracellular ANCA and activate neutrophils. ANCA-activated neutrophils release factors that generate C5a, and they can also adhere to and kill endothelial cells in vitro.17–20 Further evidence for a direct pathogenic role of ANCA was provided by transfer experiments in genetically engineered mice. In these studies, MPO knockout mice were immunized with murine MPO. Subsequent passive transfer of serum (containing high-titer anti-MPO antibodies) or lymphocytes into wild-type mice resulted in glomerulonephritis and small-vessel vasculitis.21 A similar approach was used to generate high-titer antibody to murine PR3, but passive transfer of serum containing high-titer anti-PR3 antibodies failed to produce any pathology.22 A number of clinical and laboratory observations argue against a primary role for ANCA in the pathogenesis of GPA. Patients may have active GPA in the absence of ANCA.23 Antibody titers do not correlate well with disease activity, and patients with GPA in remission may continue to have high ANCA titers without experiencing a recurrence of disease.23 In addition, it is not clear how ANCA could give rise to granulomatous inflammation. Therefore a precise role of ANCA in the pathogenesis of GPA remains to be defined.

Clinical Features Most patients with GPA initially seek medical attention because of upper or lower respiratory tract symptoms, which are commonly accompanied by generalized symptoms of fatigue, fever, or arthralgias. Patients often present with upper respiratory tract abnormalities such as paranasal sinus pain and purulent drainage or bloody nasal discharge with nasal mucosal ulceration.6 Nasal septal perforation may follow, as may erosion of the nasal cartilage leading to saddle-nose deformity. Pulmonary involvement occurs in 85% to 90% of patients and may be asymptomatic or clinically expressed as cough, hemoptysis, dyspnea, and chest discomfort. The usual form of pulmonary involvement is multiple bilateral, nodular cavitary infiltrates (Fig. 59.1A), which on biopsy reveal the typical necrotizing granulomatous vasculitis. A less common form of lung involvement in GPA is diffuse alveolar hemorrhage (Fig. 59.2; see later discussion of MPA).

Tracheal stenosis resulting from active disease or scarring occurs in approximately 20% of patients and may result in severe airway obstruction (Fig. 59.3). Typically, it is diagnosed after the onset of other disease manifestations, but it may be a presenting feature.24 The area of stenosis is usually limited to the subglottic trachea and results from a mixture of inflammation and fibrosis. Approximately 50% of patients develop symptoms of subglottic tracheal stenosis at a time when the remainder of their disease is quiescent.24 Dyspnea, with or without voice changes, is the most common presenting symptom. A similar process can also occur in the bronchial tree, giving rise to endobronchial stenosis with obstructive lung disease.6 Clinically significant disease can occur in a wide range of other organ systems.6 This can particularly include glomerulonephritis that can lead to renal failure, ocular involvement that can be visually threatening, as well as disease of the peripheral and central nervous system, skin, joints, and other sites. The similarity in clinical features between GPA and other disease processes can provide challenges not only during diagnosis but also in determining disease relapse. In patients with GPA who are still receiving immunosuppressive therapy, relapse should be based on objective evidence of disease activity, taking care to rule out other conditions that may have a similar appearance, such as infection, medication toxicity, or chronic disease sequelae.

Diagnosis The diagnosis of GPA is usually made by the demonstration of clinically compatible features together with characteristic histologic findings on tissue biopsy. Lung tissue obtained by open biopsy from a radiographically abnormal location offers the highest diagnostic yield. Biopsy of the paranasal sinuses or tracheal mucosa may reveal granulomatous inflammation with necrosis but often does not demonstrate vasculitis.25 In patients with clinical evidence of renal disease, kidney biopsy can confirm the presence of glomerulonephritis. Laboratory findings reflect systemic inflammation and include elevated erythrocyte sedimentation rate (ESR) and/or C reactive protein (CRP), anemia, leukocytosis, and thrombocytosis. Because of the potential for asymptomatic renal involvement, the urinalysis is a very important test in detecting features of glomerulonephritis. The specificity of a PR3-ANCA for GPA is very high. However, the predictive value of this test for diagnosis of GPA is highly dependent on the clinical setting. In patients with sinusitis, glomerulonephritis, and noninfectious pulmonary infiltrates, the predictive value of a positive anti–PR3-ANCA may exceed 90%.26 For other clinical presentations (e.g., pulmonary infiltrates without glomerulonephritis) in which the prevalence of GPA would be less, the predictive value is often too low to justify initiation of toxic therapy in the absence of a tissue diagnosis. In its typical presentation, GPA can usually be readily differentiated from other pulmonary-renal syndromes based on clinicopathologic features (Table 59.1). However, if the typical features are not present, the differential diagnosis is broad and includes a variety of infectious, neoplastic, and autoimmune diseases.

Pathology The distinctive triad of granulomatous inflammation, necrosis, and vasculitis of the respiratory tract distinguishes GPA from other forms of pulmonary vasculitis.27 In the lung, granulomatous inflammation usually produces solitary or multiple parenchymal nodules that may be bronchocentric, angiocentric, or interstitial. Within these nodules, an outer rim of granulomatous inflammation surrounds randomly scattered areas of necrosis or vasculitis or both (see Fig. 59.1). Although granulomatous lesions may be found outside the respiratory tract, small-vessel vasculitis is more common. The histopathology

CHAPTER 59  Immunologic Nonasthmatic Diseases of the Lung

A

B

C

D

989

Fig. 59.1  (A) Chest computed tomographic scan of a patient with granulomatosis with polyangiitis (GPA) shows typical nodular lung infiltrate with cavitation. (B) Low-power view of granulomatous inflammation and geographic necrosis (arrow) in a lung biopsy from a patient with GPA. (C) Granulomatous vasculitis involving a small pulmonary artery in the lung of a patient with GPA. The vessel wall is markedly thickened with an inflammatory infiltrate that includes multinucleated giant cells. (D) Glomeruli showing segmental necrosis with early crescent formation (arrows).

A

B

Fig. 59.2  (A) Chest computed tomographic scan of a patient with microscopic polyangiitis shows “groundglass” infiltrates typical of alveolar hemorrhage. (B) Low-power view of the lung biopsy from the same patient showing capillaritis and alveolar hemorrhage.

of vasculitis in GPA is varied and may include acute leukocytoclastic capillaritis, necrotizing arteritis, granulomatous vasculitis, and venulitis.3 The renal lesion in GPA is characterized by a focal, segmental, necrotizing glomerulonephritis (Fig. 59.1D).28 In contrast to other forms of glomerulonephritis, the glomerular pathology typically lacks evidence of immune complex deposition and is often referred to as pauciimmune. Granulomatous or vasculitic lesions are rarely found in kidney biopsy specimens.

Treatment GPA can follow a diverse clinical course that is strongly influenced by the sites of organ involvement and severity. If left untreated, generalized

GPA is usually lethal. In one series of untreated patients, the mean survival time was 5 months, and more than 90% of patients died within 2 years after diagnosis.29 The effect of treatment with glucocorticoids alone on the course of GPA has never been fully defined. The most widely quoted paper on this subject is a literature review of 26 cases of GPA treated in the 1950s and early 1960s with glucocorticoids.30 Many of these patients were seriously ill, and although glucocorticoids prolonged survival, mortality occurred in just over 1 year. In 1973, Fauci and Wolff at the National Institutes of Health reported on the induction of disease remission in 12 patients with GPA who were treated with daily cyclophosphamide and glucocorticosteroids.31 Subsequent studies of larger cohorts of patients confirmed the efficacy

990

SECTION E  Respiratory Tract 16

12 Normal 8 Patient 4 L/s

A

0 6

4

2

0

(L) –4

–8

B

–12

C Fig. 59.3  (A) Sagittal section of magnetic resonance imaging scan of the neck shows stenosis of the subglottic trachea (arrow) in a patient with granulomatosis with polyangiitis (GPA). (B) Flow-volume loop in a patient with GPA and subglottic tracheal stenosis demonstrates flattening of the inspiratory and expiratory phases, which is typical of fixed extrathoracic airway obstruction. (C) View through operative laryngoscope shows stenosis of the subglottic trachea in a patient with GPA.

of this treatment to induce remission in 80% to 100% of patients.32 Through extended follow-up, it became apparent that despite successful remission induction, relapse of disease occurred in 50% to 70% of patients.6 Furthermore, the use of repeated or prolonged courses of cyclophosphamide to prevent or treat disease relapses resulted in a high rate of serious drug-related toxicity, including major infections, infertility, myeloproliferative disorders, cystitis, and transitional cell carcinoma of the bladder.33 This prompted the investigation of alternative approaches to cyclophosphamide for the treatment of GPA. Current therapy for GPA is considered to have two phases: induction where active disease is put into remission followed by maintenance to continue remission.34,35 Treatment decisions are further influenced by the degree of disease severity. Features of severe disease include alveolar hemorrhage, glomerulonephritis, nervous system involvement, or other immediately organ- or life-threatening features. Examples of nonsevere disease include involvement of the sinus, skin, joint, or lung without respiratory compromise. Organ site is additionally important, because not all manifestations require systemic immunosuppressive therapy. In patients who present with severe active GPA, the options for remission induction include glucocorticosteroids combined with either cyclophosphamide or rituximab. Glucocorticoids are usually started as prednisone 1 mg/kg/day, continued for the first month and then tapered. Patients with life-threatening disease are typically first treated with methylprednisolone 1000 mg/day for 3 days, although there has been no data comparing this to lower doses. Clinical trials have used

different prednisone tapering schedules that have ended either with discontinuation or tapering to a stable dose of prednisone at 5 mg/ day. Although a metaanalysis suggested that continuing low-dose prednisone may lessen the risk of relapse, this remains uncertain.36 In settings where cyclophosphamide is used for induction of severe active disease, this can be given either as a daily oral dose (2 mg/kg/day) or as intravenous pulses (15 mg/kg every 3 weeks).37 Both cyclophosphamide regimens appear equally effective at inducing remission, with the daily oral regimen being found to have a lower rate of disease relapse. Cyclophosphamide is given for 3 to 6 months, after which time it is stopped and switched to a less toxic agent for remission maintenance therapy as is discussed later. The use of rituximab, a chimeric monoclonal antibody against CD20, has been a successful approach for remission induction that allows avoidance of cyclophosphamide. After an encouraging pilot experience, two comparative studies were conducted that provided evidence for the efficacy of rituximab in GPA and MPA.38–40 In the RAVE trial, rituximab 375 mg/m2/week for 4 weeks was compared with cyclophosphamide 2 mg/kg/day in a randomized double-blind placebo-controlled trial.38,39 At the primary endpoint, which was being in remission off prednisone at month 6, rituximab was as effective as cyclophosphamide and appeared to be more effective in those enrolled at the time of a relapse. These results led to the approval of rituximab by the Food and Drug Administration (FDA) for the treatment of GPA and MPA. In the RITUXVAS trial, all patients received two doses of cyclophosphamide

991

CHAPTER 59  Immunologic Nonasthmatic Diseases of the Lung

TABLE 59.1  Clinical Comparison of Pulmonary Renal Syndromesa Characteristic

Granulomatosis With Polyangiitis (%)

Microscopic Polyangiitis (%)

Eosinophilic Granulomatosis With Polyangiitis (%)

Anti-GBM Disease (%)

Upper airways disease

95

No

50-60

No

Asthma

No

No

90-100

No

Pulmonary nodules

70-85

No

40-60

No

Alveolar hemorrhage

5-10

10-20

Rare

100

Glomerulonephritis

70-80

75-90

10-40

90-100

Gastrointestinal involvement

400

Borderline

4.0–16

100–400

Mild AHR

1.0–4.0

25–100

0.25–1.0

6–25

Normal

Moderate AHR Marked AHR

100 pg/mL using nasal sponges

Elevated from >2 hours postallergen challenge in nasal fluid. Low concentrations mean levels may be undetectable by nasal lavage, but collection by filter paper or sponges is more sensitive. Reduced by intranasal corticosteroids.

137,162,163

IL-5

0–20 pg/mL (nasal lavage), Levels > 1000 pg/mL using filter strips/ sponges

Elevated from 2 hours postallergen challenge. High concentration makes IL-5 a more reliable marker than IL-4. Levels may correlate with eosinophil lavage counts. Suppressed by intranasal corticosteroids.

137,162,163,232

IL-10

0–10 pg/mL (filter strips); levels > 200 pg/mL with nasal sponges

Low levels, often below assay detection limits; but some studies have shown increase after allergen challenge. IL-10 levels on immunohistochemical staining of nasal biopsy tissue inversely correlate with allergen response, suggesting protective effect.

162,233,234

IL-13

0–2 pg/mL (nasal lavage), >500 pg/mL using filter strips/sponges

Elevated from 4 hours postallergen challenge; inhibited by intranasal corticosteroids. Levels may correlation with IL-5.

137,162,163

Others: IL-1α, IL-1β, IL-2, IL-3, IL-6, TNF-α, IFN-γ, GM-CSF

pg/mL range

Levels may increase during allergen LPR; some studies suggest evidence of steroid suppression.

137,235

0–2000 pg/mL

Increased in allergen EPR.

137,235

pg/mL range

Increased during allergen LPR; steroid suppressible.

137,163,235

Increased at 6–8 hours and 24 hours post allergen. Resting levels elevated during seasonal exposure. Reduced by anti-IL-5 monoclonal antibody.

141,162,179,182

Cytokines IL-4

Chemokines IL-8 Eotaxin, RANTES, MCP-1, MIP-1α

Markers of Eosinophil Activation 4–51 ng/mL in healthy Eosinophil cationic protein (ECP) controls; >200 ng/mL in undiluted samples postallergen; >300 ng/mL in nasal polyps Major basic protein (MBP)

0–30 ng/mL (postallergen, nasal lavage)

Increase from baseline seen during allergen EPR and LPR. Correlation between LPR levels and eosinophil influx. LPR levels reduced by oral prednisolone.

144

Eosinophil-derived neurotoxin (EDN)

0–>1000 ng/mL (postallergen, nasal lavage)

Increase from baseline seen during allergen LPR; inhibited by prednisolone. Greater variability than MBP measurements.

144

0–150 pg/mL (postallergen, nasal lavage)

Increased during allergen-induced EPR, peak at 1.5 µm, at both 15-µg (484 ± 183, 420 ± 121, and 337 ± 169, respectively) and 30-µg doses (551 ± 221, 457 ± 200, and 347 ± 172, respectively). These measurements showed a significant difference between the 6-µm and 1.5-µm aerosols at both doses, but not between other particle sizes.17 These results suggest that regional targeting of inhaled β2-agonists to the proximal airways may be more important than distal alveolar deposition for bronchodilation. Other studies confirm that targeting aerosol distribution by alterations in aerosol particle size and/or inspiratory flow rate can affect the pulmonary response to inhaled drugs.18,19 Nevertheless, further work is needed to determine whether targeting of specific lung regions is practical using these methods and if differences in deposition pattern lead to significant alterations in long-term clinical outcome measures.

Anatomic Factors: Airway Caliber and Distortion Related to Disease A reduction in airway caliber, related to disease, alters the airflow pattern in the lung and affects the distribution of an inhaled aerosol, shifting deposition toward the central or proximal airways,8 as illustrated in

Fig. 63.4  The image on the left is a gamma camera scan of the lungs (anterior view) of a healthy subject after inhaling 0.9% saline aerosol containing technetium-99m sulfur colloid (MMAD = 1.12 µm). Deposition appears uniform throughout the lung field, with good penetration to the lung periphery and no deposition in the trachea or large bronchi. The gamma camera image on the right shows deposition of the same aerosol in the lungs of a patient with asthma who had clinically severe airway obstruction at the time of inhalation (FEV1 = 36% of predicted). In this scan, deposition in the larger conducting airways and trachea appears enhanced compared with that in the healthy subject, presumably as the result of impaction of particles in obstructed airways. Deposition distal to the obstructed airways is reduced. FEV1, Forced expiratory volume in 1 second; MMAD, mass median aerodynamic diameter. (Left image, Reproduced with permission from Laube BL, Georgopoulos A, Adams GK III. Preliminary study of the efficacy of insulin aerosol delivered by oral inhalation in diabetic patients. JAMA 1993;269:2106-9. © 2012 American Medical Association. All Rights Reserved; and Laube BL, Swift DL, Wagner Jr HN, Norman PS, Adams III GK. The effect of bronchial obstruction on central airway deposition of a saline aerosol in patients with asthma. Am Rev Respir Dis 1986;133:740-3.)

Fig. 63.4. The 2D gamma camera image on the left shows the distribution of a 1.1 µm MMAD radiolabeled aerosol, generated by a jet nebulizer, in the lungs of a healthy subject with no airway obstruction.20 In contrast, the image on the right shows the distribution of the same radiolabeled aerosol in the lungs of a patient with severe asthma (FEV1 of 36% of predicted)21 and the effect of airways obstruction on the regional deposition pattern. The degree of airways obstruction at the time of aerosol administration plays a major role in determining the distribution of an inhaled medication. Several studies have shown that, in patients with severe lung disease, central airway deposition may be enhanced and peripheral deposition reduced, as mucus plugging, turbulent airflow, and airway obstruction increase.22,23 In addition, deposition at airway bifurcations will be exaggerated in the presence of airway obstruction secondary to disease, even when inhaling at low flow rates.

Patient-Related Factors: Ability to Correctly Use the Delivery System and Adherence Many patients do not use the correct inhalation technique with their inhalers, either because they have not been taught proper technique, or because they have modified the technique after instruction. Health care providers should ensure that their patients can and will use these devices correctly. This requires that the clinician be aware of the devices that are currently available to deliver the prescribed drugs, know the various techniques that are appropriate for each device, be able to evaluate the patient’s inhalation technique to confirm that he, or she, is using the devices properly, and ensure that the inhalation method is appropriate for each patient. A recent metaanalysis, spanning 40 years of publications in this area, has concluded that patient technique has not improved, even with instruction.24 The advantages and disadvantages

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SECTION E  Respiratory Tract

of each device as well as specific, detailed recommendations for the correct use of most commercially available inhalers are set forth in a Consensus Statement published in the European Respiratory Journal.25

AEROSOL DRUG DELIVERY DEVICES Therapeutic aerosols are produced by pneumatic (jet), ultrasonic, and new-generation nebulizers, pMDIs, and DPI devices. Each of these delivery systems has advantages and limitations that should be considered in selecting a device for use by an adult or a pediatric patient. Advantages and disadvantages of the different systems are reviewed in the following descriptions of each type.

Nebulizers for Liquid Formulations

Jet Nebulizers.  Conventional pneumatic, or jet, nebulizers use compressed air or oxygen to break up a thin film or jet of fluid into droplets suitable for inhalation. The nebulizer bowl is filled with drug in aqueous solution or suspension. Compressed air or oxygen is applied to the jet inlet and, traveling at a high velocity, exits through a narrow orifice, creating an area of low pressure at the outlet of the adjacent liquid feed tube. This pressure differential causes fluid from the reservoir to be drawn up into (i.e., the Venturi effect) and out of the tube. The liquid is then shattered into droplets of various sizes by the nebulizer walls or internal baffles. The larger droplets are returned to the fluid reservoir, whereas the finer droplets are carried out of the nebulizer to the patient by the flow of air. Large particles are lost on the interior surfaces of the nebulizer, in the connecting T-piece often mounted on top of the nebulizer and in the mouthpiece, or face mask, which provides the interface for the nebulizer to the patient. The MMAD of jet nebulizers used for therapy varies, but should be between 1 and 4 µm to optimize deposition in the lower respiratory tract. A jet nebulizer delivers aerosol continuously while the patient inhales and exhales. During this process, as much as 30% to 40% of the nominal dose is trapped in the nebulizer, and more than 60% of the ED is wasted to the atmosphere during exhalation, translating to an availability of less than 10% of the nebulizer contents to the patient. The advantage of these nebulizers is their low cost and the production of aerosol with little effort on the part of the patient. As a result of the high flow of gas through a jet nebulizer, solvent evaporates during nebulization, reducing the volume delivered and concentrating the aerosol. The rate of evaporation depends on the volume of fluid placed in the reservoir. With a reservoir fill volume of 3 to 5 mL, compared with the 2 mL in unit dose “nebules,” a greater total amount of drug is aerosolized and delivered to the patient, albeit for a longer treatment time.26,27 At the end of nebulization, when no further aerosol is produced, approximately 0.5 to 1.5 mL of concentrated solution is left in the nebulizer reservoir. This is referred to as the dead volume, representing drug that is unavailable to the patient.26,27 The greater the dead volume, the less the amount of drug available to the patient. The driving pressure or the flow rate of compressed air applied to the jet affects aerosol output and particle size from jet nebulizers. The higher the pressure or flow rate, the greater the output over time in terms of total solution aerosolized, and the smaller the particle size.27 The time required to deliver the medication varies with the airflow rate used to drive the nebulizer. Typical treatment for traditional jet nebulizers ranges between 10 and 25 minutes.27 In general, mouthpieces are used during nebulizer delivery. However, face masks may be necessary for treatment of acutely ill or uncooperative patients such as infants and toddlers. The face mask for a nebulizer should not incorporate a tight seal. If available, a face mask with vent holes should be used, which will reduce deposition on the face and in the eyes.28,29 Improvements in face mask design, such as reduced mask

dead space, provide for greater inhaled mass of drug while reducing facial and ocular deposition.29 When the patient does not tolerate the face mask, practitioners sometimes use the mouthpiece of a nebulizer to direct the aerosol toward the nose and mouth, usually in a child. This is called the “blow-by” technique. Data supporting this maneuver as an effective method for delivering aerosol to the lungs are lacking, however, and an NIH Expert Panel has indicated that the use of this technique is not appropriate.30 A number of jet nebulizers are currently available to aerosolize bronchodilator, steroid, antiallergy, and antimicrobial drug solutions. Performance characteristics for jet nebulizers vary in terms of the MMAD of the aerosol produced, as well as the mass of drug available for inhalation over a standard treatment time. The physician should know the performance characteristics of a given nebulizer before selecting it for a specific patient, because differences between devices can have substantial implications for clinical management.31 In addition, the physician should be aware of the treatment times required for complete nebulization of the prescribed drug dose with the different devices. The goal should be to select a nebulizer that provides sufficient therapy in as short a time as possible. Optimizing these parameters should increase patient adherence with the therapeutic regimen. The physician also should be aware that in addition to the active ingredient, therapeutic nebulizer solutions may contain excipients such as buffers and preservatives (e.g., metabisulfite, benzalkonium chloride, ethylenediaminetetraacetic acid [EDTA]) that when inhaled can produce asthma-like symptoms, such as bronchoconstriction and cough, sometimes accompanied by a marked reduction in FEV1.32 Jet nebulizers are relatively inefficient in comparison with newergeneration devices, as described further on. Despite this limitation, they are still widely used. A more detailed description of these systems can be found in the European Respiratory Society (ERS) Guidelines on nebulizers published in 2001.33

Ultrasonic Nebulizers.  Ultrasonic nebulizers incorporate a piezoelectric crystal, which is vibrated at a high frequency with sufficient intensity to create standing waves on the surface of the liquid overlying the crystal. Droplets are formed that remain within the nebulizer until they are swept out by a fan or the patient’s inspiratory breath. Most current ultrasonic nebulizers operate at frequencies above 1 MHz, producing aerosols with MMADs between 2 and 12 µm, with an output that is two to three times higher than with most jet nebulizers.34 Heat is produced along with the aerosol, however, because the ultrasonic nebulizer solution is sonicated, and the temperature can rise 10° to 15° C over a 10-minute treatment period. This may adversely affect heat-sensitive components of formulations, such as proteins. Ultrasonic nebulizers also are not suitable for nebulizing suspensions.

New-Generation Nebulizers New-generation nebulizers are shown in Fig. 63.5, and key design features are listed in Table 63.2. They include breath-enhanced nebulizers that entrain air through the nebulizer during inspiration, breath-actuated nebulizers that reduce or eliminate aerosol generation during the patient’s expiratory phase, vibrating mesh or plate systems that produce aerosols through multiple holes or apertures, systems that extrude or force liquid through precision-made nozzles, and soft mist inhalers. Mechanical generation of low-velocity (soft mist) aerosol, improved particle characteristics for enhanced lower lung deposition, augmented aerosol output, and systems that minimize residual volume of medication left in the nebulizer have substantially improved aerosol device efficiency, thereby increasing drug delivery to the patient. Some “smart” nebulizers also can monitor patient adherence with the therapeutic regimen and provide a means of management of the patient’s treatment schedule. The newer

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems

eFlow, PARI, GE

AeroEclipse, TMI

I-neb, Respironics USA

AeroNeb Go, Aerogen, IRE

MicroAir NE-U22, Omron JPN

Respimat Boehringer-Ingelheim, GE

Fig. 63.5  New-generation nebulizers that are commercially available for therapy. All designs incorporate advanced technologies and/or features that are more efficient in producing and providing aerosol.

TABLE 63.2  Design Features of New-

Generation Nebulizers Category

Device Name

Manufacturer

Vibrating mesh/ vibrating aperture

MicroAir NE-U03, NE-U22 Touchspray e-Flow I-neb AAD System AeroNeb Go

Omron, United States

Extrusion through nozzles

Respimat

Boehringer-Ingelheim, Germany

Breath-actuated

AeroEclipse

Trudell Medical, Canada

Breath-enhanced

Ventstream Pro Pari LC Plus and LC Star, LC Sprint

Respironics, United States PARI, Germany

“Smart” nebulizers: Controls dose during inhalation

I-neb AAD System

Respironics, United States

“Smart” nebulizers: Controls inhalation rate and inhalation volume

Akita system

Inamed, Munich

ODEM, United Kingdom PARI, Germany Respironics, United States Aerogen, United States

AAD, Adaptive aerosol delivery.

designs (see Fig. 63.5) are more efficient in providing aerosolized therapy, with pulmonary deposition improved from the former standard of approximately 10% to more than 60% of the nominal dose.35

Breath-Actuated Nebulizers.  Breath-actuated nebulizers generate aerosol only during inspiration, eliminating wastage of aerosol during exhalation and increasing the delivered dose threefold or higher, compared with continuous and breath-enhanced nebulizers. AeroEclipse (Trudell Medical International, London, Ontario, Canada) is an example of a

1055

breath-actuated jet nebulizer (see Fig. 63.5), in which the nebulizer baffle is moved away from the jet orifice, causing nebulization to cease. This mechanism, described by TT Mercer,36 was incorporated into the AeroEclipse design as a unique spring-loaded, one-way valve design that draws the jet to the capillary tube during inspiration and causes nebulization to cease when the patient’s inspiratory flow falls below threshold, or the patient exhales into the device. Expiratory pressure on the valve at the initiation of exhalation moves the nebulizer baffle away from its position directly above the jet orifice, reducing the pressure in this area and stopping the aerosolization of the fluid. Thus drug wastage during the expiratory phase of the breathing cycle is almost completely eliminated. The in vitro performance of the AeroEclipse has been compared with that of several other small-volume nebulizers under continuous, rather than intermittent, airflow conditions. In vitro simulation experiments using an adult breathing pattern showed that the AeroEclipse provided double the amount of aerosolized albuterol on an inhalation test filter, but nebulization time was 70% longer than the comparator systems.37 Other in vitro experiments using simulated breathing patterns representative of patients with cystic fibrosis38 showed a reduction in drug loss during nebulization with the AeroEclipse, but delivery efficiency was not significantly better than the comparator. Thus the caregiver has to weigh the advantages of recommending a potentially more efficient system in terms of drug output against prolonged treatment times.

Breath-Enhanced Nebulizers.  Other new-generation nebulizers are breath-enhanced designs (LC Plus, LC Star, and LC Sprint, PARI, Germany; Ventstream Pro, Respironics, United States). These nebulizers have a design feature that vents supplemental air into the nebulizer across the venturi jet in the nebulizer bowl. This supplemental air leads to emission of more aerosol particles from the nebulizer and increased drug output per breath,38 which results in shorter treatment times. During expiration, air flow is directed such that it avoids flow across the jet in the nebulizer bowl, which would decrease the jet flow output in traditional jet nebulizers. Plasma levels of albuterol after treatment with the Ventstream and with a conventional, constant-flow nebulizer have been compared. Measured plasma levels were higher with the Ventstream, suggesting greater deposition of drug in the lung. Greater systemic side effects also were noted.39 Thus, although inhalation with the Ventstream, or other breath-enhanced nebulizers, reduces treatment time and increases patient convenience, higher lung doses may result, potentially leading to an increase in adverse events.

Vibrating Mesh Devices: Passive.  The I-neb Adaptive Aerosol Delivery (AAD) device (Respironics, United States) and the MicroAir (Omron Healthcare, Japan) are passive vibrating mesh devices (see Fig. 63.5). The mesh is attached to a transducer horn and vibrations of the piezoelectric crystal that are transmitted via the transducer horn force the solution through the mesh to create an aerosol. The AAD also is a “smart” nebulizer system that is breath-actuated to control the inhaled dose to the patient. This is accomplished by monitoring the breathing pattern over consecutive tidal breaths of the patient and aerosolizing drug during 50% to 80% of each inspiration until the preestablished dose has been inhaled.40 Built-in electronics indicate when the treatment should be stopped. The AAD also provides a means of monitoring patient treatment schedules and delivered doses, with the intention of improving compliance with therapy. Vibrating Mesh Devices: Active.  The AeroNeb Go (Aerogen Inc, Ireland) and the eFlow (PARI) are active vibrating mesh devices. In these devices, an aperture plate vibrates at a high frequency and draws

1056

SECTION E  Respiratory Tract

the solution through multiple nozzles or apertures that are precisiondrilled into the plate. These devices are battery-operated, dispense aerosols with high FPFs and very low dead volumes, and shut off when treatment is complete (see Fig. 63.5). With the AeroNeb Go, drug is packaged in miniature containers, and the dose is dispensed when the patient pushes a button. The eFlow is designed to be used either with a very low residual volume, to reduce drug waste, or with a relatively large residual volume, so that it can replace conventional jet nebulizers with the same fill volume. Two commercial types of the eFlow are available in the United States: the Trio is available from a limited number of pharmacies that provide drugs to patients with cystic fibrosis. The Altera is available for aerosolization of the antibiotic aztreonam lysine for inhalation (Cayston). Vibrating mesh devices have a number of advantages over other nebulizer systems: They are very efficient and quiet and generally are portable. However, they are considerably more expensive than other types of nebulizers and require a significant amount of maintenance and cleaning after each use to prevent buildup of deposit and blockage of the apertures, especially when suspensions are aerosolized.

Pressurized Metered-Dose Inhalers (pMDIs)

Formulation Issues.  Whether used alone, or in conjunction with a spacer or valved holding chamber (VHC), pMDIs are the most commonly used aerosol delivery devices worldwide. These devices are portable, compact, and relatively easy to use (Table 63.3 lists drugs delivered by pMDI devices). Two key advantages of propellant-based inhalers are that: (1) a uniform dose of drug is released from the inhaler within a fraction of a second after actuation; and (2) doses are reproducible throughout the canister life. One of the major developments in recent

TABLE 63.3  Availability of Respiratory

Therapies in Different Inhaler Categoriesa Inhaler Category b

Dosimetric Systems.  The AKITA system (Activaero, Germany) is another “smart” nebulizer system. It controls the entire inhalation maneuver of the patient by applying a positive pressure delivered with a computer-controlled compressor. It can be used with conventional jet nebulizers and has been shown to improve aerosol delivery efficiency, with up to 60% deposition in the lung periphery of patients with COPD.41 Such computer-controlled systems cost significantly more than traditional nebulizer delivery systems alone. Soft Mist Inhalers.  The only soft mist inhaler that is currently commercially available is the Respimat (Boehringer-Ingelheim, Germany) (see Fig. 63.5). It uses mechanical energy to create an aerosol with a low-velocity spray (10 mm/s) and delivers a unit dose of drug in a single actuation. A detailed review of this device has been published previously.42 Similar to the pMDI, the Respimat requires hand–breath coordination on the part of the patient to dispense drug. The particle size of the aerosol ranges between 2.2 and 5.5 µm MMAD, depending on the formulation being aerosolized. Deposition studies in normal volunteers using radiolabeled drug solutions have demonstrated mean delivery efficiencies to the lower respiratory tract of between 31% and 45% of the ED.43 Some factors to consider in selecting a nebulizer for patients are listed in Box 63.3.

in Selection of Device

FPF, Fine particle fraction; MMAD, mass median aerodynamic diameter.

Drug

SABA

Albuterol/salbutamol Fenoterol Levalbuterol (R-salbutamol) Terbutaline

✓ ✓ ✓ ✓

✓

LABA

Salmeterol Formoterol Olodaterol Indacaterol maleate

✓ ✓

✓ ✓

Anticholinergic Ipratropium bromide

✓

LAMA

Tiotropium bromide Glycopyrronium bromide Umeclidinium

Corticosteroid

Budesonide Fluticasone Beclomethasone Ciclesonide Mometasone Flunisolide

Antibiotic

Tobramycin

Antiviral

Zanamivir Ribavirin

Chromones

Nedocromil sodium

Combination Products ICS/LABA Beclomethasone/ formoterol Budesonide/formoterol Fluticasone/salmeterol Mometasone/formoterol Fluticasone/vilanterol

BOX 63.3  Nebulizers: Points to Consider • MMAD • FPF • Age of patient • Assembly, cleaning, portability • Face mask versus mouthpiece • Treatment time • Dose in single actuation versus nebulization over time • Cost and convenience • Use with all drugs or customized for specific drugs • Delivery efficiency • Requirement for hand–breath coordination

Category

DPI Nebulizer

✓

✓ ✓ ✓ ✓ ✓

✓

✓ ✓

✓ ✓ ✓

✓

✓ ✓ ✓

✓ ✓ ✓

✓ ✓ ✓

✓ ✓

✓ ✓ ✓ ✓

✓ ✓ ✓ ✓

SABA/AC

Fenoterol/ipratropium bromide

✓

LAMA/LABA

Tiotropium bromide monohydrate/olodaterol hydrochloride Umeclidinium/vilanterol Glycopyrronium bromide/ indacaterol maleate

✓

ICS/LABA/ LAMA a

pMDI

Fluticasone furoate/ umeclidinium/vilanterol

✓ ✓ ✓

Does not include all drugs AC, Anticholinergic; ICS, inhaled corticosteroid; LABA, long-acting beta agonist; LAMA, long-acting muscarinic antagonist; SABA, short-acting beta agonist.

b

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems years has been the transition from CFC propellant pMDIs to HFA propellant pMDIs, as mandated by the Montreal Protocol. Salbutamol (albuterol in the United States) and all corticosteroid inhalers are now formulated with HFA.12 HFA is medically safe, is nontoxic to animals and humans, is devoid of pharmacologic activity, and can be cosolved with ethanol and corticosteroids to produce a solution formulation. One of the drawbacks of this propellant is that it is not compatible with the surfactants that were used in the CFC inhalers—namely, oleic acid, lecithin, and sorbitan trioleate. This issue presented a challenge to the pharmaceutical industry and meant a revised approach to reformulating the existing drugs used to treat asthma and COPD to accommodate this incompatibility. It also provided an opportunity to redesign and improve the MDI hardware for better performance. Fig. 63.6 shows one of the main physical differences between the HFA and CFC propellants.44 Owing to a combination of formulation changes and valve and actuator redesign, the HFA plume is “softer” than the CFC propellant, wider in shape and with a reduced forward jet velocity.12 Three different strategies have been used for reformulating and repackaging CFC pMDIs with HFA: (1) maintaining the same type of formulation (i.e., a suspension [or solution] CFC pMDI became a suspension [or solution] HFA pMDI); (2) changing the formulation

Fig. 63.6  High-speed photographs of the plume geometry of albuterol aerosols. Top, HFA albuterol (Airomir, 3M Pharmaceuticals, St. Paul, MN); bottom, CFC albuterol (Ventolin, GlaxoSmithKline, Research Triangle Park, NC). CFC, Chlorofluorocarbon; HFA, hydrofluoroalkane. (Reproduced with permission from Dolovich M, Leach C. Drug delivery devices and propellants. In: Busse W, Holgate S, editors. Asthma and rhinitis. 2nd ed. Oxford: Blackwell Science; 2000, p. 1719-31.)

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(i.e., a suspension CFC pMDI became a solution HFA pMDI); and (3) changing the formulation as well as the patient interface (i.e., a suspension CFC pMDI became a solution HFA pMDI with a redesign to provide an integral actuator or spacer for the pMDI). Some examples of inhalers in the first category are salbutamol, fluticasone, and salmeterol. Examples in the second category are BDP and ciclesonide. Flunisolide is an example of the third category. For salbutamol, fluticasone, budesonide, and beclomethasone, beclometasone the particle size of the HFA suspension formulations is similar to that of the CFC formulation, and lung deposition averages 7% to 30%. Table 63.4 compares and contrasts the commercially available HFA pMDI albuterol products. With CFC pMDIs, the doses actuated from a new pMDI canister, or from one that had been stored or unused for brief periods of time, contained less active substance than subsequent actuations. Reduction in ED per actuation under such circumstances led to the recommendation to prime these pMDIs before each use. For most albuterol HFA pMDIs, the reduction in dose is still an issue when the pMDI is first used, and priming is required (see Table 63.4 for the number of sprays that are required initially). With the exception of Xopenex HFA, after the initial priming, no additional priming is needed for the other four commercially available albuterol HFA products unless the pMDI has not been used for approximately 2 weeks. The issue of priming after inactivity is more complicated for HFA inhaled corticosteroid (ICS) pMDIs (Table 63.5). For some corticosteroid products, the transition to an HFA propellant solution formulation led to the introduction of ethanol as a cosolvent for the drug. These solution formulations yielded extrafine aerosols, with MMADs of 1 µm and increased FPFs.45 Concentrations of alcohol in the various ICS formulations ranged from 8% to 37% (w/w), with the higher amounts of alcohol causing irritation on inhalation in some patients. Fig. 63.7 shows gamma camera images of the lungs of an asthmatic patient after inhalation of HFA BDP (Qvar) (MMAD = 1 µm) and CFC BDP (MMAD = 3.2 µm). This study shows that the total lung dose with the HFA BDP, as well as the dose delivered to the lung periphery, is approximately threefold to fivefold greater than with the CFC BDP formulation.46 Of importance, mean percent deposition in the lungs for the eight patients in the study shown in Fig. 63.7 was 53.8 ± 10.2% with Qvar and 16.3 ± 8.8% with CFC BDP, indicating a wide range of variability in percent deposition among patients. Clinical trials have shown that one dose of extrafine HFA BDP is clinically equivalent to 2.6 times the dose of CFC BDP.47 Practice guidelines (Global Initiative for Asthma [GINA] and British Thoracic Society [BTS]) and regulatory agencies recommend 100 µg of beclomethasone as Qvar HFA pMDI to be clinically equivalent to 200 µg as beclomethasone CFC pMDI. Nevertheless, whenever a change is made in a device that delivers an ICS, titration to the lowest effective ICS dose should be performed.

TABLE 63.4  Similarities and Differences Between HFA pMDI Albuterol Aerosols Sprays to Prime After Days of Inactivity

Dose Counter

4

14 days: 4

Yes

No

4

14 days: 4

Yes

Yes

4

14 days: 4

No

ProAir HFA

Yes

3

14 days: 3

Yes

Xopenex HFA

Yes

4

3 days: 4

No

Product (All Suspensions)

Contains Alcohol

Proventil HFA

Yes

Ventolin HFA Airomir HFA

Initial Sprays to Prime

HFA, Hydrofluoroalkane; pMDI, pressurized metered-dose inhaler.

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SECTION E  Respiratory Tract

TABLE 63.5  Similarities and Differences Between HFA pMDI ICS Aerosols Initial Sprays to Prime

Sprays to Prime After Days of Inactivity

Product

Drug

Flovent HFA (suspension)

Fluticasone propionate

4

21 days: 4

Dose Counter Yes

Qvar (solution)

Beclomethasone dipropionate

2

10 days: 2

Yes

Aerospan HFA (solution)

Flunisolide

2

>14 days: 2

Yes

Alvesco HFA (solution)

Ciclesonide

3

10 days: 3

Yes

HFA, Hydrofluoroalkane; ICS, inhaled corticosteroid; pMDI, pressurized metered-dose inhaler.

Anterior

Posterior

Lung deposition (ex-actuator)

HFA BDP 67.9%

CFC BDP 16.9%

Fig. 63.7  Scintigraphic images of the lungs, anterior and posterior views, in a patient with asthma after inhalation of two different formulations of radiolabeled beclomethasone dipropionate (BDP) pressurized aerosol. The dose deposited in the lungs for Qvar (the HFA solution formulation of BDP) is four times greater than for Beclovent (the CFC suspension formulation), and the drug appears to be distributed more toward the periphery in this example. The xenon-133 gas outline is shown on each image to indicate the periphery of the lung. CFC, Chlorofluorocarbon; HFA, hydrofluoroalkane. (Reprinted with permission of the American Thoracic Society. Copyright © 2013 American Thoracic Society. Adapted from Figure 1 of Dolovich MB, Labiris NR. Imaging drug delivery and drug responses in the lung. Proc Am Thorac Soc 2004;1:329-37. Official journal of the American Thoracic Society. This figure has been adapted. Please visit www.atsjournals.org to view the original figure.)

Another corticosteroid, ciclesonide (Alvesco), is available as an HFA pMDI product. It also was formulated as a solution with extrafine particles and is similar in potency to extrafine beclomethasone (Qvar). Both contain ethanol as a formulation excipient. With solution HFA pMDIs, the diameter of the actuator orifice typically is smaller, and the aerosol predictably finer. When flunisolide CFC was changed to an HFA solution product (AEROSPAN HFA, ACTON Pharmaceuticals, Inc., Marlborough, MA), the actuator mouthpiece was extended to become a small open-tube spacer. This actuator helped reduce the ED to the same amount as from the CFC inhaler. The patient still needs to coordinate actuation with inhalation, however, because the redesign does not include a valve to allow the aerosol to be contained in the spacer.

Breath-Actuated pMDIs.  Breath-actuated pMDIs were developed to overcome the commonly encountered problem of poor actuationinhalation coordination with use of standard pMDIs. The Autohaler

BOX 63.4  Pressurized Metered-Dose

Inhalers: Points to Consider in Selection of Device • MMAD • FPF • Age of patient for use • Ability of patient to inhale and actuate at the same time • Delivery efficiency • Dose counter FPF, Fine particle fraction; MMAD, mass median aerodynamic diameter.

automatically actuates at inspiratory flow rates of approximately 30 L/ min, and the Easibreathe actuates at 20 L/min. For patients with poor actuation-inhalation coordination, breath-actuated pMDIs may improve lung deposition over that achievable with pMDIs alone. Some factors to consider in selecting a pMDI are listed in Box 63.4.

pMDIs and Spacers and Valved Holding Chambers. Spacer devices and valved holding chambers (VHCs) have been developed for use with pMDIs in response to difficulties encountered by adult and pediatric patients when taking their pMDI medications. The most common problems are related to timing or hand–breath coordination (i.e., coordination of actuating the pMDI and inhaling the spray at the same time).48 The use of a spacer or VHC device can overcome this problem, increasing the probability of optimal delivery of the drug to the lung. Spacers can be as simple as an open tube or as complex as a reservoir chamber with a valve that allows the aerosol to be held in the chamber until the patient inhales. Usually, the VHC interfaces with the pMDI placed at the rear of the chamber. For reverse-flow devices, the pMDI is removed from its actuator mouthpiece and placed proximally, adjacent to, or in the mouthpiece. With reverse flow, the pMDI is fired in the direction away from the patient and reentrained back into the inspiratory breath as the patient inhales (Fig. 63.8). A number of different devices are commercially available. Volumes for the various devices range from 15 to 750 mL. Some factors to consider in selecting a spacer or VHC are listed in Box 63.5. A number of factors affect the particle size distribution and the dose of drug available at the exit of spacer and VHC devices. The addition of a one-way valve to convert an open tube into a reservoir for the aerosol, the shape and volume of the device, flow of air through the device, face masks’ mouthpieces, and manufacturing materials all affect aerosol characteristics and drug yield. The inhalation valve, which is used to contain the aerosol and reduce oropharyngeal deposition (as a baffle), must be able to withstand the initial pressure from the pMDI on firing but

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems

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Tube Spacer

Open Tube Valved Holding Chamber

Holding Chamber

Valved Holding Chamber

Reverse-Flow Spacer Reverse-Flow Reverse-Flow Spacer

A

B

Fig. 63.8  Types of add-on devices for pressurized metered-dose inhalers. (A) Schematic representations. (B) Specific devices.

BOX 63.5  Spacers and Valved Holding

Chambers: Points to Consider in Selection of Device • Design of the device: open tube (spacer), valved holding chamber (VHC) with forward flow, or chamber with reverse flow • Device volume • Component material of device body: plastic with electrostatic charge, metallic coating, or nonelectrostatic material • Presence or absence of an integrated expiratory valve • Inspiratory and expiratory valve resistance level • Age of patient (infant, child, or adult) for whom the spacer or VHC is prescribed • Ability of patient to perform the necessary inhalation technique • Face mask or other interface (i.e., mouthpiece) • Fit of face mask (i.e., loose or tight) Adapted from Dolovich M. In my opinion—interview with the expert. Pediatr Asthma Allergy Immunol 2004;17:292-300.

also must have a sufficiently low resistance to open readily on inhalation, particularly when the device is to be used by children and infants. Exhalation valves in a face mask attached to a spacer device also must provide low resistance for similar reasons.49 Issues of spacer volume, tidal volume, frequency of breathing, and dead space between the spacer and mouth are of particular concern with use of these devices in children. Differences of twofold to threefold in drug available at the mouth have been measured for spacers used by infants.50 Clinicians should therefore be aware of the delivery efficiencies of spacer and VHC devices before prescribing them for their patients. Face mask design also is important and has been reviewed previously.51 Coordination is still required with all three types of spacer design, because even a 2-second delay between firing and inhaling the discharged spray will result in substantial loss of drug and reduced lung delivery.52 Although drug loss from sedimentation occurs during the interval between actuation and inhalation with VHCs, the reduction in suspended dose is not as great as with the open tube spacers. Some fraction of the aerosol also is retained in the spacer through impaction on walls and on the inhalation valve, if present. The decrease in drug output from

plastic spacers is largely due to the presence of electrostatic charge on the plastic.53 Use of a metallic-coated device, or device manufactured from a nonelectrostatic plastic, or washing the plastic device periodically with deionizing detergent, can overcome the loss of drug that is related to electrostatic charge.52,53 Despite differences in design, the initial forward velocity of the pMDI droplets is reduced during the time the aerosol traverses the length of the spacer or VHC. At the same time, the size of the particles exiting the chamber is reduced both as a result of impaction on the chamber walls and partial evaporation of propellant. The MMAD of the pMDI aerosol exiting a spacer is decreased by approximately 25% and the fraction containing particles less than 5 µm in diameter is increased.54 The increase in this fraction appears to depend on the pMDI formulation. The more concentrated the drug, the higher the FPF of aerosol exiting the device relative to the original pMDI aerosol.55 The reduction in particle velocity and size with a spacer or VHC leads to a reduction in the oropharyngeal dose of drug, but the percentage decrease will vary with the spacer or VHC and the pMDI tested.56,57 The advantage of reduced oropharyngeal deposition is fewer side effects from ICS aerosols, which has been demonstrated in a number of published clinical trials.58,59 A variety of factors have been recognized to contribute to poor aerosol delivery in both pediatric and adult patients who use spacers and VHCs. For example, with multiple consecutive actuations, or sprays, from one or more drugs into the chamber, both the total dose and the respirable dose of drug available for inhalation will be reduced. The extent of these losses varies for different drugs and spacer designs. A review of the issues contributing to poor aerosol delivery from holding chambers has been published by Mitchell and Nagel.60

Drugs in Powder Form: Dry Powder Inhalers A number of DPIs are commercially available in North America and many more in Europe. Some dispense individual doses of drug from punctured gelatin capsules such as the Aerolizer (Novartis Pharmaceuticals, United States) and HandiHaler (Boehringer Ingelheim, Germany) and others from a tape system containing multiple sealed, single doses in blisters (Diskus, Glaxo, United Kingdom). The Turbuhaler (Astra Draco, Sweden) is an example of a multidose reservoir powder system. Many DPIs hold the drug powder in bulk in a reservoir, but capsule DPI designs have increased in the last several years.

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SECTION E  Respiratory Tract

Aerosols of dry powder are created by directing air through an aliquot of loose powder. Because DPIs are breath-actuated, the need to synchronize inhalation with actuation is eliminated. However, the dispersion of the powder into respirable particles is dependent on the creation of turbulent flow in the inhaler. Creation of this turbulent flow is a function of both the patient’s ability to inhale the powder at a sufficiently high inspiratory flow rate and the design of the powder device. The design and performance characteristics of various DPIs are discussed in two recent reviews.25,61 Drugs that are available as DPIs are summarized in Table 63.3. Most powder-dispensing systems require the use of a carrier substance. This vehicle substance is mixed with the drug to enable the powder to be more readily dispensed from the device. Lactose, sorbitan trioleate, and mannitol are three sugars that are approved by the FDA for this purpose. The size and surface characteristics of the particles in the carrier/active substance powder blend affect how the formulation “flows” out of the device. The amount of these substances is substantially greater than the drug and can represent 98% or more of the weight per inhaled dose in some blends. The particle size of micronized dry powder particles is on the order of 1 to 2 µm, but the size of the carrier particles can range from approximately 20 to 65 µm. Consequently, most of the carrier deposits in the oropharynx.

Design and Performance.  DPIs that are currently commercially available are passive, or patient-driven, and rely on the patient’s inspiratory effort to dispense the dose from the device. The specific resistance of a DPI device affects the maximal inspiratory flow rate (IFR) that can be drawn through the device. Although high resistance decreases the ability to draw air through the inhaler, inhalation at the optimal IFR and a fast initial acceleration rate help to produce an aerosol with a greater FPF.62,63 Thus an advantage of high-resistance devices is the potential for increasing delivery of drug to the lower respiratory tract. The resistance of a DPI can be classified with respect to the inhalation flow required to produce a pressure drop of 4 kPa across the device.25 This value was chosen because it is the one recommended by pharmacopoeias for the in vitro characterization of the dose emitted from a DPI. A low-resistance device allows an inspiratory flow of more than 90 L/min to produce a pressure drop of 4 kPa. A medium-resistance device allows an inspiratory flow of 60 to 90 L/min, whereas a mediumto high-resistance device allows 50 to 60 L/min inspiratory flow and a high-resistance device allows flows less than 50 L/min.64 Some studies have shown that preschool-aged children with asthma65 and patients with COPD66 may have problems achieving minimum flows through some DPIs. Inhalation flow also appears to be reduced during acute exacerbations.67 Another study has shown that COPD patients with FEV1 values below 27% of predicted were able to inhale through the HandiHaler device, despite its high resistance, at the minimum target of 30 L/min.68 Wide variability has been documented in the FPF of drug powders from existing DPIs, ranging from 10% to 60% of the nominal dose. This variability may potentially result in marked differences in lung deposition between the different devices. Ambient humidity can markedly affect drug powders delivered from DPIs. The ED will decrease in a humid environment, probably as a result of powder clumping and growth resulting from the added moisture. It is therefore important to store DPIs in a cool, dry environment. Oropharyngeal and Lung Deposition With Dry Powder Inhalers.  The peak inspiratory flow rate to dispense powder from most commercially available DPIs is between 30 and 60 L/min. These flow rates result in oropharyngeal doses comparable with that received from a pMDI without an add-on device. Lung deposition from various DPIs has been measured in adults and children. Fig. 63.9 shows percent lung

Diskus/Accuhaler Clickhaler Diskhaler Easyhaler Novolizer Ultrahaler Turbuhaler

17% 27% 12% 19% 20-32% 13% 15-25%

Fig. 63.9  Percent lung deposition for most commonly used dry powder inhalers. Scintigram (left) illustrates potential deposition of aerosol in upper and lower airways. (Courtesy Henry Chrystyn, PhD.)

BOX 63.6  Dry Powder Inhalers: Points to

Consider in Selection of Device

• Specific resistance of the device (i.e., low, medium, high) • Inspiratory flow rate required for optimal delivery • Ability of the patient to perform the correct inspiratory maneuver • Amount of drug lost in the oropharynx • How the powder is stored (e.g., capsule, reservoir, blister on tape) • Dose counter (a feature of some dry-powder inhalers) • Cost Adapted from Dolovich M. In my opinion—interview with the expert. Pediatr Asthma Allergy Immunol 2004;17:292-300.

deposition for the most commonly used DPIs.69-73 All were tested in healthy volunteers using optimal inhalation technique. Variability in lung deposition exists between DPI devices, with rates ranging from 12% to 32% of the ED among the subjects who were studied. These values are similar to, or slightly better, than those observed with pMDIs. All DPIs require the patient to prepare the dose before inhalation, and the appropriate procedure is described in the package insert or patient information leaflet. Patients who do not perform these procedures correctly may receive no dose, irrespective of the inhalation maneuver they subsequently adopt, and this type of critical error occurs frequently.74 As part of their preparation, patients should be instructed to exhale into the room to functional residual capacity before inhaling through their DPI device. When they inhale, they should inhale as forcefully and as deeply from the beginning of their inspiration to optimize delivery to the lungs.25 The use of DPIs has eliminated the need for actuationinhalation coordination that is required of pMDIs. Lung deposition, however, is only slightly improved with DPI administration compared with pMDI delivery (see Fig. 63.9), and, in the United States, the cost of most DPI medications is higher than for pMDI. Some factors to consider in selecting a DPI are listed in Box 63.6.

FUTURE DIRECTIONS Systemic Drug Delivery Over the past several years, the use of inhalation devices to deliver nonrespiratory medications to the systemic circulation via the lung has been undertaken with success. Advances have been made in terms of delivering drugs to treat systemic diseases such as diabetes and osteoporosis and

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems pain associated with cancer.9 Developments in device design have been driven by the recognition that drugs used to treat these diseases can use the peripheral lung as part of the overall delivery system for these therapies. Aerosols generated for these applications must by necessity have a high deposition fraction in peripheral lung for uptake by the systemic circulation to occur.

Nebulizing Catheter The nebulizing catheter takes advantage of the bronchoscope channel to target aerosol treatment to specific sites within a lung lobe.75 This device currently is only an investigative tool.

Nanoparticle Therapy Research into the use of drugs produced as nanoparticles (i.e., particles less than 0.1 µm in diameter) is an expanding field, with promise for high-efficiency drug delivery for treatment of systemic diseases and targeted delivery to specific sites within the lung. Nanoparticles provide new formulation options for pMDIs, nebulizers, and DPIs. Compared with traditional aerosol formulations, nanoparticle formulations also have the advantage of the potential for intracellular drug delivery.76,77 Nanotechnology and nanomedicine are still in their infancy in terms of development.

Generic Devices A generic inhaler is unpatented and therefore tends to be significantly cheaper than its name-brand counterpart. Generic devices for inhalation therapies will be manufactured to replace brand name devices as their patents expire.78 Because of patent laws, however, generic drug companies have not introduced a generic albuterol HFA inhaler in the United States. However, generic HFA salbutamol (albuterol in the United States) pMDIs are available in Canada, Europe, and many other countries. Recently a generic DPI of albuterol (ProAir RespiClick, Teva Respiratory LLC) was approved in the United States.

Vaccines Delivery of vaccines by inhalation is an expanding area of research. The US Food and Drug Administration first approved FluMist, the nasal spray influenza vaccine, produced by MedImmune, a subsidiary of London-based AstraZeneca PLC, in 2003. By all accounts, the vaccine worked well in the early years. However, according to MedImmune, during the 2015 to 2016 flu season the FluMist quadrivalent vaccine was found to be only 46% effective, compared with the flu shot’s 65% effectiveness. Because of this finding, the US Centers for Disease Control (CDC) Advisory Committee on Immunization Practices decided to not recommend the spray for the 2016 to 2017 flu season because of its poor performance compared with the flu shot. In response to these findings, AstraZeneca has initiated a scientific investigation to identify potential causes of lower effectiveness. In recent years, important strides have been made toward global measles control. However, logistical and financial difficulties have slowed progress in developing countries where measles is a major cause of illness and death. Although measles virus (MeV) vaccine is usually given subcutaneously, or intramuscularly, delivery by inhalation has been proposed to help improve coverage in developing countries. Both liquid and dry powder aerosol formulations are under development for use in primary immunization of infants and for delivery of the second or “booster” dose to older children. To date, studies have shown that the aerosolized vaccine provides a stronger and more durable boosting response compared with vaccination by injection in school-age children,79 whereas it appears to be inferior to the subcutaneous vaccine in terms of the rate of seropositivity in 9.0- to 11.9-month-old infants.80 Others are testing an aerosolized vaccine to prevent Mycobacterium tuberculosis, which infects a third of the world’s population every year

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and leads to 1.3 million deaths annually.81 In the first human trial of aerosolized MVA85A, a modified vaccinia virus Ankara (MVA) that expresses the tuberculosis antigen 85A(Ag85A), Satti and colleagues recently showed that vaccine delivered by aerosol was safe and elicited an immune response in the lungs that was superior to that induced by intradermally administered vaccine.82 Additional studies are needed to determine whether this route of delivery increases the level of protection from tuberculosis.

Mannitol Dry Powder A new study has shown that adding inhaled dry powder mannitol to standard therapy for cystic fibrosis produces sustained improvement in lung function for up to 52 weeks.83 In the double-blind study, 318 patients were randomized to receive inhaled mannitol, either 400 mg twice a day as the treatment regimen or 50 mg twice a day as the control regimen, for 26 weeks, followed by an additional 26 weeks of open-label active treatment. Patients in the treatment group showed a 106.5-mL mean improvement in FEV1, representing an 8.22% improvement from baseline, compared with a 52.4-mL improvement (4.47%) in the control group. Percent improvement from baseline was statistically significantly greater in the treated patients, compared with controls (P = 0.029). Treated patients also experienced fewer pulmonary exacerbations than control subjects. Mannitol DPI also is available in some countries as a challenge substance for measuring airway hyperresponsiveness, a clinical feature of asthma.

INHALER SELECTION FOR THERAPY Myriad drug delivery devices are available for the many aerosol therapies. This wide range of devices, each with a different set of instructions for operation, can be confusing for both the physician and patient in trying to select an appropriate delivery system for the drug prescribed. Selecting an inhaler that the patient can and will use correctly is key to successful treatment outcomes with inhaled medications. The following recommendations are taken from the American College of Chest Physicians/American College of Asthma, Allergy, and Immunology (ACCP/ACAAI) Guidelines4 and the ERS/International Society for Aerosols in Medicine (ISAM) Task Force Consensus Statement25 on device selection by care providers for their patients.

Recommendations • Be aware of the devices that are available to deliver specific drugs and classes of drugs (Table 63.3). • Know the advantages and disadvantages of each device. • Choose devices that the patient can and will use effectively. • Choose devices that have been approved by the appropriate authorities. • Train health care workers in proper device technique. • Train patients about the correct inhalation maneuver that is appropriate for the device being prescribed. • Check your patient’s inhaler technique regularly. • Review the patient’s adherence to treatment at each visit. • Do not switch to a new device without the patient’s involvement and without follow-up education on how to use the device properly.

SUMMARY Medications delivered as liquid aerosols from a variety of nebulizer systems, pressurized aerosols using pMDIs, and dry powders from DPIs remain the primary options for treating asthma and other respiratory diseases by inhalation. It is important that physicians and other health care workers understand that deposition of aerosols in the lung depends

1062

SECTION E  Respiratory Tract

on the combination of aerosol-inhaler physical factors, patient-related ventilatory factors, and the nature of the airway or lung disease. Health care providers also must recognize that although many devices are available, not all such devices are appropriate for all patients. Other important considerations relate to the way devices perform, and specific inhalation techniques are necessary for the proper use of available devices. Mastering specific inhalation techniques requires differing levels of cognitive ability that will vary with age. Health care providers should educate their patients in the correct use of their inhaler and emphasize the benefits of correct technique and compliance with the prescribed regimen on treatment outcomes. If the lung anatomy and function is viewed as a black box, and we use conventional clinical measures as a guide to determine outcomes of successful therapy, clinical responses presumably will be comparable for different inhalers if enough drug is given. It is necessary, however, to move beyond this paradigm and begin to consider how to optimize delivery of aerosolized medications to the lung, which means delivering the minimum amount of therapy to provide the best possible response without side effects. This can be achieved in part by better education of both health care providers and patients about aerosol therapy, inhaler choices, and correct inhaler technique.

REFERENCES Introduction 1. Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA); 2017. Available from: http://www.ginasthma.org. 2. Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease (GOLD); 2018. Available from: http://www.goldcopd.org. 3. Brocklebank D, Ram F, Wright J, et al. Comparison of the effectiveness of inhaler devices in asthma and chronic obstructive airway disease: a systematic review of the literature. Health Technol Assess 2001;5:1–149. 4. Dolovich MB, Ahrens RC, Hess DR, et al. Device selection and outcomes of aerosol therapy: evidence-based guidelines: American College of Chest Physicians/American College of Asthma, Allergy, and Immunology. Chest 2005;127:335–71. 5. Dolovich MB, MacIntyre NR, Anderson PJ, et al. Consensus statement: aerosols and delivery devices. American Association for Respiratory Care. Respir Care 2000;45:589–96. 6. Scheuch G, Kohlhaeufl MJ, Brand P, et al. Clinical perspectives on pulmonary systemic and macromolecular delivery. Adv Drug Deliv Rev 2006;58:996–1008. 7. Yu CP, Nicolaides P, Soong TT. Effect of random airway sizes on aerosol deposition. Am Ind Hyg Assoc J 1979;40:999–1005. 8. Dolovich MB. Influence of inspiratory flow rate, particle size, and airway caliber on aerosolized drug delivery to the lung. Respir Care 2000;45:597–608. 9. Laube BL. The expanding role of aerosols in systemic drug delivery, gene therapy, and vaccination: an update. Transl Respir Med 2014;2:3. 10. Iacono AT, Johnson BA, Grgurich WF, et al. A randomized trial of inhaled cyclosporine in lung-transplant recipients. N Engl J Med 2006;354:141–50. 11. Olschewski H, Simonneau G, Galie N, et al; Aerosolized Iloprost Randomized Study Group. Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 2002;347:322–9. 12. Dolovich MB, Dhand R. Aerosol drug delivery: developments in device design and clinical use. Lancet 2011;377:1032–45.

Factors That Affect Aerosol Deposition 13. Dolovich M. Aerosols and aerosol delivery devices. In: Adkinson NF, Bochner BS, Busse WW, et al, editors. Middleton’s allergy: principles & practice. 7th ed. Philadelphia: Mosby; 2009. p. 679–700. 14. Newman SP, Chan HK. In vitro/in vivo comparisons in pulmonary drug delivery. J Aerosol Med Pulm Drug Deliv 2008;21:77–84.

15. Heyder J. Deposition of inhaled particles in the human respiratory tract and consequences for regional targeting in respiratory drug delivery. Proc Am Thorac Soc 2004;1:315–20. 16. 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 1999;104:S258–67. 17. Usmani OS, Biddiscombe MF, Barnes PJ. Regional lung deposition and bronchodilator response as a function of beta2-agonist particle size. Am J Respir Crit Care Med 2005;172:1497–504. 18. Laube BL, Norman PS, Wagner HN Jr, et al. The effect of aerosol distribution on airway responsiveness to inhaled methacholine in patients with asthma. J Allergy Clin Immunol 1992;89:510–18. 19. Ruffin RE, Dolovich MB, Wolff RK, et al. The effects of preferential deposition of histamine in the human airway. Am Rev Respir Dis 1978;117:485–92. 20. Laube BL, Georgopoulos A, Adams GK III. Preliminary study of the efficacy of insulin aerosol delivered by oral inhalation in diabetic patients. JAMA 1993;269:2106–9. 21. Laube BL, Swift DL, Wagner HN Jr, et al. The effect of bronchial obstruction on central airways deposition of a saline aerosol in patients with asthma. Am Rev Respir Dis 1986;133:740–3. 22. Ilowite JS, Gorvoy JD, Smaldone GC. Quantitative deposition of aerosolized gentamicin in cystic fibrosis. Am Rev Respir Dis 1987;136:1445–9. 23. Laube BL, Links JM, LaFrance ND, et al. Homogeneity of bronchopulmonary distribution of Tc-99m aerosol in normal subjects and in cystic fibrosis patients. Chest 1989;95:822–30. 24. Sanchis J, Gich I, Pedersen S, Aerosol Drug Management Improvement Team (ADMIT). Systematic review of errors in inhaler use: has patient technique improved over time? Chest 2016;150:394–406. 25. Laube BL, Janssens HM, de Jongh FH, et al., European Respiratory Society, International Society for Aerosols in Medicine. What the pulmonary specialist should know about the new inhalation therapies. Eur Respir J 2011;37:1308–31.

Aerosol Drug Delivery Devices 26. Dolovich MB. Assessing nebulizer performance. Respir Care 2002;47:1290–301. 27. Hess D, Fisher D, Williams P, et al. Medication nebulizer performance. Effects of diluent volume, nebulizer flow, and nebulizer brand. Chest 1996;110:498–505. 28. Erzinger S, Schueepp KG, Brooks-Wildhaber J, et al. Face masks and aerosol delivery in vivo. J Aerosol Med 2007;20(Suppl. 1):S78–84. 29. Smaldone GC, Sangwan S, Shah A. Face mask design, facial deposition, and delivered dose of nebulized aerosols. J Aerosol Med 2007;20(Suppl. 1):S66–77. 30. National Asthma Education and Prevention Program, National Institutes of Health. Expert Panel Report 3: guidelines for the diagnosis and management of asthma. Bethesda: National Institutes of Health; 2007. Available from: https://www.nhlbi.nih.gov/health-topics/guidelines-for -diagnosis-management-of-asthma. 31. O’Riordan TG. Formulations and nebulizer performance. Respir Care 2002;47:1305–12. 32. Beasley R, Fishwick D, Miles JF, et al. Preservatives in nebulizer solutions: risks without benefit. Pharmacotherapy 1998;18:130–9. 33. Smith EC, Denyer J, Kendrick AH. Comparison of 23 nebulizer/compressor combinations for domiciliary use. Eur Respir J 1995;8:1214–21. 34. Rau JL. Design principles of liquid nebulization devices currently in use. Respir Care 2002;47:1257–75. 35. Hess D. Aerosol delivery devices in the treatment of asthma. Respir Care 2008;53:699–723. 36. Mercer TT, Goddard RF, Flores RL. Effect of auxiliary air flow on the output characteristics of compressed-air nebulizers. Ann Allergy 1969;27:211–17. 37. Rau JL, Ari A, Restrepo RD. Performance comparision of nebulizer designs: constant-output, breath-enhanced and dosimetric. Respir Care 2004;49:174–9.

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems 38. Leung K, Louca E, Coates AL. Comparison of breath-enhanced to breath-actuated nebulizers for rate, consistency, and efficiency. Chest 2004;126:1619–27. 39. Newnham DM, Lipworth BJ. Nebuliser performance, pharmacokinetics, airways and systemic effects of salbutamol given via a novel nebuliser delivery system (‘Ventstream’). Thorax 1994;49:762–70. 40. Denyer J, Nikander K, Smith NJ. Adaptive aerosol delivery (AAD) technology. Expert Opin Drug Deliv 2004;1:165–76. 41. Brand P, Beckmann H, Maas Enriquez M, et al. Peripheral deposition of alpha1-protease inhibitor using commercial inhalation devices. Eur Respir J 2003;22:263–7. 42. Hochrainer D, Holz H, Kreher C, et al. Comparison of the aerosol velocity and spray duration of Respimat Soft Mist inhaler and pressurized metered dose inhalers. J Aerosol Med 2005;18:273–82. 43. Pitcairn G, Reader S, Pavia D, et al. Deposition of corticosteroid aerosol in the human lung by Respimat Soft Mist inhaler compared to deposition by metered dose inhaler or by Turbuhaler dry powder inhaler. J Aerosol Med 2005;18:264–72. 44. Dolovich M, Leach C. Drug delivery devices and propellants. In: Busse W, Holgate S, editors. Asthma and rhinitis. 2nd ed. Oxford: Blackwell Science; 2000. p. 1719–31. 45. Leach C. Effect of formulation parameters on hydrofluoroalkane-beclomethasone dipropionate drug deposition in humans. J Allergy Clin Immunol 1999;104:S250–2. 46. Dolovich M, Labiris R. Imaging drug delivery and drug responses in the lung. Proc Am Thorac Soc 2004;1:329–37. 47. 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 1999;104:1215–22. 48. Crompton GK, Barnes PJ, Broeders M, et al. The need to improve inhalation technique in Europe: a report from the Aerosol Drug Management Improvement Team. Respir Med 2006;100:1479–94. 49. Dubus JC, Rhem R, Dolovich M. Delivery of HFA and CFC salbutamol from spacer devices used in infancy. Int J Pharm 2001;222:101–8. 50. Blake K, Mehta R, Spencer T, et al. Bioavailability of inhaled fluticasone propionate via chambers/masks in young children. Eur Respir J 2012;39:97–103. 51. Nikander K, Berg E, Smaldone GC. Jet nebulizers versus pressurized metered dose inhalers with valved holding chambers: effects of the facemask on aerosol delivery. J Aerosol Med 2007;20:S46–58. 52. Lauricella S, Dolovich M. The effects of inhalation delay and spacer pretreatment on HFA-pMDI delivery from several small volume valved holding chambers. J Aerosol Med 2007;20:202. 53. Rau JL, Coppolo DP, Nagel MW, et al. The importance of nonelectrostatic materials in holding chambers for delivery of hydrofluoroalkane albuterol. Respir Care 2006;51:503–10. 54. Dolovich M. Lung dose, distribution, and clinical response to therapeutic aerosols. Aerosol Sci Technol 1993;18:230–40. 55. Dolovich MB. Aerosol delivery devices and airways/lung deposition. In: Schleimer R, O’Byrne P, Szefler S, et al, editors. Inhaled steroids in asthma: optimizing effects in the airways. New York: Marcel Dekker; 2002. p. 169–211. 56. Roller CM, Zhang G, Troedson RG, et al. Spacer inhalation technique and deposition of extrafine aerosol in asthmatic children. Eur Respir J 2007;29:299–306. 57. Kim CS, Eldridge MA, Sackner MA. Oropharyngeal deposition and delivery aspects of metered-dose inhaler aerosols. Am Rev Respir Dis 1987;135:157–64. 58. Salzman GA, Pyszczynski DR. Oropharyngeal candidiasis in patients treated with beclomethasone dipropionate delivered by metered-dose inhaler alone and with Aerochamber. J Allergy Clin Immunol 1988;81:424–8. 59. Meeran K, Burrin JM, Noonan KA, et al. A large volume spacer significantly reduces the effect of inhaled steroids on bone formation. Postgrad Med J 1995;71:156–9.

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60. Mitchell JP, Nagel MW. Valved holding chambers (VHCs) for use with pressurised metered-dose inhalers (pMDIs): a review of causes of inconsistent medication delivery. Prim Care Respir J 2007;16:207–14. 61. Hoppentocht M, Hagedoorn P, Frijlink HW, et al. Technological and practical challenges of dry powder inhalers and formulations. Adv Drug Deliv Rev 2014;75:18–31. 62. Everard ML, Devadason SG, Le Souëf PN. Flow early in the inspiratory manoeuvre affects the aerosol particle size distribution from a Turbuhaler. Respir Med 1997;91:624–8. 63. Kamin WES, Genz T, Roeder S, et al. Mass output and particle size distribution of glucocorticosteroids emitted from different inhalation devices depending on various inspiratory parameters. J Aerosol Med 2002;15:65–73. 64. Chrystyn H. Effects of device design on patient compliance: comparing the same drug in different devices. In: Dalby RN, Byron PR, Peart J, et al, editors. Respiratory drug delivery Europe 2009. River Grove, Ill.: Davis Healthcare International Publishing; 2009. p. 105–16. 65. Bentur L, Mansour Y, Hamzani Y, et al. Measurement of inspiratory flow in children. Pediatr Pulmonol 2004;38:304–7. 66. Jarvis S, Ind PW, Shiner RJ. Inhaled therapy in elderly COPD patients; time for re-evaluation? Age Ageing 2007;36:213–18. 67. Pedersen S. How to use a Rotahaler. Arch Dis Child 1986;61:11–14. 68. Al Showair RA, Tarsin WY, Assi KH, et al. Can all patients with COPD use the correct inhalation flow with all inhalers and does training help? Respir Med 2007;101:2395–401. 69. Mackie AE, McDowall JE, Falcoz C, et al. Pharmacokinetics of fluticasone propionate inhaled via the Diskhaler and Diskus powder devices in healthy volunteers. Clin Pharmacokinet 2000;39(Suppl.):22–30. 70. Warren S, Taylor G, Smith J, et al. Gamma scintigraphic evaluation of a novel budesonide dry powder inhaler using a validated radiolabeling technique. J Aerosol Med 2002;15:15–25. 71. Newman SP, Pitcairn GR, Adkin DA, et al. Comparison of beclomethasone dipropionate delivery by Easyhaler dry powder inhaler and pMDI plus large volume spacer. J Aerosol Med 2001;14:217–25. 72. Newman SP, Hirst PH, Pitcairn GR. Scintigraphic evaluation of lung deposition with a novel inhaler device. Curr Opin Pulm Med 2001;7(Sup pl.):S12–14. 73. Pitcairn GR, Lim J, Hollingworth A, et al. Scintigraphic assessment of drug delivery from the Ultrahaler dry powder inhaler. J Aerosol Med 1997;10:295–306. 74. Schulte M, Osseiran K, Betz R, et al. Handling of and preferences for available dry powder inhaler systems by patients with asthma and COPD. J Aerosol Med Pulm Drug Deliv 2008;21:321–8.

Future Directions 75. Waldrep JC, Selting KA, Reinero C, et al. Could inhaled chemotherapy be targeted to a specific lung lobe? J Aerosol Med 2007;20(175):P036. 76. Bailey MM, Berkland CJ. Nanoparticle formulations in pulmonary drug delivery. Med Res Rev 2009;29:196–212. 77. Chan VS. Nanomedicine. An unresolved regulatory issue. Regul Toxicol Pharmacol 2006;6:218–24. 78. Pirozynski M, Sosnowski TR. Inhalation devices: from basic science to practical use, innovative vs. generic products. Expert Opin Drug Deliv 2016;13(11):1559–71. 79. Dilraj A, Sukhoo R, Cutts FT, et al. Aerosol and subcutaneous measles vaccine: measles antibody responses 6 years after re-vaccination. Vaccine 2007;25:4170–4. 80. Low N, Bavdekar A, Jeyaseelan L, et al. A randomized, controlled trial of an aerosolized vaccine against measles. N Engl J Med 2015;372(16):1519–29. 81. WHO. Global tuberculosis report 2013. World Health Organization 2013. http://www.who.int/tb/publications/global_report/en/. 82. Satti I, Meyer J, Harris SA, et al. Safety and immunogenicity of a candidate tuberculosis vaccine MVA85A delivered by aerosol in BCG-vaccinated healthy adults: a phase 1, double-blind, randomized controlled trial. Lancet Infec Dis 2014;14(10):939–46. 83. Aitken ML, Bellon G, De Boeck K, et al. Long-term inhaled dry powder mannitol in cystic fibrosis: an international randomized study. Am J Respir Crit Care Med 2012;185:645–52.

CHAPTER 63  Aerosols and Aerosol Drug Delivery Systems

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SELF-ASSESSMENT QUESTIONS 1. Respirable particles are: a. Less than 1 µm in diameter b. Between 1 and 3 µm in diameter c. Less than 0.5 µm in diameter d. Less than 5 µm in diameter e. Larger than 5 µm in diameter 2. Which of the following determine where aerosol particles deposit in the lungs? a. Aerosol particle size b. Whether the patient has airway obstruction c. How fast or slowly the patient is inhaling when the aerosol is administered d. Whether the patient is nose-breathing, or breathing through the mouth e. Whether the patient holds their breath after inhaling drug 3. In patients with severe airway obstruction, aerosol particles are likely to deposit: a. Proximal to the airways that are obstructed b. In the small, peripheral airways c. Evenly throughout the lung d. In the mouth and back of the throat e. In the alveolar region of the lungs

4. When using a valved holding chamber (VHC) with a pMDI, should the patient: a. Place the VHC in the mouth, actuate the pMDI, and wait 5 sec before inhaling the aerosol b. Place the VHC in the mouth, actuate the pMDI, and wait 1 sec before inhaling the aerosol c. Place the VHC in the mouth and inhale while actuating the pMDI d. Actuate the pMDI, place the VHC in the mouth, and inhale the aerosol 5. Valved holding chambers alter the pressurized drug aerosol resident in the chamber by: a. Reducing the median size of the aerosol through impaction of large particles on the chamber walls and inspiratory valve b. Increasing the fine particle fraction of the aerosol compared with the original pMDI aerosol c. Reducing the dose of drug exiting the VHC d. Maintaining the dose of useful drug aerosol exiting the VHC

SECTION F  Gastrointestinal Tract

64  Gastrointestinal Mucosal Immunology M. Cecilia Berin, Seema S. Aceves

CONTENTS Introduction, 1065 Gastrointestinal Structure and Function, 1065 Organization of the Gastrointestinal Immune Tissue, 1068

SUMMARY OF IMPORTANT CONCEPTS • Functions of the gastrointestinal tract include motility, immunologic regulation, and nutrient absorption. • Gastrointestinal mucosal immunity is essential for tolerance and immune regulation to commensal and pathogenic microbiota. • Symbiotic interactions between the gastrointestinal immune system and the microbiome are required for proper gastrointestinal mucosal development and function. • Mucosal immunity comprises inductive and effector arms that involve both innate and adaptive immunity. • Inductive sites include intraepithelial lymphocytes, lamina propria immune cells, Peyer patches, and mesenteric lymph nodes. • Effector arm includes antimicrobial peptides, mast cells, phagocytes, cytotoxic and T helper cells, and B cells that produce neutralizing antibodies.

INTRODUCTION The gastrointestinal tract has the unique characteristic of being the largest organ exposed to external antigens (Fig. 64.1). It is multifunctional and serves as a mucosal barrier, an absorptive surface, and a site of active immunity to foreign antigens. The normal structural and functional components of the gastrointestinal immune system are the focus of this chapter. From the protective layer of mucus to the specialized mucosal epithelium and the deeper immune aggregates, the gastrointestinal tract requires precise regulation to balance immune reactions against foreign antigens while fostering the symbiotic commensal microbiota. The innate and adaptive immune systems act in concert to hold the gastrointestinal mucosal immune system in a state of tolerance to benign antigens while maintaining an appropriate capacity to respond to pathogenic insults.

Innate Immunity in the Gastrointestinal Tract, 1069 Adaptive Immunity in the Gastrointestinal Tract, 1071

GASTROINTESTINAL STRUCTURE AND FUNCTION Esophagus Structure and Function The esophagus functions as a muscular tube that coordinates food transit from the oral cavity to the stomach. The upper esophageal sphincter is the proximal boundary and the lower esophageal sphincter is the distal boundary. After the mouth, the esophageal mucosa is the next mucosal barrier to contact antigens. Contact time is typically less than 10 seconds, but the exposure may be significant, because foods have not undergone any digestive processing. The normal esophagus contains a baseline number of T cells and dendritic cells that likely participate in health and disease states.

Epithelium and Lamina Propria.  Luminal regions of the human esophagus are composed of nonkeratinized squamous epithelium. This epithelium protects the body from swallowed materials as well as acid. Esophageal submucosal glands secrete bicarbonate and mucus, which augments these functions. Salivary mucins, such as Muc5b, are water soluble, thereby providing lubrication but not a viscoelastic protective barrier.1 The epithelium is composed of the stratum basale, which abuts the lamina propria and provides a constant renewal source for the more luminal epithelial cells, the stratum intermedium, and the stratum superficialis. Esophageal epithelial cells use desmosomes, adherens junctions, and tight junctions for intercellular attachment and to control intercellular permeability. Tight junction proteins belong to the families of claudins, occludins, and junctional adhesion molecules. Tight junction proteins such as zonula occludins protein-1 (zo-1) perform the additional function of linking claudins and occludins to the actin cytoskeleton. E-cadherin is a component of the adherens junction, which surrounds cell membranes and supports adhesion of tight junction proteins. Junctional proteins that are expressed in normal esophageal mucosae include claudins-1, -4, and -7.2 Functional loss 1065

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SECTION F  Gastrointestinal Tract

Gastric Structure.  The gastric cardia consists of a narrow strip of

Mouth

Esophagus

Stomach (cardia, corpus, antrum)

Small intestine (duodenum, ileum, jejunum) Large intestine Cecum Appendix Rectum Anus Fig. 64.1  Overview of gastrointestinal organs.

of E-cadherin leads to decreased esophageal epithelial integrity and increased permeability.3 The lamina propria (LP) lies beneath the epithelium and is normally made up of a nonfibrotic, reticular network connecting the epithelium to the muscularis mucosa. Whereas the normal esophageal epithelium is usually devoid of eosinophils, the LP contains T and B cells, eosinophils, and mast cells.4,5 In addition, lymphoid aggregates exist within the lamina propria of the esophagus, but their role in antigen presentation and tolerance is unclear. Projections of the lamina propria into the epidermal space are known as vascular papillae and can increase the contact surface area between the basal zone and the epithelium.

Esophageal Muscle.  The most superficial layer of esophageal smooth muscle is the muscularis mucosa. The muscularis mucosal layer thickens from the proximal to the distal esophagus. The outer esophageal muscle layer, the muscularis propria, comprises two layers. Longitudinally oriented muscle fibers are external, and concentric muscle fibers are internal. Actions of the proximal esophagus are coordinated by striated muscle, whereas actions of the mid and distal esophagus are coordinated by smooth muscle.

Stomach Structure and Function The stomach is composed of at least three anatomically distinct sections, the cardia; the body/corpus/fundus; and the antrum, each of which is designed to initiate stages of digestion. The stomach’s primary function is to prepare ingested food products for digestion and absorption that will occur in the small intestine. This highly regulated function occurs as a result of three closely related processes that are controlled by hormonal and neural mediators. The stomach initially acts to mechanically and biochemically disrupt large pieces of food and serves as a reservoir for residual chyme, the semisolid mass of partially digested foodstuffs. In a highly regulated fashion, the stomach releases chyme into the small intestine.

cells immediately distal to the gastroesophageal junction. Epithelia of the cardia are primarily composed of mucous cells that are believed to protect the esophagus from gastric acidity. The corpus, which encompasses most of the stomach’s surface area, participates in the mechanical breakdown of food through churning actions of rugal folds and in the biochemical breakdown initiated by secretion of acid and pepsin. A layer of secreted molecules that includes mucus, trefoil factors, bicarbonate, acid, defensins, and prostaglandins protects epithelial surfaces. The cellular interface is composed of columnar epithelia and a series of tightly packed tubular glands that contain acid-secreting parietal cells and pepsin-secreting chief cells. Resident cells include lymphocytes, endothelia, fibroblasts, myocytes, nerve cells, and a scattering of eosinophils. Distinctly absent are neutrophils. Structural features include a matrix of collagen that is intertwined between these resident cells. The remainder of the stomach is composed of the antrum; a smooth surface located just after the gastric body and just before the pylori os. The antrum functions as a reservoir for chyme that has completed its initial digestive process in the gastric body. The antrum releases chyme into the small intestine in a highly regulated fashion, where it will complete digestive and absorptive processes. To facilitate this process, the antrum contains a series of glands that are composed of mucous and endocrine cells. Resident cells.  In addition to the columnar epithelia, mucosal surfaces are composed of a variety of surface cells including goblet, enterochromaffin, parietal, and chief cells. Parietal or oxyntic cells.  Parietal cells produce acid through the hydrogen-ATPase pump that is located in tubulovesicles near the apical surface of the cell. Three stimulatory and one inhibitory receptor are present on the basal surface of parietal cells. Muscarinic (M3) receptors are stimulated by parasympathetic vagal nerve–derived acetylcholine. Cholecystokinin (CCKB) receptors are stimulated by gastrin that is released from duodenal G cells after exposure to protein. Finally, gastrin and acetylcholine can stimulate production and release of histamine from enterochromaffin-like (ECL) cells. The end product of stimulation of these receptors is activation of the hydrogen-ATPase pump leading to acid secretion and an intraluminal gastric pH of 2. The superiority of hydrogen-ATPase pump inhibitors (proton pump inhibitors) is made clear because antagonism of all three stimulatory receptors on the basal surface would be required to completely inhibit acid secretion. Prostaglandin E2 receptors are inhibitory to proton secretion. These receptors are stimulated by somatostatin that is produced by D cells located in the corpus, antral, and small intestinal mucosa. Whether or not lack of acid contributes to allergic diseases has not been determined, but some evidence suggests that host sensitization to specific food proteins may be increased in the absence of acid.6 Mucous foveolar (pit) cells.  These cells continually secrete mucus that functions as a mechanical barrier, antimicrobial shield, and lubrication for foods. Mucus contains a number of structurally distinct glycoproteins, antimicrobial peptides, bicarbonate, and water that function to form a distinct separation of luminal contents from epithelial surface. Estimates in murine systems suggest that the thickness of mucus ranges from 100 to 200 micrometers. Whereas acetylcholine is the primary physiologic stimulant of mucus production prostaglandins also can also stimulate secretion. Acid does not break down mucus, but pepsin, bile salts, ethanol, and nonsteroidal antiinflammatory drugs (NSAIDs) can penetrate the mucosal layer and cause mucosal injury. Chief cells.  The primary function of chief cells is the synthesis and release of the proenzyme pepsinogen. Located throughout the gastric mucosa, as well as duodenum, chief cells produce pepsinogen in response to stimulation by acetylcholine, histamine, or cholecystokinin. Acid

CHAPTER 64  Gastrointestinal Mucosal Immunology

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cleaves N-terminal amino acid sequences, which leaves the active enzyme pepsin to initiate protein digestion.

TABLE 64.1  Structural Cells and Function

Small Intestine Structure and Function

Location

Structural Cells

Function

Esophagus

Squamous epithelium

Motility Barrier

Cardia

Columnar epithelial cells Mucous cells Chief cells

Barrier Acid protection

Corpus (Body, Fundus)

Columnar epithelial cells D cells Chief cells

Mechanical food breakdown Acid and pepsin secretion

Antrum

Columnar epithelial cells Goblet cells Enterochromaffin Parietal/oxyntic cells Chief cells Acid secretion D cells Mucous foveolars/pit cells Chief cells

Complete digestion Chyme reservoir and release Mucus secretion Histamine release Prostaglandin E2 secretion Pepsinogen secretion

Small Intestine

Absorptive columnar cells D cells Chief cells M cells Intraepithelial lymphocytes Goblet cells Crypt stem cells Paneth cells

Digestion Absorption Processing/presentation of antigens

Colon

Columnar epithelial cells Crypts Goblet cells Endocrine cells Stem cells

Water absorption

The small intestine represents the largest portion of the gastrointestinal tract. The small intestine’s surface area, which is amplified by folds, villi, and microvilli, is approximately the size of a singles tennis court. Its length, ranging from 6 to 10 meters, is separated from the stomach and colon by two sphincters, the pylorus and ileocecal valve. The small intestine consists of three parts: the duodenum, jejunum, and ileum. Although the division point between the jejunum and ileum is not entirely clear, morphologic features help distinguish these two sections. Compared with the ileum, the jejunum is thicker and contains larger folds (valvulae conniventes) that are thought to increase its absorptive surface. The ileum contains the organized lymphoid follicles Peyer patches (PP) and isolated lymphoid follicles (ILF). Digestion and absorption are the most recognized functions of the small intestine. Release of pancreatic enzymes and bile into the small intestinal lumen initiates protein, carbohydrate, and fat absorption. In addition, minerals and vitamins are absorbed here through a number of well-defined transport systems. The other major function of the small intestine is presentation and processing of antigenic material. What defines which antigens will adhere to the apical epithelial surface and undergo further presentation and processing is not certain. In the sections that follow, processes that define how mucosal surfaces are innately protected from and actively respond to antigens will be described. Table 64.1 provides an overview of the function of structural cells of the gastrointestinal tract.

of the Gastrointestinal Tract

Structure.  Mucosal surfaces are composed of villi and crypts (crypts of Lieberkuhn), which are spaced adjacent to each other with a 3 : 1 ratio of length (villi:crypt). The bulk of epithelia present on villous surfaces comprise absorptive columnar cells. Goblet cells, intraepithelial lymphocytes, and endocrine cells are also present. Crypts are also lined with endocrine, Paneth cells, and stem cells that serve to renew the villous epithelia. Overlying lymphoid follicles and PP is a single layer of columnar cells termed the follicle-associated epithelium (FAE). Centrally located within the FAE are specialized microfold (M) cells. M cells are different from absorptive epithelia; they do not contain microvilli or membrane-associated hydrolytic enzymes and contain less glycocalyx. M cells contain a characteristic feature, the invaginated subdomain within the basolateral membrane that forms an intraepithelial “pocket,” which is thought to function in antigen processing and presentation (Fig. 64.2). (See Adaptive Immunity, later). Underlying the epithelium is a robust set of immune cells including lymphocytes, eosinophils, and mast cells. The submucosa is equipped with an efficient blood supply from the celiac and superior mesenteric arteries, a venous drainage to the inferior vena cava, and a lymphatic system through the celiac and mesenteric nodes that permits communications with other mucosal surfaces (Figs. 64.3 and 64.4).

Colon Structure and Function As with the rest of the gastrointestinal tract, the colon begins and ends with clearly demarcated boundaries; the proximal end begins with the ileocecal valve, and the distal side ends at the anal sphincter. The colon reaches a length of 1.5 meters in adults and is divided into the cecum, ascending, transverse, descending, and sigmoid colons and the rectum. The colon’s primary function is to absorb water so that a formed stool can be expelled. With a remarkable absorptive capacity, the colon receives more than 1.5 liters of fluid per day from the small intestine and excretes an average of 200 mL of excrement per day. In addition,

Goblet cell

M cell

DC

A

T cell

B

C

Fig. 64.2  Uptake of antigens across the epithelial surface. Antigens can cross the epithelial surface by at least three routes. (A) Passage through the M cells that overlie Peyer’s patches. (B) Uptake across goblet cell– associated passages. (C) Capture by dendritic cell processes that extend between epithelial cells.

the colon harbors the bulk of the intestinal microbiome. Bacteria ferment remaining stool components, and acetate, propionate, and butyrate are created as byproducts. Absorptive columnar epithelial cells line mucosal surfaces and are interspersed by crypts that contain goblet, endocrine, and stem cells.

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SECTION F  Gastrointestinal Tract A layer of secreted molecules that includes mucus, trefoil factors, and defensins protect epithelial surfaces. As in the rest of the gastrointestinal tract, underlying cells and molecules form structural and metabolic frameworks integral to supporting mucosal integrity. Other cells include lymphocytes, endothelia, fibroblasts, myocytes, endothelia, nerve cells, mast cells, and a scattering of eosinophils. Neutrophils are distinctly absent.

Intestinal Lumen

Villi

ORGANIZATION OF THE GASTROINTESTINAL IMMUNE TISSUE

Epithelium Lymphatic

Lamina propria

Mucus

Crypt

To mesenteric lymph node

Fig. 64.3  Lymphatic structures in the intestinal mucosa. Lymphatic vessels in the subepithelial space transport cells and mediators that regulate immunologic function.

The digestive tract mucosa from mouth to anus is variably populated with cells that participate in innate and adaptive immunity against foreign antigens. The mucosal immune system comprises intraepithelial lymphocytes, a dense population of resident immune cells in the lamina propria, as well as organized lymphoid structures such as PP, isolated lymphoid follicles, and mesenteric lymph nodes that drain the small and large intestine (Fig. 64.4). The mucosal immune system can be further divided into effector and inductive sites. The resident population of cells forms the effector arm of the mucosal immune system and comprises phagocytes that engulf and kill microbes, cytotoxic T cells that kill infected cells, B cells that produce neutralizing antibodies, and T helper cells that support these effector functions through production of cytokines. Inductive sites of the gastrointestinal tract are organized lymphoid structures that bring together naïve T cells, B cells, and antigen-presenting MHC class II

Toll-like receptors (TLR)

Mucus-trefoil factors, antimicrobial peptides, slgA

CD4 or CD8+

Antigen

DC

Antigen Microbiome

M cell SED Peyer patch (PP)

B cell

DC

IEL Intestinal lumen

Th0

Lamina propria

Th1 Th17

T cell

Tr

Nerve

Th2 Crypt Afferent lymphatic

Cytotoxic lymphocyte Mθ

MC

EOS

Paneth cells Mesenteric lymph node

Naïve CD4+

Gut wall Efferent lymphatic

Tolerant/primed CD4+

Systemic distribution

Fig. 64.4  Intestinal mucosa overview. The epithelial surface is exposed to an abundant and diverse microbiome. To protect itself, it is coated with mucus that contains trefoil proteins, sIgA, and antimicrobial peptides. Antigens may pass through the epithelial surface and undergo processing in Peyer patches. Within the subepithelial dome (SED) reside a number of lymphocytes, including Th0 cells, which can differentiate into Th1, Th2, T regulatory (Treg), or Th17 cells. Other resident cells include macrophages, cytotoxic lymphocytes, mast cells (MC), and eosinophils (EOS). (Adapted from Atkins D, Furuta GT. Mucosal immunology, eosinophilic esophagitis, and other intestinal inflammatory diseases. J Allerg Clin Immunol 2010;125(2 Suppl 2):S255-61.)

CHAPTER 64  Gastrointestinal Mucosal Immunology cells. These include the draining lymph nodes and specialized lymphoid structures within the gastrointestinal mucosa. The latter include PPs in the small intestine and structurally similar lymphoid tissues in the rectum, as well as smaller isolated lymphoid follicles and cryptopatches (precursor to isolated lymphoid follicles) scattered throughout the intestine. After activation in these organized tissues, antigen-specific T and B cells migrate to effector sites of the gastrointestinal mucosa.

INNATE IMMUNITY IN THE GASTROINTESTINAL TRACT The normal flora of the GI tract varies from mouth to anus. The oral cavity contains primarily streptococci. The stomach is largely sterile, as is the proximal small intestine. In contrast, the terminal ileum and colon contain about 1014 resident bacterial flora representing more than 1000 species of anaerobes and aerobes. Close interactions between the microflora and the innate and adaptive immune systems forms a system of checks and balances for gastrointestinal mucosal immune homeostasis.

Antimicrobial Peptides The innate immune system provides a functional host defense barrier and a bridge to the adaptive immune system. In the esophagus, the first protective barrier is the mucous layer comprising mucin-2 and glycoproteins.7 The small intestine has a single layer mucus layer of glycocalyx that can be penetrated by bacteria. The colon’s glycocalyx has two mucus layers of which the inner layer is impermeable to bacteria. Goblet cells produce trefoils that are secreted into the mucous layer and are critical for immune defense and epithelial barrier function. Trefoils promote epithelial cell survival and migration. In multiple models of mucosal injury, recombinant trefoil therapy can decrease the severity of epithelial injury and decrease the time to epithelial reconstitution.8 The small intestine contains a mucous layer capable of neutralizing acid by actively secreting bicarbonate. In the small intestine, the mucous layer contains both secretory IgA and antimicrobial peptides. Mucins play an important role in normal intestinal immune regulation, and mucin-2 deficiency causes spontaneous inflammation and cancer in murine models.9 In the gastrointestinal tract, antimicrobial peptides are in contact with the microbiota and help balance the appropriate bacterial load and diversity while controlling bacterial translocation. The small molecule families defensins (α and β), cathelicidins, and cryptdins are composed of antimicrobial peptides with bactericidal capacity. The defensin family includes human β-defensin (HBD) -1 and -2. The α-defensins comprise human neutrophil peptide (HNP)-1 through -6 and the cryptdins 1 to 6. Human LL-37 (cathelicidin) is another small antimicrobial peptide. α-and β-defensins as well as cathelicidins are normally expressed in the intestinal and colonic epithelial mucosa as well as in intestinal leukocytes. The significant difference in normal bacterial flora from mouth to anus is reflective of and maintained by the antibacterial spectrum of the antimicrobial peptides. HBD3 is expressed in the esophagus and oral cavity. HBD4 is expressed in the gastric antrum. Paneth cells express the α-defensins HD-5 and -6 as well as the antimicrobial enzymes phospholipase A2 and lysozyme.10 Generally, β-defensins are expressed throughout the intestine by epithelial cells. HBD1 is expressed in the epithelial cells of multiple gastrointestinal locations, including the small intestine and colon. In contrast, HBD2 is expressed only at low levels at baseline in the small intestine but can be upregulated in both the intestine and stomach during inflammatory states. Normal human colonic mucosa expresses the cathelicidin LL-37, and expression increases in response to bacterial CpG motifs.11 Cryptdin 4, which has antibacterial activity against Escherichia coli, is not expressed in the small intestine

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but is expressed at high levels in the colon consistent with bacterial load being higher in the colon. The importance of antimicrobial peptides in host defense has been shown by experimentally modulating their expression. For example, mice functionally deficient in Paneth cell α-defensins are susceptible to Salmonella infection, whereas transgenic expression of human defensin HD-5 in mice is protective against Salmonella infection compared with wild type littermate controls.12,13 It is possible that regulation of α-defensins plays a role in the dysbiosis that predisposes to inflammatory bowel disease because genetic variants in genes such as transcription factor 4 (TCF) that regulate α-defensin expression can associate with an increased risk of Crohn disease.14 In addition to having antibacterial properties, the defensins and cathelicidins have chemotactic properties for neutrophils, dendritic cells, and memory T cells.10,15,16

Toll-Like Receptors and Nod-Like Receptors Toll-like receptors (TLRs) were first characterized in the fruit fly Drosophila melanogaster as innate immune system IL-1-like receptors. Human TLRs 1 to 9 have since been well characterized. TLRs 1, 2, 4, 5, 6 are expressed on the cell surface. TLRs 3, 7, 8, 9 are expressed intracellularly and have ligands that are similar to host antigens and require endosomal internalization. TLRs and their signaling pathways are important for mucosal homeostasis and limit the penetration of commensal microbiota into the intestine and associated lymph nodes.9 Generally, in healthy adults, TLRs are expressed ubiquitously in myeloid cells, leukocytes, and epithelium.17 However, in the gut, where the microflora is dense and diverse, TLR expression is regulated to avoid immune overreaction and to maintain gastrointestinal homeostasis. TLR-3 and -5 are expressed in intestinal epithelial cells. TLR-2 and -4 are not typically expressed in the esophagus or small intestine, but both can be induced during disease states.17,18 Epithelial cell–specific deletions in TLR signaling such as MyD88 and TNF receptor–associated factor 6 lead to spontaneous inflammation due to loss of epithelial barrier integrity that separates the intestinal epithelium from commensal bacteria.19,20 Like the TLRs, the nod-like receptors (NLRs) are pattern recognition receptors in the GI tract. They respond to bacterial and endogenous pathogen and damage-associated molecular patterns. Nod1 and Nod2 are cytosolic receptors that detect muropeptides derived from bacterial peptidoglycan and play a role in shaping the innate immune system, controlling the microflora, and protecting against inflammation and cancer. For example, NLRC4 is important for expulsion of infected epithelial cells and for protection from malignancy.19,21,22 Nods are expressed by epithelial cells, stromal cells, neutrophils, and dendritic cells. NLRP proteins interact with pyrin-containing proteins to form inflammasomes that cleave pro-IL-1β and pro-IL-18 into their mature forms by using the caspase-1 pathway. Activation of the caspase pathway leads to pyroptosis, a form of cell death that causes cellular release of mediators that instigate and propagate inflammatory pathways. NLRPs recognize both bacterial products such as flagellin and toxins, and crystals such as urea.23 In animal models, NLRs and the IL-1β pathway are important in innate gastrointestinal host defense. Nod signaling in the stromal compartment is required for formation of intestinal lymphoid follicles.24 Interactions between Nods and the microbiome are also essential. For example, gram-negative bacteria induce Nod-dependent CCL20 and β-defensin secretion to promote development of intestinal lymphoid follicles from cryptopatches.25 Nods are also required for appropriate microflora control. Nod2 deficiency increases bacterial load and decreases the clearance of bacteria from intestinal crypts.26,27 In addition, mice deficient in Nod2 or transgenic for the Nod2 frame shift mutation have increased gut permeability, which also occurs in human Crohn disease; the etiology of increased permeability is likely related to bacterial

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SECTION F  Gastrointestinal Tract

defection in response to increased T cell and TLR responses.28 NLRP6 deficiency associates with dysbiosis both for bacteria and viruses and increases colitis risk.29,30 A complex interplay between the resident microflora and the innate and adaptive immune system is critical for controlling gut permeability, bacterial load, and inflammation.

Intestinal Microbiome Several studies have evaluated the interaction between the normal commensal gut flora, the intestinal epithelium, and the mucosal immune system. The first pivotal observation pointing to a symbiotic relationship between the microbiome and gut immunity was that germ-free mice have very few immune cells and are nearly absent of IgA. Prenatal treatment of mice with antibiotics also reduces bone marrow neutrophils, documenting that the microbiome and its metabolites such as short chain fatty acids are likely critical for proper local and systemic immune development.31 Reconstitution with specific flora revealed that although mesenteric lymph nodes and PP were genetically programmed, the development of isolated lymphoid follicles was dependent on gut flora. Despite the diversity of symbiotic bacteria, four families predominate in the human large intestine: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. These symbionts and the mucosal epithelium have evolved in a mutually beneficial coexistence that can be altered to cause dysbiosis. The presence of microbiota is essential for proper epithelial rejuvenation. For example, when only half of the usual microbiota is present, epithelial damage is increased, but resultant epithelial cell proliferation is decreased.32 Dysbiosis is likely to be both a result of and a direct cause of local and systemic allergy and/or autoimmunity, and it is clear that pathogenic states are associated with distinct patterns of microbial dysbiosis. The interplay between the microbiome and innate immunity is complex. The numbers of basophils, the transcription profile and trafficking of resident macrophages, and interactions between enteric neurons and macrophages in the intestinal muscularis that influence gastrointestinal motility are regulated by the microbiota.33-35 The mechanisms of cross-talk between the microbiome and the innate immune system include transcriptional reprogramming of intestinal cells and epigenetic changes in innate and structural cells.19

Innate Immune Cells: Eosinophils, Mast Cells, Macrophages, Innate Lymphoid Cells

Eosinophils.  The distribution of eosinophils in the GI tract varies from mouth to anus via a general gradient of increasing baseline eosinophilia from proximal to distal. Eosinophils populate the fetal intestinal tract in early embryogenesis, and their intestinal accumulation is independent of bacterial colonization and presence of lymphocytes.36,37 Whereas the normal esophageal epithelium is devoid of eosinophils, the lamina propria and muscularis mucosa normally have eosinophils.5,38,39 The lower gastrointestinal tract has significant numbers of eosinophils that traffic in a CCL-11 (eotaxin-1)-dependent manner.37,39 Within the colonic lamina propria, eosinophil numbers are relatively constant but are highest in the cecum and rectosigmoid.39,40 Eosinophils are rarely found in the surface and crypt epithelium, and, as such, their presence in these locations may indicate a pathologic process. Increasing evidence suggests that the eosinophil serves a homeostatic function in the gastrointestinal tract. Insights from eosinophil-deficient mice show a decrease in mucosal secretory IgA and smaller Peyer patches with decreased numbers of CD103+ T cells and dendritic cells.41-43 With some parasitic infections, parasite expulsion on chronic or repeat infection can be dysregulated in the absence of eosinophils/ IL-5.44 Eosinophils are also important for homeostasis of inflammation and metabolic regulation. Visceral adipose tissue eosinophils inversely correlate with obesity. Phenotypic reversal of increased adiposity and

poor insulin and glucose tolerance can occur with eosinophil repletion.45 Small intestinal eosinophils decrease IL-17 production by CD4+ cells via the production of IL-Rα and inhibition of IL-1β.46 In addition, eosinophils can be required to restrict inflammatory responses to infection by intestinal nematodes by decreasing the production of IL-4 and maintaining mucosal IgA,47 underscoring their role in immune balance.

Mast Cells.  The gastrointestinal tract is one of the largest reservoirs of mast cells in the body. Mast cells are found in the human gastrointestinal tract from the esophagus to the colon. The esophageal and colonic epithelium is normally entirely devoid of tryptase-positive cells, but tryptase-positive mast cells are found in varying numbers (14.5 to 17.6 per hpf) through the colonic lamina propria, and connective tissue mast cells (tryptase-chymase double positive mast cells) predominate in the esophageal lamina propria.5,40 Mast cells can play a protective role in the intestine, and increased mast cell numbers and degranulation are found in bowel disorders such as Crohn disease and irritable bowel syndrome. During helminth infection in mice, mast cell mobilization from their normal location in the lamina propria into the epithelium depends on intact notch signaling. Mast cell deficient mice have delayed expulsion of Trichinella spiralis and Strongyloides as well as impaired Th2 responses.48 Therefore, although gastrointestinal mast cells are induced during immune responses to certain pathogens, their normal function in gastrointestinal immune homeostasis remains unclear. Innate Lymphoid Cells.  Natural killer (NK) and lymphoid tissue inducer (LTi) cells were the first described members of the innate lymphoid cell family. LTi cells, which have now been reclassified as a constituent population of CCR6+ ILC3s, are required for lymph node formation during embryogenesis and produce IL-17 and IL-22. Over the last 7 years, a rapidly growing body of literature has underscored the importance of innate lymphoid cells (ILCs) in gastrointestinal homeostasis.49 These cells do not express lineage markers found on B, T, NK or NKT cells, mast cells, basophils, granulocytes, dendritic cells, or macrophages and are thus referred to as lineage negative. With the exception of FoxP3 regulatory T cells, an ILC compartment has been described to match each of the adaptive CD4+ T cell compartments including ILC1s (IFN-γ producing, proinflammatory cells), ILC2s (GATA3hi, IL-5, IL-13 secreting cells), and ILC3s (RORγt+, Th17-like cells). ILC3s are crucial for proper lymphoid organ development. Unlike adaptive T cells, there is a relative paucity of ILCs in lymphoid tissue but an abundance of ILC1s, ILC2s, and CCR6-ILC3s in the intestine. The expression of α4β7 and CXCR6 on ILCs explains their natural ability to effectively home to intestinal tissues, and parabiosis studies demonstrate that ILCs populate mucosal surfaces early in their development, can proliferate in situ, and continue to reside in the mucosa through their lifetime. During active disease states, there can be increased numbers of ILCs at the site of injury, such as an increased number of ILC2s in active pediatric eosinophilic esophagitis.50 ILCs are important for pathogen resistance. ILC2s are required for clearance of N. brasiliensis as well as potentially for Strongyloides and Trichuris expulsion. ILC2-derived IL-4, IL-5, and IL-13 is important for protection from helminth infection. ILC3s are important for protection of infection from bacteria such as C. rodentium and Salmonella typhimurium, as well as from rotavirus.51 These effects are mediated through ILC3 production of IL-22 and the induction of antimicrobial responses in epithelial cells. Current data also suggest that in both humans and mice, ILC2s could be important for IL-17–mediated clearance of Candida infections.52 Lastly, ILC3s may play a role in protection from tumorigenesis in the colon.53

CHAPTER 64  Gastrointestinal Mucosal Immunology

Macrophages.  The gastrointestinal mucosa is the largest reservoir of mononuclear phagocytes in the body, with the highest numbers in the colon and a morphologically unique population in the smooth muscle. Muscularis macrophages express a specific tissue-protective gene profile compared with lamina propria macrophages.54 Macrophages regulate the inflammatory response to the normal flora, respond to pathogens, and scavenge debris and dead cells. Intestinal macrophages are derived from and replenished by circulating monocytes. A novel population of macrophages derived from the pro-B cell/B cells can also occupy the intestinal tract.55 Once in intestinal tissue, macrophages have a lifespan of weeks to months. Resident macrophages are highly phagocytic and can ingest microbes and function as scavengers without generating an inflammatory response that could damage surrounding tissue. Responses to innate signals via the TLR and NOD pathways are blunted in intestinal macrophages, likely to protect from aberrant response to commensal microflora. Macrophage responses to IL-10 are critical to intestinal mucosal immune regulation, and loss of macrophage IL-10 receptor leads to spontaneous colitis.56,57 Despite their anergic phenotype, intestinal macrophages do participate in host defense through phagocytosis and microbial killing. They express an array of innate recognition receptors, including TLRs that allow microbial recognition; lipoprotein and phospholipid recognition receptors that facilitate uptake of apoptotic cells; and complement receptors. After uptake, intestinal macrophages efficiently kill microbes through mechanisms such as generation of reactive oxygen and nitrogen species and autophagy.

Dendritic Cells.  The intestinal lamina propria contains a dense network of dendritic cells (DCs) that sample antigen and are responsible for the initiation of adaptive immune responses, as discussed in detail later. Through their expression of TLRs and other pattern-recognition receptors, these resident cells also participate in innate immune responses. Lamina propria DCs are responsive to flagellin, and transport captured Salmonella typhimurium to draining lymph nodes in a TLR5-dependent manner.58 Expression of the TLR adaptor molecule MyD88 in DCs is essential for induction of cytokine responses from DCs and ILCs and for generation of appropriate antimicrobial immunity.59

ADAPTIVE IMMUNITY IN THE GASTROINTESTINAL TRACT Structural barriers as well as innate immune barriers limit the exposure of the mucosal immune system to antigenic material, but protein antigens penetrating through these barriers can be presented to T lymphocytes to generate an adaptive immune response. T cells reactive to exogenous antigens from food or flora are not deleted during thymic development, as self-reactive T cells are, and therefore homeostatic mechanisms are necessary to suppress inappropriate immune reactivity to nonpathogenic material in the gastrointestinal mucosa. Impairment of these homeostatic mechanisms can lead either to localized inflammation triggered by the microbial flora or food antigens or to systemic reactivity to food allergens.

Antigen Uptake The gastrointestinal tract is exposed to a significant antigen burden derived from food and microbial flora. Different regions of the gut face different antigenic challenges; there is a decreasing gradient of intact food allergens and an increasing microbial load from mouth to anus. It has been thought the small intestine is the most relevant site of antigenic exposure because it is the site of nutrient absorption and therefore the most “leaky” from an electrophysiology perspective. The epithelium of the mouth and esophagus also has a dense network of

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antigen-presenting cells, and their presence indicates a readiness to absorb and present antigens. Soluble and particulate antigens are handled by distinct mechanisms in the small intestine (Fig. 64.2). Particulate antigens, such as viruses and bacteria, are preferentially taken up into the gut-associated lymphoid tissue (GALT), including PP and isolated lymphoid follicles (ILF). This is related to the presence of a specialized subset of epithelial cells termed M cells in the follicle-associated epithelium overlying the PP and ILF. Antigens taken up by M cells are rapidly transported to the organized lymphoid tissue below, which includes a network of subepithelial DCs in the PP. Another mechanism of uptake of particulate antigens includes direct sampling by resident phagocytes that extend dendrites between enterocytes and can directly engulf bacteria from the intestinal lumen.60 Soluble antigens, including many food antigens, are taken up across epithelial cells lining the intestinal villus. Electron microscopy studies of antigen uptake have shown transcytosis across enterocytes as a major pathway of entry. Two-photon microscopy has identified goblet cells as a unique entry point for luminal antigens in the small intestine,61 and this pathway has been termed goblet cell–associated passages (GAPs). Similar to M cells these cells deliver antigen to closely associated DCs. GAPs are regulated by tonic cholinergic signals,62 which promote the formation of GAPs and antigen delivery across the epithelium. The colonic microbiota normally suppresses formation of colonic GAPs. The net result of homeostatic antigen uptake is that an immunologically significant quantity of food antigen reaches the systemic circulation intact after a meal in humans.63 When there has been prior exposure to an antigen, uptake of that antigen can be modified by the presence of immunoglobulins. IgA primarily functions in immune exclusion; however, secretory IgA can also facilitate antigen uptake via M cells of the Peyer patches by a Dectin-1-dependent mechanism.64 Similarly, IgG and IgE can facilitate uptake of antigen by transcytosis after binding to epithelial immunoglobulin receptors (FcRn65 and CD23,66 respectively).

Antigen Presentation Antigen-presenting cells (APCs) in the gastrointestinal tract include professional APCs including DCs, B cells, and macrophages. DCs have the capacity to migrate to lymph nodes, where they can interact with naïve T cells and induce an immune response. Common DC progenitors give rise to a population of classical DCs (cDCs) that express CD103; these can be further subdivided based on expression of CD11b. CD103+CD11b+ DCs, also called cDC2s, are dependent on the transcription factor IRF4 for their development, whereas CD103+CD11b- DCs, also called cDC1s, are dependent on the transcription factors IRF8 and BATF3 for their development. Functional specialization has been described for these subsets, with CD103+CD11b+ cells specialized for induction of immune responses to extracellular pathogens (Th2, Th17) and CD103+CD11b- cells specialized for immune responses to intracellular pathogens (Th1, cross-presentation to CD8 T cells).67-69 In addition to cDCs, there is a population of monocyte-derived mononuclear phagocytes (MPs) that express high levels of CX3CR1 and are transcriptionally similar to macrophages. CD103+ DCs migrate to the draining lymph node in a CCR7-dependent mechanism, whereas CX3CR1+ MPs do not migrate under steady state conditions. CX3CR1+ MPs extend dendrites into the lumen and are highly phagocytic. Although they do not migrate, they can present antigen to T cells that migrate back to the lamina propria after priming in the mesenteric lymph node, and therefore contribute to recall responses in tissues.70 Within the PP, subepithelial DCs migrate to T cell areas of the PP upon appropriate stimulation, such as in response to adjuvant. As in the lamina propria, distinct lineages of DCs corresponding to cDC1 and cDC2 have been described in the subepithelial dome.71 Within the stratified epithelium of the oral and esophageal mucosa are subsets of DCs that are distinct from those

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SECTION F  Gastrointestinal Tract

in the lower gastrointestinal tract and bear greater similarity to those described in the skin. These include the Langerhans cells found in the most superficial epithelial layers, interstitial DCs, and langerin+ interstitial DCs.72 Oral DCs acquire and present antigen to T cells and can generate either tolerance or protective immunity, depending on the context of antigen administration. Whether esophageal DCs can acquire and present ingested food antigens in vivo is unknown. MPs without migratory activity in the gastrointestinal mucosa interact with resident T cells of the lamina propria and participate in the reactivation of memory T cells. Local activation of effector T cells has been thought to be a major role of the nonmigrating CX3CR1+ phagocytes of the intestine that express MHC II and process and present acquired antigens.70 CD11cneg macrophages of the mouth and small intestine can also present antigen to T cells and preferentially induce the development of regulatory T cells through an IL-10- and retinoic acid-dependent mechanism.70,73

Adaptive Immunity to Food Antigens: Immune Tolerance The average daily protein intake in young children in the United States is greater than 50 g per day. Although the majority of protein is digested and absorbed as amino acids or immunologically inert peptides, intact antigen can be readily detected in the blood after a meal in healthy volunteers. The immune system is not ignorant of these proteins, as shown by the presence of food-specific IgG and IgA antibodies in the serum of healthy individuals. The clinical tolerance to these exogenous proteins is believed to be related to an active regulatory immune response termed oral tolerance. Wells and Osborne first described oral tolerance in 1911, when they found that guinea pigs could not be induced to undergo anaphylaxis to proteins that were already present in the diet.74 Subsequent studies showed that oral administration of antigen resulted in the generation of CD4+ and CD8+ T cells with regulatory or suppressive activity in mice and humans.75 Transfer of CD4+ or CD8+ T cells from fed mice to naïve mice could transfer tolerance in an antigen-specific manner. Deletion of peripherally induced Foxp3+ CD4+ CD25+ regulatory T cells (Tregs) can reverse oral tolerance.70 Although immune tolerance can be generated at sites other than the gastrointestinal tract (skin, airways), dendritic cells migrating from the small intestinal lamina propria have unique features that promote responder T cells to develop into Tregs. Migratory CD103+ DCs isolated from the MLN of mice or humans induce Tregs with gut-homing capacity through a mechanism dependent on retinoic acid and TGF-β. An investigation into the role of DC subsets in oral tolerance showed that cDCs were necessary for tolerance induction, but there was redundancy in the cDC1 and cDC2 subset in the induction of oral tolerance.76 Tregs primed in the MLN migrate to the lamina propria, where they are further expanded by CX3CR1+ MPs before seeding the periphery and suppressing systemic antigen-specific immunity through TGF-β-dependent mechanisms.70 There is evidence that the liver can also participate in the induction of oral tolerance mediated through plasmacytoid DCs that induce the deletion of antigen-specific CD8+ T cells.77 In breastfeeding infants, oral tolerance can be conferred by antigen-IgG complexes present in the milk. Facilitated uptake of antigen-IgG complexes by FcRn expressed on the intestinal epithelium promotes the development of immune tolerance and prevents development of experimental asthma or food allergy.78,79 Interestingly, although oral tolerance can be demonstrated in humans,80,81 food-specific Tregs identified by MHC-II tetramer staining are markedly absent compared with aeroallergen-specific Tregs in peripheral blood.82

Adaptive Immunity to Microbial Antigens: Immune Exclusion The gastrointestinal immune system is exposed to a significant microbial antigen burden. The density of organisms is highest in the large intestine,

but the mouth, esophagus, and small intestine also contain a normal commensal flora. The innate and adaptive immune system work in a coordinated manner to keep the flora compartmentalized to the gastrointestinal mucosa. In the absence of signaling through MyD88 and TRIF, the downstream signaling molecules of the TLRs, there is a compensatory systemic antibody response against the intestinal flora that protects against systemic dissemination of bacteria.20 Mice lacking both innate signaling and immunoglobulin production develop failure to thrive and protein-losing enteropathy triggered by the gut flora. Commensal bacteria are normally sampled by the mucosal immune system and are carried by migratory DCs to the draining MLN, where they induce an IgA response that is partially dependent on T cell help.83 T cell–independent IgA class-switching has also been documented in the intestinal lamina propria.84 IgA induced by the flora is secreted into the intestinal lumen by epithelial cells expressing the polymeric immunoglobulin receptor (pIgR). Although the body expends considerable energy in IgA production, IgA deficiency is associated with a relatively mild phenotype, and humans with IgA deficiency can be asymptomatic. This is likely due in part to compensation by secretory IgM.

Microbial Shaping of Immunity Microbial colonization plays a major role in setting the tone of the adaptive immune response not only locally in the gastrointestinal tract, but systemically. This has been shown through a variety of model systems in mice. Bacillus fragilis was the first commensal organism shown to modify the T helper cell profile through the production of polysaccharide A when introduced into germ-free mice. B. fragilis induces Foxp3+ Tregs that prevent experimental colitis.85 Expansion of colonic Tregs has also been shown with mouse and human Clostridia species via the production of the short chain fatty acid butyrate.86 Segmental filamentous bacteria (SFB) were identified as Th17-inducing bacteria.87 The induction of Th17 responses by SFB has systemic consequences, including contribution to nonintestinal autoimmunity.88 Human IL17-inducing commensal microorganisms have also been identified.89 Although less well studied than the influence of bacterial species, commensal protozoa also shape the immune tone and disease susceptibility. The mouse commensal protozoa Tritrichomonas musculis promotes Th1 and Th17 responses in the intestine through activation of the inflammasome.90 The virome is also emerging as an important contributor to the pathogenesis of inflammatory bowel disease91 and celiac disease.92 In summary, despite major efforts to exclude microorganisms through the production of secretory immunoglobulins, the commensal microbiota has profound immunomodulatory effects through surface polysaccharides or metabolites that shape the immune system and lead to susceptibility or resistance to a wide range of inflammatory, allergic, or autoimmune disease.

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53. Eisenring M, vom Berg J, Kristiansen G, et al. IL-12 initiates tumor rejection via lymphoid tissue-inducer cells bearing the natural cytotoxicity receptor NKp46. Nat Immunol 2010;11(11):1030–8. 54. Gabanyi I, Muller PA, Feighery L, et al. Neuro-immune interactions drive tissue programming in intestinal macrophages. Cell 2016;164(3): 378–91. 55. Audzevich T, Bashford-Rogers R, Mabbott NA, et al. Pre/pro-B cells generate macrophage populations during homeostasis and inflammation. Proc Natl Acad Sci USA 2017;114(20):E3954–63. 56. Zigmond E, Bernshtein B, Friedlander G, et al. Macrophage-restricted interleukin-10 receptor deficiency, but not IL-10 deficiency, causes severe spontaneous colitis. Immunity 2014;40(5):720–33. 57. Shouval DS, Biswas A, Goettel JA, et al. Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function. Immunity 2014;40(5): 706–19. 58. Uematsu S, Jang MH, Chevrier N, et al. Detection of pathogenic intestinal bacteria by Toll-like receptor 5 on intestinal CD11c + lamina propria cells. Nat Immunol 2006;7(8):868–74. 59. Friedrich C, Mamareli P, Thiemann S, et al. MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium. PLoS Pathog 2017;13(5):e1006357. 60. Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001;2(4):361–7. 61. McDole JR, Wheeler LW, McDonald KG, et al. Goblet cells deliver luminal antigen to CD103 + dendritic cells in the small intestine. Nature 2012;483(7389):345–9. 62. Knoop KA, McDonald KG, McCrate S, et al. Microbial sensing by goblet cells controls immune surveillance of luminal antigens in the colon. Mucosal Immunol 2015;8(1):198–210. 63. Husby S, Jensenius JC, Svehag SE. Passage of undegraded dietary antigen into the blood of healthy adults. Quantification, estimation of size distribution, and relation of uptake to levels of specific antibodies. Scand J Immunol 1985;22(1):83–92. 64. Rochereau N, Drocourt D, Perouzel E, et al. Dectin-1 is essential for reverse transcytosis of glycosylated SIgA-antigen complexes by intestinal M cells. PLoS Biol 2013;11(9):e1001658. 65. Yoshida M, Masuda A, Kuo TT, et al. IgG transport across mucosal barriers by neonatal Fc receptor for IgG and mucosal immunity. Springer Semin Immunopathol 2006;28(4):397–403. 66. Li H, Nowak-Wegrzyn A, Charlop-Powers Z, et al. Transcytosis of IgE-antigen complexes by CD23a in human intestinal epithelial cells and its role in food allergy. Gastroenterology 2006;131(1):47–58. 67. Schlitzer A, McGovern N, Teo P, et al. IRF4 transcription factor-dependent CD11b + dendritic cells in human and mouse control mucosal IL-17 cytokine responses. Immunity 2013;38(5):970–83. 68. Tussiwand R, Everts B, Grajales-Reyes GE, et al. Klf4 expression in conventional dendritic cells is required for T helper 2 cell responses. Immunity 2015;42(5):916–28. 69. Cerovic V, Houston SA, Westlund J, et al. Lymph-borne CD8alpha + dendritic cells are uniquely able to cross-prime CD8+ T cells with antigen acquired from intestinal epithelial cells. Mucosal Immunol 2015;8(1):38–48. 70. Hadis U, Wahl B, Schulz O, et al. Intestinal tolerance requires gut homing and expansion of FoxP3 + regulatory T cells in the lamina propria. Immunity 2011;34(2):237–46. 71. Bonnardel J, Da Silva C, Wagner C, et al. Distribution, location, and transcriptional profile of Peyer’s patch conventional DC subsets at steady state and under TLR7 ligand stimulation. Mucosal Immunol 2017;10(6):1412–30.

72. Nudel I, Elnekave M, Furmanov K, et al. Dendritic cells in distinct oral mucosal tissues engage different mechanisms to prime CD8+ T cells. J Immunol 2011;186(2):891–900. 73. Mascarell L, Saint-Lu N, Moussu H, et al. Oral macrophage-like cells play a key role in tolerance induction following sublingual immunotherapy of asthmatic mice. Mucosal Immunol 2011;4(6):638–47. 74. Wells HG, Osborne TB. The biological reactions of the vegetable proteins. I. Anaphylaxis. J Infect Dis 1911;8:66–124. 75. Weiner HL, da Cunha AP, Quintana F, et al. Oral tolerance. Immunol Rev 2011;241(1):241–59. 76. Esterhazy D, Loschko J, London M, et al. Classical dendritic cells are required for dietary antigen-mediated induction of peripheral T(reg) cells and tolerance. Nat Immunol 2016;17(5):545–55. 77. Dubois B, Joubert G, Gomez de Aguero M, et al. Sequential role of plasmacytoid dendritic cells and regulatory T cells in oral tolerance. Gastroenterology 2009;137(3):1019–28. 78. Mosconi E, Rekima A, Seitz-Polski B, et al. Breast milk immune complexes are potent inducers of oral tolerance in neonates and prevent asthma development. Mucosal Immunol 2010;3(5):461–74. 79. Ohsaki A, Venturelli N, Buccigrosso TM, et al. Maternal IgG immune complexes induce food allergen-specific tolerance in offspring. J Exp Med 2018;215(1):91–113. 80. Du Toit G, Sayre PH, Roberts G, et al. Effect of avoidance on peanut allergy after early peanut consumption. N Engl J Med 2016;374(15):1435–43. 81. Kraus TA, Toy L, Chan L, et al. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology 2004;126(7):1771–8. 82. Bacher P, Heinrich F, Stervbo U, et al. Regulatory T cell specificity directs tolerance versus allergy against aeroantigens in humans. Cell 2016;167(4):1067–78.e16. 83. Macpherson AJ, Uhr T. Induction of protective IgA by intestinal dendritic cells carrying commensal bacteria. Science 2004;303(5664): 1662–5. 84. He B, Xu W, Santini PA, et al. Intestinal bacteria trigger T cell-independent immunoglobulin A(2) class switching by inducing epithelial-cell secretion of the cytokine APRIL. Immunity 2007;26(6):812–26. 85. Round JL, Mazmanian SK. Inducible Foxp3 + regulatory T-cell development by a commensal bacterium of the intestinal microbiota. Proc Natl Acad Sci USA 2010;107(27):12204–9. 86. Atarashi K, Tanoue T, Oshima K, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 2013;500(7461):232–6. 87. Ivanov II, Atarashi K, Manel N, et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 2009;139(3):485–98. 88. Lee YK, Menezes JS, Umesaki Y, et al. Proinflammatory T-cell responses to gut microbiota promote experimental autoimmune encephalomyelitis. Proc Natl Acad Sci USA 2011;108(Suppl. 1):4615–22. 89. Tan TG, Sefik E, Geva-Zatorsky N, et al. Identifying species of symbiont bacteria from the human gut that, alone, can induce intestinal Th17 cells in mice. Proc Natl Acad Sci USA 2016;113(50):E8141–50. 90. Chudnovskiy A, Mortha A, Kana V, et al. Host-protozoan interactions protect from mucosal infections through activation of the inflammasome. Cell 2016;167(2):444–56.e14. 91. Cadwell K, Patel KK, Maloney NS, et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell 2010;141(7):1135–45. 92. Bouziat R, Hinterleitner R, Brown JJ, et al. Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease. Science 2017;356(6333):44–50.

CHAPTER 64  Gastrointestinal Mucosal Immunology

1074.e1

SELF-ASSESSMENT QUESTIONS 1. The mucosal immune system can be divided into inductive and effector sites. An example of an effector site is: a. Peyer patch b. Mesenteric lymph node c. Isolated lymphoid follicle d. Lamina propria 2. The epithelium senses microbial products through which of the following: a. Nod-like receptors b. Chemokines c. Defensins d. Mucins

3. Commensal bacteria or bacterial products that drive Th17 responses in the gastrointestinal tract include: a. Bacteroides fragilis b. Segmented filamentous bacteria c. Clostridia species d. Butyrate

65  Eosinophilic Gastrointestinal Disorders Chen E. Rosenberg, Marc E. Rothenberg

CONTENTS Introduction, 1075 Overview of Disorders, 1075 Clinical Evaluation, 1077 Eosinophilic Esophagitis, 1078

SUMMARY OF IMPORTANT CONCEPTS • Eosinophils are constituents of the gastrointestinal tract at baseline, which means that their mere presence is not pathognomonic for a particular disease. • Eosinophilic gastrointestinal disorders consist of eosinophilic esophagitis, eosinophilic gastritis, eosinophilic enteritis, and eosinophilic colitis, as well as eosinophilic gastroenteritis (involving a combination of gastrointestinal segments). • Eosinophilic gastrointestinal disorders are primarily eosinophilic inflammatory conditions involving mechanisms that fall between pure immunoglobulin E (IgE)–mediated and T helper cell type 2 (Th2) responses. • Therapy for eosinophilic gastrointestinal disorders consists of a combination of approaches including elimination or elemental diets, antiinflammatory agents, immunosuppression, and an emerging class of anti-Th2 response biologic agents.

INTRODUCTION Eosinophilic gastrointestinal disorders (EGIDs) are defined as disorders that selectively affect the gastrointestinal (GI) tract with eosinophil-rich inflammation in the absence of known causes of eosinophilia. These disorders are being increasingly recognized and include eosinophilic esophagitis (EoE), eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis, and eosinophilic colitis (Box 65.1). Eosinophils are integral members of the GI mucosal immune system, and EGIDs are primarily polygenic eosinophilic inflammatory disorders involving mechanisms that fall between pure immunoglobulin E (IgE)–mediated and T helper cell type 2 (Th2) responses. Studies have identified contributory roles for food antigens, cytokines interleukin 5 (IL-5) and interleukin 13 (IL-13), chemokines (specifically, the eotaxin family of chemokines), and polarization of Th2 immunity in the pathogenesis of EGIDs, and these findings provide a rationale for disease-specific therapies.

OVERVIEW OF DISORDERS Patients with EGIDs present with a variety of symptoms, including abdominal pain, nausea, vomiting, diarrhea, gastric dysmotility,

Eosinophilic Gastritis and Gastroenteritis, 1082 Eosinophilic Colitis, 1083 Conclusion, 1084

dysphagia, growth failure, bleeding, and anemia; notably, these symptoms are shared by many GI conditions. EGIDs can have a significant impact on quality of life, often resulting in school and work absenteeism, and they are also associated with extensive dietary restrictions, which can lead to dramatic lifestyle alterations. Evidence is accumulating that supports the concept that EGIDs arise secondarily to the interplay of genetic, immune, and environmental factors. Notably, about 10% of patients with EGIDs have an immediate family member affected by the disorder.1 Additionally, several lines of evidence suggest shared mechanisms between EGIDs and allergic diseases: About 75% of patients with EGIDs have atopy2; disease activity is often reversed by elimination of dietary antigens3; histologic evidence suggests mast cell activation and degranulation; Th2 cytokines are overexpressed in the diseased tissue; and anti–Th2 cytokine therapy (e.g., anti–IL-13 and anti–IL-4-Rα) show promising efficacy in patients. Although patients with EGIDs usually demonstrate sensitization to multiple foods with positive food-specific serum IgE levels and/or skin tests, they often have high levels of total and food-specific IgG4, and only a minority have IgE-mediated clinical reactions to these foods, such as food-induced anaphylactic responses. Thus, EGIDs have properties that fall between pure IgE-mediated food allergy and Th2-mediated inflammatory disorders. The prevalence of primary EGIDs has been the subject of numerous studies over the last two decades. Eosinophilic esophagitis (EoE) is the most common of these conditions with a recent prevalence estimate of 57 cases per 100,000 persons in the United States.4 Overall, epidemiologic results indicate that EGIDs are increasing in incidence and prevalence, with EoE being the most common, followed by eosinophilic gastroenteritis, eosinophilic gastritis, and eosinophilic colitis, respectively. The estimated prevalence of eosinophilic gastroenteritis, eosinophilic gastritis, and eosinophilic colitis is 8.4/100,000, 6.3/100,000, and 3.3/100,000, respectively.5 In more than 50% of patients, EGIDs occur independent of peripheral blood eosinophilia, indicating the potential significance of GI-specific mechanisms for regulating eosinophil levels; indeed, previous work has demonstrated the importance of the eotaxin pathway in this process.6-9 However, some patients with EGIDs have substantially elevated levels of peripheral blood eosinophils and meet the diagnostic criteria for hypereosinophilic syndrome (HES); this syndrome is defined by sustained, severe peripheral blood eosinophilia (1500 eosinophils/mm3 or

1075

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SECTION F  Gastrointestinal Tract

BOX 65.1  Differential Diagnosis for Eosinophilic Gastrointestinal Disorders Eosinophilic Esophagitis Gastroesophageal reflux disease Celiac disease Eosinophilic gastroenteritis Crohn disease Infection Hypereosinophilic syndrome Achalasia Adverse drug reaction Vasculitis Pemphigus Hypermobility syndromes (e.g., Ehlers-Danlos syndrome and Loeys-Dietz syndrome) Netherton syndrome Hyper-IgE syndrome Graft-versus-host disease Eosinophilic Gastritis and Eosinophilic Gastroenteritis Hypereosinophilic syndrome Celiac disease Inflammatory bowel disease (e.g., Crohn disease or ulcerative colitis) Adverse drug reaction Connective tissue disease (e.g., scleroderma) Rheumatologic disease (e.g., systemic lupus erythematosus) Vasculitis (e.g., eosinophilic granulomatosis with polyangiitis or polyarteritis nodosa) Malignancy

more) and the presence of end-organ involvement in the absence of known causes for eosinophilia.10 Notably, although HES commonly involves the GI tract, the other end organs typically associated with HES (e.g., heart, skin) are rarely involved in EGIDs.

Gastrointestinal Eosinophils Under Homeostatic Healthy States Eosinophils are present at low levels in numerous tissues. In biopsy and autopsy specimens, the only organs that normally demonstrate tissue eosinophils at substantial levels are the GI tract, spleen, lymph nodes, adipose tissue, and thymus. In the healthy pediatric GI tract, eosinophil levels progressively increase from the proximal to the distal intestine.11 In most cases, eosinophils can be considered to represent a normal finding, provided that they are evenly dispersed in the lamina propria and do not infiltrate the epithelium in more than occasional numbers, coalesce into aggregates or microabscesses, or show extensive degranulation.12 Table 65.1 lists the eosinophil levels in the GI tract in apparently normal endoscopic biopsies. A search for eosinophils throughout the GI tracts of mice and humans has revealed that these cells are normally present in the lamina propria of the stomach, small intestine, cecum, and colon.11,13 Unlike intestinal lymphocytes and mast cells, eosinophils are not normally present in Peyer patches or intraepithelial locations, although they frequently infiltrate these regions in EGIDs.14 Fetal mice have eosinophils located in similar regions and concentrations to adult mice,13 which indicates that eosinophil homing into the GI tract occurs independent of endogenous flora. Indeed, germ-free mice and mice deficient in innate signaling responses (MYD88 deficient) have normal levels of GI eosinophils.13 These data suggest that eosinophils respond to distinct stimuli compared with other intestinal leukocytes. Indeed, constitutive expression of eotaxin-1 provides the unique signal that promotes localization of eosinophils into the murine GI tract at baseline. Tissue-dwelling eosinophils have

Gastric outlet obstruction Infection Ancylostoma caninum (hookworm) Anisakis Ascaris Enterobius vermicularis (pinworm) Epstein-Barr virus Eustoma rotundatum Giardia lamblia Helicobacter pylori Schistosomiasis Strongyloides stercoralis Toxocara canis Trichinella spiralis Eosinophilic Colitis Infection Ancylostoma caninum (hookworm) Enterobius vermicularis (pinworm) Inflammatory bowel disease (e.g., Crohn disease or ulcerative colitis) Adverse drug reaction Vasculitis Posttransplant infiltrates Lymphocytic colitis Collagenous colitis Systemic mastocytosis

distinct cytokine expression patterns under inflammatory and noninflammatory conditions, illustrated by esophageal eosinophils in patients with EoE expressing relatively high levels of Th2 cytokines.

Proinflammatory Role of Eosinophils Eosinophils are pleiotropic cells that respond to a variety of triggers. In vitro studies show that eosinophil granule constituents are toxic to a variety of tissues, including intestinal epithelium. Eosinophil granules contain a crystalloid core composed of major basic proteins MBP-1 and MBP-2 and a matrix composed of eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase (EPX). These cationic proteins share certain proinflammatory properties but differ in other ways. For example, MBP, EPX, and ECP have cytotoxic effects on epithelium at concentrations similar to those in biologic fluids from patients with eosinophilia. Also, ECP and EDN belong to the ribonuclease A superfamily and possess antiviral and ribonuclease activity. Circulating levels of EDN are elevated in patients with EoE and can distinguish patients with active and inactive disease.15 MBP triggers degranulation of mast cells and basophils. Triggering of eosinophils by engagement of receptors for cytokines, immunoglobulins, and complement can lead to the generation of a wide range of inflammatory cytokines, including IL-1, -3, -4, -5, and -13; granulocyte macrophage colony–stimulating factor (GM-CSF); transforming growth factors (TGFs); tumor necrosis factor α (TNF-α); RANTES (CCL5); macrophage inflammatory protein 1α (MIP-1α, or CCL3); vascular endothelial cell growth factor (VEGF); and eotaxin-1 (CCL-11). Although these cytokines are mostly found in modest quantities per eosinophil compared with other cells, they have the potential to modulate multiple aspects of the immune response. In fact, eosinophilderived TGF-β is linked with epithelial growth, fibrosis, and tissue remodeling. Additionally, eosinophils can “catapult” their mitochondrial DNA16 and release their chromosomal DNA17 in response to activation,

CHAPTER 65  Eosinophilic Gastrointestinal Disorders

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TABLE 65.1  Gastrointestinal Eosinophil Levels in Normal Pediatric Endoscopy Biopsiesa VILLOUS LAMINA PROPRIA

LAMINA PROPRIA

CRYPT/ GLANDULAR EPITHELIUM

SURFACE EPITHELIUM

Mean

Max

Mean

Max

Mean

Max

Mean

Max N/A

N/A

N/A

N/A

N/A

0.03 ± 0.10

1

N/A

Antrum

1.9 ± 1.3

8

N/A

N/A

0.0

0

0.02 ± 0.04

Fundus

2.1 ± 2.4

11

N/A

N/A

0.0

0

0.008 ± 0.03

1

Duodenum

9.6 ± 5.3

26

2.1 ± 1.4

9

0.06 ± 0.09

2

0.26 ± 0.36

6

Esophagus

1

Ileum

12.4 ± 5.4

28

4.8 ± 2.8

15

0.47 ± 0.25

4

0.80 ± 0.51

4

Ascending colon

20.3 ± 8.2

50

N/A

N/A

0.29 ± 0.25

3

1.4 ± 1.2

11

Transverse colon

16.3 ± 5.6

42

N/A

N/A

0.22 ± 0.39

4

0.77 ± 0.61

4

8.3 ± 5.9

32

N/A

N/A

0.15 ± 0.13

2

1.2 ± 1.1

9

Rectum

N/A, Not applicable. a Values indicate the mean number of eosinophils per high-power field, plus or minus the standard deviation, for each anatomic region of the GI tract and each region of the mucosa. From Debrosse CW, Case JW, Putnam PE, et al. Quantity and distribution of eosinophils in the gastrointestinal tract of children. Pediatr Dev Pathol 2006;9:210-8.

thus resulting in DNA nets that may have a role in trapping intestinal pathogens. Eosinophils express major histocompatibility complex (MHC) class II molecules and relevant costimulatory molecules (CD28, CD40, CD80 [B7-1], CD86 [B7-2]) and secrete an array of cytokines capable of promoting lymphocyte proliferation and activation and Th1 or Th2 polarization (IL-2, -4, -6, -12, -10). Further eosinophil-mediated damage is caused by toxic hydrogen peroxide and halide acids generated by EPX and by superoxide generated by the respiratory burst oxidase enzyme pathway in eosinophils. Clinical investigations demonstrate extracellular deposition of MBP and ECP in the small bowel of patients with eosinophilic gastroenteritis and a correlation between the level of eosinophils and disease severity. Electron microscopy studies have revealed ultrastructural changes in the secondary granules (indicative of eosinophil degranulation and mediator release) in duodenal samples from patients with eosinophilic gastroenteritis. Furthermore, Charcot-Leyden crystals, remnants of eosinophil degranulation, are often found on microscopic examination of stool obtained from patients with eosinophilic gastroenteritis.

CLINICAL EVALUATION Patients with EGIDs present with a variety of clinical problems, most often failure to thrive, abdominal pain, irritability, gastric dysmotility, vomiting, diarrhea, dysphagia, microcytic anemia, and hypoproteinemia. A diagnostic evaluation for EGIDs should be performed on all patients with these refractory problems, especially in individuals with a strong history of atopy, peripheral blood eosinophilia, or a family history of EGIDs. Depending on the GI segment involved, the frequency and type of specific symptoms vary. For example, abdominal pain and dysphagia are most common in eosinophilic gastroenteritis and EoE, respectively. However, there are no pathognomonic symptoms or laboratory tests for diagnosing EGIDs. Peripheral blood eosinophil counts are generally in the normal range in most patients, and when levels are above normal, they are typically mild (G mutation of the untranslated RMRP. Eur J Hum Genet 2002;10(7): 439–47. 77. Buckley RH, Wray BB, Belmaker EZ. Extreme hyperimmunoglobulinemia E and undue susceptibility to infection. Pediatrics 1972;49:59–70. 78. Buckley RH. The hyper-IgE syndrome. Clin Rev Allergy Immunol 2001;20(1):139–54. 79. Grimbacher B, Holland SM, Gallin JI, et al. Hyper-IgE syndrome with recurrent infections—an autosomal dominant multisystem disorder. N Engl J Med 1999;340(9):692–702. 80. Gernez Y, Freeman AF, Holland SM, et al. Autosomal dominant Hyper-IgE syndrome in the USIDNET registry. J Allergy Clin Immunol Pract 2018;6(3):996–1001.

81. Holland SM, Deleo FR, Elloumi HZ, et al. STAT3 mutations in the Hyper-IgE syndrome. N Engl J Med 2007;357:1608–19. 82. Milner JD, Brenchley JM, Laurence A, et al. Impaired T(H)17 cell differentiation in subjects with autosomal dominant hyper-IgE syndrome. Nature 2008;452(7188):773–6. 83. 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 1991;88:713–21. 84. Renner ED, Puck JM, Holland SM, et al. Autosomal recessive hyperimmunoglobulin E syndrome: a distinct disease entity. J Pediatr 2004;144(1):93–9. 85. Zhang Q, Davis JC, Lamborn IT, et al. Combined immunodeficiency associated with DOCK8 mutations. N Engl J Med 2009;361(21): 2046–55. 86. Minegishi Y, Karasuyama H. Hyperimmunoglobulin E syndrome and tyrosine kinase 2 deficiency. Curr Opin Allergy Clin Immunol 2007;7(6):506–9. 87. Zhang Y, Yu X, Ichikawa M, et al. Autosomal recessive phosphoglucomutase 3 (PGM3) mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment. J Allergy Clin Immunol 2014;133(5):1400–9.e1–5. 88. Stray-Pedersen A, Backe PH, Sorte HS, et al. PGM3 mutations cause a congenital disorder of glycosylation with severe immunodeficiency and skeletal dysplasia. Am J Hum Genet 2014;95(1):96–107. 89. Renner ED, Hartl D, Rylaarsdam S, et al. Comel-Netherton syndrome defined as primary immunodeficiency. J Allergy Clin Immunol 2009;124(3):536–43. 90. Engelhardt KR, Gertz ME, Keles S, et al. The extended clinical phenotype of 64 patients with dedicator of cytokinesis 8 deficiency. J Allergy Clin Immunol 2015;136(2):402–12. 91. Hanson EP, Monaco-Shawver L, Solt LA, et al. Hypomorphic nuclear factor-kappaB essential modulator mutation database and reconstitution system identifies phenotypic and immunologic diversity. J Allergy Clin Immunol 2008;122(6):1169–77. 92. Petersheim D, Massaad MJ, Lee S, et al. Mechanisms of genotypephenotype correlation in autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency. J Allergy Clin Immunol 2018;141(3):1060–73.e3. 93. Mousallem T, Yang J, Urban TJ, et al. A nonsense mutation in IKBKB causes combined immunodeficiency. Blood 2014;124(13): 2046–50. 94. Orange JS, Brodeur SR, Jain A, et al. Deficient natural killer cell cytotoxicity in patients with IKK-gamma/NEMO mutations. J Clin Invest 2002;109(11):1501–9. 95. Miot C, Imai K, Imai C, et al. Hematopoietic stem cell transplantation in 29 patients hemizygous for hypomorphic IKBKG / NEMO mutations. Blood 2017;130(12):1456–67. 96. Feske S, Gwack Y, Prakriya M, et al. A mutation in Orai1 causes immune deficiency by abrogating CRAC channel function. Nature 2006;441(7090):179–85. 97. Lacruz RS, Feske S. Diseases caused by mutations in ORAI1 and STIM1. Ann N Y Acad Sci 2015;1356:45–79. 98. Broome CB, Graham ML, Saulsbury FT, et al. Correction of purine nucleoside phosphorylase deficiency by transplantation of allogeneic bone marrow from a sibling. J Pediatr 1996;128(3):373–6. 99. Notarangelo LD. Multiple intestinal atresia with combined immune deficiency. Curr Opin Pediatr 2014;26(6):690–6. 100. Cheon CK, Ko JM. Kabuki syndrome: clinical and molecular characteristics. Korean J Pediatr 2015;58(9):317–24. 101. Vetrie D, Vorechovsky I, Sideras P, et al. The gene involved in X-linked agammaglobulinaemia is a member of the src family of protein-tyrosine kinases. Nature 1993;361:226–33. 102. Tsukada S, Saffran DC, Rawlings DJ, et al. Deficient expression of a B cell cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia. Cell 1993;72:279–90. 103. Smith CIE, Satterthwaite AB, Witte ON. X-linked agammaglobulinemia: a disease of Btk tyrosine kinase. In: Ochs HD, Smith CIE, Puck JM,

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126. Schubert D, Bode C, Kenefeck R, et al. Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nat Med 2014;20(12):1410–16. 127. Agarwal S, Cunningham-Rundles C. Autoimmunity in common variable immunodeficiency. Curr Allergy Asthma Rep 2009;9(5):347–52. 128. Cunningham-Rundles C, Cooper DL, Duffy TP, et al. Lymphomas of mucosal-associated lymphoid tissue in common variable immunodeficiency. Am J Hematol 2002;69(3):171–8. 129. Burks AW, Sampson HA, Buckley RH. Anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia. N Engl J Med 1986;314:560–4. 130. Chapel H, Cunningham-Rundles C. Update in understanding common variable immunodeficiency disorders (CVIDs) and the management of patients with these conditions. Br J Haematol 2009;145(6):709–27. 131. Clark JA, Callicoat PA, Brenner NA. Selective IgA deficiency in blood donors. Am J Clin Pathol 1983;80:210–13. 132. Bronson PG, Chang D, Bhangale T, et al. Common variants at PVT1, ATG13-AMBRA1, AHI1 and CLEC16A are associated with selective IgA deficiency. Nat Genet 2016;48(11):1425–9. 133. 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 1991;2:265–81. 134. Shackelford PG, Granoff DM, Polmar SH, et al. Subnormal serum concentrations of IgG2 in children with frequent infections associated with varied patters of immunologic dysfunction. J Pediatr 1990;116: 529–38. 135. Perez E, Bonilla FA, Orange JS, et al. Specific antibody deficiency: controversies in diagnosis and management. Front Immunol 2017;8:586. 136. Tiller TL Jr, Buckley RH. Transient hypogammaglobulinemia of infancy: review of the literature, clinical and immunologic features of 11 new cases, and long-term follow- up. J Pediatr 1978;92(3):347–53. 137. Dorsey MJ, Orange JS. Impaired specific antibody response and increased B-cell population in transient hypogammaglobulinemia of infancy. Ann Allergy Asthma Immunol 2006;97(5):590–5. 138. Agarwal S, Cunningham-Rundles C. Thymoma and immunodeficiency (good syndrome): a report of 2 unusual cases and review of the literature. Ann Allergy Asthma Immunol 2007;98(2):185–90. 139. Durandy A, Revy P, Fischer A. Autosomal hyper-IgM syndromes caused by an intrinsic B cell defect. In: Ochs HD, Smith CIE, Puck JM, editors. Primary immunodeficiency diseases: a molecular and genetic approach. New York: Oxford University Press; 2007. p. 269–78. 140. Revy P, Muto T, Levy Y, et al. Activation-induced cytidine deaminase (AID) deficiency causes the autosomal recessive form of the Hyper-IgM syndrome (HIGM2). Cell 2000;102:565–75. 141. Minegishi Y, Lavoie A, Cunningham-Rundles C, et al. Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome. Clin Immunol 2000;97(3):203–10. 142. Imai K, Catalan N, Plebani A, et al. Hyper-IgM syndrome type 4 with a B lymphocyte-intrinsic selective deficiency in Ig class-switch recombination. J Clin Invest 2003;112(1):136–42. 143. Kracker S, Di VM, Schwartzentruber J, et al. An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex. J Allergy Clin Immunol 2015;135(4):998–1007. 144. Gardes P, Forveille M, Alyanakian MA, et al. Human MSH6 deficiency is associated with impaired antibody maturation. J Immunol 2012;188(4):2023–9. 145. Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001;27(1):20–1. 146. Bacchetta R, Passerini L, Gambineri E, et al. Defective regulatory and effector T cell functions in patients with FOXP3 mutations. J Clin Invest 2006;116(6):1713–22. 147. Baud O, Goulet O, Canioni D, et al. Treatment of the immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) by allogeneic bone marrow transplantation. N Engl J Med 2001;344(23):1758–62.

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148. Peltonen-Palotie L, Halonen M, Perheentupa J. Autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy. In: Ochs HD, Smith CIE, Puck JM, editors. Primary immunodeficiency diseases: a molecular and genetic approach. New York: Oxford University Press; 2007. p. 342–53. 149. Kisand K, Boe Wolff AS, Podkrajsek KT, et al. Chronic mucocutaneous candidiasis in APECED or thymoma patients correlates with autoimmunity to Th17-associated cytokines. J Exp Med 2010;207(2):299–308. 150. Pedroza LA, Kumar V, Sanborn KB, et al. Autoimmune regulator (AIRE) contributes to Dectin-1-induced TNF-alpha production and complexes with caspase recruitment domain-containing protein 9 (CARD9), spleen tyrosine kinase (Syk), and Dectin-1. J Allergy Clin Immunol 2012;129(2):464–72.e1–3. 151. Schuster V, Terhorst C. X-linked lymphoproliferative disease due to defects of SH2D1A. In: Ochs HD, Smith CIE, Puck JM, editors. Primary immunodeficiency diseases: a molecular and genetic approach. New York: Oxford University Press; 2007. p. 470–84. 152. Nichols KE, Harkin DP, Levitz S, et al. Inactivating mutations in an SH2 domain-encoding gene in X-linked lymphoproliferative syndrome. Proc Natl Acad Sci USA 1998;95(23):13765–70. 153. Sayos J, Wu C, Morra M, et al. The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM [see comments]. Nature 1998;395(6701):462–9. 154. Tangye SG, Phillips JH, Lanier LL, et al. Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome. J Immunol 2000;165(6): 2932–6. 155. Morra M, Silander O, Calpe-Flores S, et al. Alterations of the X-linked lymphoproliferative disease gene SH2DIA in common variable immunodeficiency syndrome. Blood 2001;98(5):1321–5. 156. Sieni E, Cetica V, Hackmann Y, et al. Familial hemophagocytic lymphohistiocytosis: when rare diseases shed light on immune system functioning. Front Immunol 2014;5:167. 157. Henter JI, Horne A, Arico M, et al. HLH-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatr Blood Cancer 2007;48(2):124–31. 158. Nagle DL, Karim MA, Woolf EA, et al. Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. Nat Genet 1996;14:307–11. 159. Dinauer MC, Orkin SH, Brown R. The glycoprotein encoded by the X-linked chronic granulomatous disease locus is a component of the neutrophil cytochrome b complex. Nature 1987;327:717. 160. Winkelstein JA, Marino MC, Johnston RB Jr, et al. Chronic granulomatous disease. Report on a national registry of 368 patients. Medicine (Baltimore) 2000;79(3):155–69. 161. Greenberg DE, Ding L, Zelazny AM, et al. A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease. PLoS Pathog 2006;2(4):e28. 162. Vowells SJ, Fleisher TA, Sekhsaria S, et al. Genotype-dependent variability in flow cytometric evaluation of reduced nicotinamide adenine dinucleotide phosphate oxidase function in patients with chronic granulomatous disease. J Pediatr 1996;128:104–7. 163. Gallin JI, Alling DW, Malech HL, et al. Itraconazole to prevent fungal infections in chronic granulomatous disease. N Engl J Med 2003;348(24):2416–22. 164. International Chronic Granulomatous Disease Cooperative Study Group. A controlled trial of interferon gamma to prevent infection in chronic granulomatous disease. N Engl J Med 1991;324:509–16. 165. Seger RA. Hematopoietic stem cell transplantation for chronic granulomatous disease. Immunol Allergy Clin North Am 2010;30(2): 195–208. 166. Ott MG, Schmidt M, Schwarzwaelder K, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 2006;12(4):401–9. 167. 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 2001;28(1):73–6.

168. Bustamante J, Boisson-Dupuis S, Abel L, et al. Mendelian susceptibility to mycobacterial disease: genetic, immunological, and clinical features of inborn errors of IFN-gamma immunity. Semin Immunol 2014;26(6):454–70. 169. Jouanguy E, Altare F, Lamhamedi S, et al. Interferon-gamma-receptor deficiency in an infant with fatal bacille Calmette-Guerin infection. N Engl J Med 1996;335:1956–61. 170. Altare F, Durandy A, Lammas D, et al. Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 1998;280:1432–5. 171. de Jong R, Altare F, Haagen I, et al. Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Sci 1998;280: 1435–8. 172. Dupuis S, Dargemont C, Fieschi C, et al. Impairment of mycobacterial but not viral immunity by a germline human STAT1 mutation. Science 2001;293(5528):300–3. 173. Picard C, von Bernuth H, Ghandil P, et al. Clinical features and outcome of patients with IRAK-4 and myd88 deficiency. Medicine (Baltimore) 2010;89(6):403–25. 174. Puel A, Cypowyj S, Bustamante J, et al. Chronic mucocutaneous candidiasis in humans with inborn errors of interleukin-17 immunity. Science 2011;332(6025):65–8. 175. van de Veerdonk FL, Plantinga TS, Hoischen A, et al. STAT1 mutations in autosomal dominant chronic mucocutaneous candidiasis. N Engl J Med 2011;365(1):54–61. 176. Glocker EO, Hennigs A, Nabavi M, et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N Engl J Med 2009;361(18):1727–35. 177. Orange JS. Natural killer cell deficiency. J Allergy Clin Immunol 2013;132(3):515–25. 178. Mace EM, Hsu AP, Monaco-Shawver L, et al. Mutations in GATA2 cause human NK cell deficiency with specific loss of the CD56(bright) subset. Blood 2013;121(14):2669–77. 179. Cottineau J, Kottemann MC, Lach FP, et al. Inherited GINS1 deficiency underlies growth retardation along with neutropenia and NK cell deficiency. J Clin Invest 2017;127(5):1991–2006. 180. Mace EM, Bigley V, Gunesch JT, et al. Biallelic mutations in IRF8 impair human NK cell maturation and function. J Clin Invest 2017;127(1): 306–20. 181. Fleisher G, Starr S, Koven N, et al. A non-x-linked syndrome with susceptibility to severe Epstein-Barr virus infections. J Pediatr 1982;100(5):727–30. 182. Cuellar-Rodriguez J, Gea-Banacloche J, Freeman AF, et al. Successful allogeneic hematopoietic stem cell transplantation for GATA2 deficiency. Blood 2011;118(13):3715–20. 183. Oda H, Kastner DL. Genomics, biology, and human illness: advances in the monogenic autoinflammatory diseases. Rheum Dis Clin North Am 2017;43(3):327–45. 184. Sonmez HE, Ozen S. A clinical update on inflammasomopathies. Int Immunol 2017;29(9):393–400. 185. Torrelo A. CANDLE syndrome as a paradigm of proteasome-related autoinflammation. Front Immunol 2017;8:927. 186. Vece TJ, Watkin LB, Nicholas S, et al. Copa syndrome: a novel autosomal dominant immune dysregulatory disease. J Clin Immunol 2016;36(4):377–87. 187. Sullivan KE, Winkelstein JA. Genetically- determined deficiencies of the complement system. In: Ochs HD, Smith CIE, Puck JM, editors. Primary immunodeficiency diseases: a molecular and genetic approach. New York: Oxford University Press; 2007. p. 589–608. 188. Westberg J, Fredrikson GN, Truedsson L, et al. Sequence-based analysis of properdin deficiency: identification of point mutations in two phenotypic forms of an X-linked immunodeficiency. Genomics 1995;29:1–8. 189. Hartmann D, Fremeaux-Bacchi V, Weiss L, et al. Combined heterozygous deficiency of the classical complement pathway proteins C2 and C4. J Clin Immunol 1997;17:176–84. 190. Saijo T, Chen J, Chen SC, et al. Anti-granulocyte-macrophage colonystimulating factor autoantibodies are a risk factor for central nervous system infection by Cryptococcus gattii in otherwise immunocompetent patients. MBio 2014;5(2):e912–14.

CHAPTER 69  Primary Immunodeficiency Diseases

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SELF-ASSESSMENT QUESTIONS 1. According to the IUIS classification, which of the following does not represent a broad category of conditions that are now included as primary immunodeficiency diseases (PIDs)? a. Antibody deficiencies b. Immune dysregulatory diseases c. Autoinflammatory diseases d. Hyper-IgE syndromes e. Phenocopies of PIDs 2. Which of the following gene defects causes a primary immunodeficiency because of its inherent impact on T cell function? a. ATM b. PIK3CD c. CD40LG d. IKBKG (NEMO) e. RAG1

3. Patients with common variable immunodeficiency (CVID) not having a known genetic defect causing the disease are at substantively increased risk for all of the following conditions except: a. Colitis b. Mycobacterial infections c. Autoimmune hemolytic anemia d. Granulomatous disease e. Lymphoma

70  Treatment of Primary Immunodeficiency Diseases Ivan Chinn, Rebecca A. Marsh

CONTENTS Introduction, 1153 General Management Strategies in Primary Immunodeficiency Diseases, 1153 Immunizations, 1153 Immunoglobulin G Replacement Therapy, 1153

SUMMARY OF IMPORTANT CONCEPTS • Evolving treatment strategies for primary immunodeficiency diseases have led to ongoing improvement in survival and quality of life for affected individuals over time. • Universal precautions and proper handwashing should be taught and practiced, and infections must be identified early and treated aggressively in patients with primary immunodeficiency diseases. • Immunizations, IgG replacement therapy, and antimicrobial prophylaxis should be used appropriately to minimize the development of infections in immunodeficient patients. • Targeted biologic modifiers can be used effectively to treat a variety of conditions in patients with primary immunodeficiency diseases but must be applied with awareness concerning established adverse effects. • Hematopoietic cell transplantation provides definitive cure for many primary immunodeficiency diseases, but current approaches continue to offer room for improvement. • Gene therapy has demonstrated success for sustained treatment of some primary immunodeficiency diseases but remains experimental as efforts continue toward overcoming historical barriers, such as insertional mutagenesis and loss of gene-corrected cells over time. • Implementation of best practice demands individualized treatment of primary immunodeficiency diseases with attention to the needs of each patient and the disease-specific considerations inherent to each immunodeficient condition.

INTRODUCTION Treatment options for primary immunodeficiency diseases (PIDs) have both advanced and continued to develop over time, resulting in greater survival and quality of life for affected individuals. An excellent example lies in chronic granulomatous disease (CGD), which at one time was known as “fatal granulomatous disease of childhood,” but which now boasts approximately 90% survival to adulthood.1 Greater understanding of disease pathology and advances in medical technology have led to similar improvements in many other conditions. In this chapter, important therapies and management strategies for PIDs are reviewed, and disease-specific considerations for key conditions within the nine

Antimicrobial Prophylaxis, 1155 Targeted Biologic Modifiers, 1155 Allogeneic Hematopoietic Cell Transplantation, 1157 Gene Therapy, 1157 Disease-Specific Considerations, 1158

International Union of Immunological Societies (IUIS) categories2 are discussed further.

GENERAL MANAGEMENT STRATEGIES IN PRIMARY IMMUNODEFICIENCY DISEASES Important precautionary measures can reduce or minimize infections in patients with PIDs. First, exposure to infected individuals should be avoided as much as possible. Patients with severe immunologic defects (e.g., severe combined immunodeficiency disease [SCID]) require strict isolation, but in most cases implementation of excessive measures becomes unnecessary. The need to encourage routines that will maximize quality of life for the patient cannot be overstated. Universal precautions and proper handwashing should be practiced by the patient, family members, and all others who come into contact with the immunodeficient individual. Finally, acute infections must be identified early and treated aggressively. A low threshold should be maintained for hospital admission.

IMMUNIZATIONS Infections should be prevented through the use of immunizations in patients who are capable of mounting an immune response. Live vaccines should be avoided in several PIDs because of the risk of severe infection. A summary of recommended guidelines is presented in Table 70.1.3,4 Household members should generally be vaccinated to facilitate herd immunity, with the exception of live influenza vaccine in individuals with close contact with SCID patients and oral polio vaccine in all cases.5 It should be noted that patients receiving immunoglobulin G (IgG) replacement therapy (IgRT) do not require routine immunizations.

IMMUNOGLOBULIN G REPLACEMENT THERAPY IgRT has remained an integral component of the field of PID for decades.6 The concept of transferring immune protection through serum was first reported by von Behring and Kitasato in 1890. IgRT was first applied to a PID by Bruton, as reported in 1952, for the successful treatment of his patient with X-linked agammaglobulinemia (XLA). IgG was infused

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TABLE 70.1  Immunizations in Patients with Primary Immunodeficiency Diseases EXPERT RECOMMENDATIONS Primary Defect

Not Recommended

Recommended

B cell function

Severe antibody deficiency Mild antibody deficiency

Live attenuated vaccines (excluding BCG) Live attenuated influenza, OPV, adenovirus, typhoid, yellow fever

Inactivated or recombinant influenza, human papillomavirus All others, especially pneumococcal vaccine and Hib

T cell function

Severe deficiency Mild deficiency

All Live attenuated influenza, OPV, rotavirus, adenovirus, smallpox, typhoid, yellow fever, BCG

None Inactivated or recombinant vaccines, live attenuated MMR, varicella, and herpes zoster (if adequate T cell quantity and function)

Complement

Live attenuated influenza, adenovirus, typhoid

All others, especially pneumococcal, meningococcal, and Hib vaccines

Phagocytic function

Live attenuated influenza, adenovirus, typhoid, BCG

All inactivated and recombinant vaccines, other live attenuated viral vaccines

BCG, Bacillus Calmette-Guérin; Hib, H. influenzae type b; MMR, measles-mumps-rubella; OPV, oral poliovirus. Adapted from Shearer WT, Fleisher TA, Buckley RH, et al. Recommendations for live viral and bacterial vaccines in immunodeficient patients and their close contacts. J Allergy Clin Immunol 2014;133:961-6; and Sobh A, Bonilla FA. Vaccination in primary immunodeficiency disorders. J Allergy Clin Immunol Pract 2016;4:1066-75.

TABLE 70.2  Infections and Trough

Serum IgG Levels for Patients with Agammaglobulinemia Who Are Receiving Intravenous Immunoglobulin Replacement Therapy Trough Serum IgG Levels (mg/dL) Annual incidence of bacterial infections requiring hospitalization  Pneumonia  Septicemia

800

0.16

0.05

0.00*

0.12 0.02

0.05 0.00

0.00* 0.00

*p < 0.001 compared with LTC4 = LTE4. Signaling through cysLT1 elicits bronchoconstriction, mucus secretion, and airway edema. Therapeutic cysLT1 antagonists include montelukast, pranlukast, and zafirlukast. CysLT1 expression is increased at the transcriptional level by type 2 cytokines, including IL-4 and IL-13, thus likely explaining why cysLT1 expression is increased in subjects with allergic diseases. CysLT2 is expressed on lung macrophages, airway smooth muscle, peripheral blood leukocytes, mast cells, and

brain tissue.29 A recent mouse study showed that cysLT2 is also expressed in the lung on bronchial smooth muscle, alveolar macrophages, conventional dendritic cells, and eosinophils.30 In this study, a cysLT2 antagonist inhibited multiple antigen challenge–induced increases in eosinophils and mononuclear cells into the lung. CysLT2 in humans maps to chromosome 13 (13q14).31,32 The binding affinity for cysLT2 is LTD4 = LTC4 > LTE4. There are currently no selective cysLT2 inhibitors that are in clinical use, thus the function of cysLT2 is inferred from animal studies. Studies in cysLT2-deficient mice suggest that signaling through cysLT2 contributes to vascular permeability, inflammation, and tissue fibrosis, but not bronchoconstriction. Gemilukast (ONO-6950), which reportedly inhibits both cysLT1 and cysLT2, is being evaluated in phase-2 trials for the treatment of asthma.33 In a trial of nonsmoking subjects with asthma, gemilukast significantly reduced the maximum percent fall in forced expiratory volume in 1 second (FEV1) during the early and late phase responses compared with placebo; however, there was no difference in outcomes between gemilukast and montelukast.34 GPR99 has recently been identified as the third cysteinyl LT receptor and is now termed cysLT3. The predominant ligand for cysLT3 is LTE4, and there are no known human cysLT3 antagonists. Animal studies using cysLT3 knockout mice revealed that these mice had a dosedependent loss of LTE4-mediated vascular permeability, whereas LTC4 or LTD4 had no effect, suggesting a preference of cysLT3 for LTE4 even when cysLT1 is present.35 CysLT3 was detected on lung and nasal epithelial cells in mice.36 After either Alternaria alternata or LTE4 airway challenge in mice, cysLT3-deficient mice were fully protected against profound epithelial cell mucin release and swelling. CysLT3-deficient mice have reduced baseline numbers of goblet cells, suggesting a function of this receptor in regulating epithelial cell homoeostasis.36 There is evidence that LTD4 and PGE2 may combine to synergistically promote vascular inflammation. Combined injection of LTD4 and PGE2 potentiated vascular permeability and edema in the ears of mice and was mediated by signaling through cysLT1 and the PGE2 receptor EP2.37 LTD4 plus PGE2-potentiated vascular inflammation was partially sensitive to cysLT1 or EP3 antagonists but was completely blocked by simultaneous treatment of both cysLT1 and EP3 antagonists in vivo and in vitro. This interplay between PG and LT receptor signaling could have clinical significance in that both may need to be blocked for therapeutic purposes to inhibit inflammatory responses. There are two LTB4 receptors, BLT1 and BLT2.38 The high-affinity receptor for LTB4 is BLT1, and this receptor mediates most of the chemoattractant and proinflammatory actions of LTB4. Signaling through BLT1 enhances leukocyte recruitment to infected sites and is involved in the elimination of pathogen. BLT2 has much less affinity for LTB4 compared with BLT1. BLT2 is highly expressed in epithelial cells in various tissues including intestine and skin. BLT2 signaling promotes cell-cell junctions, protects against transepidermal water loss, and prevents entry of environmental substances into the body.

THERAPEUTIC STRATEGIES TO INHIBIT LEUKOTRIENES There have been two successful strategies to antagonize LTs for therapeutic benefit. The first is to antagonize the production of the LTs by blocking their synthesis through 5-LO inhibition. The second strategy is to inhibit LT binding on target tissues through LT receptor antagonists. Blocking 5-LO would have the combined benefit of inhibiting production of both LTB4 and the cysteinyl LTs. Theoretically, 5-LO inhibition would inhibit cysteinyl LT-induced bronchoconstriction and vascular permeability, in addition to LTB4-mediated neutrophil and eosinophil chemotaxis. 5-LO is composed of two domains, a C-terminal catalytic domain and an N-terminal C2-like β-barrel domain.39 The C-terminal

CHAPTER 97  Antileukotriene Therapy in Asthma catalytic domain contains a nonheme iron in the active site that functions as an electron acceptor or donor during catalysis. When 5-LO is activated, the iron is oxidized from the ferrous state (Fe2+) into the ferric state (Fe3+), by which 5-LO can enter the catalytic cycle. The C2-like domain interacts with lipids such as phosphatidylcholine, diacylglycerides, or lipid membranes, in addition to Ca2+ or Mg2+ ions. Ca2+, in addition to glycerides, stimulate 5-LO activity through reduction in the requirement for activating lipid hydroperoxides. There are three main groups of 5-LO inhibitors: redox-active compounds, non-redoxtype inhibitors, and iron-ligand inhibitors that have weak redox properties. Zileuton is the only 5-LO inhibitor that has been approved for the treatment of asthma and is an iron-ligand type inhibitor. Zileuton inhibits an estimated 26% to 86% of endogenous leukotriene production.40 Zileuton has several therapeutic shortcomings that has limited its widespread acceptance for asthma treatment. First, it has the potential for liver toxicity, likely because of alkylation and irreversible inhibition of glutathione S-transferase M1 (GSTM-1). Second, the half-life is very short. Therefore 5-LO use requires liver function testing and multiple daily doses. There is an extended release form of zileuton available, which may be prescribed twice daily as opposed to four times a day; however, the total daily dose is the same at 2400 mg. Another approach to block LT generation is to inhibit FLAP, which transfers free arachidonic acid to 5-LO. Although there have been several clinical trials of FLAP inhibitors in asthma and in cardiovascular diseases, none have been approved for human use. Development of 5-LO and FLAP inhibitors continue, because there have been more than 20 that have been or are being studied for therapeutic use in asthma, cardiovascular diseases, or rheumatologic conditions.41 Therapeutic cysLT1 antagonists include montelukast, pranlukast, and zafirlukast. The bulk of the clinical trials of examining the therapeutic effect of LTs in asthma and allergic diseases have been in this class of medications, and these will be discussed in great detail in the next sections of this chapter. The LTB4 pathway is also a possible therapeutic target for asthma treatment.42 Two different avenues to inhibit the biologic effect of LTB4 have been pursued. The first is to antagonize LTB4 binding to its receptors (BLT1 and BLT2). The second is to decrease the production of LTB4 by blunting the activity of LTA4H, the rate-limiting enzyme in the production of LTB4. There is a large body of scientific evidence linking LTB4 to human diseases and demonstrating efficacy of LTB4 antagonists in a wide array of preclinical models. As a result, at least five BLT antagonists and six LTA4H inhibitors have reached Phase 2 clinical trials. However, despite the extensive efforts to discover and develop drugs targeting the LTB4 pathway, only one, the LTA4H antagonist ubenimex, has reached the market to date. Ubenimex is a naturally occurring dipeptide that inhibits a large spectrum of aminopeptidases, including aminopeptidase N, leucine aminopeptidase, and LTA4H. Although ubenimex is approved in Japan for use as an adjunct to chemotherapy in acute nonlymphocytic leukemia, there are no published trials for its use in asthma or allergic diseases.

LEUKOTRIENE ANTAGONISM IN ASTHMA Although the presence of elevated levels of LTs in biologic fluids after challenges suggested a potential role for these mediators in disease pathogenesis, only antagonizing the LTs with subsequent improvement in physiologic and immunologic parameters, as well as symptom improvement, would prove their actual contribution to disease processes. As mentioned in the preceding section, the only pharmacologic options currently available for antagonizing the LT pathway are 5-LO inhibition and cysLT1 receptor blockade (Table 97.1). 5-LO inhibition blocks the production of both LTB4 and cysteinyl LTs, therefore blunting all

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TABLE 97.1  Leukotriene Antagonists and

Pathway Inhibitors

Leukotriene Receptor Antagonists

Leukotriene Pathway Inhibitors

Mechanism of action

Block the actions of cysteinyl leukotrienes (cysLTs)

Block the production of leukotriene B4 and cysLTs

Specific agents

Zafirlukast (Accolate) Montelukast (Singulair) Pranlukast (Onon)

Zileuton (ZyfloCR)

downstream activity of these mediators. In contrast, cysLT1 receptor blockade has little impact on the production of the LTs.

Allergen-Induced Bronchoconstriction and Inflammation The finding that cysLT1 blockade inhibited LTD4-induced bronchospasm was an important proof of concept experiment that cysLT1 antagonists could be used to determine the effect of this class of medications on the therapeutic and biologic effects of cysteinyl LTs in asthma pathogenesis.43 Ensuing studies substantiated that cysteinyl LTs contributed to a major portion of the bronchospasm that resulted from allergen challenge. Administration of the cysLT1 antagonist zafirlukast before allergen challenge inhibited immediate phase bronchospasm by approximately 80% and reduced the late phase by 50%.44,45 Although zafirlukast treatment markedly reduced airway obstruction, it resulted in a small, but statistically significant, reduction in allergen-induced airways responsiveness.46 The effect of cysLT1 blockade on airways responsiveness was the same as corticosteroids.47 Montelukast pretreatment also blocked late phase bronchoconstriction by 50%, yet it did not reduce sputum eosinophils compared with placebo, suggesting that the physiologic effects of cysLT1 blockade was not linked to eosinophil-driven physiologic mechanisms.48,49 The effect of LT antagonism on allergen-induced airway inflammation has been examined in BAL fluid and tissue biopsy. CysLT1 antagonists and 5-LO inhibitors blunted the influx of airway inflammatory cells after segmental allergen challenge, a procedure where an allergen to which a subject is sensitized is administered via bronchoscopy into the lower airways.50,51 Zafirlukast treatment for 1 week before segmental allergen challenge significantly reduced the number of basophils and lymphocytes in BAL fluid 48 hours compared with placebo, whereas there was only a nonsignificant numerical reduction in airway eosinophils.50 5-LO blockade with zileuton treatment resulted in similar findings.51 One study suggested that the effectiveness of LT antagonism in reducing allergen-induced inflammation was proportional to the LT levels produced as a result of allergen challenge.52

Exercise-Induced Bronchospasm Exercise-induced bronchospasm (EIB) is consistently blunted by both cysLT1 antagonists and 5-LO inhibitors by approximately 30% to 60% (Fig. 97.2).53-56 The ability of LT antagonism to reduce EIB in children is not as great as in adults, with inhibition in the 30% to 40% range.57,58 LT antagonists reduced the decrement in pulmonary function to cold-air hyperventilation in 3- to 5-year-old children by approximately 60%.59 It is unknown whether the mechanism by which cold air–induced bronchospasm is different from that caused by exercise, or whether there is a difference in LT antagonism response in children based on age. Administration of the cysLT1 antagonist montelukast in adults with EIB for 12 weeks provided continued protection against decrements in pulmonary function when the drug was administered 16 to 18 hours before exercise. The effect of montelukast in this setting was significantly

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SECTION H Therapeutics

15

Placebo Zileuton

10 10 FEV1 % change [mean ± SE]

Percent Change in FEV1

5 0 −5

−10 −15 −20

8 6 4 2

0

−25 0 3 5 7 9 10−15 16−20 21−25 26−30 31−35 36−40 41−45 Minutes after Exercise Fig. 97.2  Montelukast significantly reduced the maximal fall in forced expiratory volume in 1 second (FEV1) after exercise and shortened the recovery time. Mean (±standard error) percent change in FEV1 over time after exercise, after treatment with montelukast (open squares) or placebo (solid squares). Measurements were made in all subjects immediately after exercise, 3 minutes later, and every 2 minutes thereafter until the FEV1 began to improve. (From Manning PJ. Inhibition of exercise-induced bronchoconstriction by MK-571, a potent leukotriene D4-receptor antagonist. N Engl J Med 1990;323:1736-9.)

superior to the long-acting β-agonist (LABA) salmeterol over the same time frame in two 8 week trials.60,61 Although there was no difference in the ability of montelukast and salmeterol to block EIB early in the studies, extended use of salmeterol resulted in a blunted protective effect, suggesting tachyphylaxis to the LABA, whereas there was no diminished therapeutic effect in EIB over time with montelukast treatment.60,61

Aspirin-Exacerbated Respiratory Disease Approximately 10% of adults with asthma will have asthma symptoms and reduction in pulmonary function after ingesting aspirin or other nonsteroidal antiinflammatory drugs (NSAIDs), and this is termed AERD.62 The exact mechanisms causing this adverse response are not fully defined; however, there is increased expression of LTC4 synthase and cysLT2 by mast cells and eosinophils in patients with AERD. It is unknown whether the increase in LTC4 synthase expression is an underlying mechanism leading to AERD. Treatment with both cysLT1 antagonists and 5-LO inhibitors reduced AERD pulmonary function abnormalities and symptoms, strongly suggesting that LTs are involved in AERD pathogenesis. Several studies revealed that there is almost 100% inhibition of aspirin-induced bronchospasm by LT antagonists.63-65 Blunting the AERD response is dependent upon the dose of aspirin administered, and there was loss of inhibition of the effect of LT antagonists when standard doses of aspirin were ingested.66 5-LO inhibition decreased aspirin-induced urinary LTE4 and mast cell produced tryptase in nasal secretions.64,67 These results substantiate that LT antagonists impact mast cells and their activation, but the importance of LT generation as a result of mast cell activation in this setting needs further definition. Extended studies of LT antagonism in subjects with AERD reinforce the use of these drugs in this patient population.54,68 In general, patients with AERD demonstrate more severe asthma and have additive gains in both FEV1 and symptom improvement with the inclusion of a 5-LO

0

1

2 Hours

3

4

Fig. 97.3  Zileuton significantly increased forced expiratory volume in 1 second (FEV1) compared with standard therapy, including inhaled corticosteroids, after lysine aspirin challenge. Percent change in FEV1 (treatment mean ± standard error) after a single dose of zileuton (600 mg or matching placebo). The increase in FEV1 was significant compared with placebo at 1, 3, and 4 hours (p < 0.05, p < 0.05, and p < 0.05, respectively).

inhibitor or a cysLT1 antagonist, in addition to existing treatment with inhaled or oral corticosteroids (Fig. 97.3).68 Patients with AERD also have improved rhinitis and sinus symptoms with the addition of a 5-LO inhibitor.68 There are no data to suggest that LT antagonism controls asthma in patients with AERD to a greater degree than in other forms of asthma.

Inflammation in Chronic Asthma There is evidence that long-term LT antagonism may reduce inflammation in asthma. For instance, both cysLT1 antagonism and 5-LO inhibition reduced peripheral blood eosinophils.69,70 Pranlukast, a cysLT1 antagonist, decreased tissue eosinophils after 4 weeks of therapy, although there was no change in forced FEV1.71 Montelukast decreased eosinophils in tissue biopsies compared with pretreatment baseline; however, there was no statistically significant difference compared with placebo. However, another study did not show a reduction in tissue eosinophils with montelukast treatment compared with pretreatment baseline.72 In patients with increased sputum eosinophils, montelukast decreased sputum eosinophils after 4 weeks of therapy. In this study, a subgroup of subjects who had greater baseline sputum eosinophils had improvement in morning peak flow with montelukast treatment, and this alteration in pulmonary function was greater than the asthma population as a whole. However, in subjects treated with high-dose inhaled corticosteroids, montelukast did not reduce sputum eosinophils.73 Exhaled nitric oxide is commonly used as a surrogate for airway inflammation. Three studies reported that montelukast reduced exhaled nitric oxide to a small, but statistically significant, degree.74-76

Nocturnal Asthma Persons with nocturnal asthma have an increase in airway eosinophilia at 4 A.M. These subjects had an increase in urine LTE4 levels and BAL levels of LTB4 and cysteinyl leukotrienes.77 5-LO inhibition with zileuton inhibited BAL eosinophils and LT levels and decreased urine LTE4, while improving both asthma symptoms and pulmonary function in patients with nocturnal asthma.78 Interestingly, the increase in FEV1 in patients

CHAPTER 97  Antileukotriene Therapy in Asthma with nocturnal asthma was related to a reduction in the BAL levels of LTB4, but not BAL cysteinyl LTs.

TREATMENT TRIALS IN CHRONIC ASTHMA Treatment with oral zileuton, zafirlukast, and montelukast induced a rapid and statistically significant bronchodilator effect of 10% to 30%.79-81 Intravenous montelukast increased FEV1 by 15% within 15 minutes, whereas this degree of bronchodilator response was not seen until 2 hours after oral administration.79 These data suggest that LTs are constitutively expressed in asthma and mediate baseline bronchoconstriction that can be reversed quickly by LT antagonism. Treatment of patients with a β-agonist induced further bronchodilation, implying that these two classes of medications function through separate bronchodilating pathways.80 The rapid effect of LT antagonists, and the additive effect of a β-agonist on pulmonary function, propose that LT-modifying agents could be effective in treating severe, acute asthma exacerbations. Two clinical trials assessing intravenous montelukast in subjects experiencing acute exacerbations reported significantly increased FEV1 and a nonsignificant decrease in hospitalizations.82,83 Trials of oral cysLT1 agents have not shown consistent benefit in acute asthma exacerbations, and a Cochrane review did not support acute anti-LT therapy.84 Several longer term (3 to 6 month) controlled trials of zafirlukast, montelukast, and zileuton consistently revealed statistically significant improvement in asthma patients, who in general had moderate disease.69,85-87 Use of these medications induced sustained and statistically significant gains in FEV1 (7% to 15%), symptom scores, and decreased rescue β-agonist use compared with placebo. Nighttime asthma symptom scores decreased by 30% to 40%. Cessation of treatment with either cysLT1 antagonists or 5-LO inhibitors have not resulted in rebound increase in symptoms or worsened pulmonary function.85 Montelukast and zafirlukast both improved asthma quality-of-life scores.85 Zileuton, zafirlukast, and montelukast all decrease the need for steroid therapy secondary to asthma exacerbations.69, 85 These classes of medications have also significantly reduced days missed from school or work compared with placebo and have also reduced asthma exacerbation rates.69,70,87 Longer term treatment trials have also shown that LT antagonists are efficacious in children. Montelukast treatment resulted in significantly reduced rescue β-agonist use, asthma exacerbations, and improved quality of life compared with placebo. In another study of 6- to 14-yearold children who were concomitantly taking inhaled corticosteroids, montelukast therapy improved symptoms and provided for increased tapering of inhaled corticosteroids.88 Montelukast has also been examined as treatment for early life wheezing. In children aged 2 to 5 years, montelukast significantly reduced wheezing episodes compared with placebo.89 In a study of 10- to 26-month-old children, montelukast treatment improved FEV1 and symptoms, while reducing the fraction of exhaled nitric oxide (FeNO).90 Montelukast significantly reduced the asthma exacerbation rate by 32% in 2- to 5-year-old children compared with placebo and also reduced the need for inhaled corticosteroids.91 Although one study suggested that montelukast reduced symptoms from RSV-induced bronchiolitis, later trials did not confirm those results.92-94 In another study examining the effect of montelukast prophylaxis for symptoms related to upper respiratory tract infections, there was no benefit compared with placebo.95 A recent Cochrane Database review of five studies that examined the effect of cysLT antagonists in the management of episodic viral wheeze in children aged 1 to 6 years, reported there was no evidence of benefit associated with maintenance of intermittent cysLT1 antagonist treatment, compared with placebo, for reducing the number of children with one or more viral-induced episodes requiring rescue oral corticosteroids.96

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As mentioned earlier, early challenge studies revealed that SRS, LTC4, and LTD4 caused regional contraction of guinea pig airways when administered intravenously, with greater contraction of small airways compared with large airways.11 These results led to studies examining the impact of LT antagonism in the small airways of humans. In adult asthma subjects who had never been prescribed inhaled corticosteroids, montelukast reduced regional air trapping on CT scan compared with placebo.97 The reduction in regional air trapping with montelukast treatment correlated with improved quality of life and symptoms, yet there was only a slight change in FEV1 and no difference in lung volume. In adults with asthma who were preselected for increased residual volume by plethysmography, residual volume measurements increased with montelukast therapy, suggesting improvement in small airway function.98 In both of these previously two mentioned studies, the decreased air trapping seen on CT scan with montelukast treatment correlated with improved symptoms, but not an increase in FEV1.97,98 In a pediatric study, montelukast increased the residual volume percent predicted and the ratio of residual volume to total lung capacity but failed to improve FEV1.99 These results strongly suggest that cysLT1 antagonism does improve regional airflow, perhaps specifically to the small airway, because FEV1 is more a measure of large airway function.

Leukotriene Antagonism Compared with Inhaled Corticosteroids Double-blind trials revealed that inhaled low-dose beclomethasone or fluticasone improved FEV1 by twofold compared with the cysLT1 antagonists pranlukast, zafirlukast, and montelukast, whereas there was no difference in exacerbation rates between these classes of medication.70,100,101 Both classes of drugs had similar effects on peripheral blood eosinophil counts.70 In general, there were also improvements in symptom scores and reduced rescue β-agonist usage in asthma patients treated with inhaled corticosteroids compared with cysLT1 antagonists.70,101 Although there was more rapid improvement in pulmonary function with cysLT1 antagonists, by the end of week 2 of therapy, the increase in FEV1 was significantly greater with inhaled corticosteroids.101 Similarly, in a randomized, double-blind, double-dummy, placebo-controlled parallelgroup trial of patients with chronic asthma, montelukast increased peak expiratory flow rate (PEFR) more quickly than inhaled corticosteroids, but over time, the improvement in PEFR was greater with inhaled corticosteroid treatment compared with montelukast (Fig. 97.4).70 In trials in children age 6 to 14 years, inhaled corticosteroid treatment resulted in greater pulmonary function, improved symptom scores, and reduced exacerbation rate compared with cysLT1 antagonists.102,103 A Cochrane review that included 56 trials that compared cysLT1 antagonists versus inhaled corticosteroids, mostly at low doses, reported that inhaled corticosteroids were superior for pulmonary function, exacerbation rate, and symptom scores.104 A subsequent Cochrane review that included 37 studies examined the efficacy of cysLT1 antagonists added to inhaled corticosteroids compared with the same dose, an increasing dose, or a tapering dose of inhaled corticosteroids in each arm for adults and adolescents 12 years of age and older with persistent asthma.105 This analysis revealed that for adolescents and adults with persistent asthma and who had suboptimal asthma control with daily use of inhaled corticosteroids, the addition of cysLT1 antagonists was beneficial for reducing moderate to severe asthma and for improving lung function and asthma control compared with the same dose of inhaled corticosteroids. Trials of cysLT antagonists in asthma revealed that there is significant heterogeneity in response to this class of medications. Approximately 40% to 55% of subjects will have what is deemed a significant clinical response.70,106 In a head-to-head comparison of responsiveness to montelukast versus inhaled corticosteroids, 42% of the montelukasttreated subjects had improvement in FEV1 by 11% or more, whereas

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SECTION H Therapeutics 50 Beclomethasone Mean change in morning PEFR from baseline, L/min

40 30 20 Montelukast 10 0 -10

Placebo 1

3

5

7

9

11

13

15

17

19

21

Time receiving active treatment, d Fig. 97.4  Onset of action of montelukast, beclomethasone, and placebo. Treatment effect for peak expiratory flow rate (PEFR) in the morning over the first 21 days of treatment. Vertical lines represent standard error.

50% of the inhaled corticosteroid-treated subjects had the same degree of improvement.70 This study implies that there are fewer responders to LT antagonism compared with inhaled corticosteroids. The same trend existed in children with asthma aged 6 to 17 years in whom an improvement in FEV1 of greater than 7.5% was noted in 22% of montelukast-treated subjects, whereas 40% of children had the same level of FEV1 increase when treated with inhaled corticosteroids.107 Of note, there was a greater percentage of subjects treated with either cysLT1 antagonists or inhaled corticosteroids who had symptom improvement compared with the percentage that had gains in pulmonary function.108 Currently, there are no firm predictors, either clinical or biochemical, that conclusively determine which patients will have a significant improvement with LT antagonist therapy. There are two studies in pediatric patients that implied that children who had elevated urinary LT levels, especially in those with lower FeNO, had greater responses to montelukast.107,109 Although clinical trials revealed that the efficacy of inhaled corticosteroids is superior to LT antagonists, the question as to whether these results translate into “real-world” effectiveness is important to determine optimal treatment strategies for asthma patients. This is particularly critical in populations where compliance is an issue and desirability of taking an oral drug may be greater than inhaled medication. In a study performed in Great Britain that incorporated a large outpatient population, at 2 years there was a small, but statistically significant, improvement in outcomes in the patients treated with inhaled corticosteroids compared with montelukast that was not seen at the 2-month mark.110 An effectiveness study performed in children revealed no differences in hospitalizations for asthma between the cysLT1 antagonist and inhaled corticosteroid–treated groups.111 In a retrospective cohort analysis of pharmacy and medical claims evaluated after 12 months of therapy with either montelukast or zafirlukast compared with inhaled fluticasone, there was a significant reduction in hospitalizations and emergency department visits for asthma in the fluticasone-treated group compared with those treated with the cysLT1 antagonists. One caveat of this report is that there was no control for the severity of asthma at the beginning of the study.112

Leukotriene Antagonists Compared with Theophylline As reviewed previously, there are many studies that compared LT antagonists with inhaled corticosteroids, because LT modifiers were originally placed in the controller category and inhaled corticosteroids

were considered first-line controller medications. However, there are far fewer studies comparing LT antagonists with other classes of asthma medications. In a double-blind, placebo-controlled trial of zileuton (400 or 600 mg four times daily) compared with twice daily theophylline that was titrated to therapeutic levels, the theophylline-treated subjects had increased trough-level pulmonary function.113 Sixty minutes after morning dosing, there was no difference in pulmonary function between the treatment groups. There were no significant differences in symptom scores or rescue β-agonist use between the zileuton- and theophylline-treated groups. There were also no differences between the groups in adverse events or number of exacerbations; however, there was a reduction in peripheral eosinophilia with zileuton treatment.

Leukotriene Antagonists as Add-On Therapy in Patients Not Controlled with Inhaled Corticosteroids Several trials have compared the addition of a cysLT1 antagonist to inhaled corticosteroids in patients who are not well controlled on inhaled corticosteroids alone. Compared with the addition of placebo to inhaled corticosteroids, the combination of montelukast and inhaled corticosteroids increased both symptom scores and pulmonary function in a 12-week double-blind, double-dummy trial.114 Although the increase in FEV1 with the addition of montelukast to inhaled corticosteroids was modest (5%) compared with placebo (1%), as shown in Fig. 97.5, there was an additive reduction of peripheral blood eosinophils with the addition of montelukast compared with placebo.114 In patients with severe asthma, the addition of zafirlukast to inhaled corticosteroids improved pulmonary function, increased symptom scores, and reduced exacerbations compared with placebo.115 It is important to note that in this trial, the dose of zafirlukast was fourfold greater than the recommended dose, and there was also increased liver function abnormalities. In a trial where doubling the dose of budesonide was compared with the addition of montelukast to inhaled corticosteroids in patients whose asthma was not well controlled at 800 µg of budesonide, there was improvement in both groups in FEV1 and symptom scores compared with the original inhaled corticosteroid treatment.116 However, there was no difference in outcomes between the groups at the end of the trial. In a study of severe asthma patients who were taking on average three controller medications, and approximately 50% of which were taking oral corticosteroids, the addition of montelukast did not improve outcomes.117

CHAPTER 97  Antileukotriene Therapy in Asthma 8

Montelukast + Beclomethasone

4 0 Percent change –4 from baseline (Mean ± SE) –8

Beclomethasone Montelukast AM PM removal removal

–12 –16

0

2

4

Placebo 6

9

13

16

Weeks in active treatment Fig. 97.5  Effects of adding montelukast to inhaled corticosteroids. The addition of montelukast (10 mg) to inhaled beclomethasone (200 µg twice daily) resulted in improved FEV1 over the 16 week treatment period.

The use of cysLT1 antagonists allowed for weaning of high-dose inhaled corticosteroids in two double-blind, placebo-controlled studies. The addition of pranlukast permitted a decrease in beclomethasone dosing from 1600 µg to 800 µg per day in patients with moderately severe disease, without ceding asthma control.118 Adding pranlukast maintained morning and evening peak flow rate, despite the reduction in beclomethasone dosing. Although patients taking placebo who reduced inhaled corticosteroids had increased peripheral blood eosinophils and FeNO, there were no increases in these parameters in the patients randomized to pranlukast. In the other study, the dose of inhaled corticosteroid was reduced from 1600 µg to 1000 µg per day during a single-blind, placebo-controlled run-in period, after which patients were randomized to either montelukast or placebo. The patients treated with montelukast further weaned their inhaled corticosteroid dose by 47%, whereas the placebo group reduced their dosage by 30%, a statistically significant difference.119

CysLT1 Antagonists Compared to Long-Acting β-Agonists Multiple trials have compared the addition of montelukast or the LABA salmeterol to inhaled corticosteroids when additional controller therapy was necessary because of poor symptom control.120-125 In each trial, although both groups benefitted compared with before add-on therapy was instituted, the groups randomized to LABA had greater improvement compared with the addition of the cysLT1 antagonist in terms of pulmonary function and symptom scores. Improvement in peak flow ranged from 25 to 35 L/min with LABA, compared with 15 to 25 L/min with montelukast. It is important to note that the entry criteria for the definition of asthma included a postbronchodilator response of 12% or more after short-acting β-agonist, therefore perhaps selecting for patients who might have greater responsiveness to β-agonist at baseline. In several of the trials, asthma symptoms improved to a greater degree with the addition of a LABA compared with montelukast, although this was not universal.122-125 The addition of LABA versus cysLT1 antagonists to inhaled corticosteroids was evaluated in a Cochrane analysis with the conclusion that LABA reduced exacerbations (17% improvement with LABA compared with cysLT1 antagonists).126 Furthermore, the use of LABAs resulted in greater improvement in most other endpoints, although the rate of hospitalizations was no different, likely owing to the low occurrence of this measure. Similar studies examining the effect of LABA to cysLT1 antagonists when added to inhaled corticosteroids have also been performed in pediatric patients with asthma. A trial conducted by the National Heart, Lung, and Blood Institute’s Childhood Asthma Research and Education

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(CARE) Network examined the addition of either montelukast or LABA to inhaled corticosteroid versus a doubling of the inhaled corticosteroid dose in children aged 6 to 17 years who were not well controlled on low-dose inhaled corticosteroids (Fig. 97.6). The group treated with LABA and inhaled corticosteroids had significant improvements in symptoms, lung function, and exacerbation rate compared with the other two groups, between which there were no differences in these endpoints.127 A recent study compared the effectiveness and cardiovascular safety of LABA to cysLT1 antagonists in older adults with asthma who were already taking inhaled corticosteroids.128 In this study, Medicare beneficiaries aged 66 and older were enrolled who were treated exclusively with inhaled corticosteroids plus LABA or inhaled corticosteroids plus a cysLT1 antagonist. CysLT1 antagonist add-on treatment was associated with greater odds of asthma-related hospitalizations or emergency department visits (odds ratio = 1.4; p < 0.001), in addition to exacerbations requiring oral corticosteroids or antibiotics, than LABA treatment. CysLT1 antagonist add-on therapy was also less effective in controlling acute symptoms, as defined by rescue β-agonist use compared with LABA. However, cysLT1 antagonist treatment was associated with lower odds of experiencing a cardiovascular event than LABA treatment (odds ratio 0.86; p = 0.006). Although symptoms and pulmonary function were improved when LABA was added to inhaled corticosteroids compared with the addition of cysLT1 antagonists to inhaled corticosteroids, the opposite effect occurred on inflammatory endpoints. In three trials, the addition of a cysLT1 antagonist to inhaled corticosteroids significantly decreased peripheral blood eosinophils and FeNO to a greater degree than the addition of a LABA to inhaled corticosteroids.120,129,130 Furthermore, the addition of a cysLT1 antagonist decreased bronchial responsiveness more than the addition of a LABA.130

Leukotriene Antagonists for Allergic Rhinitis and Atopic Dermatitis Montelukast is approved in the United States for treatment of seasonal allergic rhinitis. Several trials reported that cysLT1 antagonists improved outcomes for seasonal and perennial rhinitis compared with placebo.131-134 Trials comparing nasal corticosteroids to montelukast universally reported that nasal corticosteroids significantly improved outcomes compared with the cysLT1 antagonist.135-139 In a study comparing the addition of montelukast or nasal fluticasone to the combination of inhaled fluticasone/salmeterol in subjects with both rhinitis and asthma, the group randomized to nasal fluticasone had significant improvement in nasal symptoms, whereas neither therapy had an impact on asthma outcomes.137 Montelukast did not improve atopic dermatitis symptoms compared with placebo in two trials.140, 141

Candidate Genes That May Regulate Responses to Leukotriene Antagonists Pharmacogenetic studies have largely targeted two aspects of LT biology that likely impact an individual’s ability to respond to LT antagonists; these include the generation of LTs by the 5-LO pathway and the ability of LTs to signal through their receptors. The gene encoding 5-LO (ALOX5) and the regulatory promoter region contains a tandem repeat polymorphism that modulated the response to zileuton.142 A follow-up study examining the importance of this tandem repeat polymorphism in ALOX5 revealed that patients treated with montelukast who had the wild type allele with five tandem repeats had fewer asthma exacerbations, decreased rescue albuterol usage, and increased FEV1 versus subjects who were homozygous for the minor allele with four tandem repeats.143 Ensuing candidate gene studies identified single nucleotide polymorphisms (SNPs) in ALOX5 (rs4987105 and rs4986832) that corresponded with lung function

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Pairwise comparisons LABA vs. ICS LABA better Neutral ICS better LABA vs. LTRA LABA better Neutral LTRA better ICS vs. LTRA ICS better Neutral LTRA better 0

10

20

A

30

40

50

60

0.4

0.5

0.6

Percent of patients Probability of best response

LABA

ICS

LTRA

0.0

B

0.1

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Probability of best response

Fig. 97.6  Pairwise comparison of three step-up therapies and the overall probability of best response. (A) Pairwise comparisons of the three step-up therapies. The proportion of patients with a best response to a long-acting β-agonist (LABA) step-up was higher that the proportion with a best response to a leukotriene receptor antagonist (LTRA) step-up (52% vs. 34%, P = 0.02) or an inhaled corticosteroid (ICS) step-up (54% vs 34%, P = 0.004). The best response results for LTRA step-up were similar to those for an ICS step-up (B) The pattern of differential response according to the probability that the best response to a treatment occurred during the period in which that treatment was received; LABA step-up was most likely to provide the best response. Covariate-adjusted estimates were obtained from rank-ordered logistic regression models; I bars indicate bootstrap-based 95% confidence intervals. (From Lemanske RF Jr. Step-up therapy for children with uncontrolled asthma receiving inhaled corticosteroids. N Engl J Med 2010;362:975-85.)

alterations in clinical trials of subjects treated with montelukast and zileuton.144-146 Further candidate gene studies have confirmed that ALOX5 polymorphisms regulate the response to montelukast.145 Pharmacogenetic associations exist for SNPs in ALOX5 (rs2115819) and genes encoding LTA4H (rs2660845) and LTC4S (rs730012) and the gene for MRP1 (rs119774). SNPs in the corticotropin-releasing hormone receptor 1 gene (CRHR1) (rs739645, rs1876831, and rs1876829) and a SNP in histone deacetylase 2 (HDAC2) (rs3757016) were associated with contradictory therapeutic effects on lung function between montelukast and inhaled corticosteroids.147 An association between montelukast levels and symptom control was noted in a gene locus coding for a solute carrier organic anion transporter family member 2B1 (SLCO2B1).148 There have been two genome-wide association studies (GWAS) that examined therapeutic responsiveness to LT antagonists.148 In the first

GWAS, DNA and phenotypic information from two placebo-controlled trials (total N = 526) of zileuton response were queried. Using a gene– environment GWAS model, the 12-week change in FEV1 after LT antagonist treatment was evaluated.149 The top 50 single-nucleotide polymorphism associations were replicated in an independent zileuton treatment cohort and two additional cohorts of montelukast response. In a combined analysis, rs12436663 in mitochondrial ribonuclease P (MRPP3) achieved genome-wide significance (P = 6.28 × 10−8). Homozygous rs12436663 carriers had a significant reduction in mean change in FEV1 after zileuton treatment. MRPP3 encodes a product that regulates transfer RNA processing and maturation and is located in a cluster of genes on chromosome 14 q proposed to regulate IgE phenotypes and autoimmune disease. Furthermore, rs517020 in glycosyltransferase 1 domain containing 1 (GLT1D1) was associated with worsening

CHAPTER 97  Antileukotriene Therapy in Asthma responses to both montelukast and zileuton (combined P = 1.25 × 10-7). This gene encodes a member of the glycosyltransferase family, but the function of the gene product is currently unknown. In the second GWAS, genome-wide genotype and phenotypic data available from an American Lung Association-Asthma Clinical Research Center cohort were used to evaluate 8-week change in FEV1 associated with montelukast administration in a discovery of 133 asthmatics.150 The top 200 SNPs from the discovery GWAS were then tested in 184 additional samples from two independent cohorts. Twenty-eight SNP associations from the discovery GWAS were replicated, and myeloid/lymphoid or mixed-lineage leukemia translocated to chromosome 3 protein (MLLT3) (rs6475448) achieved genome-wide significance. Subjects for all four studies who were homozygous for rs6475448 had increased FEV1 from baseline in response to montelukast. Other genes that are involved in LT production of signaling have also been identified as being associated with asthma. A 444A>C SNP in the LTC4S gene was associated with severe asthma and an altered response to zafirlukast.151 The 601 A>G variant of the cysLT2 gene, encoding the Met201Val receptor variant, was associated with atopic asthma in the general European population, where the allelic frequency is approximately 2.6%. Further studies will undoubtedly lead to more insight in the role of genetic polymorphisms to the contribution of the LT synthesis and receptor signaling pathways to asthma pathogenesis.

DOSING Montelukast is dosed at 10 mg every evening for patients older than 16 years, and the recommended dose for children between the ages of 6 and 14 years is 5 mg per day. For children 2 to 5 years, the dose is 4 mg per day.152 In all clinical trials, montelukast was dosed at bedtime. Zafirlukast is dosed at 20 mg twice daily in adults, with the recommendation that doses be taken either 1 hour before a meal or 2 hours after eating. This same dosing recommendation holds for children older than 12 years, whereas children between the ages of 5 and 12 years are dosed at 10 mg twice per day.153 Safety and efficacy have not been established for children younger than 5 years. Zileuton is indicated for the prophylaxis and chronic treatment of asthma in adults and for children 12 years of age and older. The controlled release formulation of zileuton is dosed as 1200 mg twice daily, for a total daily dose of 2400 mg per day.154

SAFETY There is a 4.4% incidence of elevated liver function tests with zileuton treatment, and the majority of the cases of increased liver enzyme values occurred within the first 3 months of therapy.155 Increased doses of zafirlukast (80 mg daily) may also increase liver enzyme values, whereas no increase in liver function tests have been seen with montelukast treatment compared with placebo. All of the LT antagonists are metabolized by the liver; therefore there is a risk of significant interactions with other drugs metabolized by the cytochrome P-450 enzyme system. As a result, doses of other medications metabolized by the cytochrome P-450 system need to be reduced. For instance, warfarin doses need to be reduced by 50% if this medication is taken concomitantly with zafirlukast.156 Zileuton and theophylline are both metabolized by the same cytochrome P-450 system, and as a result, it is recommended that the theophylline dose be lowered by 50% and that the blood theophylline level be monitored.157 There are no known drug interactions with montelukast. There have been reports of eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg-Strauss syndrome) in patients who were taking LT antagonists. For instance, eight patients treated with zafirlukast and who had previously been taking oral corticosteroids

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were diagnosed with EGPA once the corticosteroids had been tapered.158 EGPA has also been reported to occur in patients who were concomitantly treated with montelukast, pranlukast, and zileuton.159-161 It is not certain whether there was a direct causal effect of anti-LT therapy, or whether the EGPA manifested as a result of oral corticosteroid tapering. Although most patients who were newly diagnosed with EGPA were taking oral corticosteroids, not all were. It is important to note that EGPA has also been diagnosed in patients taking other medications, including inhaled corticosteroids, when oral corticosteroids were being withdrawn.161 A Dutch group retrospectively examined all adverse drug reactions for montelukast in children and adults that were reported to the Netherlands Pharmacovigilance Center and the World Health Organization (WHO) Global database, VigiBase through 2016.162 Eight patients with EGPA were reported to the Dutch database and 563 patients with EGPA to the VigiBase. It is important to consider a diagnosis of EGPA when asthma patients present with new rashes, increased respiratory symptoms, and especially neurologic signs and/or symptoms. In this situation, peripheral eosinophil count and chest X-ray should be pursued with further testing considered if warranted. The FDA added a precaution of neuropsychiatric disorders to the use of LT antagonists in 2009.163 The association of neuropsychiatric disorders with these medications is controversial, because having a diagnosis of asthma is associated with an increased rate of suicide and depression;164 therefore the neuropsychiatric adverse events may not be a result of the drug, but an effect of the unresolved asthma. In a Dutch retrospective study of adverse events to montelukast mentioned in the immediately preceding paragraph, depression was the symptom most frequently reported in the entire population to VigiBase (reporting odds ratio [ROR] 6.93; 95% confidence interval 6.5-7.4), whereas aggression was most commonly reported in children (ROR 29.77; 95% CI: 27.5-32.2).162 Suicidal ideation was also a prominent adverse event reported to VigiBase (ROR 20.43; 95% CI: 19.18-21.76). Nightmares were often reported for both children and adults to both the Dutch and WHO databases. In an uncontrolled Spanish study of children who experienced adverse reactions with montelukast therapy, nightmares were reported within 1 week of starting this medication and resolved rapidly after the drug was discontinued.165 A recent systematic review reported that observational studies did not find a significant association between neuropsychiatric events and LT antagonists.166 These authors suggest that the pharmacovigilance studies that detected a signal for neuropsychiatric events may have been influenced by the 2009 FDA warning, and that high-quality epidemiologic studies should be conducted to evaluate the association and to quantify the risk, in both children and adults.

LEUKOTRIENE ANTAGONISTS IN ASTHMA TREATMENT GUIDELINES The current treatment guidelines, published in 2007, suggest that LT antagonists can be used as an alternative to inhaled corticosteroids in the treatment of persistent asthma, especially in patients who either do not respond to or who do not wish to take inhaled corticosteroids.167,168 As mentioned earlier, in those patients who do not have an adequate therapeutic response to inhaled corticosteroids, outcomes with the addition of a LABA to inhaled corticosteroids are improved compared with the addition of an LT antagonist. There are no data that support the addition of an LT antagonist to the combination of an inhaled corticosteroid and LABA, although the treatment guidelines do not recommend against this. In patients with AERD, LT antagonists may be helpful,169 although there are no data to support that this patient population has increased response to these medications compared with the general population

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with asthma. LT antagonists may be helpful in patients with EIB, although there are no guidelines for this condition. As in the treatment of any disease, it is important to recognize that each patient is unique, and that some persons will respond to some drugs, but perhaps not others. This is particularly true in asthma where the heterogeneity of therapeutic responses to different classes of medications is well documented, as was highlighted earlier in this chapter. Although there are no validated biomarkers that predict the response to LT antagonists, a therapeutic trial to a specific medication is a reasonable approach to determine efficacy. Hopefully, the recent emphasis on funding precision medicine studies will yield important information that will assist in early and directed prescribing of asthma medications that have the optimal therapeutic effect with minimal adverse effects.

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CHAPTER 97  Antileukotriene Therapy in Asthma 40. Dube LM, Swanson LJ, Awni W. Zileuton, a leukotriene synthesis inhibitor in the management of chronic asthma. Clinical pharmacokinetics and safety. Clin Rev Allergy Immunol 1999;17(1–2):213–21. 41. Bruno F, Spaziano G, Liparulo A, et al. Recent advances in the search for novel 5-lipoxygenase inhibitors for the treatment of asthma. Eur J Med Chem 2018;153:65–72. 42. Bhatt L, Roinestad K, Van T, et al. Recent advances in clinical development of leukotriene B4 pathway drugs. Semin Immunol 2017;33:65–73. 43. Drazen JM. Anti-leukotrienes as novel anti-inflammatory treatments in asthma. Adv Exp Med Biol 2002;507:217–21. 44. Findlay SR, Barden JM, Easley CB, et al. Effect of the oral leukotriene antagonist, ICI 204,219, on antigen-induced bronchoconstriction in subjects with asthma. J Allergy Clin Immunol 1992;89(5):1040–5. 45. 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 1991;337(8743):690–4. 46. Hui KP, Barnes NC. Lung function improvement in asthma with a cysteinyl-leukotriene receptor antagonist. Lancet 1991;337(8749):1062–3. 47. Dempsey OJ, Kennedy G, Lipworth BJ. Comparative efficacy and anti-inflammatory profile of once-daily therapy with leukotriene antagonist or low-dose inhaled corticosteroid in patients with mild persistent asthma. J Allergy Clin Immunol 2002;109(1):68–74. 48. Diamant Z, Grootendorst DC, Veselic-Charvat M, et al. The effect of montelukast (MK-0476), a cysteinyl leukotriene receptor antagonist, on allergen-induced airway responses and sputum cell counts in asthma. Clin Exp Allergy 1999;29(1):42–51. 49. Wenzel SE. Inflammation, leukotrienes and the pathogenesis of the late asthmatic response. Clin Exp Allergy 1999;29(1):1–3. 50. Calhoun WJ, Lavins BJ, Minkwitz MC, et al. Effect of zafirlukast (Accolate) on cellular mediators of inflammation: bronchoalveolar lavage fluid findings after segmental antigen challenge. Am J Respir Crit Care Med 1998;157(5 Pt 1):1381–9. 51. Kane GC, Pollice M, Kim CJ, et al. A controlled trial of the effect of the 5-lipoxygenase inhibitor, zileuton, on lung inflammation produced by segmental antigen challenge in human beings. J Allergy Clin Immunol 1996;97(2):646–54. 52. Hasday JD, Meltzer SS, Moore WC, et al. Anti-inflammatory effects of zileuton in a subpopulation of allergic asthmatics. Am J Respir Crit Care Med 2000;161(4 Pt 1):1229–36. 53. Finnerty JP, Wood-Baker R, Thomson H, et al. Role of leukotrienes in exercise-induced asthma. Inhibitory effect of ICI 204219, a potent leukotriene D4 receptor antagonist. Am Rev Respir Dis 1992;145(4 Pt 1):746–9. 54. Leff JA, Busse WW, Pearlman D, et al. Montelukast, a leukotriene-receptor antagonist, for the treatment of mild asthma and exercise-induced bronchoconstriction. N Engl J Med 1998;339(3):147–52. 55. Meltzer SS, Hasday JD, Cohn J, et al. Inhibition of exercise-induced bronchospasm by zileuton: a 5-lipoxygenase inhibitor. Am J Respir Crit Care Med 1996;153(3):931–5. 56. Reiss TF, Hill JB, Harman E, et al. Increased urinary excretion of LTE4 after exercise and attenuation of exercise-induced bronchospasm by montelukast, a cysteinyl leukotriene receptor antagonist. Thorax 1997;52(12):1030–5. 57. 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 1998;133(3):424–8. 58. Pearlman DS, Ostrom NK, Bronsky EA, et al. The leukotriene D4-receptor antagonist zafirlukast attenuates exercise-induced bronchoconstriction in children. J Pediatr 1999;134(3):273–9. 59. Bisgaard H, Nielsen KG. Bronchoprotection with a leukotriene receptor antagonist in asthmatic preschool children. Am J Respir Crit Care Med 2000;162(1):187–90. 60. Edelman JM, Turpin JA, Bronsky EA, et al. Oral montelukast compared with inhaled salmeterol to prevent exercise-induced

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bronchoconstriction. A randomized, double-blind trial. Exercise Study Group. Ann Intern Med 2000;132(2):97–104. 61. Villaran C, O’Neill SJ, Helbling A, et al. Montelukast versus salmeterol in patients with asthma and exercise-induced bronchoconstriction. Montelukast/Salmeterol exercise Study Group. J Allergy Clin Immunol 1999;104(3 Pt 1):547–53. 62. Sladek K, Szczeklik A. Cysteinyl leukotrienes overproduction and mast cell activation in aspirin-provoked bronchospasm in asthma. Eur Respir J 1993;6(3):391–9. 63. Christie PE, Smith CM, Lee TH. The potent and selective sulfidopeptide leukotriene antagonist, SK&F 104353, inhibits aspirin-induced asthma. Am Rev Respir Dis 1991;144(4):957–8. 64. Fischer AR, Rosenberg MA, Lilly CM, et al. Direct evidence for a role of the mast cell in the nasal response to aspirin in aspirin-sensitive asthma. J Allergy Clin Immunol 1994;94(6 Pt 1):1046–56. 65. Israel E, Fischer AR, Rosenberg MA, et al. The pivotal role of 5-lipoxygenase products in the reaction of aspirin-sensitive asthmatics to aspirin. Am Rev Respir Dis 1993;148(6 Pt 1):1447–51. 66. Stevenson DD, Simon RA, Mathison DA, et al. Montelukast is only partially effective in inhibiting aspirin responses in aspirin-sensitive asthmatics. Ann Allergy Asthma Immunol 2000;85(6 Pt 1):477–82. 67. Kumlin M, Dahlen B, Bjorck T, et al. Urinary excretion of leukotriene E4 and 11-dehydro-thromboxane B2 in response to bronchial provocations with allergen, aspirin, leukotriene D4, and histamine in asthmatics. Am Rev Respir Dis 1992;146(1):96–103. 68. Dahlen B, Nizankowska E, Szczeklik A, et al. Benefits from adding the 5-lipoxygenase inhibitor zileuton to conventional therapy in aspirin-intolerant asthmatics. Am J Respir Crit Care Med 1998;157(4 Pt 1):1187–94. 69. Liu MC, Dube LM, Lancaster J. Acute and chronic effects of a 5-lipoxygenase inhibitor in asthma: a 6-month randomized multicenter trial. Zileuton Study Group. J Allergy Clin Immunol 1996;98(5 Pt 1):859–71. 70. 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 1999;130(6):487–95. 71. Nakamura Y, Hoshino M, Sim JJ, et al. Effect of the leukotriene receptor antagonist pranlukast on cellular infiltration in the bronchial mucosa of patients with asthma. Thorax 1998;53(10):835–41. 72. Overbeek SE, O’Sullivan S, Leman K, et al. Effect of montelukast compared with inhaled fluticasone on airway inflammation. Clin Exp Allergy 2004;34(9):1388–94. 73. Jayaram L, Duong M, Pizzichini MM, et al. Failure of montelukast to reduce sputum eosinophilia in high-dose corticosteroid-dependent asthma. Eur Respir J 2005;25(1):41–6. 74. Bisgaard H, Loland L, Oj JA. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am J Respir Crit Care Med 1999;160(4):1227–31. 75. Sandrini A, Ferreira IM, Gutierrez C, et al. Effect of montelukast on exhaled nitric oxide and nonvolatile markers of inflammation in mild asthma. Chest 2003;124(4):1334–40. 76. Straub DA, Minocchieri S, Moeller A, et al. The effect of montelukast on exhaled nitric oxide and lung function in asthmatic children 2 to 5 years old. Chest 2005;127(2):509–14. 77. Martin RJ, Cicutto LC, Ballard RD. Factors related to the nocturnal worsening of asthma. Am Rev Respir Dis 1990;141(1):33–8. 78. Wenzel SE, Trudeau JB, Kaminsky DA, et al. Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma. Am J Respir Crit Care Med 1995;152(3): 897–905. 79. Dockhorn RJ, Baumgartner RA, Leff JA, et al. Comparison of the effects of intravenous and oral montelukast on airway function: a double blind, placebo controlled, three period, crossover study in asthmatic patients. Thorax 2000;55(4):260–5. 80. 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 1992;146(2):358–63.

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81. 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 1993;119(11):1059–66. 82. Camargo CA Jr, Gurner DM, Smithline HA, et al. A randomized placebo-controlled study of intravenous montelukast for the treatment of acute asthma. J Allergy Clin Immunol 2010;125(2):374–80. 83. Camargo CA Jr, Smithline HA, Malice MP, et al. A randomized controlled trial of intravenous montelukast in acute asthma. Am J Respir Crit Care Med 2003;167(4):528–33. 84. Watts K, Chavasse RJ. Leukotriene receptor antagonists in addition to usual care for acute asthma in adults and children. Cochrane Database Syst Rev 2012;(5):CD006100. 85. Reiss TF, Chervinsky P, Dockhorn RJ, et al. Montelukast, a once-daily leukotriene receptor antagonist, in the treatment of chronic asthma: a multicenter, randomized, double-blind trial. Montelukast Clinical Research Study Group. Arch Intern Med 1998;158(11):1213–20. 86. 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 1994;150(3):618–23. 87. Suissa S, Dennis R, Ernst P, et al. Effectiveness of the leukotriene receptor antagonist zafirlukast for mild-to-moderate asthma. A randomized, double-blind, placebo-controlled trial. Ann Intern Med 1997;126(3):177–83. 88. Phipatanakul W, Greene C, Downes SJ, et al. Montelukast improves asthma control in asthmatic children maintained on inhaled corticosteroids. Ann Allergy Asthma Immunol 2003;91(1):49–54. 89. 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 2001;108(3):E48. 90. Straub DA, Moeller A, Minocchieri S, et al. The effect of montelukast on lung function and exhaled nitric oxide in infants with early childhood asthma. Eur Respir J 2005;25(2):289–94. 91. Bisgaard H, Zielen S, Garcia-Garcia ML, et al. Montelukast reduces asthma exacerbations in 2- to 5-year-old children with intermittent asthma. Am J Respir Crit Care Med 2005;171(4):315–22. 92. Bisgaard H, Flores-Nunez A, Goh A, et al. Study of montelukast for the treatment of respiratory symptoms of post-respiratory syncytial virus bronchiolitis in children. Am J Respir Crit Care Med 2008;178(8):854–60. 93. Bisgaard H, Study Group on M Respiratory Syncytial V. A randomized trial of montelukast in respiratory syncytial virus postbronchiolitis. Am J Respir Crit Care Med 2003;167(3):379–83. 94. Proesmans M, Sauer K, Govaere E, et al. Montelukast does not prevent reactive airway disease in young children hospitalized for RSV bronchiolitis. Acta Paediatr 2009;98(11):1830–4. 95. Kozer E, Lotem Z, Elgarushe M, et al. RCT of montelukast as prophylaxis for upper respiratory tract infections in children. Pediatrics 2012;129(2):e285–90. 96. Brodlie M, Gupta A, Rodriguez-Martinez CE, et al. Leukotriene receptor antagonists as maintenance and intermittent therapy for episodic viral wheeze in children. Cochrane Database Syst Rev 2015;(10):CD008202. 97. Zeidler MR, Kleerup EC, Goldin JG, et al. Montelukast improves regional air-trapping due to small airways obstruction in asthma. Eur Respir J 2006;27(2):307–15. 98. Kraft M, Cairns CB, Ellison MC, et al. Improvements in distal lung function correlate with asthma symptoms after treatment with oral montelukast. Chest 2006;130(6):1726–32. 99. Spahn JD, Covar RA, Jain N, et al. Effect of montelukast on peripheral airflow obstruction in children with asthma. Ann Allergy Asthma Immunol 2006;96(4):541–9. 100. Bleecker ER, Welch MJ, Weinstein SF, et al. Low-dose inhaled fluticasone propionate versus oral zafirlukast in the treatment of persistent asthma. J Allergy Clin Immunol 2000;105(6 Pt 1):1123–9. 101. Busse W, Raphael GD, Galant S, et al. Low-dose fluticasone propionate compared with montelukast for first- line treatment of persistent asthma: a randomized clinical trial. J Allergy Clin Immunol 2001;107(3):461–8.

102. Garcia Garcia ML, Wahn U, Gilles L, et al. Montelukast, compared with fluticasone, for control of asthma among 6- to 14-year-old patients with mild asthma: the MOSAIC study. Pediatrics 2005;116(2):360–9. 103. Ostrom NK, Decotiis BA, Lincourt WR, et al. Comparative efficacy and safety of low-dose fluticasone propionate and montelukast in children with persistent asthma. J Pediatr 2005;147(2):213–20. 104. Chauhan BF, Ducharme FM. Anti-leukotriene agents compared to inhaled corticosteroids in the management of recurrent and/or chronic asthma in adults and children. Cochrane Database Syst Rev 2012;(5):CD002314. 105. Chauhan BF, Jeyaraman MM, Singh Mann A, et al. Addition of anti-leukotriene agents to inhaled corticosteroids for adults and adolescents with persistent asthma. Cochrane Database Syst Rev 2017;(3):CD010347. 106. DuBuske LM, Grossman J, Dube LM, et al. Randomized trial of zileuton in patients with moderate asthma: effect of reduced dosing frequency and amounts on pulmonary function and asthma symptoms. Zileuton Study Group. Am J Manag Care 1997;3(4):633–40. 107. Szefler SJ, Phillips BR, Martinez FD, et al. Characterization of within-subject responses to fluticasone and montelukast in childhood asthma. J Allergy Clin Immunol 2005;115(2):233–42. 108. Zeiger RS, Szefler SJ, Phillips BR, et al. Response profiles to fluticasone and montelukast in mild-to-moderate persistent childhood asthma. J Allergy Clin Immunol 2006;117(1):45–52. 109. Rabinovitch N, Graber NJ, Chinchilli VM, et al. Urinary leukotriene E4/ exhaled nitric oxide ratio and montelukast response in childhood asthma. J Allergy Clin Immunol 2010;126(3):545–51.e1–4. 110. Price D, Musgrave SD, Shepstone L, et al. Leukotriene antagonists as first-line or add-on asthma-controller therapy. N Engl J Med 2011;364(18):1695–707. 111. Ducharme FM, Noya FJ, Allen-Ramey FC, et al. Clinical effectiveness of inhaled corticosteroids versus montelukast in children with asthma: prescription patterns and patient adherence as key factors. Curr Med Res Opin 2012;28(1):111–19. 112. Stempel DA, Meyer JW, Stanford RH, et al. One-year claims analysis comparing inhaled fluticasone propionate with zafirlukast for the treatment of asthma. J Allergy Clin Immunol 2001;107(1):94–8. 113. Schwartz HJ, Petty T, Dube LM, et al. A randomized controlled trial comparing zileuton with theophylline in moderate asthma. The zileuton Study Group. Arch Intern Med 1998;158(2):141–8. 114. Laviolette M, Malmstrom K, Lu S, et al. Montelukast added to inhaled beclomethasone in treatment of asthma. Montelukast/ Beclomethasone additivity group. Am J Respir Crit Care Med 1999;160(6):1862–8. 115. Virchow JC Jr, Prasse A, Naya I, et al. Zafirlukast improves asthma control in patients receiving high-dose inhaled corticosteroids. Am J Respir Crit Care Med 2000;162(2 Pt 1):578–85. 116. Price DB, Hernandez D, Magyar P, et al. Randomised controlled trial of montelukast plus inhaled budesonide versus double dose inhaled budesonide in adult patients with asthma. Thorax 2003;58(3):211–16. 117. Robinson DS, Campbell D, Barnes PJ. Addition of leukotriene antagonists to therapy in chronic persistent asthma: a randomised double-blind placebo-controlled trial. Lancet 2001;357(9273):2007–11. 118. Tamaoki J, Kondo M, Sakai N, et al. Leukotriene antagonist prevents exacerbation of asthma during reduction of high-dose inhaled corticosteroid. The Tokyo Joshi-Idai Asthma Research Group. Am J Respir Crit Care Med 1997;155(4):1235–40. 119. Lofdahl CG, Reiss TF, Leff JA, et al. Randomised, placebo controlled trial of effect of a leukotriene receptor antagonist, montelukast, on tapering inhaled corticosteroids in asthmatic patients. BMJ 1999;319(7202):87–90. 120. Bjermer L, Bisgaard H, Bousquet J, et al. Montelukast and fluticasone compared with salmeterol and fluticasone in protecting against asthma exacerbation in adults: one year, double blind, randomised, comparative trial. BMJ 2003;327(7420):891. 121. Busse W, Nelson H, Wolfe J, et al. Comparison of inhaled salmeterol and oral zafirlukast in patients with asthma. J Allergy Clin Immunol 1999;103(6):1075–80.

CHAPTER 97  Antileukotriene Therapy in Asthma 122. Fish JE, Israel E, Murray JJ, et al. Salmeterol powder provides significantly better benefit than montelukast in asthmatic patients receiving concomitant inhaled corticosteroid therapy. Chest 2001;120(2):423–30. 123. Ilowite J, Webb R, Friedman B, et al. Addition of montelukast or salmeterol to fluticasone for protection against asthma attacks: a randomized, double-blind, multicenter study. Ann Allergy Asthma Immunol 2004;92(6):641–8. 124. 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 2000;106(6):1088–95. 125. Ringdal N, Eliraz A, Pruzinec R, et al. The salmeterol/fluticasone combination is more effective than fluticasone plus oral montelukast in asthma. Respir Med 2003;97(3):234–41. 126. Ram FS, Cates CJ, Ducharme FM. Long-acting beta2-agonists versus anti-leukotrienes as add-on therapy to inhaled corticosteroids for chronic asthma. Cochrane Database Syst Rev 2005;(1):CD003137. 127. Lemanske RF Jr, Mauger DT, Sorkness CA, et al. Step-up therapy for children with uncontrolled asthma receiving inhaled corticosteroids. N Engl J Med 2010;362(11):975–85. 128. Altawalbeh SM, Thorpe CT, Zgibor JC, et al. Antileukotriene agents versus Long-Acting Beta-Agonists in older adults with persistent asthma: a comparison of add-on therapies. J Am Geriatr Soc 2016;64(8):1592–600. 129. Lipworth BJ, Dempsey OJ, Aziz I, et al. Effects of adding a leukotriene antagonist or a long-acting beta(2)-agonist in asthmatic patients with the glycine-16 beta(2)-adrenoceptor genotype. Am J Med 2000;109(2):114–21. 130. Wilson AM, Dempsey OJ, Sims EJ, et al. Evaluation of salmeterol or montelukast as second-line therapy for asthma not controlled with inhaled corticosteroids. Chest 2001;119(4):1021–6. 131. Busse WW, Casale TB, Dykewicz MS, et al. Efficacy of montelukast during the allergy season in patients with chronic asthma and seasonal aeroallergen sensitivity. Ann Allergy Asthma Immunol 2006;96(1):60–8. 132. Donnelly AL, Glass M, Minkwitz MC, et al. The leukotriene D4-receptor antagonist, ICI 204,219, relieves symptoms of acute seasonal allergic rhinitis. Am J Respir Crit Care Med 1995;151(6):1734–9. 133. Meltzer EO, Malmstrom K, Lu S, et al. Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: a randomized, placebo-controlled clinical trial. J Allergy Clin Immunol 2000;105(5):917–22. 134. van Adelsberg J, Philip G, Pedinoff AJ, et al. Montelukast improves symptoms of seasonal allergic rhinitis over a 4-week treatment period. Allergy 2003;58(12):1268–76. 135. Di Lorenzo G, Pacor ML, Mansueto P, et al. Randomized placebo-controlled trial comparing desloratadine and montelukast in monotherapy and desloratadine plus montelukast in combined therapy for chronic idiopathic urticaria. J Allergy Clin Immunol 2004;114(3):619–25. 136. Martin BG, Andrews CP, van Bavel JH, et al. Comparison of fluticasone propionate aqueous nasal spray and oral montelukast for the treatment of seasonal allergic rhinitis symptoms. Ann Allergy Asthma Immunol 2006;96(6):851–7. 137. Nathan RA, Yancey SW, Waitkus-Edwards K, et al. Fluticasone propionate nasal spray is superior to montelukast for allergic rhinitis while neither affects overall asthma control. Chest 2005;128(4):1910–20. 138. Pullerits T, Praks L, Ristioja V, et al. Comparison of a nasal glucocorticoid, antileukotriene, and a combination of antileukotriene and antihistamine in the treatment of seasonal allergic rhinitis. J Allergy Clin Immunol 2002;109(6):949–55. 139. Ratner PH, Howland WC 3rd, Arastu R, et al. Fluticasone propionate aqueous nasal spray provided significantly greater improvement in daytime and nighttime nasal symptoms of seasonal allergic rhinitis compared with montelukast. Ann Allergy Asthma Immunol 2003;90(5):536–42.

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140. Friedmann PS, Palmer R, Tan E, et al. A double-blind, placebo-controlled trial of montelukast in adult atopic eczema. Clin Exp Allergy 2007;37(10):1536–40. 141. Veien NK, Busch-Sorensen M, Stausbol-Gron B. Montelukast treatment of moderate to severe atopic dermatitis in adults: a randomized, double-blind, placebo-controlled trial. J Am Acad Dermatol 2005;53(1):147–9. 142. Drazen JM, Yandava CN, Dube L, et al. Pharmacogenetic association between ALOX5 promoter genotype and the response to anti-asthma treatment. Nat Genet 1999;22(2):168–70. 143. Telleria JJ, Blanco-Quiros A, Varillas D, et al. ALOX5 promoter genotype and response to montelukast in moderate persistent asthma. Respir Med 2008;102(6):857–61. 144. Klotsman M, York TP, Pillai SG, et al. Pharmacogenetics of the 5-lipoxygenase biosynthetic pathway and variable clinical response to montelukast. Pharmacogenet Genomics 2007;17(3):189–96. 145. Lima JJ, Zhang S, Grant A, et al. Influence of leukotriene pathway polymorphisms on response to montelukast in asthma. Am J Respir Crit Care Med 2006;173(4):379–85. 146. Tantisira KG, Lima J, Sylvia J, et al. 5-lipoxygenase pharmacogenetics in asthma: overlap with Cys-leukotriene receptor antagonist loci. Pharmacogenet Genomics 2009;19(3):244–7. 147. Mougey EB, Chen C, Tantisira KG, et al. Pharmacogenetics of asthma controller treatment. Pharmacogenomics J 2013;13(3):242–50. 148. Mougey EB, Feng H, Castro M, et al. Absorption of montelukast is transporter mediated: a common variant of OATP2B1 is associated with reduced plasma concentrations and poor response. Pharmacogenet Genomics 2009;19(2):129–38. 149. Dahlin A, Litonjua A, Irvin CG, et al. Genome-wide association study of leukotriene modifier response in asthma. Pharmacogenomics J 2016;16(2):151–7. 150. Dahlin A, Litonjua A, Lima JJ, et al. Genome-Wide Association study identifies novel pharmacogenomic loci for therapeutic response to montelukast in asthma. PLoS ONE 2015;10(6):e0129385. 151. Thompson MD, Capra V, Clunes MT, et al. Cysteinyl leukotrienes pathway genes, atopic asthma and drug response: from population isolates to large Genome-Wide Association studies. Front Pharmacol 2016;7:299. 152. US National Library of Medicine. Montelukast dosing. Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=ed2caffa -a75c-1c27-ae30-2d8280eaea7d. 153. US National Library of Medicine. Zafirlukast dosing. Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=4bf90772 -58d5-456d-ad1c-fc448cc5a231. 154. US National Library of Medicine. Zileuton dosing. Available from: https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=9bc08d7b -13da-444f-ac8a-3714a05176cc. 155. Watkins PB, Dube LM, Walton-Bowen K, et al. Clinical pattern of zileuton-associated liver injury: results of a 12-month study in patients with chronic asthma. Drug Saf 2007;30(9):805–15. 156. Hendeles L. Dose selection and dosing interval determination for LTRA use in asthma. Postgrad Med 2000;108(4 Suppl.):12–21. 157. Granneman GR, Braeckman RA, Locke CS, et al. Effect of zileuton on theophylline pharmacokinetics. Clin Pharmacokinet 1995;29(Suppl. 2):77–83. 158. Wechsler ME, Garpestad E, Flier SR, et al. Pulmonary infiltrates, eosinophilia, and cardiomyopathy following corticosteroid withdrawal in patients with asthma receiving zafirlukast. JAMA 1998;279(6):455–7. 159. Wechsler ME, Finn D, Gunawardena D, et al. Churg-Strauss syndrome in patients receiving montelukast as treatment for asthma. Chest 2000;117(3):708–13. 160. Wechsler ME, Pauwels R, Drazen JM. Leukotriene modifiers and Churg-Strauss syndrome: adverse effect or response to corticosteroid withdrawal? Drug Saf 1999;21(4):241–51. 161. Weller PF, Plaut M, Taggart V, et al. The relationship of asthma therapy and Churg-Strauss syndrome: NIH workshop summary report. J Allergy Clin Immunol 2001;108(2):175–83.

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162. Haarman MG, van Hunsel F, de Vries TW. Adverse drug reactions of montelukast in children and adults. Pharmacol Res Perspect 2017;5(5). 163. US Food and Drug Administration. Updated Information on Leukotriene Inhibitors: Montelukast (marketed as Singulair), Zafirlukast (marketed as Accolate), and Zileuton (marketed as Zyflo and Zyflo CR). Available from: https://wayback.archive-it.org/7993/20170404172438/ https://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafety InformationforPatientsandProviders/DrugSafetyInformationfor HeathcareProfessionals/ucm165489.htm. 164. Kuo CJ, Chen VC, Lee WC, et al. Asthma and suicide mortality in young people: a 12-year follow-up study. Am J Psychiatry 2010;167(9):1092–9. 165. Cereza G, Garcia Dolade N, Laporte JR. Nightmares induced by montelukast in children and adults. Eur Respir J 2012;40(6):1574–5.

166. Law SWY, Wong AYS, Anand S, et al. Neuropsychiatric events associated with Leukotriene-Modifying agents: a systematic review. Drug Saf 2018;41(3):253–65. 167. Global Initiative for Asthma. Global Strategy for Asthma Management and Prevention (GINA); 2018. Available from: http://ginasthma.org/. 168. National Heart, Lung, and Blood Institute. Guidelines for the diagnosis and management of asthma (EPR-3); 2007. Available from: https://www.nhlbi.nih.gov/files/docs/guidelines/asthgdln.pdf. 169. Lazarinis N, Bood J, Gomez C, et al. Leukotriene E4 induces airflow obstruction and mast cell activation via the CysLT1 receptor. J Allergy Clin Immunol 2018;142(4):1080–9.

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1598.e1

SELF-ASSESSMENT QUESTIONS 1. The leukotriene receptors are a member of which family? a. G protein–coupled receptor family b. Hematopoietin receptor family c. Interferon receptor family d. TNF receptor family e. IL-1 receptor family 2. Which protein is essential for the formation of LTA4? a. Cyclooxygenase b. LTA4 hydrolase c. LTC4 synthase d. Lipid peroxidase e. 5-lipoxygenase activating protein 3. Which is an effect of cysteinyl leukotrienes on group 2 innate lymphoid cells? a. Augments interferon gamma production b. Decreases amphiregulin c. Increases eotaxin production d. Induces IL-4 production e. Causes apoptosis

4. Which pharmacologic agent inhibits leukotriene synthesis? a. Indomethacin b. Montelukast c. Pranlukast d. Zafirlukast e. Zileuton 5. In asthma clinical trials, which strategy most consistently improves outcomes in persons whose asthma is not well controlled on lowdose inhaled corticosteroids? a. Doubling the dose of the inhaled corticosteroid b. Addition of a long-acting β-agonist c. Addition of a leukotriene synthesis inhibitor d. Addition of a leukotriene receptor antagonist e. Addition of theophylline

98  Unconventional Theories and Unproven Methods in Allergy Gailen D. Marshall, Jr., Stephen B. LeBlanc CONTENTS Introduction, 1599 Unconventional Theories of Allergic Disease, 1600 Allergic Toxemia or Tension Fatigue Syndrome, 1600 Multiple Food and Chemical Sensitivities: Idiopathic Environmental Intolerance, 1600 Candida Hypersensitivity Syndrome, 1602

SUMMARY OF IMPORTANT CONCEPTS • The optimal care for patients with allergy-based diseases is evolving to a precision based methodology. • Not all commonly used allergy-related techniques and therapies are highly evidence-based and demonstrate varying degrees of efficacy and safety. • Medical theories that are often controversial may lead to numerous treatments and procedures that lack strong evidence-based rationale. • Allergy practitioners should be knowledgeable about both conventional and unconventional methods to provide optimal management of patients with allergic disease.

INTRODUCTION The traditional roles of standard medical practices are increasingly blurred by the growing availability of large volumes of information via social media. Concurrently, the emergence of alternative practitioners and therapies that do not adhere to the classic tenets of allergy care continues to escalate. These alternative approaches may or may not incorporate more classical allergy therapy and may or may not have any sound scientific or clinical rationale for their development or use. This chapter provides an overview of diagnostic tests, treatment methods and therapies that lack the traditional evidence basis for conventional allergy care. The provider who treats patients with allergic disease should be knowledgeable about both conventional and alternative techniques and theories. This will allow a dialog with the patient to be established whereby the provider makes best evidence-based recommendations while remaining empathetic, sensitive, and nonjudgmental to patient preferences. By establishing such a rapport with patients, the provider can more easily provide information and recommendations and, based upon a sound understanding of the theory and/or rationale for some of these diagnostic and therapeutic practices, gain trust to counsel the patient about the questionable benefits of and risks associated with unconventional methods. Unproven methods of diagnosis and treatment include those subjected to clinical trials with negative results and those not evaluated. It should be acknowledged that the quality and validity of some of these negative trials can be legitimately questioned in terms of investigator bias, sample size and composition, and even study design. Those techniques that

Food Additive Sensitivity, 1603 Unproven Diagnostic Tests, 1603 Inappropriate Diagnostic Tests, 1605 Unproven Treatment Methods, 1605 Therapies Based on Controlled Environmental Exposures, 1607 Current Versus Historical Practices, 1608 have not been evaluated can range from a traditional basis for their utility to a rationale that is largely based upon commercial potential of the test or product. They are often labeled controversial methods when they lack reliable scientific rationale and/or credibility and significant clinical efficacy has not been demonstrated. Experimental methods are those that show greater promise of clinical utility based on currently accepted scientific rationale or empirical observations by experts (a lower but defined level of evidence) but lack sufficient evidence to be recommended for general use. It should be acknowledged that some currently used therapies were at one time controversial but became more accepted as scientific understanding improved and/or clinical data were collected. Standard of care encompasses various methods of diagnosis and treatment that are widely used by most reliable providers in a particular specialty who are knowledgeable about the disease. Trained and experienced allergists typically use methods that are scientifically based and have documented effectiveness and safety. They select procedures for each patient based on personal knowledge of that patient’s history and examination. Within the range of acceptable methods, often more than one validated option is available. Factors such as patient preference, lifestyle, and payer restrictions may be applicable in deciding among several options. Within the totality of unproven tests, techniques, and therapies are those with a different basis for utility. Often these are considered to be more “natural” or “traditional,” because they may have emerged from cultural medical practices (such as traditional Chinese medicine, Ayurvedic medicine, Native American medicine, etc.) and, although they may have been used for hundreds or thousands of years, still lack the stringent evidence basis currently demanded to prove efficacy (or in some cases even their safety). These practices often are referred to collectively as “alternative” or “complementary.”1 Complementary and alternative medicine (CAM) modalities are currently used globally for treatment of some allergy-based diseases but generally lack rigorous research basis for their use. Traditional CAM techniques are more thoroughly described and discussed in Chapter 99. Generally speaking, many of the modalities, both diagnostic and therapeutic, described are of primarily historical interest, because it has become more difficult to find practitioners who use them. The primary intent of this chapter is to provide background with relevant citations on how allergy has been practiced both conventionally and unconventionally and, for those controversial techniques still being used, an

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overview for understanding so that the practitioner can discuss options with patients in an open, respectful environment that will optimize the likelihood of ultimately doing what is best for the patient.

UNCONVENTIONAL THEORIES OF ALLERGIC DISEASE Allergy is a term that has vastly different meanings to different individuals. To practitioners who are extensively trained in the principles and practices of this medical specialty, the term defines a spectrum of clinical maladies initiated and propagated by the inappropriate production of antigen (allergen)-specific IgE with subsequent downstream activation of cells, particularly mast cells and eosinophils, in the immune cascade. The clinical consequences of allergic illnesses are characteristic of the immune cascades involved. However, to much of the public and even non–allergy trained practitioners, the term allergy connotes myriad clinical symptoms that occur as a result of exposure to any of a number of environmental substances, most of which are incapable of eliciting the formation of IgE by the afflicted host. As a result of this misunderstanding of the immune pathophysiology, many unconventional allergy practices have emerged over the years. Descriptions of these conditions are strikingly similar. Patients typically report a long-standing pattern of multiple symptoms suggesting the involvement of many different organ systems. They also often have difficulties with various psychologic processes including memory, cognitive function, personality changes, and attentiveness. Physical examinations are usually normal or, if other comorbidities exist, do not show any consistent abnormalities among patients with similar symptoms. Conventional tests of allergic sensitivities are usually unrevealing and, accordingly, claimed to be unreliable by advocates of the unconventional theory. Unproven diagnostic methods are used to establish the existence of these diseases, although the patient’s belief that the symptoms are caused by environmental exposures is often sufficient for diagnosis. Extensive laboratory testing fails to reveal any consistent organ dysfunction, and there is no consistent physical evidence of pathology to account for the symptomatic complaints. The perceived allergens include foods, food additives, drugs, environmental chemicals, hormones, microorganisms, and even electromagnetic radiation. The reported sensitivities usually are multiple. A succession of theories for the etiology and pathogenesis of these conditions based on alternative forms of hypersensitivity all lack experimental proof that the presumed sensitivity to environmental substances is immunologically mediated. Anecdotal case reports and clinical experience are often cited as evidence for the existence of the specific illness.

ALLERGIC TOXEMIA OR TENSION FATIGUE SYNDROME Allergic toxemia is the prototype for a certain category of environmental sensitivity-related illnesses. This entity is difficult to define, because descriptions vary widely, even though it is claimed to be associated with a specific symptom complex.2 The most commonly reported clinical manifestations are various combinations of recurrent headache, abdominal pain, fatigue, musculoskeletal pain, various respiratory complaints, and pallor. Fatigue is the most prominent and common symptom. The term allergic tension fatigue syndrome has been applied to the same condition, especially when diagnosed in children.3 Less frequent complaints are lymph node enlargement, low-grade fever, urinary frequency, bladder discomfort, excessive coryzal episodes, tachycardia, urticaria, difficulty concentrating, and lower eyelid pallor. The condition is almost always attributed to sensitivity to multiple foods, especially milk, chocolate, corn, and wheat. It is claimed that reactions to foods occur more commonly in the winter than in summer. Diagnosis is based

solely on symptoms as there are no pathognomonic tests available. Findings on physical examination are usually unrevealing even though the reported clinical manifestations that could be verified objectively such as lymphadenopathy, fever, and tachycardia are rarely confirmed by examination. Routine laboratory testing shows no abnormalities.3 The onset of symptoms and signs after ingestion of foods often is delayed by hours, days, or even weeks. Improvement from an elimination diet is typically inconsistent, intermittent, and temporary. Accordingly, additional foods are usually eliminated from the diet, making it even more restrictive. Lack of response to elimination diets have been attributed to poor cooperation by the patient, trace quantities of “offending” foods in the diet, or concurrent infections. No definitive double-blind, placebo-controlled clinical trials supporting the existence of food-induced allergic toxemia have been reported. The symptoms of patients diagnosed with allergic toxemia are common in the general healthy population. In the absence of objective physical or pathologic signs, this illness does not suggest an allergic mechanism. The pattern of symptoms in these patients is variable, and it has yet to be shown that they are consistently reproduced by the ingestion of specific foods in any sort of controlled study. Patients with active allergic rhinitis and asthma or other chronic diseases also may experience fatigue, difficulty in concentration, and other nonspecific symptoms, but the concept that these occur as the direct result of defined allergic mechanisms is highly suspect. No evidence has emerged showing that elimination diets can consistently control symptoms in patients diagnosed with allergic toxemia.

MULTIPLE FOOD AND CHEMICAL SENSITIVITIES: IDIOPATHIC ENVIRONMENTAL INTOLERANCE The concept that certain persons suffer a specific illness caused by multiple food and chemical sensitivities is the basis of a medical tradition known as clinical ecology,2,4 which largely evolved from the theory of food-induced allergic toxemia. The illness was originally termed environmental illness and has also has been called environmentally induced disease,5 chemical hypersensitivity syndrome,6 multiple chemical sensitivities (MCS),7 cerebral allergy, chemically induced immune dysregulation, 20th century disease,8 total allergy syndrome, ecologic illness, food and chemical sensitivities, and toxic chemical encephalopathy. All of these terms refer to the same clinical malady. For the purpose of this discussion, all of these will be combined into the term idiopathic environmental intolerance (IEI). The syndrome of IEI was first described as MCS in patients who attributed their illness to workplace exposures.9,10 It was defined as an acquired disorder characterized by recurrent symptoms, referable to multiple organ systems, occurring in response to demonstrable exposure to many chemically unrelated compounds at doses far below those established in the general population to cause toxic effects. No validated laboratory or clinical testing has been shown to correlate with symptoms. A workshop of the International Programme on Chemical Safety of the World Health Organization11 recommended the term idiopathic environmental intolerances, because the term multiple chemical sensitivities “makes an unsupported judgment on causation” (i.e., environmental chemicals), does not refer to a clinically defined disease, and is not based on “accepted theories of underlying mechanisms [or] validated clinical criteria for diagnosis.” Furthermore, the “relationship between exposures and symptoms is unproven.” IEI is described by clinical ecologists as a polysymptomatic condition, which suggests pathologic changes in numerous areas and systems of the body. Nevertheless, no abnormal physical findings or laboratory test results, gross or microscopic evidence of inflammation, or other objective signs of a pathophysiologic disorder can be found with

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy appropriate investigations. The most common complaints in IEI—fatigue, headache, nausea, malaise, pain, mucosal irritation, disorientation, and dizziness—vary tremendously among affected patients and are commonly reported in the population at large in the absence of physical illness. Some defined physical and psychiatric disorders also have been attributed by clinical ecologists to multiple food and chemical sensitivities. However, significant psychologic and even psychiatric profiles are consistently found in these patients. The debate is cause and effect (i.e., does the IEI cause the psychologic dysfunction or is the psychologic dysfunction primarily responsible for the somatic complaints?).12 The list of items purported to cause this condition is broad. Patients often complain of difficulty in tolerating exposure to certain buildings, often making this an occupational health issue. Despite the emphasis on synthetic chemicals, causes of illness also have included natural gas, electromagnetic radiation, viruses, fungi, yeast, and wood dust. In certain cases, the presumed causative chemical is an endogenous hormone, especially progesterone in women.13 Patients almost always attribute their illness to a combination of foods, environmental chemicals, and pharmaceuticals. The presumed causes are believed to act both in the induction of disease pathophysiology (even though it is not defined) and in provoking recurrent symptoms once disease occurs. No consistent dose-response relationship in the provocation of symptoms is evident. The duration of exposure to environmental agents required to induce the disease has ranged from seconds to years, with no correlation of presumed dose and exposure duration with the severity of illness.14 The diagnostic procedure most commonly used by clinical ecologists is provocation-neutralization (see description later). After diagnostic testing, many of these patients develop an extended set of causes corresponding to the items used in the test that were interpreted as having positive results. Randolph and Moss4 first proposed a theory that failure of the human body to adapt to industrial synthetic chemicals resulted in the development of a state of hypersensitivity to these agents. The mechanism of the hypersensitivity was unexplained, and no supporting empirical evidence for the theory has been offered. Various immunologic mechanisms have been proposed to explain IEI pathophysiology. They are based on the theory that environmental chemicals function as haptens to induce formation of immunoglobulin G (IgG) antibodies, IgE antibodies, or sensitized T cells.15 The typical clinical illness described by patients with IEI is not clinically consistent with immune-based hypersensitivity reactions (I-IV). Additionally, even accounting for the more limited laboratory technology available when most studies were conducted, evidence of increased specific antibodies or of cellular immune responses to any of the environmental agents identified by these patients as a cause of their illness is lacking. An autoimmune theory was proposed from an uncorroborated report of circulating immune complexes or autoantibodies in a single series of patients with IEI.5 Immune response dysregulation as a basis for the ill-defined hypersensitivity state of the affected patient also has been suggested, but evidence for abnormal immune function or immune deficiency in IEI has not been reported in any controlled study. Additional concepts unique to clinical ecology are used to explain the lack of a consistent dose-response relationship between the perceived chemical exposure and the symptoms, and they are inconsistent with modern concepts of immunology. Total body load and chemical overload assume that the immune system has a fixed capacity to handle a limited quantity of environmental antigen and that exceeding this capacity provokes symptoms.15 Masked food sensitivity is used to explain an adverse reaction to a food when it is eaten after several days of abstention, whereas frequent ingestion of the same food causes symptoms to disappear (apparently a form of tolerance induction). These concepts sometimes are referred to collectively as “adaptation-de-adaptation,”

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but they have never been explained physiologically or even documented in controlled settings. Spreading phenomenon is a putative mechanism, whereby exposure to one substance induces or predisposes to develop immune responses to other, unrelated ones. It is used to explain periods of increasing symptoms in the absence of environmental changes. Nonimmunologic theories for IEI include: (1) deficiency of anti­ oxidants induced by the generation of free radicals by environmental chemicals, drugs, and foods6; (2) neurotoxicity caused by endogenous β-endorphin “sensitization” induced by environmental stimuli,16 odorinduced kindling of olfactory-limbic neural pathways, or immunologicto-neurogenic inflammation “switching” mediated by neuropeptides; (3) end-organ dysesthesia involving the nasal mucosa, the lung, the entire respiratory tree, or multiple organs17; and (4) hereditary coproporphyria.18 None of these theories have been supported by experimental evidence. Finally, some practitioners support a primary psychologic/ psychiatric etiology for the IEI. This approach is discussed later. Diagnosis of IEI most often rests on an environmental history and results of provocation-neutralization testing. The patient’s self-report of symptoms after exposure to odors or fumes, or after eating certain foods, usually suffices. The testing protocols are rarely blinded or controlled with placebo. Some practitioners may supplement provocationneutralization testing with other laboratory tests, including total serum immunoglobulins and complement components; detection of circulating autoantibodies and immune complexes; lymphocyte subset analysis; assays to detect a variety of environmental chemicals in blood; urine, fat, and/or hair samples; and quantitation of circulating hormones, amino acids, and mediators. Data from both the clinical ecology literature and independent reviews of series of patients with IEI do not support the diagnostic usefulness of any of these tests.2,3,19 Practitioners who accept the validity of the IEI concept typically use various combinations of three treatment modalities: avoidance of environmental chemical exposure, special elimination diets, and neutralization therapy. Drugs are usually regarded as harmful synthetic chemicals that themselves can cause environmental illness yet patients are often instructed to take vitamin and mineral supplements. Some are treated with certain salts, such as sodium bicarbonate to neutralize allergic reactions and with amino acids, intravenous γ-globulin, or supplemental oxygen to relieve symptoms. The espoused rationale for the treatment program is to eliminate or reduce symptoms and to strengthen the immune system. Complete avoidance of multiple environmental chemicals is consistently recommended,2 which often requires a significant change in lifestyle. Many patients remodel their homes in an attempt to make them safe from exposure to toxic chemicals. Alternatively, they may relocate to isolated, more rural communities. They often wear filter masks in public and even within their own homes. Elimination diets typically include avoidance of all food additives and a “rotary diversified diet” in which foods are rotated on a 4- to 5-day cycle. No prospective, controlled clinical trials have been performed to evaluate the effectiveness of the combination of food and chemical avoidance with neutralization therapy. An unblinded evaluation of 21 patients with a variety of symptoms treated by food and chemical avoidance and with neutralizing injections for food sensitivity showed improvement in both patients and control subjects.20 A retrospective review of 50 cases showed that after an average of 2 years of treatment, the condition had been ameliorated in only 4% of the patients, whereas it was unchanged in 44% and had worsened in 52%.21 Published results of several critical evaluations of patients claiming to have IEI do not support a role for food and chemical sensitivities as a cause of the reported symptoms, nor do they indicate immunologic dysfunction. Psychologic evaluations of patients with IEI confirm the impression that their perceived reactions to environmental chemicals and foods are often manifestations of preexisting psychologic

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dysfunction. By including normal control subjects in their study, Black and colleagues22 showed that the number of reported symptoms and the prevalence of mood disorder, anxiety disorder, and somatoform illness were all significantly higher in patients with IEI than in a population of normal people. Unfortunately, most patients with IEI are reluctant to accept any sort of psychiatric diagnosis or treatment, opting instead for an extreme avoidant lifestyle, to which they may adhere for years. Brodsky23 has studied the social consequences of extreme environmental chemical avoidance that leads to social isolation centered on the illness. The importance of preexisting psychiatric illness is made particularly clear by the study of Simon and associates,24 who evaluated 37 aircraft workers exposed to phenol-formaldehyde causing transient mucosal irritation that resolved on cessation of exposure. Thirteen of these workers had chronic disabling symptoms provoked by common environmental agents and were diagnosed by a clinical ecologist as having IEI. The prevalence of preexisting psychiatric morbidity caused by anxiety, depression, somatization, and medically unexplained symptoms was significantly higher in these patients than in the 24 patients who had transient symptoms only. Two studies have examined the clinical and laboratory evidence for immunologic illness in patients with multiple food and chemical sensitivities. In 50 cases studied by Terr,21 levels of circulating immunoglobulins, complement components, and lymphocyte subsets were mostly within the normal expected range and did not correlate with severity of reported illness or length of reported exposure to the foods or chemicals thought to cause the illness. None of the patients had clinical evidence of immunodeficiency or autoimmune disease, and the prevalence of allergic disease was similar to that found in the general population. Thus the diagnosis of multiple food and/or chemical sensitivities manifested by numerous symptoms in the absence of objective physical findings lacks a demonstrable scientific foundation. There is no available evidence that these patients suffer from allergic or other immunologic abnormalities. The rationale for the illness is based on anecdotal reports with no verification using properly controlled, blinded challenges. Diagnosis comes from the subjective history alone or in combination with results from provocation-neutralization testing, which has been shown to be unreliable. The treatment and theories underlying this symptom complex are neither well defined nor well understood. Yet, for anyone who has provided care for these patients, there is little doubt that the majority do have some illness that often has significant morbidity and adversely impacts their functional status and quality of life. Medical and psychologic evaluations suggest that most of these patients have significant psychologic/psychiatric dysfunction as a major component of their illness. Whether this is the cause of their symptoms or results from the severity/duration of their symptoms is often not clear. Regardless, practitioners have an obligation to be respectful, compassionate, and empathetic of these patients’ compromised clinical state. Even if a particular practitioner cannot provide help for a given patient, good faith attempts to direct the patient to someone who may be able to help is part of the Hippocratic process for all medical professionals. A dismissive or condescending attitude has little place in the care of these or any other patients.

CANDIDA HYPERSENSITIVITY SYNDROME Candida hypersensitivity syndrome (also called candidiasis hypersensitivity syndrome, chronic systemic candidiasis, and yeast hypersensitivity syndrome) has been proposed as an illness related to hypersensitivity to a toxin released from Candida albicans and results from excessive amounts of Candida spp. found in the body of otherwise immunocompetent hosts.25 The illness is described in terms of numerous wideranging symptoms without specific physical findings or laboratory

abnormalities other than assessments for the presence of Candida in stool, vaginal secretions, oral swabs, and even blood samples (not viable organisms from blood in immunocompetent individuals). Many patients are provided a diagnosis that combines food, chemical, and Candida sensitivity. Popular books written for lay persons have promoted selfdiagnosis. C. albicans is detectable as normal flora on the skin and mucosa of respiratory, gastrointestinal, and female genitourinary tracts. Because it is a commensal organism for humans, chronic exposure to Candida antigens is virtually universal, and low levels of antibody- and cell-mediated immunity are detectable in the majority of immunologically normal, healthy persons. Opportunistic candidiasis is found in conditions involving impaired innate or acquired immunity. Thrush or localized infection of the buccal mucosa is not unusual in infants. In children and adults, it may result from antibiotic or topical inhaled corticosteroid therapy. Thrush or Candida vulvovaginitis can be a complication of diabetes mellitus, pregnancy, or progesterone therapy and may occur transiently during antibiotic therapy. Systemic candidiasis is seen most commonly in states of cell-mediated and/or neutrophil dysfunction/deficiency.26 Chronic mucocutaneous candidiasis also accompanies certain states of impaired cell-mediated immunity. These pathologic forms of candidiasis must not be confused with the unproven condition of Candida hypersensitivity. Proponents of the Candida hypersensitivity syndrome typically invoke the diagnosis in patients with numerous subjective symptoms, but they also claim that it is a cause of, or a potentiating factor for, numerous other diseases, including multiple sclerosis, arthritis, psoriasis, schizophrenia, cancer, acquired immunodeficiency syndrome (AIDS), depression, and various behavioral and emotional problems. The syndrome lacks a specific definition, and there are no diagnostic physical or laboratory test abnormalities or evidence of C. albicans overgrowth, either locally or systemically in affected patients. Predisposing factors are said to include current or past use of antibiotics, corticosteroids, or birth control pills and diets of yeast (not Candida)-containing foods, sugars, and other carbohydrates such as baked goods. No evidence has demonstrated that affected patients differ from healthy individuals in their exposure to any of these factors. Theoretical explanations include both hypersensitivity and toxicity. A presumed C. albicans toxin is claimed to have a suppressive effect on the immune system, although clinical or experimental data indicating immunotoxicity in these patients are lacking. The diagnosis is based entirely on history, often established by questionnaire. No published reports have indicated that antibody levels of any immunoglobulin isotype specific to Candida antigens are higher in patients with this diagnosis than in control subjects. Recommended treatment consists of diet, nutritional supplements, vitamins, minerals, and certain medications (particularly systemic antifungal therapies). The diet restricts intake of sugar, dietary yeast, and foods believed to contain mold. A rotary diet often is advised. Antifungal drugs are recommended, usually in exceedingly low oral doses. To date, no clinical trials have been conducted to evaluate the effectiveness of diet or nutritional supplements in these patients. In a randomized, double-blind trial of nystatin for patients classified as suffering from Candida hypersensitivity syndrome, oral or vaginal nystatin, or a combination of the two, was no different from placebo in altering symptoms.27 No data are available on the natural course of the condition. Candida hypersensitivity syndrome has not been definitively confirmed as a distinct clinical entity, and a sound theoretical basis for the syndrome has not been identified. The utility of the diagnostic questionnaire and of serum antibody levels to C. albicans antigens is not established. The single reported therapeutic trial of nystatin indicates that any benefit is no different from that obtained with placebo. The other recommended therapeutic measures are untested.

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy

FOOD ADDITIVE SENSITIVITY Attention deficit–hyperactivity disorder (ADHD) in children and some adults, previously called hyperactivity, hyperkinesis, and minimal brain dysfunction, is considered to be a psychiatric condition characterized by excessive activity, inattention that is inappropriate to the stage of development, increased level of impulsivity, difficulty in discipline, and poor school/work performance. It is not an uncommon problem of childhood and is increasingly recognized in adolescents and adults as well. The natural history is unpredictable, with some children losing all symptoms during puberty and others continuing to exhibit some or all manifestations into adulthood. The cause is unknown, but the disorder is thought to encompass some combination of constitutional, genetic, environmental, and psychosocial factors. Feingold first suggested in 1973 that ADHD can be caused by sensitivity to aspirin and other salicylates.28 Accordingly, elimination of dietary salicylates as treatment for this and other psychiatric conditions was the recommendation, and initial reports claimed improvement in up to 50% of children with ADHD. Because other investigators had reported that asthma attacks in patients with aspirin-sensitive asthma could be triggered by ingestion of the yellow food coloring dye tartrazine,29 it was recommended that all food dyes and other additives, regardless of their chemical structure, toxicity, or pharmacologic properties, be eliminated. Thus the “Feingold diet” evolved as a salicylate-free, additive-free diet. Most plants contain some salicylate, making it unlikely that any plant food is truly salicylate-free. Small amounts of salicylates are found in certain animal products and even in drinking water. In fact, a true salicylate-free diet would be limited to plain meat, egg, and distilled water only.30 In practice, adherents of the Feingold diet focus on eliminating food additives, particularly dyes and preservatives, so the role of natural salicylates in foods—the issue on which the diet was based—eventually lost importance to the proponents. Scientific evidence for direct effects on the central nervous system caused by toxicity, idiosyncrasy, or allergic hypersensitivity to food additives is lacking. Acute asthma attacks precipitated by the ingestion of aspirin and other nonsteroidal antiinflammatory drugs are discussed in Chapter 78. Several published controlled clinical trials have used different protocols that have produced conflicting results. A consensus conference by the Office of Medical Applications for Research of the National Institutes of Health concluded that only a few children might benefit from improvement in behavior from a defined diet.31 Thus the hypothesis that naturally occurring food salicylates and artificial food additives cause ADHD is unproved. An additive-free diet cannot be recommended as definitive therapy for children with this condition. No evidence is available at this time to confirm that any food additive affects behavior in children through an immunologic or allergic mechanism.

UNPROVEN DIAGNOSTIC TESTS The unproven diagnostic procedures discussed in this chapter fall into two primary categories: (1) those that have no documented diagnostic value for allergy or other diseases; and (2) those that are intrinsically capable of a valid measurement that could be effective and appropriate for diagnosing certain diseases but not for allergy. Also described are other scientifically valid tests and procedures that are capable of giving diagnostic information relevant to an allergic disease but are not commonly used in current clinical practice because of lack of validation of research tools for individual patient use, inadequate or untested sensitivity, specificity, and/or cost considerations. Generally, caution should be exercised in obtaining and/or interpreting results whenever a test is only offered at one location or by one vendor.

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The methods described in this section are based on outdated, questionable, or even disproven scientific rationale. Typically, these procedures are not in accord with current knowledge of allergy pathophysiology and have been largely superseded by evidence-based test methodology.

Serial End-Point Titration Since the pioneering work of Noon and Freeman, allergy skin testing has occupied a central position in the allergist’s diagnostic toolkit. The techniques used today have been refined but still resemble the original methodology of Noon and Freeman. The currently accepted prick and intradermal skin testing methods for diagnosis of IgE antibody–mediated diseases are discussed in Chapter 67. Serial end-point titration refers to a method of testing and treatment of inhalant allergy introduced by Rinkel32 for aeroallergens and later adopted for use with food allergens. The rationale uses the procedure for initial diagnosis and also to establish a “safe” dose for initiating immunotherapy and for the “neutralization” of symptoms. The method described by Rinkel32 and its subsequent modifications are complicated and based primarily on empirical observations. Increasing fivefold serial concentrations of allergen are injected intradermally, usually in 0.01-mL doses. The test site is observed for the presence of a wheal after 10 minutes without regard for the presence or absence of any sort of flare.33 Observed results are reported as end points, defined as a clinical pattern associated with the test dose that initiates serial 2-mm or greater incremental increases in wheal diameter with increasing fivefold concentrations. Because every allergen is tested with up to nine serial intradermal injections, the total number of injections is high and the cost of the procedure is considerable. In addition to establishing the initial dose of allergen immunotherapy (AIT) that can be used safely, the optimal (or maintenance) dose—defined as the dose at which clinical symptoms are eliminated—is set at 25 to 50 times the quantity of allergen producing the end point. This paradigm can vary from allergen to allergen.33 Serial end-point titration evolved into the concept of symptom neutralization at a particular injected dose of allergen. This was based upon observations that patients reported less symptoms at a specific dose of allergen presumably predicted by end-point titration results. Several controlled clinical trials provided evidence that serial intradermal end-point titration of pollen allergens produces increasing wheal diameter with increasing doses of the allergen, and that the end point—as defined by Rinkel and others (who used both wheal and flare data)—is a safe method for establishing the starting dose for immunotherapy. The method is extremely conservative, because it usually underestimates a safe starting dose, particularly in patients who demonstrate high skin sensitivity to the allergen. Accordingly, this can prolong the length of time required to reach the maintenance level for immunotherapy shown by numerous controlled studies to be effective for ameliorating allergic rhinitis or asthma. These studies further show that a calculated optimal maintenance AIT dose based on the end point is usually too low with little more effectiveness than placebo.34 The technique of skin testing by intradermal serial end-point titration using the method of Rinkel and his followers is highly questionable as a reliable test for establishing IgE-mediated sensitivity because the test responses are read too soon and the presence or absence of concomitant erythema is not considered—both of which decrease the sensitivity of the test. In addition, the method requires an unnecessarily large number of intradermal injections, which both are uncomfortable to the patient and have greater potential for significant systemic side effects. The use of a starting dose based upon the less sensitive end point for initiating AIT may unnecessarily prolong the course of treatment.

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Provocation-Neutralization

Antigen Leukocyte Cellular Antibody Test

Provocation-neutralization is a procedure that was intended to test for allergy to foods, inhalants, and environmental chemicals by exposing the patient to test doses of these substances administered intradermally, subcutaneously, or sublingually, with the aim of either producing (provocation) or preventing (neutralization) subjective allergy symptoms. It evolved from serial end-point titration skin testing, initially to diagnose food allergy in patients with multiple subjective symptoms and later using inhalant32 and chemical allergens. The method is based on the concept that extremely small quantities of allergen can make symptoms disappear (neutralization). The publications on this procedure frequently discuss testing and treatment as a single entity. Intracutaneous provocation testing is performed similarly to skin end-point titration. Increasing serial fivefold dilutions of allergen or chemical extracts was administered into the arm while the patient recorded subjective sensations in an unblinded manner. Neither the nature nor the intensity of the symptoms constituting a positive test result is standardized, nor were the testing measures necessarily the same as those reported by the patient for the illness being tested. Once a test result was considered positive, a progressive series of decreasing allergen concentrations is administered until a dose is reached at which the patient reported no new clinical sensations. This dose was considered the neutralizing dose, which was then used for subsequent treatment. Provocation-neutralization testing has been used primarily by practitioners who subscribe to the concept of multiple food and chemical sensitivities as a cause of patient symptoms. Because the procedure requires provoking and neutralizing symptoms one allergen at a time, and the theory was based on illness caused by exposure to multiple foods and environmental substances, the procedure often involved many individual tests that often required weeks or months of full-day testing sessions—time intensive for the patient and typically extremely expensive. Published reports on provocation-neutralization testing (not treatment) reached various conclusions, depending on methodology. A variety of illnesses were investigated using different end-point measurements, reflecting the lack of standardization for the procedure and the numerous diseases for which it is recommended. Rigorous evaluation using double-blind, placebo-controlled clinical trials showed provocation or neutralization of symptoms using this technique and allergen were not different from those seen with placebo.35 Provocation-neutralization testing should not be confused with recognized forms of target organ provocative testing discussed elsewhere in this book. Bronchial provocation testing with allergens (Chapter 61); provocative nasal inhalation testing (Chapter 61); the oral, doubleblind, placebo-controlled food challenge test (Chapter 81); and patch testing for contact dermatitis (Chapter 34) all are validated, wellstandardized techniques with defined, objective physical measurements or observations.

The antigen leukocyte cellular antibody test (ALCAT) is a modification of the cytotoxic test that uses electronic instrumentation and computerized data analysis to examine and monitor changes in leukocyte cell volumes.38 Like the cytotoxic test, it also has been promoted as a diagnostic test for food, inhalant, and chemical allergy or intolerance in a host of conditions, including arthritis, urticaria, bronchitis, gastroenteritis, childhood hyperreactivity, rhinitis, and atopic dermatitis. Typically, the results are used to establish elimination diets for these diseases. There are multiple vendors for this test, many which can be found with a simple internet search. Examination of language on these sites reveals a careful avoidance of any specific health claims or even claims of accuracy (covered by the “individual results may vary” phrase). Common utility of the results involves such measures as dietary elimination, avoidance, and “rotation” diets in which different dietary regimens based upon test results are “rotated” over time to eliminate symptoms. The reports claiming utility have not been from well-controlled, blinded clinical trials.38

Cytotoxic Test The cytotoxic test (also known as the leukocytotoxic test) for food allergy was first described in 1956 and later modified.36 The test was based on belief that morphologic changes in peripheral blood leukocytes exposed to an allergen in vitro indicated sensitization to that particular allergen. Allergic sensitization diagnosed by this method encompassed rhinitis, asthma, headache, gastrointestinal symptoms, skin disease, hearing disorders, genitourinary diseases, and obesity. Largely anecdotal reports of symptomatic improvement from dietary changes based on the cytotoxic test have been reported.37 All of the reports were open label, meaning the patient knew what they were (and were not) being fed.

Electrodermal Testing Electrodermal testing (electroacupuncture) uses a technique that claimed to identify substances, especially foods, that cause allergy and to provide information about optimal dilution of treatment extracts in immunotherapy. It uses a device, often referred to as a Voll machine (named after its inventor Reinhold Voll, a German physician and acupuncturist),39 that measures the electrical impedance of the skin at designated acupuncture points in response to a 1.5-V electric current while the patient holds the negative electrode in one hand. Vials of food or inhalant extracts are placed in contact with an aluminum plate in the circuit. A change in impedance supposedly detects allergy to that particular food. The positive electrode is used to probe selected points on the skin. The lower extremities are said to relate to food allergy, the trunk and upper extremities to inhalant allergies, and the scalp to allergies that localize in the nose and sinuses. Proponents of this procedure claim that it has been used successfully for many years in Europe to diagnose and treat allergy. They admit that the theory for the procedure is unknown and that results are empirical. The equipment is expensive, and the manufacturer declares that its use in the United States is for investigational purposes only. A double-blind, randomized block design study of atopic patients and controls tested repeatedly showed that the procedure was unable to confirm the presence or absence of skin test reactivity, and the test could not distinguish atopic from nonatopic individuals.40

Applied Kinesiology Applied kinesiology claims to identify specific allergic sensitizations by measuring the patient’s muscle strength with manual muscle testing in the presence of a suspected allergen. Practitioners assert that pathologic conditions in individual organ systems are reflected in corresponding muscle groups (called viscerosomatic relationships) maintained through energy fields communicating throughout the body. In allergic patients, particularly those with suspected food allergy, this communication presumably can be influenced by the allergen when present in direct proximity to the patient. Typically, allergens are placed in closed containers that the patient holds in one hand while a technician subjectively estimates muscle strength in the opposite arm. A decrease in muscle power is said to indicate a positive test result. Variations in the procedure include placing the allergen container on the chest while the patient is supine with the arms outstretched, or even testing while the allergen is placed near but not in actual contact with the body. For uncooperative infants, a

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy surrogate is tested, first alone and then while holding the child’s hand, with the results based on subtraction of the two results of subjectively measuring the muscle strength of the surrogate. Proponents of applied kinesiology claim efficacy and reliability for this procedure without any support from properly controlled studies. A single published report of blinded testing to multiple foods in 20 subjects did not show consistency or reproducibility in the results.41

INAPPROPRIATE DIAGNOSTIC TESTS The methods discussed in this section are not recommended for allergy diagnosis because they are ineffective when used for that purpose, although they may be valid and effective for other suspected clinical conditions. Some of these methods promoted for allergy diagnosis are based on an incorrect concept of allergic disease pathogenesis; others were derived from unconfirmed observations of patients.

Specific Immunoglobulin G Antibodies Some clinical laboratories offer quantitative measurements of circulating IgG antibodies to food and mold allergens. Antibodies are measured by solid-phase immunoassay methods, discussed in Chapter 72. Lowlevel IgG antibodies to foods are commonly found in plasma, but they are of no known pathogenic significance in atopic disease. Although some practitioners have postulated that these antibodies may be responsible for delayed symptoms or vague intolerance to foods, data that support this hypothesis are still lacking.42 Allergen immunotherapy can induce IgG4 allergen-specific “blocking” antibodies. These can be measured in standardized assays. Increased allergen-specific (not total) titers occur with successful AIT and are associated with sustained clinical benefit.43 High titer, antigen-specific IgG antibodies can be involved in the pathogenesis of serum sickness and possibly in certain phases of hypersensitivity pneumonitis. Measurement of specific IgG antibodies to the relevant antigen may be diagnostically helpful in these particular diseases, especially to confirm that exposure to a suspect antigen (such as an infectious agent) has occurred.

Chemical Analysis of Body Fluids The diagnosis of “chemical sensitivities” by those who subscribe to the concept of IEI, discussed earlier in this chapter, may involve tests for the presence of environmental chemicals in samples of whole blood, serum, erythrocytes, urine, fat, and hair from afflicted individuals. These individuals may present to an allergist’s office because of concern that, when exposed to some combination of these substances, their symptoms ensue. IEI (also referred to as chemical intolerance or multiple chemical sensitivities) is a subjective illness marked by multiple nonspecific symptoms related to exposure to various chemicals, biologics, and physical agents.10,44 The most common chemicals measured are organic solvents, other hydrocarbons, pesticides, and metals. In similar fashion, some providers have recommended quantitative measurements of vitamins, minerals, and amino acids in blood and urine in a search for environmental chemical sensitivities. Modern analytic techniques and instrumentation such as mass spectroscopy are available today to detect and quantify exceedingly small amounts of most chemical substances from samples of biologic material. These sensitive methods have revealed that many environmental chemicals are detectable in normal as well as ill individuals because of their ubiquitous presence in the environment of today’s industrialized world. Under certain circumstances it may be proper and appropriate to consider suspected chemical toxicity by the detection of potentially toxic quantities of a specific substance. However, widespread screening of large numbers of chemicals has not yielded data that lend any significant credibility to the concept of chemical sensitivities.

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Food Immune Complex Assay Food intolerance causing delayed symptoms (i.e., more than 2 hours after ingestion) is a vexing problem, especially because patients presumably so affected often describe vague and subjective symptoms not typical for known mast cell–mediated allergic manifestations. Given that pathology caused by circulating immune complexes typically takes longer to develop than IgE-mediated causes, studies were done to show the presence of circulating immune complexes to various food proteins and that these complexes might be associated with clinical symptoms in afflicted individuals. Accordingly, food immune complex assays (FICA) were developed and evaluated for their diagnostic value in atypical food allergy presentations.45 The procedure involved a radioimmunoassay in which an antibody to a food was coupled covalently to the solid phase immunosorbent and then incubated with the test serum to permit the insolubilized antibody to react with the food allergen in circulating immune complexes. Radiolabeled antiimmunoglobulin (usually anti– human IgE or anti–human IgG) was used to quantitate the amount of circulating immune complexes containing the food allergen.45 Several published studies suggested that circulating food proteins, food antibodies, and food immune complexes46 are common findings in normal individuals. Other studies showed evidence of circulating immune complexes of several different antibody isotypes in the serum of both normal and food atopic persons.47 Patients with food-induced symptoms were not demonstrated to differ from normal control subjects in the quantity or quality of these complexes. Finally, food immune complex assays based on the solid-phase radioimmunoassay methodology were never fully characterized for sensitivity and specificity to validate their value in diagnostic testing.48 Clinical studies to date indicate that food–antibody immune complexes can be found in normal individuals with no reported symptoms of food intolerance. No well-defined clinical disease has yet been identified as being causally linked to food immune complexes.

UNPROVEN TREATMENT METHODS This section describes a group of procedures advocated for treating patients with allergic diseases, some of which are directed specifically toward allergic conditions and others promoted for many chronic conditions, including allergy. All are without established efficacy in patients with allergic conditions, although many have been widely used. Their demonstrated benefits were no different from placebo, typically involving an improved sense of well-being unrelated to a pharmacologic action.

Neutralization Therapy Neutralization therapy, also called “symptom-relieving” therapy and “tolerance,” is a therapeutic extension of provocation-neutralization testing. Specified doses of allergen extracts are self-administered to relieve symptoms. After provocation-neutralization testing, doses of one or more tested substances identified as “neutralizing” by the methodology described previously are self-administered by the intracutaneous,49 subcutaneous,20 or sublingual route.50 No consistent protocols have been reported. Patients use the neutralizing solution whenever symptoms appear, or before anticipated exposure to the substance believed to induce the symptoms. Treatment also can be given on a regular maintenance schedule, usually daily or twice weekly. The published literature on neutralization therapy indicates that it has been used in a variety of conditions, including atopy, rheumatologic, infectious, and many others. Neutralizing substances have included atopic allergens, environmental chemicals, hormones, viral vaccines, food extracts, histamine or serotonin, and even saline or distilled water. The literature can be confusing, because published reports do not usually

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separate provocation-neutralization testing from neutralization treatment. Support rests largely on a single reported double-blind, placebocontrolled, crossover study of daily subcutaneous injections of food extracts in eight patients.20 Improvement occurred with both placebo and food extract, but more frequently with the latter. The study, labeled by the author as preliminary, has been criticized for inappropriate statistical analysis, and no final report has ever been published. A report that 20 patients with perennial rhinitis treated with sublingual dust extract improved both subjectively and objectively is questionable, because five of the patients did not have house dust mite allergen sensitivity.51 Accordingly, neutralization therapy is an unproven procedure of unknown value. Its advocates recommend its use for such a wide variety of illnesses and subjective conditions. To achieve its stated clinical goals, typical prescriptions use a large number of different substances administered by three different routes. To date, no sound clinical studies have been published to support its use in any condition.

increasing doses to produce a flush. Water and salts of sodium, potassium, calcium, magnesium, and other minerals are given to correct for water and salt depletion from sweating. Polyunsaturated oils, such as a mixture of soybean, walnut, peanut, and safflower oils, which are said to be “essential,” are consumed orally. A planned schedule of balanced meals and adequate sleep is recommended, along with avoidance of medications and alcohol. This routine is repeated daily, usually for 30 days. All reports to date by proponents of this detoxification procedure are anecdotal. Results are measured by the patient’s self-report of symptomatic improvement. Some of the therapeutic components (diet, elimination of unnecessary medications, aerobic exercise, and adequate sleep) may be the primary reasons for subjective improvement. No controlled studies of this form of detoxification have yet to be reported for the treatment of allergy, IEI, drug addiction, or any other illness. The ability of the procedure to remove chemicals from fat has not been confirmed.

Enzyme-Potentiated Desensitization

Autogenous Urine Therapy

Enzyme-potentiated desensitization (EPD) is a procedure in which allergen is mixed with the enzyme β-glucuronidase immediately before injection. It is promoted as an improvement over conventional allergen immunotherapy, because it requires fewer injections and uses a lower concentration of allergen. This method was developed by McEwen and colleagues in the 1960s and was studied for several decades.52 A very low dose of allergen, approximately equivalent to the amount administered in a standard skin-prick test, was mixed with partially purified β-glucuronidase in a dose equivalent to the amount of enzyme present in 4 mL of human blood. Immediately after mixing, 0.125 mL of the mixture was injected intradermally. A single preseasonal dose was considered sufficient to produce a therapeutic effect lasting for an entire pollen season,53 and a dose every 2 to 6 months for perennial allergy. Proponents have claimed success in treating allergic rhinitis, asthma, sinusitis, nasal polyposis, eczema, urticaria, migraine headaches, ulcerative colitis, irritable bowel syndrome, rheumatoid arthritis, and petit mal seizures. The effectiveness of this form of treatment and the presumed effect of β-glucuronidase on the immune system was never established. In a double-blind study in which a single injection was given preseasonally to 44 patients allergic to grass pollen, significant improvement over a placebo effect was evident when measured by overall patient preference and reduced requirement for drug therapy, but daily symptom records showed no effect.53 Several reports of double-blind studies in children with mite-allergic asthma and pollen-allergic rhinitis showed clinical improvement,54 but a similar trial of grass pollen–allergic rhinitis in England showed no treatment effect.55 EPD was based on a notion that the enzyme suppresses the immune response to ambient allergen exposure. The minute amount of enzyme used (far less than what is normally found in situ) is unlikely to have significant pharmacologic effects. It is extremely unlikely that any form of lasting desensitization would be effective with the extremely limited treatment protocol.

A belief that human urine is therapeutic has existed since ancient times. In the early 1930s, several medical publications claimed that a specific substance called proteose, one of various water-soluble compounds produced during hydrolysis of proteins, is excreted in allergic disease.57 Injections of extracts of this substance were recommended for treatment of allergy, with reports of successes in many other diseases as well. Other reports could not reproduce these findings. The practice subsided after several years but then resurfaced.58 A search of the internet provides information on several practitioners and centers (mostly naturopathic) that practice what has now been termed autogenic urine immunotherapy (AUIT). The published reports consist mostly of uncontrolled anecdotal histories of apparently successful treatment of a variety of allergic conditions, including asthma, rhinitis, anaphylaxis, urticaria, angioedema, and serum sickness. Other reports have failed to prove efficacy. No well-controlled studies have been published to date.

Detoxification Chemical detoxification is recommended by a small group of practitioners to remove undesirable chemicals from patients believed to have IEI. The putative basis for this modality is that the clinical sensitivity is caused by toxic effects of low-levels of synthetic chemicals in the body.56 Treatment consists of several steps. Aerobic physical exercise is performed for 20 to 30 minutes, followed immediately by forced sweating in a sauna at 140° to 180° F for 2.5 to 5 hours and then physical exercise, a cooling shower, and additional exercise. Niacin is then given in

Acupuncture Acupuncture is an ancient Oriental form of medical practice that is recommended for virtually any disease. Over the centuries, its popularity has waxed and waned. Today, some medical and nonmedical practitioners use acupuncture exclusively or in connection with other forms of treatment, including medications, Chinese herbal therapy, homeopathy, naturopathy, and psychotherapy. In moxibustion, heated ground mugwort plant is burned into the patient’s skin with acupuncture needles.59 Modern versions of acupuncture include the use of laser beams, electrical stimulation, and injections of medications or supplements at acupuncture sites.60 Acupuncture is commonly used to treat allergic diseases (see Chapter 99 for more details). Some proponents claim that needling of the skin activates or inhibits various cell mediators and enzymes, although no significant support for this hypothesis has been published in the peer reviewed literature. Randomized controlled clinical trials and metaanalyses on acupuncture treatment for various allergic diseases have been performed. Some trials use sham acupoints as placebo, but others use no treatment or “standard therapy.” A few regimens have included adjunctive treatment when necessary. A wide variety of measures have been used to determine effectiveness, including symptom reports, pulmonary function testing, laboratory data, and quality of life questionnaires. A significant challenge to properly evaluating efficacy is the apparent lack of consistency in selection of skin sites, depth of penetration, and other factors in the placement of needles. Reported results include improvement in some measures to no improvement at all compared with controls. Metaanalyses reflect these uncertainties, diverse

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy methodology, limited quality of data reporting, and evidence of patient selection bias.61 In most cases, the potential value of acupuncture for treatment of allergic disease would be complementary to rather than as an alternative to conventional pharmacotherapy.

Homeopathy Homeopathy is both a philosophy and an alternative medical practice. The theory that “like cures like” is translated into the administration of exceedingly minute quantities of substances believed to cause disease as a method of curing the disease. The substances are called remedies and usually consist of extensive dilutions of various plant and/or animal organ extracts. The exceedingly small quantities used for treatment are prepared by serial dilution of extracts through the violent shaking of a container of diluted extract. The remedies are given orally, and patients sometimes take as many as 50 different extracts daily. An alternative theory proposes that disease is cured by the induction of protective immunity. It is questionable, on theoretical grounds, whether exceedingly minute quantities of ingested material could have any therapeutic effect.62 The materials used for treatment are unstandardized. Adverse effects are unlikely because of the small amount of material administered. The real dangers are psychologic dependence and delay in the institution of effective therapy. In common with other practitioners of “alternative” disease treatment, homeopathic practitioners claim to cure numerous diseases, including all forms of allergy. Several published controlled clinical trials have compared homeopathic treatment with placebo or standard therapy for allergic rhinitis and asthma. Both allergen extracts in vanishingly low concentrations and standard homeopathic remedies are used. These trials report both positive63 and negative64 results. Metaanalyses conclude that homeopathic treatment of respiratory allergy cannot be recommended.65

Laser Therapy Laser treatment for asthma delivers energy to specific targets, including endobronchial tissue, tympanic membrane, blood, and skin. This treatment is a distinct entity from bronchial thermoplasty, an approved procedure for treatment of severe asthma using radiofrequency ablation of smooth muscle in bronchial airways.66 The laser therapy modalities are variously named laser ablation, photodynamic therapy, helium-neon laser irradiation, noninvasive hemolaserotherapy, low-intensity or lowenergy laser radiation, pulsed- and continuous-wave low-energy infrared laser radiations, and laseropuncture (laser acupuncture). The theoretical rationale is based on the notion that when a patient’s tissue (variable depending upon the specific methodology used) is exposed to a light energy source, a stimulatory effect is induced. This effect is based on the direct exposure to tissue, exposure at acupuncture points, or indirect exposure of blood, which then induces a specific change in pulmonary function, including bronchial hyperreactivity, immune status, and symptoms.67 Cochrane database reviews reflect the absence of common endpoint measurements and limited reporting quality, with little insight into the long-term efficacy and safety profiles of these treatment modalities. Assessment of validity of laser therapy will require additional welldesigned trials.68

Vitamin and Nutrient Supplements Nutritional supplementation has a long and complex history in Western society. Their proposed utility are either to promote or maintain health or treat specific diseases, even though true nutritional deficiencies are relatively uncommon in individuals with an adequate dietary food intake. Vitamins, minerals, amino acids, enzymes, and other nutritional supplements have been recommended by some practitioners for numerous medical and psychiatric conditions, including allergy. These may be prescribed singly or in combination, often in very high doses, commonly

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known as orthomolecular or mega therapy. Some allergy practitioners who recommend vitamin therapy have the notion that a deficiency of these substances causes or worsens various allergic diseases. Antioxidant supplements, such as vitamin C, vitamin E, and glutathione, are typically recommended according to a theory that allergic inflammation generates free radicals, which cause oxidative damage to tissues.69 This theory has been widely criticized based primarily on the evidence that the amount of these vitamins necessary to reverse or prevent oxidative damage could not be practically achieved in a clinical setting. The data exploring the clinical efficacy of vitamin supplementation have been variable in terms of efficacy in specific populations with specific supplement preparations.70,71 Most of these reports are from studies with small sample size or having other limitations such as selection bias, study design lacking randomization or control population; all concerns that may limit any overall conclusions from being formed. Of note, however, excessive intake of fat-soluble vitamins can result in their accumulation in the body and subsequent toxicity. Supplementation with specific vitamins, minerals, or other nutrients is standard treatment for the corresponding deficiency diseases. However, the data available that attempt to link nutritional deficiencies (including vitamins) in the pathophysiology of allergy and asthma risk are not strong. Additionally, evidence for clinical efficacy in allergy of ingesting supraphysiologic doses of vitamins (mega therapy) is lacking. A possible exception is the more recent epidemiologic evidence that suggests a causal association between vitamin D deficiency and morbidity from asthma and other allergic diseases.72 As with other vitamin supplements, the data using vitamin D supplementation are not yet definitive. This is likely related not only to differences in protocols but also differences in patient study populations—race, age, gender, asthma phenotype, and other variables. Even with this confusion, there appears to be a signal that warrants further investigation with carefully designed and controlled clinical trials.

THERAPIES BASED ON CONTROLLED ENVIRONMENTAL EXPOSURES Avoiding allergens whenever possible is a cornerstone of allergy therapy. Patients who have experienced anaphylaxis on exposure to a specific drug or food or Hymenoptera venom must be particularly vigilant in avoiding contact with the inciting substance unless or until such time that sensitization can be altered therapeutically. Controlling exposure to indoor house dust mite, indoor molds, and animal pet danders is the standard recommendation for patients with these specific allergies. Allergen avoidance is thus part of an overall management program, with the goal to reduce or eliminate allergic disease without undue disruption of the patient’s daily activities. In sharp contrast with these well-recognized and well-established principles of environmental control–based allergy management, practitioners who subscribe to concepts such as those of IEI may recommend extreme measures to reduce or eliminate environmental chemical exposures. The nature of these exposures are vast and often ubiquitous and include many plastic materials, synthetic clothing, various fragrances, detergents, pesticides, synthetic carpeting, gasoline or diesel fumes, vehicle exhaust emissions, smoke, natural gas fumes, chlorine, alcohol, and virtually any synthetic chemical. Patients adherent to this form of treatment typically undergo profound lifestyle changes including alterations in family life (unable to live in their houses/apartments) and inability to continue employment. These environmental restrictions are often accompanied by extreme dietary restrictions. The physical and emotional challenges of such extraordinary but unproven avoidance measures are apparent. To date, no controlled studies have addressed the need for or efficacy of such treatment approaches.73

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Salt-Based Treatments Speleotherapy (Greek speleos, “cave”) is a treatment regimen based in underground environments said to be of clinical benefit for patients with cutaneous, asthma, and other respiratory disorders. Salt cave spas arose in northern Mediterranean and eastern European countries and later near the Dead Sea because of the high content of mineral salts in certain geologic formations and/or seawater. Treatment is carried out in specifically designated caves or mines, sometimes with patients performing physical or breathing exercises. Benefits are variously attributed to the underground climate (temperature, relative humidity, absence of air pollutants, high aerosol salt content) or radiation, which differ among the caves and mines. No evidence from randomized controlled trials has been found for these conditions.74 Halotherapy (Greek halos, “salt”) is designed to replicate the conditions of speleotherapy in an indoor clinic. Most trials are reported in Russian language journals, examining the impact of the treatment on asthma, chronic bronchitis, and other upper and lower respiratory tract diseases. Halotherapy also is recommended as adjuvant therapy to accompany conventional or “alternative” treatments.75

Dietary Therapy Although active research is ongoing to develop tolerogenic food antigen exposure therapies for patients with documented IgE-mediated food allergies, these have not yet been fully established in terms of safety and efficacy.76 Accordingly, elimination of the offending food currently remains the standard of care. Fortunately, in most documented cases, the number of foods to be avoided is small, so that highly restrictive diets are unnecessary. In contrast, recommendations to eliminate multiple foods, resulting in excessive dietary restriction and potential malnutrition, has been a major component of certain unconventional allergy treatment programs. In general, two theories for multiple food eliminations are recognized: (1) the concept of multiple food allergy, based on incorrect testing (such as IgG-based RAST) and/or lack of understanding about specificity limitations of food based allergy skin tests; and (2) the concept that many foods can inhibit the proper function of the immune system (a central tenet of clinical ecology).2 Treatment based on provocation-neutralization testing required the elimination of all foods and environmental chemicals that provoke symptoms. Because such a diet was typically deficient in supplying proper nutritional content, a “rotary diversified diet” was recommended,2 in which the foods believed to cause sensitivity are permitted in the diet once every 4 or 5 days. This diet also is claimed to prevent the development of new food sensitivities. No studies showing efficacy of these dietary strategies have been published. Historically, multiple theories have claimed that certain foods, food additives, or food contaminants can alter, disturb, or inhibit the normal functioning of the immune system. Highly restrictive diets, often accompanied by supplemental vitamins, minerals, amino acids, or other nutrients, have been recommended to boost the immune system. Diets and supplements, including Chinese herbs which have been used for centuries, also have been recommended as treatment for atopic diseases.77 These theories and recommendations are not originally based on current scientific evidence, some being thousands of years old.76 Recent controlled trials have heralded efforts to objectively examine Chinese herbal remedies for various allergic manifestations, and some hold promising potential.78–80

Alternative Routes of Immunotherapy Administration Since the initial days of Noon and Freeman’s first successful immunizations for aeroallergens, researchers have been searching for safer, more efficacious, convenient, and cost-effective strategies to deliver allergen

immunotherapy. Although the most common route remains subcutaneous, other routes have been explored, such as intralymphatic and epicutaneous.81 Although these routes may be more invasive, they also offer the benefit of inducing tolerance much more rapidly, in as few as three injections via the intralymphatic route.

Vitamin D and Asthma Although the role and importance of vitamin D in bone metabolism has been known for many years, more recently deficiency of this fat-soluble vitamin has also been linked to many atopic conditions, most notably asthma. Although results from observational and longitudinal studies exploring the link between vitamin D deficiency and asthma in both children and adults have often been contradictory, one large study by Wu et al involving 1024 children in the Childhood Asthma Management Program (CAMP) demonstrated those with vitamin D deficiency had less improvement in FEV1 to inhaled corticosteroids over the course of a year.82 However, in two recent trials (VIDA and ViDiA), supplementation of vitamin D in deficient individuals did not improve asthma control.83,84 Further studies are needed to better elucidate the role of vitamin D deficiency in both pathogenesis and potential treatment target in patients with asthma.

CURRENT VERSUS HISTORICAL PRACTICES A number of the unproven and controversial methods described and referenced in this chapter are currently still being espoused by certain practitioners, whereas other described techniques now are of mostly historical interest. A quick search of the internet indicates there are many advertisements, from patient blogs to commercial products, that still espouse and market some of the ideas and products described in this chapter, suggesting that some of these may be revived eventually in a similar or altered form. In some cases, notably that of sublingual allergen immunotherapy (see Chapter 86), methods previously considered unproven are undergoing intensive controlled trials that may or may not establish their usefulness in allergy practice. Numerous studies and abundant anecdotal information attest to the interest in and use of unconventional diagnostic and therapeutic methodologies and theories worldwide. Some modalities are especially prevalent in Europe; Traditional Chinese medicine, Ayurveda, native American medicine, and others have gained a significant following in Western culture. Other alternative clinical fields including naturopathy, chiropractic, reflexology, and others target allergic diseases in their therapeutic programs. The evidence for these approaches is highly variable. Some is discussed in Chapter 99 on Complementary and Alternative Medicine. The current status of these methods and theories in the United States is summarized in Box 98.1.

Perspective Scientifically unproven and medically illogical methods of diagnosis and treatment that are controversial and unorthodox in the view of most practicing physicians are not unique to the field of clinical allergy. The overview of such methods and concepts presented in this chapter provides a background against which relevant issues likely to be encountered in clinical practice can be identified. Allergists and immunologists must be familiar with these methods to counsel patients appropriately. It is important to note that patients may present for care to clinical allergists-immunologists and espouse some of these concepts because of their own research (often internet-based), listening to other patients through online blogs, list serves, etc. or having been seen by any of a number of different types of alternative practitioners. Typically, these patients do have some sort of illness. The fact that such illness is unlikely to be due to any of these described mechanisms does not lessen their suffering or the allergy specialist’s responsibility to behave toward them

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy

BOX 98.1  Current Status of Unorthodox

and Unproven Diagnostic and Therapeutic Practices in Allergy in the United States Procedures Currently Being Actively Practiced Diagnosis Skin end-point titration Provocation-neutralization Antigen leukocyte cellular antibody test Electrodermal testing Applied kinesiology Various serum assays for immunoglobulins, cytokines, receptors, others Blood lymphocyte counts and functions Food immune complex assay Treatment Acupuncture Homeopathic “remedies” Herbal medicine Chiropractic manipulation Diets and dietary supplements Neutralization (subcutaneous, intradermal) therapy and variations

Newer Variations of Established but Unproven Practices Treatment Laser acupuncture (with or without medication or allergen insertion at acupoints) Electrical transcutaneous nerve stimulation (TNS) Procedures Currently of Minor Interest Treatment Enzyme-potentiated desensitization Detoxification Procedures of Largely Historical Interest Diagnosis Cytotoxic test Treatment Autogenous urine injection Procedures Undergoing Current Controlled Clinical Trials Treatment Sublingual immunotherapy

in a respectful, compassionate, nonjudgmental fashion. In some cases, sound information or even testing can be provided that does accurately define the illness and its etiology, allowing more effective therapy to be prescribed. Even if such therapy is not within the boundaries of what clinical allergists practice (i.e., psychologic assessment and therapy), compassion rather than condescension or contempt is deserved by the patient who seeks our care.

REFERENCES Introduction 1. Silvers WS, Bailey HK. Integrative approach to allergy and asthma using complementary and alternative medicine. Ann Allergy Asthma Immunol 2014;112:280–5.

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Unconventional Theories of Allergic Disease 2. Bell IR. Clinical ecology: a new medical approach to environmental illness. Bolinas, CA: Common Knowledge Press; 1982. 3. Speer F. The allergic tension fatigue syndrome. Pediatr Clin North Am 1954;1:1029–37. 4. Randolph TG, Moss RW. An alternative approach to allergies. New York: Lippincott & Crowell; 1980. 5. Rea WJ. Environmentally triggered cardiac disease. Ann Allergy 1978;40:243–51. 6. Arnold E, Clark CE, Lasserson TJ, et al. Herbal interventions for chronic asthma in adults and children. Cochrane Database Syst Rev 2008;(1):CD005989. 7. Cullen MR. The worker with multiple chemical hypersensitivities: an overview. State Art Rev Occup Med 1987;2:655–61. 8. Stewart DE, Raskin J. Psychiatric assessment of patients with “20th-century disease” (“total allergy syndrome”). Can Med Assoc J 1985;133:1001–6. 9. Spencer TR, Schur PM. The challenge of multiple chemical sensitivity. J Environ Health 2008;70:24–7. 10. Dantoft TM, Andersson L, Nordin S, et al. Chemical intolerance. Curr Rheumatol Rev 2015;11:167–84. 11. International Program on Chemical Safety. Conclusions and recommendations of a workshop on multiple chemical sensitivities, Berlin, Germany, February 21-23. Reg Toxicol Pharmacol 1996;24: 5188–9. 12. Weiss EM, Singewald E, Baldus C, et al. Differences in psychological and somatic symptom cluster score profiles between subjects with Idiopathic environmental intolerance, major depression and schizophrenia. Psychiatry Res 2017;249:187–94. 13. Mabray CR, Burditt ML, Martin TL, et al. Treatment of common gynecologic-endocrinologic symptoms by allergy management procedures. Obstet Gynecol 1982;59:560. 14. Terr AI. Clinical ecology in the workplace. J Occup Med 1989;31:257–61. 15. Ashford NA, Miller CS. Clinical exposures. New York: Van Nostrand Reinhold; 1991. 16. Bell IR, Bootzin RR, Davis TP, et al. Time-dependent sensitization of plasma beta-endorphin in community elderly with self-reported environmental chemical odor intolerance. Biol Psychiatry 1996;40:134–43. 17. Cohn JR. Multiple chemical sensitivity or multi-organ dysesthesias? [editorial]. J Allergy Clin Immunol 1994;93:953–4. 18. Hahn M, Bonkovsky HL. Multiple chemical sensitivity syndrome and porphyria: a note of caution and concern. Arch Intern Med 1997;157:281–6. 19. Terr AI. ‘Multiple chemical sensitivities’: immunologic critique of clinical ecology theories and practice. State Art Rev Occup Med 1987;3:683–94. 20. Miller JB. A double-blind study of food extract injection therapy: a preliminary report. Ann Allergy 1977;38:185–91. 21. Terr AI. Environmental illness: a clinical review of 50 cases. Arch Intern Med 1986;146:145–9. 22. Black DW, Rathe A, Goldstein RB. Environmental illness: a controlled clinical trial of 26 subjects with ‘20th century disease.’. JAMA 1990;264:3166–70. 23. Brodsky CM. ‘Allergic to everything’: a medical subculture. Psychosomatics 1983;24:731. 24. Simon GE, Katon WJ, Sparks PJ. Allergic to life: psychological factors in environmental illness. Am J Psychiatry 1990;147:901–6. 25. Truss CO. The role of Candida albicans in human illness. J Orthomol Psychiatry 1981;10:228. 26. Singh S, Fatima Z, Hameed S. Predisposing factors endorsing Candida infections. Infez Med 2015;23:211–23. 27. Dismukes WE, Wade JS, Lee JY, et al. A randomized, double-blind trial of nystatin therapy for the candidiasis hypersensitivity syndrome. N Engl J Med 1990;323:1717–23. 28. Feingold BF. Why your child is hyperactive. New York: Random House; 1975. 29. Chafee FH, Settipane GA. Asthma caused by FD&C approved dyes. J Allergy 1967;40:65–72.

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30. South MA. The so-called “salicylate-free diet”. Cutis 1976;18:183. 31. National Institutes of Health Consensus Development Panel. Defined diets in childhood hyperactivity. Bethesda, MD: Office of Medical Applications for Research; 1982.

Unproven Diagnostic Tests 32. Rinkel HJ. Inhalant allergy; the whealing response of the skin to serial dilution testing. Ann Allergy 1949;7:625–30. 33. Willoughby JW. Serial dilution titration skin tests in inhalant allergy: a clinical quantitative assessment of biologic skin reactivity to allergenic extracts. Otolaryngol Clin North Am 1974;7:579–615. 34. Van Metre TE. Critique of controversial and unproven procedures for diagnosis and therapy of allergy disorders. Pediatr Clin North Am 1983;30:807. 35. Jewett DL, Fein G, Greenberg MH. A double-blind study of symptom provocation to determine food sensitivity. N Engl J Med 1990;323:429–33. 36. Bryan WT, Bryan MP. Allergy in otolaryngology. In: Paparella MM, Shumrick DA, editors. Otolaryngology, vol. 3. Philadelphia: WB Saunders; 1973. 37. Ulett GA, Perry SC. Cytotoxic testing and leukocyte increase: an index of food sensitivity. II. Coffee and tobacco. Ann Allergy 1975;34:150–60. 38. Pasula MJ. The ALCAT test: in vitro procedure for determining food sensitivities. Folia Med Cracov 1993;34:153–7. 39. Voll R. The phenomenon of medicine testing in electroacupuncture according to Voll. Am J Acupuncture 1980;8:87. 40. Semizzi M, Senna G, Crivellaro M, et al. A double-blind, placebo-controlled study on the diagnostic accuracy of an electrodermal test in allergic subjects. Clin Exp Allergy 2002;32:928–32. 41. Garrow JS. Kinesiology and food allergy. BMJ 1988;296:1573–4.

Inappropriate Diagnostic Tests 42. Mullin GE, Swift KM, Lipski L, et al. Testing for food reactions: the good, the bad, and the ugly. Nutr Clin Pract 2010;25:192–8. 43. Shamji MH, Durham SR. Mechanisms of allergen immunotherapy for inhaled allergens and predictive biomarkers. J Allergy Clin Immunol 2017;140:1485–98. 44. Hetherington L, Battershill J. Review of evidence for a toxicological mechanism of idiopathic environmental intolerance. Hum Exp Toxicol 2013;32:3–17. 45. Sheffer AL, Lieberman PL, Aaronson DW, et al. Measurement of circulating IgG and IgE food-immune complexes. J Allergy Clin Immunol 1988;81:758–60. 46. Paganelli R, Levinsky RJ, Brostoff J, et al. Immune complexes containing food proteins in normal and atopic subjects after oral challenge and effect of sodium cromoglycate on antigen absorption. Lancet 1979;1:1270–2. 47. Paganelli R, Cavagni G, Pallone F. The role of antigenic absorption and circulating immune complexes in food allergy. Ann Allergy 1986;57:330–6. 48. Leary HL, Halsey JF. An assay to measure antigen-specific immune complexes in food allergy patients. J Allergy Clin Immunol 1984;74:190–5.

Unproven Treatment Methods 49. Kailin EW, Collier R. “Relieving” therapy for antigen exposure. JAMA 1971;217:78. 50. Rapp D. Double-blind confirmation and treatment of milk sensitivity. Med J Aust 1978;1:571–2. 51. Scadding GK, Brostoff J. Low dose sublingual therapy in patients with allergic rhinitis due to house dust mite. Clin Allergy 1986;16:483–502. 52. McEwen LM. Enzyme potentiated desensitization. V. Five case reports of patients with acute food allergy. Ann Allergy 1975;35:98–103. 53. Fell P, Brostoff J. A single dose desensitization for summer hayfever: results of a double blind study—1988. Eur J Clin Pharmacol 1990;38:77–9. 54. Galli E, Bassi MS, Mora E, et al. A double-blind randomized placebo-controlled trial with short-term beta-glucuronidase therapy in

children with chronic rhinoconjunctivitis and/or asthma due to dust mite allergy. J Investig Allergol Clin Imunol 2006;16:345–50. 55. Radcliffe MJ, Lewith GT, Turner RG, et al. Enzyme-potentiated desensitization of seasonal allergic rhinitis: double-blind randomized controlled study. BMJ 2003;327:251–4. 56. Root DE, Katzin DB, Schnare DW. Diagnosis and treatment of patients presenting subclinical signs and symptoms of exposure to chemicals which bioaccumulate in human tissue. Proceedings of the National Conference on Hazardous Wastes and Environmental Emergencies, May 14-6, 1985, Cincinnati. 57. Liberman J, Bigland AD. Autogenous urinary proteose in asthma and other allergic conditions. BMJ 1937;1:62–5. 58. Plesch J. Urine therapy. Med Press 1994;218:128. 59. Park JE, Lee SS, Lee MS, et al. Adverse events of moxibustion: a systematic review. Complement Ther Med 2010;18:215–23. 60. Stockert K, Schneider B, Porenta G, et al. Laser acupuncture and probiotics in school age children with asthma: a randomized, placebo-controlled pilot study of therapy guided by principles of Traditional Chinese Medicine. Pediatr Allergy Immunol 2007;18:160–6. 61. Passalacqua G, Bousquet PJ, Carlsen KH, et al. ARIA update: 1. Systematic review of complementary and alternative medicine for rhinitis and asthma. J Allergy Clin Immunol 2006;117:1054–62. 62. Banerjee K, Mathie RT, Costelloe C, et al. Homeopathy for allergic rhinitis: a systematic review. J Altern Complement Med 2017;23: 426–44. 63. Kim LS, Riedlinger JE, Baldwin CM, et al. Treatment of seasonal allergic rhinitis using homeopathic preparation of common allergens in the southwest region of the US: a randomized, controlled clinical trial. Ann Pharmacother 2005;39:617–24. 64. Taylor MA, Reilly D, Llewellyn-Jones RH, et al. Randomised controlled trial of homoeopathy versus placebo in perennial allergic rhinitis with overview of four trial series. BMJ 2000;321:471–6. 65. Ernst E. Homeopathy: what does the “best” evidence tell us? Med J Aust 2010;192:458–60. 66. Dombret MC, Alagha K, Boulet LP, et al. Bronchial thermoplasty: a new therapeutic option for the treatment of severe, uncontrolled asthma in adults. Eur Respir Rev 2014;23:510–18. 67. Aimbire F, Ligeiro de Oliveira AP, Albertini R, et al. Low level laser therapy (LLLT) decreases pulmonary microvascular leakage, neutrophil influx and IL-1 beat levels in airway and lung from rat subjected to LPS-induced inflammation. Inflammation 2008;31:189–97. 68. Dabbous OA, Soliman MM, Mohamed NH, et al. Evaluation of the improvement effect of laser acupuncture biostimulation in asthmatic children by exhaled inflammatory biomarker level of nitric oxide. Lasers Med Sci 2017;32:53–9. 69. Levine SA, Reinhardt JH. Biochemical pathology initiated by free radicals, oxidant chemicals, and therapeutic drugs in the etiology of chemical hypersensitivity disease. Orthomol Psychiatry 1983;12:166–83. 70. Milan SJ, Hart A, Wilkinson M. Vitamin C for asthma and exercise-induced bronchoconstriction. Cochrane Database Syst Rev 2013;(10):CD010391. 71. Strait RT, Camargo CA. Vitamin E and the risk of childhood asthma. Expert Rev Respir Med 2016;10:881–90. 72. Hall SC, Agrawal DK. Vitamin D and bronchial asthma: an overview of data from the past 5 years. Clin Ther 2017;39:917–29. 73. Terr AI. Environmental sensitivity. Immunol Allergy Clin North Am 2003;23:311–28. 74. Beamon S, Falkenbach A, Fainburg G, et al. Speleotherapy for asthma. Cochrane Database Syst Rev 2001;(2):CD001741. 75. Gyorik SA, Brutsche MH. Complementary and alternative medicine for bronchial asthma: is there new evidence? Curr Opin Pulm Med 2004;10:37–43. 76. Gernez Y, Nowak-Węgrzyn A. Immunotherapy for food allergy: are we there yet? J Allergy Clin Immunol Pract 2017;5:250–72. 77. Jaber R. Respiratory and allergic diseases: from upper respiratory tract infections to asthma. Prim Care 2002;29:231–61. 78. Li XM, Srivastava K. Traditional Chinese medicine for the therapy of allergic disorders. Curr Opin Otolaryngol Head Neck Surg 2006;14:191–6.

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy 79. DiNicola C, Kekevian A, Chang C. Integrative medicine as adjunct therapy in the treatment of atopic dermatitis–the role of traditional Chinese medicine, dietary supplements, and other modalities. Clin Rev Allergy Immunol 2013;44:242–53. 80. Li J, Zhang F, Li J. The immunoregulatory effects of traditional Chinese medicine on treatment of asthma or asthmatic inflammation. Am J Chin Med 2015;43:1059–81. 81. Hylander T, Latif L, Petersson-Westin U, et al. Intralymphatic allergen-specific immunotherapy: an effective and safe alternative treatment route for pollen-induced allergic rhinitis. J Clin Immunol 2013;131(2):412–20.

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82. Wu AC, Tantisira K, Li L, et al. Effect of vitamin D and inhaled corticosteroid treatment on lung function in children. Am J Respir Crit Care Med 2012;186:508–13. 83. Castro M, King TS, Kunselman SJ, et al. Effect of vitamin D3 on asthma treatment failures in adults with symptomatic asthma and lower vitamin D levels: the VIDA randomized clinical trial. J Am Med Assoc 2014;311:2083–91. 84. Martineau AR, MacLaughlin BD, Hooper RL, et al. Double-blind randomized placebo-controlled trial of bolus-dose vitamin D3 supplementation in adults with asthma (ViDiAs). Thorax 2015;70:451–7.

CHAPTER 98  Unconventional Theories and Unproven Methods in Allergy

1611.e1

SELF-ASSESSMENT QUESTIONS 1. Which of the following is the most common and prominent symptom in allergic toxemia or tension fatigue syndrome? a. Respiratory complaints b. Pallor c. Musculoskeletal pain d. Fatigue e. Headache 2. Which of the following hormones has been associated with idiopathic environmental intolerance (IEI)? a. Aldosterone b. Testosterone c. Estrogen d. Progesterone e. Serotonin

3. Which of the following observes for a morphologic change in peripheral blood leukocytes exposed to particular allergens in vitro to indicate possible sensitivity to the allergens? a. Antigen leukocyte cellular antibody test (ALCAT) b. Cytotoxic test c. Neutralization d. Enzyme-potentiated desensitization e. Detoxification

99  Complementary and Alternative Medicine Renata J.M. Engler, Kamal Srivastava, Ozlem Sahin, Xiu-Min Li

CONTENTS Background, 1612 Overview of Complementary and Alternative Medicine Therapy for Allergic Rhinitis and Asthma, 1618 Alternative Medical Systems, 1621 Manipulation and Body-Based Therapies, 1624

SUMMARY OF IMPORTANT CONCEPTS • Complementary and alternative medicine (CAM) comprises medical practices that are used to augment (complementary) or replace (alternative) allopathic or conventional medicine. • Integrative health care represents a shifting focus toward bringing conventional and complementary approaches together in a systematic way for optimum outcomes to include quality of life and patient perspectives while incorporating a focus on evidence for safety and efficacy as well as legal considerations. • Representing most of the global health care services, CAM therapies use natural products, mind-body techniques, mechanical body-based practices, and other methods to enhance wellness and healing. • Both the 2007 and 2012 US National Health Interview Survey (NHIS) identified many CAM therapies (other than vitamins and minerals) commonly used by adults and children with the most common mind and body practices, including chiropractic or osteopathic manipulation, massage, meditation, and yoga. • The ten most common complementary health approaches among adults include natural products, deep breathing, yoga (including tai chi, qi gong), chiropractic or osteopathic manipulation, meditation, massage, special diets, homeopathy, progressive relaxation, and guided imagery. • The use of CAM by the US population is estimated at 33.2% of adults and 11.6% of children based on the 2012 NHIS results and accounts for 9.2% of the total out-of-pocket expenditures on health care (greater than $30 billion annually). • Guidelines for integrating patient preferences into a comprehensive medical management plan focus on ensuring that both efficacy and safety considerations are addressed to avoid adverse effects or interactions with other treatments.

BACKGROUND Conventional, allopathic, or Western medicine (i.e., mainstream medicine, orthodox medicine, regular medicine, or biomedicine) includes health care delivered by licensed physicians or doctors of osteopathy, other providers (e.g., nurse practitioners, physician assistants), and allied health professionals (e.g., physical therapists, psychologists, registered nurses).1 On a global basis, conventional medicine represents less than

1612

Mind-Body Therapies, 1625 Yoga, 1626 Biologically Based Therapies, 1630 Summary, 1634

one half of available medical care delivery systems but consistent studies of use prevalence by country is limited with the most stable estimates over time reported for Australia (49% to 52%) and the United States.2 Table 99.1 describes the complementary and alternative medicine (CAM) practitioners and therapies that were included in the 2012 NHIS (National Health Information Survey) with findings in general comparable with the NHIS results of 2007.3,4 The selected listings represent the four major categories of CAM modalities: alternative medical systems with various forms of licensing for competency (e.g., acupuncture, Ayurveda, homeopathy); biologically based therapies (e.g., chelation therapy, natural products such as herbal and nutritional supplements); manipulation and body-based therapies (e.g., chiropractic and osteopathic manipulation, massage, movement therapies); and mind-body therapies (e.g., biofeedback, meditation, guided imagery, progressive relaxation, deepbreathing exercises, hypnosis, yoga). In 2012, 33.2% of US adults and 11.6% of children (4 to 17 years of age) used complementary health approaches without significant changes from the 2007 survey, and the most commonly used were natural products (dietary supplements other than vitamins and minerals).3,4 For the US market alone, the 2012 estimate for CAM use represented total annual spending over $30 billion and 9.2% of all out-of-pocket spending on health care.3–5 Table 99.2 summarizes the US population use of complementary and integrative health approaches in age-adjusted percentages with standard errors based on 2012 survey results for adults and children.3,4 The NHIS of 2012 demonstrated consistent patterns (compared with 2007) of use with prevalence higher among women, adults aged 30 to 69 years with higher levels of education and income, living in the West, former smokers, and adults hospitalized in the last year.4 In children overall there were no significant changes in use (except for a reduction in use among Hispanic children) with similar patterns to include the following: older children (age 12 to 17 years); parents with more than a high school education (seven times as likely to use); and girls more likely to use yoga, tai chi, or qi gong.3 The use of CAM therapies presents major challenges for optimizing the quality of care and ensuring efficacy and safety when combined into complex care plans.6–8 With a focus shift toward integrative medicine by the renamed National Center for Complementary and Integrative Health (previously Complementary and Alternative Medicine) at the National Institutes of Health, there is a growing commitment to “bringing conventional and complementary approaches together in a coordinated way.”7

1613

CHAPTER 99  Complementary and Alternative Medicine

TABLE 99.1  Complementary and Alternative Medicine Practitioners, Practices, and Therapies Therapy Practitioner Required Acupuncture

Description of Medical Practice or Therapy Procedures involve stimulation of anatomic points on the body by several techniques. American acupuncture incorporates medical traditions from China, Japan, Korea, and other countries. Classic acupuncture uses fine needles to penetrate the skin with manual or electrical stimulation, but newer approaches use direct cutaneous stimulation without a needle.

Ayurveda

Originating in India, the system of medicine focuses on prevention and treatment of health problems through integration and balance of body, mind, and spirit (i.e., holistic approach). Proposed treatments aim to cleanse the body of substances that cause disease, helping to reestablish harmony and balance.

Biofeedback

Simple electronic devices are used to teach clients how to consciously regulate body functions such as breathing, heart rate, and blood pressure to improve health and well-being. Applications include stress reduction, headache elimination, reconditioning of injured muscles, pain relief, and control of asthma attacks.

Chelation therapy

A chemical is used to bind molecules of a substance (e.g., metals, minerals) and remove them from the body. In conventional medicine, the process is used to remove toxic metals such as lead, and in complementary and alternative medicine, it is used to treat conditions such as cardiovascular and cerebrovascular disease.

Chiropractic manipulation

Adjustment of the spine and joints influences the body’s nervous system and natural defense mechanisms to alleviate pain and improve general health. Chiropractic care is a form of health care that focuses on the relationship between the body’s structure, primarily the spine, and function.

Osteopathic manipulation

Like doctors of chiropractic medicine, osteopathic physicians also use hands-on therapy (i.e., manipulation or adjustment) as their core clinical procedure. Osteopathic manipulation is a full-body system of hands-on techniques to alleviate pain, restore function, and promote health and well-being.

Energy healing therapy, Reiki

Reiki is an energy medicine practice that originated in Japan. The practitioner places his hands on or near the person receiving the treatment with the intent of transmitting ki, which is thought to be life-force energy.

Hypnosis

Induction of an altered state of consciousness is characterized by increased responsiveness to suggestion. The hypnotic state is attained by first relaxing the body and then shifting attention to a narrow range of objects or ideas suggested by the hypnotherapist or hypnotist. The procedure is used to effect positive changes and to treat conditions such as chronic pain, respiratory ailments, stress, and headache.

Massage

Massage therapists manipulate muscle and connective tissue to enhance the function of those tissues and promote relaxation and well-being.

Naturopathy

An alternative medical system, naturopathic medicine proposes that there is a healing power in the body that establishes, maintains, and restores health. Practitioners work with patients toward a goal of supporting this power through the use of nutrition and lifestyle counseling, dietary supplements, medicinal plants, exercise, homeopathy, and treatments from traditional Chinese medicine.

Traditional healing

Traditional healers use ancient medical practices that are based on indigenous theories, beliefs, and experiences handed down through generations. Their methods reflect different philosophical backgrounds, cultural origins, and regional natural products adapted to healing.

Traditional Chinese medicine

Traditional Chinese medicine is the current name for a comprehensive, ancient system of health care from China that is based on the concept of a vital energy or life force, called qi or chi, that flows throughout the body along channels known as meridians, regulating a person’s spiritual, emotional, mental, and physical balance. This energy is influenced by the opposing forces of yin (negative energy) and yang (positive energy). Disease is thought to result from the disrupted flow of qi and imbalanced yin and yang. The focus is on the notion of harmony and balance, including ideas of moderation and prevention. It incorporates herbal (complex mixtures) and nutritional therapy, restorative physical exercises, meditation, acupuncture, and remedial massage.

Practitioner Not Required Deep-breathing exercises

Slow and deep inhalation through the nose, usually to a count of 10, is followed by slow and complete exhalation for a similar count. The process may be repeated 5 to 10 times for several times each day.

Diet-based therapies

These dietary practices are designed to promote health and wellness, and they have various degrees of evidence for efficacy and safety. The high-fiber, low-fat, vegetarian Ornish diet promotes weight loss and health by controlling what is eaten rather than counting calories. The South Beach diet distinguishes between good and bad carbohydrates, promoting low glycemic index foods and enhanced intake of vegetables and whole grains (without counting calories). The Atkins diet emphasizes a drastic reduction in daily intake of carbohydrates (≤40 g) coupled with an increase in protein and fat. Other special approaches include the macrobiotic and Zone diets.

Guided imagery

A series of relaxation techniques is followed by visualization of detailed, usually peaceful images. If used for treatment, the individual visualizes his or her body free of the specific problem or condition. Sessions are usually 20 to 30 min long and practiced several times each week. Continued

1614

SECTION H Therapeutics

TABLE 99.1  Complementary and Alternative Medicine Practitioners, Practices, and

Therapies—cont’d Therapy Homeopathic treatment

Description of Medical Practice or Therapy Homeopathy is a system of medical practices based on the theory that any substance that can produce disease symptoms in a healthy person can cure those symptoms in a sick person. For example, someone with insomnia may be given a homeopathic dose of coffee. Administered in diluted form, homeopathic remedies are derived from many natural sources, including plants, metals, and minerals. Toxic metals such as arsenic are diluted to a point at which the concentration is almost undetectable.

Meditation

Most of these techniques started in Eastern religious or spiritual traditions. During meditation, a person learns to focus his or her attention and suspend the stream of thoughts that normally occupy the mind. This practice is thought to result in a state of greater physical relaxation, mental calmness, and psychologic balance. Practicing meditation can change how a person responds to the flow of emotions and thoughts in the mind.

Movement therapies

Motion therapies and exercises are designed to promote health and well-being. Alexander technique teaches how to use muscles more efficiently to improve posture and overall function. It is used for low back pain and symptoms of Parkinson disease.

Natural products

Supplemental dietary ingredients other than vitamins and minerals include single ingredients or mixtures of herbal and botanical products (e.g., soy, flax seeds), enzymes, and glandular extracts. Among the most popular are echinacea, ginkgo biloba, ginseng, feverfew, garlic, kava kava, and saw palmetto.

Progressive relaxation

Tension and stress are relieved by systematically contracting and relaxing successive muscle groups.

Qi gong

The ancient Chinese discipline combines gentle physical movements, mental focus, and deep breathing directed toward specific parts of the body. Performed in repetitions, the exercises are normally performed alone or in a group for two or more times per week for 30 minutes each time.

Tai chi

The mind-body practice originated in China as a martial art. Tai chi involves slow, gentle, and controlled body movements while breathing deeply and meditating. Also known as moving meditation, tai chi is believed to help the flow throughout the body of a proposed vital energy called qi. The relaxed and graceful series of movements are done in sets called forms or routines, which are performed alone or in a group.

Yoga

Breathing exercises, physical postures, and meditation are combined to calm the nervous system and balance body, mind, and spirit. Sessions usually are 45 minutes long and are conducted one or more times each week alone or in a group.

Traditional Chinese Medicine definition. Available from: http://medical-dictionary.thefreedictionary.com/traditional+Chinese+medicine (accessed November 1, 2018); and the National Cancer Institute. NCI dictionary of cancer terms. Available from: http://www.cancer.gov/ dictionary?cdrid=449722 (accessed November 1, 2018). Data from Barnes PM, Bloom B, Nahin RL. Complementary and alternative medicine use among adults and children: United States, 2007. Natl Health Stat Report 2008(12):1-23.

As patient-centric personalized medicine with greater acknowledgement of patients as active members of their own care team continues to expand, the need to promote research that clarifies how to optimally combine modalities of care has become an even greater need. According to a 2017 Consumer Reports survey of 1003 adults, one-third of Americans admit to the use of alternative treatments in the previous year. More than half prefer natural remedies over mainstream medicine, with 21% describing mainstream medicine therapies as ineffective and 29% having their doctors recommend the complementary approach.9 From a survey of 44,743 individuals aged 4 years and older, the 2012 NHIS estimated that 59 million Americans purchased at least one complementary health approach with a total out-of-pocket expenditure exceeding 30.2 billion dollars.10 Another report on expenditures estimated an annual cost for dietary supplements alone of $42 billion dollars.11 With widespread access to and globalization of CAM therapies by the Internet, there is a growing need for education and reliable information about CAM therapies to avoid unnecessary harm and to balance benefits with risks, particularly in the context of an individual patient’s disease and medical history.8 CAM therapies are commonly used in low- and middle-income countries because they are affordable and available. International surveys of CAM consumption among patients with allergies reported prevalence rates ranging from 18% to 65%.8,10,12–15 Studies of people with allergies show that CAM users usually are younger,

are female, and have a higher level of education than those who do not use CAM.13 Patients with allergies who seek out CAM treatments tend to have a better quality of life, are dissatisfied with conventional medications, have a perception of fewer side effects with CAM, and are influenced by social networks.16,17 Patients with allergies often fail to inform their physicians about the use of CAM.12,14,18,19 Natural (nonvitamin, nonmineral) product use varies widely and represents a vast arena of potential candidates. The 2012 survey highlighted this category of use at 17.7% for adults and 4.9% for children.3,4 Table 99.3 outlines the major categories of CAM modalities used by adults and children by age-adjusted percent (with standard error) in the year prior to the 2012 national survey. These modalities generally involve a practitioner, a class of instruction, or some form of training for the user. The data demonstrates the substantial use of chiropractic or osteopathic manipulation as well as deep breathing exercises, yoga–tai chi–qi gong mind-body therapies, massage, and meditation (all more than 5% for adults).3,4 Table 99.4 outlines the reported use of selective nonvitamin, nonmineral dietary supplements during the preceding 30 days from the 2012 NHIS for adults and children (age-adjusted percent with standard error from a survey population of 88,962 American adults and 17,321 children).3,4 Nonmineral, nonvitamin supplement products represent a large market of commercially available products (in the tens of thousands with estimates of more than 60,000 in the United States alone).

1615

CHAPTER 99  Complementary and Alternative Medicine

TABLE 99.2  2012 Reported Use of

Complementary Health Approaches in AgeAdjusted Percentages Among Adults Aged ≥18 and Children Aged 4–17 Years by Gender, Age, Ethnicity/Race, Poverty and Health Insurance and Education Level (Adults or Parents) Complementary and Integrative Health Approaches

Adults (≥18 Years) Age-Adjusted % (Standard Error)

Children (4–17 Years) Age-Adjusted % (Standard Error)

33.2 (0.42)

11.6 (0.46)

Population Use Total: Any Modality Gender and Age Range Men/Boys

28.9 (0.54)

9.7 (0.56)

Women/Girls

37.4 (0.54)

13.5 (0.67)

Ages 18–44 and 4–11

34.6 (0.57)

9.3 (0.56)

Ages 45–64 and 12–17

36.8 (0.63)

14.7 (0.75)

Ages 65 and over (adult only)

29.4 (0.73)

Hispanic Origin and Race Hispanic 22.0 (0.76)

6.1 (0.54)

Non-Hispanic white, single race

37.9 (0.53)

14.9 (0.70)

Non-Hispanic Black or African American, single race

19.3 (0.75)

5.5 (0.77)

Non-Hispanic all other races

37.3 (1.21)

14.2 (1.50)

TABLE 99.3  2012 United States Reported

Use of Complementary and Integrative Health Approaches in Age-Adjusted Percentages Among Adults Aged ≥18 and Children Aged 4–17 Years by Medical Systems or Therapy Categories Complementary and Integrative Health Approaches

Adults (≥18 Years) Age-Adjusted % (Standard Error)

Alternative Medical Systems Acupuncture 1.5 (0.08)

5.7 (0.69)

0.1 (0.05)

Ayurveda

0.1 (0.02)

Homeopathic treatment

2.2 (0.11)

1.8 (0.19)

Naturopathy

0.4 (0.04)

0.2 (0.07)

Energy healing therapy

0.5 (0.05)

0.2 (0.05)

Traditional healer

0.1 (0.05)

Biologically Based Therapies Dietary supplements: 17.7 (0.37) nonmineral, nonvitamin Diet-based therapies

3.0 (0.13)

Manipulation and Body-Based Therapies Chiropractic or 8.4 (0.22) osteopathic manipulation Craniosacral therapy

Poverty Status & Health Insurance Poor 20.6 (0.76)

Children (4–17 Years) Age-Adjusted % (Standard Error)

Massage Mind-Body Therapies Deep-breathing exercises

4.9 (0.29)

0.7 (0.12) 3.3 (0.26)

0.02 (0.07) 6.9 (0.15)

0.7 (0.10)

10.9 (0.26)

2.7 (0.21)

10.1 (0.25)

3.2 (0.24)

Near Poor

25.5 (0/79)

9.1 (0.81)

Not Poor

38.4 (0.53)

14.8 (0.67)

Yoga, tai chi and qi gong

Insurance: Private

38.0 (0.50)

14.6 (0.66)

Meditation

8.0 (0.21)

1.6 (0.16)

Insurance: Public

24.8 (0.84)

7.6 (0.55)

Progressive relaxation

2.1 (0.10)

0.4 (0.07)

Guided imagery

1.7 (0.10)

0.4 (0.08)

Hypnosis

0.1 (0.03)

Biofeedback

0.1 (0.02)

2012 National Health Interview Survey, conducted by the National Center for Health Statistics (part of the Centers for Disease Control and Prevention), gathered information on 88,962 American adults and 17,321 children. Percentages (%) were age adjusted using the projected 2000 US population provided by US Census Bureau as the standard population. Age adjustment used age groups 18–24, 25–44, 45–64, 66 and over for adults and 4–11 and 12–17 for children. Data from Black LI, Clarke TC, Barnes PM, Stussman BJ, Nahin RL. Use of complementary health approaches among children aged 4-17 years in the United States: National Health Interview Survey, 2007-2012. Natl Health Stat Report 2015(78):1-19 and Clarke TC, Black LI, Stussman BJ, Barnes PM, Nahin RL. Trends in the use of complementary health approaches among adults: United States, 2002-2012. Natl Health Stat Report 2015(79):1-16.

Movement therapies

0.4 (0.09)

2012 National Health Interview Survey, conducted by the National Center for Health Statistics (part of the Centers for Disease Control and Prevention), gathered information on 88,962 American adults and 17,321 children. Percentages (%) were age adjusted using the projected 2000 US population provided by US Census Bureau as the standard population. Age adjustment used age groups 18–24, 25–44, 45–64, 66 and over for adults and 4–11 and 12–17 for children. Data from Black LI, Clarke TC, Barnes PM, Stussman BJ, Nahin RL. Use of complementary health approaches among children aged 4–17 years in the United States: National Health Interview Survey, 2007-2012. Natl Health Stat Report 2015(78):1-19 and Clarke TC, Black LI, Stussman BJ, Barnes PM, Nahin RL. Trends in the use of complementary health approaches among adults: United States, 2002-2012. Natl Health Stat Report 2015(79):1-16.

1616

SECTION H Therapeutics

TABLE 99.4  2012 Reported Use of Selective Nonvitamin, Nonmineral Dietary Supplements

During the Past 30 Days in Age-Adjusted Percentages Among Adults Aged ≥18 and Children Aged 4–17 Years in the United States Adults (>18 Years) Age-Adjusted %b (Standard Error)

Children (4–17 Years) Age-Adjusted %b (Standard Error)

Fish oil (Omega-3 or DHA fatty acid)

7.8 (0.22)

1.1 (0.13)

Prebiotics or probiotics

1.6 (0.09)

0.5 (0.09)

Melatonin

1.3 (0.08)

0.7 (0.11)

Echinacea

0.9 (0.06)

0.4 (0.08)

Garlic

0.8 (0.06)

0.1 (0.04)

Ginseng

0.7 (0.06)

0.1 (0/05)

Combination herb pill

0.6 (05)

0.1 (0/04)

Ginkgo biloba

0.7 (0.06)

Cranberry (pills, gelatin capsules)

0.8 (0.06)

Garlic supplements

0.8 (0.06)

Glucosamine or chondroitin

2.6 (0.11)

Coenzyme Q-10

1.3 (0.08)

Green tea pills

0.6 (0.05)

Saw palmetto

0.4 (0.04)

Methylsulfonylmethane (MSM)

0.4 (0.04)

Milk thistle (silymarin)

0.4 (0.04)

Valerian

0.3 (0.04)

Complementary and Integrative Health Use: Nonvitamin, Nonmineral Natural Productsa

0.1 (0.02) 0.1 (0.03)

a

Respondents might have used more than one nonvitamin, nonmineral natural product. The denominator used in the calculation of percentages was the number of adults or children who used nonvitamin, nonmineral, natural products within the past 30 days, excluding persons without usable information. Estimates are based on household interviews of a sample of the civilian, noninstitutionalized population. Data from Black LI, Clarke TC, Barnes PM, Stussman BJ, Nahin RL. Use of complementary health approaches among children aged 4–17 years in the United States: National Health Interview Survey, 2007-2012. Natl Health Stat Report 2015(78):1-19 and Clarke TC, Black LI, Stussman BJ, Barnes PM, Nahin RL. Trends in the use of complementary health approaches among adults: United States, 2002-2012. Natl Health Stat Report 2015(79):1-16. b

Table 99.4 lists some of the commonly used products by adults and children as identified in the 2012 NHIS.3,4 The Natural Medicine Database (NMD) provides independent listings of hundreds of commercial products for each generic product with thousands of combination products and provides evidence-based information efficacy, safety, and manufacturing quality of commercial products.20 The most common medical conditions of adults and children that are associated with higher frequencies of CAM use include (but are not limited to) back pain, insomnia, head or chest colds, depression, anxiety and stress, and musculoskeletal complaints.1,3,4 Multiple chronic medical conditions were also associated with increased CAM use.21 CAM use for asthma was in the range of 1% to 2% for children in both the 2007 and 2012 NHIS.1,3 Economic evaluations of complementary medicine that incorporate differences in health care systems, including estimates of cost effectiveness and net health benefits, are needed.22 Among those with chronic diseases such as asthma, a better understanding of CAM use patterns in subpopulations may improve provider recognition and management. In a study of behavioral risk factors for adult patients with asthma from 37 states and the District of Columbia, 56.6% of those with work-related asthma used CAM, compared with 27.9% of those who had non–workrelated asthma.23 Higher CAM use was associated with a greater likelihood of adverse asthma attacks in the preceding month, more emergency department visits and overnight hospital stays, and poorly controlled

asthma.23 The 2009 and 2010 Behavioral Risk Factor Surveillance System (BRFSS) survey and Asthma Callback Survey (ACBS) provided information about CAM use to include the following observations: CAM use was associated with poorer health-related quality of life and greater asthma severity but not comorbid conditions like diabetes, stroke, or cardiovascular disease.24,25 Among 7685 older asthmatics (over 55 years), 39% reported CAM use with breathing techniques commonly used, particularly among those with decreased asthma control.26 In counseling patients about CAM therapies, providers should use an evidence-based approach that considers the relevance, validity, and limitations of the available literature on efficacy and safety. The NMD, with independent health professional reviewers, has developed a level of evidence ranking as part of its editorial principles and processes.20 The NMD provides educational resources for consumers, patients, and medical professionals in a format that parallels information provided regarding traditional pharmaceuticals.6,20 The categories for safety and efficacy (as tools for balanced risk-benefit communication) are detailed in Tables 99.5 and 99.6. The NCCIH offers an online guide as well as portable app to common herbs that was adapted from the NMD approach.27 The US Food and Drug Administration (FDA) regulates herbal and other over-the-counter dietary supplements differently from conventional medicines. It has less oversight of good manufacturing practices and fewer evidence standards related to safety and efficacy. The standards for supplements are found in the Dietary Supplement Health and

CHAPTER 99  Complementary and Alternative Medicine

1617

TABLE 99.5  Criteria for Evidence-Based Safety Ratings

a

Rating

Definition

Criteria for the Rating

Likely safe

Very high level of reliable clinical evidence shows it is safe when used appropriately. Products rated likely safe are generally considered appropriate to recommend.

Safety data are available from two or more randomized clinical trials, a metaanalysis, or large-scale postmarketing surveillance that includes several hundred patients (level of evidence A),a or the product has undergone a safety review consistent with or equivalent to passing a review by the US Food and Drug Administration (FDA), Health Canada, or a similarly rigorous approval process. Studies have a low risk of bias and a high level of validity by meeting stringent assessment criteria (quality rating A).b Studies adequately measure and report safety and adverse outcomes data and consistently show no significant serious adverse effects without valid evidence to the contrary.

Possibly safe

Some clinical evidence shows it is safe when used appropriately; however, the evidence is limited by quantity, quality, or contradictory findings. Products rated possibly safe appear to be safe but do not have enough high-quality evidence to recommend for most people.

Safety data are available from one or more randomized clinical trials, a metaanalysis (level of evidence A or B), case series, two or more population-based or epidemiologic studies (level of evidence B), or limited postmarketing surveillance data. Studies have a low to moderate risk of bias and moderate to high level of validity by meeting or partially meeting assessment criteria (quality rating A or B). Studies adequately measure and report safety and adverse outcomes data and show no significant serious adverse effects without substantial evidence to the contrary. Some contrary evidence may exist; however, valid evidence supporting safety outweighs contrary evidence.

Possibly unsafe

Some clinical evidence shows safety concerns or significant adverse outcomes; however, the evidence is limited by quantity, quality, or contradictory findings. People should be advised not to take products with a possibly unsafe rating.

Safety data are available from one or more randomized clinical trials, metaanalysis (level of evidence A or B), two or more populationbased or epidemiologic studies (level of evidence B), or limited postmarketing surveillance data, or multiple, reliable case reports show a potential causal relationship between a product and serious adverse outcome. Studies have a low to moderate risk of bias and moderate to high level of validity by meeting or partially meeting assessment criteria (quality rating A or B). Studies adequately measure and report safety and adverse outcomes data and show significant serious adverse effects without substantial evidence to the contrary. Some contrary evidence may exist; however, valid evidence supporting potential safety concerns outweighs contrary evidence.

Likely unsafe

Very high level of reliable clinical evidence shows safety concerns or significant adverse outcomes. People should be discouraged from taking products with a likely unsafe rating.

Safety data are available from two or more randomized clinical trials, a metaanalysis, or large-scale postmarketing surveillance that include several hundred to several thousand patients (level of evidence A). Studies have a low risk of bias and high level of validity by meeting stringent assessment criteria (quality rating A). Studies adequately measure and report safety and adverse outcomes data and consistently show significant serious adverse effects without valid evidence to the contrary.

Unsafe

Very high level of reliable clinical evidence shows safety concerns or significant adverse outcomes. People should be discouraged from taking products with an unsafe rating.

Safety data are available from two or more randomized clinical trials, a metaanalysis, or large-scale postmarketing surveillance that includes several hundred to several thousand patients (level of evidence A). Studies have a low risk of bias and high level of validity by meeting stringent assessment criteria (quality rating A). Studies adequately measure and report safety and adverse outcomes data and consistently show significant serious adverse effects without valid evidence to the contrary.

Level of evidence is assessed using the following scale: A, high-quality, randomized, controlled trial (RCT) or high-quality metaanalysis (quantitative systematic review); B, nonrandomized clinical trial, nonquantitative systematic review, lower-quality RCT, clinical cohort study, case-control study, historical control, or epidemiologic study; C, consensus or expert opinion; D, anecdotal evidence, in vitro research, or animal research. b Study quality is assessed using the following scale: A, meets assessment criteria and has a low risk of bias; B, partially meets assessment criteria and has a low to moderate risk of bias; C, does not meet assessment criteria and has a moderate to high risk of bias. Adapted from Natural Medicines Database. Safety ratings.20

1618

SECTION H Therapeutics

TABLE 99.6  Criteria for Evidence-Based Efficacy Ratings Rating

Definition

Criteria for the Rating

Effective

Very high level of reliable clinical evidence supports its use for a specific indication. Products rated effective generally are considered appropriate to recommend.

Evidence is consistent with or equivalent to passing a review by the U.S. Food and Drug Administration (FDA), Health Canada, or similarly rigorous approval process. Evidence from two or more randomized clinical trials or a meta-analysis that includes several hundred to several thousand patients (level of evidence = A).* Studies have a low risk of bias and high level of validity by meeting stringent assessment criteria (quality rating = A).† Evidence consistently shows positive outcomes for a given indication without valid evidence to the contrary.

Likely effective

Very high level of reliable clinical evidence supports its use for a specific indication. Products rated likely effective generally are considered appropriate to recommend.

Evidence from two or more randomized clinical trials or a meta-analysis that includes several hundred patients (level of evidence = A). Studies have a low risk of bias and high level of validity by meeting stringent assessment criteria (quality rating = A). Evidence consistently shows positive outcomes for a given indication without significant valid evidence to the contrary.

Possibly effective

Some clinical evidence supports its use for a specific indication; however, the evidence is limited by quantity, quality, or contradictory findings. Products rated possibly effective may be beneficial but do not have enough high-quality evidence to recommend for most people.

One or more randomized clinical trials or a meta-analysis (level of evidence = A or B) or two or more population-based or epidemiologic studies (level of evidence = B). Studies have a low to moderate risk of bias and moderate to high level of validity by meeting or partially meeting assessment criteria (quality rating = A or B). Evidence shows positive outcomes for a given indication without substantial valid evidence to the contrary. Some contrary evidence may exist; however, valid positive evidence outweighs contrary evidence.

Possibly ineffective

Some clinical evidence shows ineffectiveness for a specific indication; however, the evidence is limited by quantity, quality, or contradictory findings. People should be advised that the product is possibly ineffective.

One or more randomized clinical trials or a meta-analysis (level of evidence = A or B) or two or more population-based or epidemiologic studies (level of evidence = B). Studies have a low to moderate risk of bias and moderate to high level of validity by meeting or partially meeting assessment criteria (quality rating = A or B). Evidence shows positive outcomes for a given indication without substantial valid evidence to the contrary. Some contrary evidence may exist; however, valid positive evidence outweighs contrary evidence.

Likely Ineffective

Very high level of reliable clinical evidence shows ineffectiveness for its use for a specific indication. People should be warned about a product that is likely ineffective.

Evidence from two or more randomized clinical trials or a meta-analysis that includes several hundred to several thousand patients (level of evidence = A). Studies have a low risk of bias and high level of validity by meeting stringent assessment criteria (quality rating = A). Evidence consistently shows negative outcomes for a given indication without valid evidence to the contrary.

*Level of evidence is assessed using the following scale: A, high-quality, randomized, controlled trial (RCT) or high-quality meta-analysis (quantitative systematic review); B, nonrandomized clinical trial, nonquantitative systematic review, lower-quality RCT, clinical cohort study, case-control study, historical control, or epidemiologic study; C, consensus or expert opinion; D, anecdotal evidence, in vitro research, or animal research. † Study quality is assessed using the following scale: A, meets assessment criteria and has a low risk of bias; B, partially meets assessment criteria and has a low to moderate risk of bias; C, does not meet assessment criteria and has a moderate to high risk of bias. Adapted from TRC Natural Medicines: Editorial Principles and Process. https://naturalmedicines.therapeuticresearch.com/about-us/editorialprinciples-and-process.aspx.

Education Act (DSHEA), a federal law that defines dietary supplements and sets product-labeling standards and health claim limits.28 It continues to be a daunting task for conventional providers to incorporate the expanding information about CAM into patient management. Table 99.7 outlines a published approach to addressing and documenting CAM use in the context of a medical visit, along with consideration of medicolegal issues.6

OVERVIEW OF COMPLEMENTARY AND ALTERNATIVE MEDICINE THERAPY FOR ALLERGIC RHINITIS AND ASTHMA Natural medicines or CAM are extensively used by patients with allergic rhinitis and asthma but few are well defined in relation to their potential value for allergic rhinitis and asthma.29,30 Allergic rhinitis is commonly

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CHAPTER 99  Complementary and Alternative Medicine treated with antihistamines, decongestants, topical corticosteroids, mast cell stabilizers, leukotriene inhibitors, and immunotherapy. Within the NMD, a search for herbal and supplements used for allergic rhinitis identified 43 ingredients (in thousands of combination trade name products) marketed to the public for treating allergic rhinitis.29

Commonly advertised product ingredients and their presumed mechanism of action are detailed in Table 99.8. The NMD efficacy rating system (Table 99.6) has listed the following products as possibly effective for allergic rhinitis: beta-glucans, butterbur, fermented milk/lactobacillus, maritime pine, milk thistle (silymarin), nasal irrigation, Phleum pratense

TABLE 99.7  Best Practice Principles for Integrating Complementary and Alternative

Therapies Into Management Plans Step

Physician-Patient Interactions

Actions and Special Considerations

1

Explore factors driving interest in complementary and alternative medicine (CAM) therapy or therapist. Document instigating factors: current use, intent for future use, considering future use, seeking information, and desire to access alternative provider. Discuss reasons for choice, such as desire for greater efficacy and safety and the acceptability of CAM compared with traditional approaches.

Explore whether to engage. Support request for CAM but indicate limitations of that support, payment issues, and time limits. Refer to another provider for in-depth consultation (if possible).

2

Document clinical reasons for seeking CAM, such as exhausted conventional medicine options with unsatisfactory outcomes, disease with bad prognosis or disability impact, and fear of conventional therapies.

Physician-patient perceptions can be shared, including clinical status, options, and understanding; need for enhanced care or diagnostics; and need for other therapeutic options.

3

Assess current disease and health status and the previous therapies used. Use a symptom diary or validated quality-of-life survey tools. Use an objective measure of disease control such as blood pressure or peak expiratory flow. Consider duration of treatment trial.

Educate the patient about options. Discuss efficacy and safety data (if available), disease evaluation, and treatment options and risks. Address acceptance or refusal of partnership (may refer elsewhere if possible).

4

Document the patient’s preferences, factors in CAM therapy choice (if defined by patient), attitude toward CAM and conventional medicine, level of trust in exploring all therapeutic options.

Consider caveats that influence patient’s comfort with discussion and disclosure. Evaluate neutrality of questions asked. Assess for a caring, supportive, and respectful atmosphere.

5

Assess and document the adequacy of the medical evaluation. Consider the need for additional testing or consultation. Explain all options to optimize diagnosis and treatment. Assess quality-of-life impact of medical concerns.

Consider provider options or recommendations. Discuss overall risks and benefits of using treatments for which data are limited. Provide an opportunity for questions and a dialogue.

6

Define a plan for follow-up visits, including provider-patient agreement regarding therapeutic partnership (with referral rather than refusal of care). Consider providing a fact sheet that includes reliable information and other resources about CAM therapy. Provider may opt out of further involvement when available evidence indicates serious risk or lack of efficacy of product. The patient may agree but continue use anyway.

Out-of-pocket costs for patient care may be required. Visits may not be covered by existing health insurance plan; future visits may be required at nonreimbursed payment rates (per hour rate). The patient’s choice may result in the provider suggesting a move to a new provider. Avoiding patient abandonment is a challenge.

7

Provide good communication by defining respectfully and clearly what the physician can do about the patient’s request, including additional visit options. Determine whether insurance will cover the therapy or time for consultation on CAM therapy. Define the provider’s perception of liability risk concerns.

Communication is optimized if the physician is honest and caring about the patient’s issues. Explain limitations such as time, the need for separate visits, cost and time needed for imparting information, and risks for the provider. Review alternatives for how to proceed, and build a consensus plan.

8

Acknowledge evolving expectations. At each visit, review shared goals for disease or symptom control, detail shared responsibility to gather more information, establish the provider’s ability to support a request or validate the safety and efficacy of the CAM therapy of interest, and clarify the roles of the therapy team involved in care management.

Consider the level of the patient’s suffering, fear, and trust. Assess psychological factors such as depression and the need for validation. Share and document disclaimers that are needed in the context of ethical considerations in the care plan.

9

Educate about new safety and efficacy issues. Monitor the patient and literature concerning specific CAM therapy. Define guidelines for stopping the trial and any ethical concerns about supporting continuation of treatment. Consider an ethics committee consultation for complex benefit-risk analyses if controversial. Document the patient’s preferences and understanding.

Explain safety and efficacy levels of evidence. Communicate balanced clinical considerations, including defined and undefined risks and the levels of evidence and uncertainty. Educate the patient about evaluating individualized benefit-risk ratio versus alternatives. Continued

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SECTION H Therapeutics

TABLE 99.7  Best Practice Principles for Integrating Complementary and Alternative

Therapies Into Management Plans—cont’d Step 10

Physician-Patient Interactions Discuss factors in choosing a CAM-qualified provider, and address questions of suitability, licensure, and competencies. Document how the provider or practice was identified by the patient. Educate the patient about questions for a CAM therapy consultation. Support the patient seeking input for an informed choice, and empower the patient to critically assess a treatment trial. Communicate and document the referring provider’s responsibilities, permission to release medical information, and disclaimer regarding any conflict of interest. The patient’s choice is tempered by risk-benefit considerations and the concept of “do no harm”; discuss reporting of adverse events and unethical or untested practices.

Actions and Special Considerations Resources are available to assess providers, including state certification requirements for CAM therapists, objective reviews, and checklists for patients during a CAM visit. Before starting unconventional treatment, educate about patient choice and establish ground rules for appointments and the level of continued physician engagement. Discuss reporting adverse events for CAM through MedWatch (www.fda.gov/Safety/MedWatch/default. htm).

Adapted from Engler RJ, With CM, Gregory PJ, Jellin JM. Complementary and alternative medicine for the allergist-immunologist: where do I start? J Allergy Clin Immunol 2009;123:309-16.

TABLE 99.8  Herbal or Food-Derived

Ingredients Found in Products Used for Allergic Rhinitis Mechanisms of Action

Common Name

Botanical Name

Antihistamine

Grape seed extract

Vitis vinifera

Decongestants

Ephedra

Ephedra spp.

Mast cell stabilizers

Quercetin Spirulina Stinging nettle

Spirulina spp. Urtica dioica

Leukotriene modifiers

Butterbur

Petasites hybridus

Potential immunomodulators

Echinacea Thymus extract

Echinacea spp. Tinospora cordifolia

Vitamin C

Bitter orange

Citrus aurantium

Other natural therapies

Capsaicin Cat’s claw

Goldenseal Methylsulfonylmethane

Uncaria guyanensis, Uncaria tomentosa Hydrastis canadensis

Adapted from Natural Medicines in the Clinical Management of Allergic Rhinitis.29

(Timothy grass), pycnogenol (Maritime pine), thymus extract, Tinospora cordifolia (herbaceous vine), and turmeric (curcumin). As of July 2018, no ingredients were listed with a rating of likely effective or effective.29 The benefits of nasal irrigation particularly with buffered hypertonic saline solution are noteworthy particularly in children.31 Only bifidobacteria, lactobacillus, and nasal irrigation were identified with likely safe and possibly effective status. Echinacea remained likely safe but with insufficient evidence for efficacy with comments that its role in prevention rather than during illness remains under study.29 An NMD search in January 2018 identified 69 potential natural ingredients or practices marketed to the public for use in asthma.30 Commonly used agents are detailed in Table 99.9. Using the NMD

efficacy rating system (Table 99.6) within the database, the following products were listed as possibly effective for asthma: black seed, caffeine, choline, magnesium, Maritime pine (pycnogenol), thymus extract, and yoga. As of June 2012, none of the ingredients in the database were listed with a rating of likely effective or effective for asthma. Fish oil, previously listed as possibly effective, is currently rated as “insufficient reliable evidence to rate” for asthma and allergic rhinitis. Asthma CAM therapies such as acupuncture, chiropractic manipulation, yoga, and movement therapies (e.g., Alexander technique) are advertised as potential asthma therapies. If all CAM modalities are considered, up to 89% of persons with asthma use some form of CAM, and one-fourth use herbal or natural medicine.30,32 In a 2010 Canadian review of cases in which asthma was considered unresponsive to usual care, the following additional CAM therapeutic options were listed33: homeopathy; chiropractic care; acupuncture; hypnosis and relaxation; herbal medicine; Chinese medicine; ayurvedic medicine; and supplements such as vitamin E, magnesium, and fish oil. None of these CAM options have sufficient evidence to merit routine use in clinical practice at this time, although some modalities merit further study and are perceived as beneficial for some patients. If used based on individual patient preferences, then a focus on safety considerations and education on monitoring response to guide cessation may be the best approach. What role these diverse therapies may have in personalized medicine and how to validate efficacy and safety for individual patients remains an ongoing challenge for clinicians. Saline nasal irrigations have considerable literature supporting its use for patients with allergic rhinitis and sinusitis.29,31–36 There is evidence that buffered hypertonic saline nasal irrigations reduce oral antihistamine use and markers of inflammation (e.g., histamine, leukotrienes) and that they improve quality of life.35 Results of a 2012 study indicate that buffered hypertonic saline irrigations are more effective than normal saline alone in reducing symptoms in children with allergic rhinitis.36 This therapeutic modality is increasingly incorporated in care plans within conventional medicine. Although vitamin and mineral supplements, including antioxidants, are not always included in reviews of CAM, these agents further complicate and challenge the practice of evidence-based integrative medicine. Often considered safe and effective in the absence of objective data, these health adjuvants and their widespread use have raised concerns described in the peer-reviewed literature. An example is β-carotene

CHAPTER 99  Complementary and Alternative Medicine

TABLE 99.9  Herbal or Food-Derived

Ingredients Found in Products Used for Asthma Mechanism of Action

Common Name

Botanical Name

Mast cell stabilizers

Indian frankincense Picrorhiza Quercetin Spirulina Stinging nettle

Boswellia serrata Picrorhiza kurroa Urtica dioica

Leukotriene modifiers

Butterbur Fish oil Indian frankincense New Zealand green-lipped mussel Perilla Pycnogenol

Petasites hybridus Boswellia serrata Perna canaliculus Perilla frutescens Pinus pinaster

Antioxidants

Grapefruit Kiwi Noni juice Sweet orange Vitamin C, E

Citrus paradisi Actinidia chinensis Morinda citrifolia Citrus sinensis

Other agents

Choline Eucalyptus Magnesium Pyridoxine (vitamin B6) Soybean

Eucalyptus globulus Glycine max

Adapted from Natural medicines in the clinical management of asthma.30

supplementation beyond standard dietary intake. Some evidence indicates that mixtures of β-carotene isomers prevent exercise-induced asthma,37 but there are growing concerns that as little as 20 mg/day for 5 to 8 years may increase the risk of lung and prostate cancer, cardiovascular mortality, and total mortality for individuals who smoke or have exposure to asbestos.38,39 This agent has been removed from the list of possible effective agents at this time.28 Vitamin E supplementation ranging between 50 and 600 IU (daily or every other day, depending on sex) has been promoted as beneficial for preventing strokes, improving heart and lung health, and decreasing health care costs. A metaanalysis of nine randomized controlled trials (RCTs) reported that vitamin E supplementation increased the risk of hemorrhagic stroke by 22% and decreased the risk of ischemic stroke by 10%.40 A second metaanalysis of 13 RCTs that included more than 166,000 subjects found no benefit from vitamin E supplementation at any dose (i.e., less than or greater than 300 IU/day) for any stroke type, nor for any preparation type (i.e., synthetic or natural source).41 A 2012 Cochrane review of 78 RCTs with more than 296,000 participants found no evidence to support antioxidant supplements for primary or secondary prevention of stroke.42 This metaanalysis suggested that a small increase in the overall mortality rate was associated with β-carotene and vitamin E and with higher doses of vitamin A. The report suggested that antioxidant supplements be considered medicinal products that should undergo “evaluation before marketing” and that no recommendations could be made for their use in “the general population or patients with various diseases.” Dietary supplementation use, even with a placebo, may encourage other health-risk behaviors (e.g., smoking) by creating an illusion of invulnerability.43

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The Second National Report on Biochemical Indicators of Diet and Nutrition in the US population indicates that no more than 10% of the general population has nutritional deficiencies of selected vitamins and minerals.44 Fewer than 1% of them have deficiencies of vitamin E, vitamin A (with 2% to 4%, depending on age, at risk for excess vitamin A and associated hepatotoxicity risk), or folate. Vitamin C deficiency (i.e., serum levels of ascorbic acid less than 11.4 mmol/L) is estimated at 6%, with a higher incidence in men (7%) than in women (5%). The incidence of vitamin D deficiency (with levels below 12 ng/mL) is relatively high at 8.1%. Vitamin D insufficiency (with levels of 12 to 20 ng/mL) is not rare (24%) and occurs at higher rates among nonHispanic blacks (31%) and Mexican Americans (12%) compared with non-Hispanic whites (3%). However, the higher rates in African Americans has been described as the vitamin D paradox in that the incidence of fall fractures is lower than in White Americans with comparable levels.45 The Childhood Asthma Management Program (1024 subjects) identified higher rates of vitamin D insufficiency (using 30 ng/mL or less rather than 20 ng/mL or less as the cutoff point) among African Americans (35%).46 In an analysis adjusted for age, sex, body mass index (BMI), income, and treatment group, vitamin D insufficiency was associated with higher odds of severe asthma exacerbation over a 4-year period.46 Definitive studies incorporating biodiversity considerations are needed to define the optimal use of supplements and complementary foods as modulators of allergic and immunologic disease in different populations.47–49 It is noteworthy that the international community has published a plan for vitamin D food fortification given the cumulative health issues associated with lower levels to include several epidemiologic studies demonstrating increased mortality risk as well as associations with respiratory tract infections and asthma exacerbations. However, there are increasing concerns about the risks of too-high vitamin D levels and supplementation doses that exceed 1000 to 2000 IU per day.49

ALTERNATIVE MEDICAL SYSTEMS Alternative medical systems involve a distinct field of study and/or licensure based on principles that differ from allopathic medical practice. Acupuncture, Ayurveda, and homeopathy practices are widely prevalent globally and in the United States.4,5

Acupuncture Acupuncture has several described forms: acupuncture, auricular acupuncture, Chinese acupuncture, ear acupuncture, foot acupuncture, hand acupuncture, Japanese acupuncture, Korean acupuncture, needle moxibustion, single point acupuncture, trigger point acupuncture, and Western acupuncture. Acupressure uses some of the principles of acupuncture but does not use needles. Acupuncture is part of Kampo medicine and traditional Chinese medicine (TCM). Acupuncture is used for a wide variety of medical conditions and is most commonly used to treat pain in the forms of neuropathy, low back pain, labor pain, temporomandibular joint dysfunction, and migraine headache. 50 Acupuncture is used for a broad range of conditions and has been the focus of controlled clinical studies of its use for allergic rhinitis, asthma, and skin itching (i.e., eczema).51,52

Acupuncture for Allergic Rhinitis.  A study of 5237 allergic rhinitis patients included 487 randomly assigned to acupuncture and 494 to control therapy, and 4256 were in a nonrandomized acupuncture group (i.e., two groups receiving acupuncture).53 The results of this and other trials suggest that treating patients with allergic rhinitis in routine care with acupuncture may enhance clinically relevant quality-of-life

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parameters and reduce antihistamine requirements.52–56 In a 2011 prospective, randomized, sham-controlled trial of semi–self-administered ear acupressure for persistent allergic rhinitis, acupressure resulted in beneficial changes in global symptom scores, sneezing scores, and activities related to quality of life.54 Other than some ear discomfort, the ear acupressure was considered safe. A 2018 randomized controlled trial of acupuncture versus sham (plus rescue medication) and rescue medication alone in 414 patients with seasonal allergic rhinitis demonstrated reduction in the number of days requiring rescue antihistamine use while improving rhinitisspecific quality of life and symptoms. The commonly used acupuncture points include large intestine (LI) meridian points LI4, LI11, LI20 bilateral, and Yintang.55 A 2015 systematic review and metaanalysis included 13 full papers and a total of 2365 participants, including 1126 as treatment group and 1239 as control group. Compared with the control group, the acupuncture treatment group exerted a significant reduction in nasal symptom scores (p = 0.03), medication scores (p = .0005), and serum immunoglobulin E (IgE) (p < 0.00001).56 No serious reactions were related to the acupuncture treatment. This metaanalysis supported the potential role of acupuncture as a safe complementary therapy in the treatment of allergic rhinitis particularly if other therapies are not well tolerated. Questions remain regarding added time and cost and how to compare the cost-effectiveness of different therapies.

Acupuncture for Asthma.  As acupuncture use grows in Europe and the United States, there are increasing numbers of publications addressing its use in asthma. Scheewe and colleagues57 conducted a RCT to evaluate the immediate effects of acupuncture as an add-on therapy to inpatient rehabilitation of children and adolescents with bronchial asthma. The intervention group (n = 46) received acupuncture; the control group (n = 47) did not. Both groups received asthma sports, climate therapy, and behavioral training. In the acupuncture group, peak expiratory flow measurement variability improved significantly (P < .01) more than that of the control patients. Moreover, the acupuncture group had significant reduction of perceived anxiety. However, the other lung function tests did not show differences between the groups. A similar finding was reported by Choi and coworkers58 in a randomized pilot study. Ngai and colleagues59 examined the effect of transcutaneous electrical nerve stimulation applied over acupuncture points or acupoints (Acu-TENS) on forced expiratory volume in patients with asthma, after exercise. Adjunctive Acu-TENS therapy appeared to reduce the decline of forced expiratory volume in 1 second (FEV1) after exercise training in patients with asthma.59 A 2011 sham-acupuncture controlled study did not show any significant benefit for acupuncture, but many questions were raised about technique, and this study did not address the applicability of these findings to variations of acupuncture-like methods.60 Although controversy continues regarding the cost-benefit ratio for acupuncture therapy integrated with traditional therapies, there is a 2014 study from Germany that randomized patients to a waiting list or immediate acupuncture supplemental care (10.8 mean sessions) and demonstrated improvement in the patient quality of life (quality-adjusted life years [QALYs]) despite additional cost.61 Despite higher initial costs for the acupuncture group (which also had more medical visits prior to treatment), the incremental cost-effectiveness ratio (ICER), reflecting the additional costs associated with realizing 1 additional QALY, improved and supported a cost-effective treatment assessment with a probability of 86.5% to 88.5%, particularly since benefits persisted 3 months beyond the treatment course completion.61

Atopic Dermatitis and Acupuncture.  Itch is a major symptom of atopic dermatitis. In 2010, Pfab and colleagues reported results of a

blinded, randomized, placebo-controlled, crossover trial of 30 subjects with atopic eczema showing that acupuncture significantly reduced the mean itch intensity and mean wheal and flare size in response to house dust mite or grass pollen skin-prick testing compared with the control group.62 In 2012, the same group reported a patient- and examinerblinded, randomized, placebo-controlled crossover trial of acupuncture compared with oral antihistamine for type 1 hypersensitivity itch and skin response in 20 adults with atopic dermatitis.63 The mean itch intensity and flare size after acupuncture was significantly lower compared with the antihistamine group receiving sham acupuncture (P