Endocrine and Organ Specific Autoimmunity (Molecular Biology Intelligence Unit) [1st ed.] 1570595380, 9781570595387, 9780585408767

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R.G. LANDES

C O M PA N Y

EISENBARTH MIU

MEDICAL INTELLIGENCE UNIT

13

George S. Eisenbarth

Molecular Mechanisms of Endocrine and Organ Specific Autoimmunity

13

Molecular Mechanisms of Endocrine and Organ Specific Autoimmunity R.G. LANDES CO M PA N Y

MEDICAL INTELLIGENCE UNIT 13

Endocrine and Organ Specific Autoimmunity George S. Eisenbarth, M.D., Ph.D. University of Colorado Health Sciences Center Denver, Colorado, U.S.A.

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MEDICAL INTELLIGENCE UNIT 13 Endocrine and Organ Specific Autoimmunity R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1999 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-538-0

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Endocrine and organ specific autoimmunity / [edited by] George S. Eisenbarth. p. cm. -- (Medical intelligence unit) Includes biographical references and index ISBN 1-57059-538-0 (alk. paper) 1. Autoimmunity--molecular aspects. 2. Autoimmune diseases--Molecular aspects. 3. Endocrine glands--Diseases--Immunological aspects. I. Series. [DNLM: 1. Autoimmune Diseases--immunology. 2. Autoimmunity. 3. Autoantigens. 4. Autoantibodies. 5. Organ Specific. 6. Endocrine Diseases-immunology. WD 305 M7184 1998] QR188.3.M675 1998 616.97'8--dc21 DNLM/DLC 98-40838 for Library of Congress CIP

MEDICAL INTELLIGENCE UNIT 13 PUBLISHER’S NOTE

Endocrine and Organ Specific Autoimmunity

R.G. Landes Company produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are publishedof within 90 to 120 days of receipt of University Colorado the manuscript. WeHealth wouldSciences like to thank Centerour readers for their continuing interestDenver, and welcome any comments Colorado, U.S.A. or suggestions they may have for future books.

George S. Eisenbarth, M.D., Ph.D.

Michelle Wamsley Production Manager R.G. Landes Company

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

CONTENTS 1. Immunobiology of Autoimmunity .......................................................... 1 Donald Bellgrau and George S. Eisenbarth Introduction ............................................................................................. 1 Genetics of the Immune Response ......................................................... 2 Antigen Presentation ............................................................................... 4 Antigen Recognition ................................................................................ 5 Tolerance .................................................................................................. 6 Cytokines/Th1 and Th2 T Cells .............................................................. 7 Apoptosis ................................................................................................. 8 Unusual T Cells and T Cell Ligands ....................................................... 9 “Etiologic” Classification of Autoimmunity .......................................... 9 “Effector” Classification of Autoimmunity .......................................... 10 “Natural” History of Autoimmunity .................................................... 11 Therapy of Autoimmunity .................................................................... 12 Conclusion ............................................................................................. 14 2. Autoimmune Polyendocrine Syndrome Type I (APECED) ................. 19 Jaakko Perheentupa and Aaro Miettinen Disease Components ............................................................................. 19 What Are the Genes Determining Susceptibility? ............................... 23 Nature of the Immune Defect ............................................................... 23 Target Autoantigens .............................................................................. 25 What Activates or Inhibits Autoimmunity? ......................................... 33 Are There Assays Which Allow for Prediction of the Disorder or Its Components? ........................................................................... 34 Therapy .................................................................................................. 36 3. Autoimmune Polyendocrine Syndrome Type II ................................... 41 Maria J. Redondo and George S. Eisenbarth Introduction ........................................................................................... 41 Autoimmune Polyendocrine Syndrome Type II ................................. 41 Other Endocrine Syndromes ................................................................ 52 Conclusion ............................................................................................. 54 4. Oncogenic Autoimmunity ...................................................................... 63 Robert P. Friday and Massimo Pietropaolo Introduction ........................................................................................... 63 Paraneoplastic Autoimmune Disorders of the Peripheral Nervous System .................................................... 65 Paraneoplastic Autoimmune Disorders of the Central Nervous System ......................................................... 69 Other Paraneoplastic Disorders Associated with Oncogenic Autoimmunity ....................................................... 71 Theoretical Aspects and Basics Questions on Oncogenic Autoimmunity .......................................................... 72 Anti-Tumor Therapy in Autoimmune Paraneoplastic Disorders ...... 76 Concluding Remarks ............................................................................. 77

5. Celiac Disease .......................................................................................... 85 Fei Bao, Marian Rewers, Fraser Scott and George S. Eisenbarth Introduction ........................................................................................... 85 Pathology ............................................................................................... 85 What Genes Determine Susceptibility? ................................................ 86 What Environmental Factors Initiate or Inhibit Development of Celiac Disease? ............................................................................... 88 What Are the Effector Molecules? ........................................................ 90 How Does Avoidance of Dietary Gluten Interrupt Disease Pathogenesis? ..................................................................................... 91 Conclusion ............................................................................................. 92 6. Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases ................................................... 97 Horia Vlase and Terry F. Davies Introduction ........................................................................................... 97 Genetics .................................................................................................. 99 Immunopathogenesis .......................................................................... 101 Potential Pathogenic Mechnisms ....................................................... 112 Apoptosis ............................................................................................. 114 Other Precipitating Factors ................................................................. 115 New Insights into Immunologic Diagnosis and Treatment .............. 116 Conclusions ......................................................................................... 118 7. Insulin Autoimmune Syndrome (IAS, Hirata Disease) ...................... 133 Yasuko Uchigata and Yukimasa Hirata Introduction ......................................................................................... 133 Insulin Autoimmune Syndrome as the Third Leading Cause of Spontaneous Hypoglycemia in Japan ........................................ 133 Onset Age, Sex Distribution, and Duration of Hypoglycemia of 226 Japanese IAS Patients Registered in Japan from 1970 to 1996 ........................................................................... 133 Drug Exposure Ahead of Development of IAS and Associated Diseases .................................................................. 135 Clinical Features of IAS Patients Out of Japan .................................. 135 Insulin in the Sera of the Patients with IAS ....................................... 135 Two Groups of IAS Defined by Clonality of Insulin Autoantibodies ............................................................... 135 Critical Amino Acids for IAS Polyclonal Responder and Importance of DR Gene Products in the Presentation of Human Insulin Antigen ............................. 137 Patients with Graves’ Disease Who Developed IAS Possess HLA-B62/Cw4/DR4 Carrying DRB1*0406 .................................... 138 Different Amino Acids for IAS Monoclonal Responder ................... 138

Possible Role of the Specific Amino Acids on the DR b-chain in IAS Pathogenesis ......................................................................... 139 Natural History of IAS ........................................................................ 146 A Novel Concept of Type VII Hypersensitivity Introduced by Insulin Autoimmune Syndrome (Hirata’ s Disease) ................ 146 8. Type I Diabetes Mellitus ....................................................................... 149 Eiji Kawasaki, Ronald G. Gill and George S. Eisenbarth Introduction to Diabetes ..................................................................... 149 What Are the Genes for Autoimmune Type 1 Diabetes? .................. 152 What Are the Triggering/Preventive Factors? .................................... 159 What Are the Target Autoantigens? ................................................... 160 What Are the Effector Mechanisms? .................................................. 165 Therapies for the Prevention of Beta Cell Destruction ..................... 167 Conclusion ........................................................................................... 172 9. Etiopathogenesis of Myasthenia Gravis (MG)..................................... 183 Jean-François Bach, Ana Maria Yamamoto, Farid Djabiri and Henri-Jean Garchon Introduction ......................................................................................... 183 Anti-AChR Autoantibodies ................................................................ 183 Genetics ................................................................................................ 185 The Driving Role of the AChR ............................................................ 188 Mechanisms of the Loss of Self Tolerance to AChR: The Role of the Thymus .................................................................. 190 10. Multiple Sclerosis .................................................................................. 195 Konstantin Balashov and Howard L. Weiner Introduction ......................................................................................... 195 Epidemiology and Genetics ................................................................ 195 Magnetic Resonance Imaging (MRI) ................................................. 196 Oligoclonal Immunoglobulins in the CSF ......................................... 197 Viruses .................................................................................................. 197 Immunopathological Mechanisms (Fig. 10.1) ................................... 197 Immune-Mediated Mechanisms of Myelin Destruction .................. 202 Immunotherapy ................................................................................... 202 Remyelination ...................................................................................... 204 Conclusion ........................................................................................... 204 11. Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy ........................................................................ 213 Aaron I. Vinik, Gary L. Pittenger, Zvonko Milicevic, Jadranka Knezevic-Cuca Introduction ......................................................................................... 213 The Normal Barrier to Immune-Mediated Nerve Destruction ........ 214

12. Ocular Autoimmunity .......................................................................... 249 Luiz V. Rizzo and Robert B. Nussenblatt Introduction ......................................................................................... 249 The Animal Model of Uveitis .............................................................. 251 Immunopathogenesis .......................................................................... 251 Immunogenetics .................................................................................. 254 Immunotherapy ................................................................................... 256 Index ................................................................................................................ 269

EDITORS George S. Eisenbarth, M.D., Ph.D. University of Colorado Health Sciences Center Denver, Colorado, U.S.A. Chapters 1, 3, 5, 8

CONTRIBUTORS Jean-François Bach, M.D., D.Sc. Hôpital Necker Paris, France Chapter 9

Farid Djabiri, Ph.D. Hôpital Necker Paris, France Chapter 9

Konstantin Balashov Center for Neurologic Disease Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts Chapter 10

Robert P. Friday Division of Immunogenetics Department of Pediatrics University of Pittsburgh School of Medicine Rangos Research Center Children’s Hospital of Pittsburgh Pittsburgh, PA Chapter 4

Feí Bao, M.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado Chapter 5

Henri-Jean Garchon, M.D. Ph.D. Hôpital Necker Paris, France Chapter 9

Donald Bellgrau, Ph.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado Chapter 1

Ronald G. Gill, Ph.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado Chapter 8

Terry F. Davies, M.D., F.R.C.P. Division of Endocrinology and Metabolism Department of Medicine Mount Sinai School of Medicine New York, New York Chapter 6

Yukimasa Hirata, M.D. Diabetes Center Tokyo Women’s Medical College Tokyo, Japan Chapter 7

Eiji Kawasaki, M.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado, U.S.A. Chapter 8 Jadranka Knezevic-Cuca, M.S. The Diabetes Institutes Departments of Internal Medicine and Pathology/Anatomy Eastern Virginia Medical School Norfolk, Virginia, U.S.A. Aaro Miettinen, M.D., Ph.D. The Haartman Institute, and HD-Diagnostics University of Helsinki and Helsinki University Hospital Helsinki, Finland Chapter 2 Zvonko Milicevic, M.D. Institut Vuk Vrhovac Zagreb, Croatia Chapter 11 Robert B. Nussenblatt, M.D. Clinical Immunology Section Laboratory of Immunology National Eye Institute, NIH Bethesda, Maryland, U.S.A. Chapter 12 Jaakko Perheentupa, M.D. The Hospital for Children and Adolescents University of Helsinki and Helsinki University Hospital Helsinki, Finland Chapter 2

Massimo Pietropaolo, M.D. Division of Immunogenetics Department of Pediatrics University of Pittsburgh School of Medicine Rangos Research Center Children’s Hospital of Pittsburgh Pittsburgh, Pennsylvania, U.S.A. Chapter 4 Gary L. Pittenger, Ph.D. The Diabetes Institutes Departments of Internal Medicine and Pathology/Anatomy Eastern Virginia Medical School Norfolk, Virginia, U.S.A. Chapter 11 Maria J. Redondo, M.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado, U.S.A. Chapter 3 Marian Rewers, M.D., Ph.D. Barbara Davis Center for Childhood Diabetes University of Colorado Health Sciences Center Denver, Colorado, U.S.A. Chapter 5 Luiz V. Rizzo, M.D., Ph.D. Clinical Immunology Section Laboratory of Immunology National Eye Institute, NIH Bethesda, Maryland, U.S.A. Chapter 12

Fraser Scott, Ph.D. Health Canada University of Ottawa Ottawa, Ontario,Canada Chapter 5 Yasuko Uchigata, M.D. Diabetes Center Tokyo Women’s Medical College Tokyo, Japan Chapter 7 Horia Vlase, M.D. Division of Endocrinology and Metabolism Department of Medicine Mount Sinai School of Medicine New York, New York, U.S.A. Chapter 6

Aaron I. Vinik, M.D., F.A.C.P., Ph.D. The Diabetes Institute Departments of Internal Medicine and Pathology/Anatomy Eastern Virginia Medical School Norfolk, Virginia, U.S.A. Chapter 11 Howard L. Weiner, M.D. Center for Neurologic Disease Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 10 Ana Maria Yamamoto, Ph.D. Hôpital Necker Paris, France Chapter 9

CHAPTER 1

Immunobiology of Autoimmunity Donald Bellgrau and George S. Eisenbarth

Introduction

A

utoimmunity can be defined as immune responses directed against self-antigens and an autoimmune disorder as a disease which results from autoimmunity.1,2 The cells of the immune system with “antigenic specificity” are B and T lymphocytes.3 An essential feature of the above definition of autoimmunity is the targeting of self molecules by B and T lymphocytes. These B and T lymphocytes have on their surface similar receptors (immunoglobulin or T cell receptors) and both cell types are clonally expanded during an active immune response. Thus at the molecular level, autoimmune disorders are likely to depend upon clonally expanded self reactive B or T lymphocytes and/or normal numbers of such clones with disordered regulation. A variety of pathogenic mechanisms can contribute to both clonal expansion and disordered regulation, or both, but for the above restricted definition of autoimmune disease, reactivity with self is an essential disease feature. In contrast a number of inflammatory diseases appear to not depend upon specific recognition of self-antigens, but rather to result from immunologic reactivity to, for example, pathogens with collateral damage of self-tissues. In that a number of these conditions can resemble autoimmune diseases, there is always the possibility for any autoimmune disorder that such a pathogen remains to be discovered. Distinctions can certainly be blurred. An immune response to a pathogen or an inciting agent such as nickel (nickel allergy) may result in specific responses to self, and these self-responses can be responsible for tissue damage or dysfunction, rather than the response to the “pathogen”. In this latter case either elimination of the self-response or the pathogen, might “cure” the disease, one of the major goals of autoimmunity research. Further blurring occurs in that potential “pathogens” can also be a component of “self” as for example in the case of retroviruses or transgenic models where “foreign” antigen expression is molecularly induced. An immune response to self-antigens is not an infrequent occurrence.4 Following subcutaneous administration of a series of proteins, ranging from a small molecule such as human insulin to human Factor VIII, most individuals respond with the production of self-reactive antibodies.5,6 In the case of insulin, the antibodies rarely interfere with the biologic function of insulin, while in the case of Factor VIII, blocking of bioactivity is a significant clinical problem. Thus, the current view is that everyone possesses B and T lymphocytes capable of reacting with self-antigens. In autoimmune diseases, this self-reactivity is of a magnitude and quality (e.g. affinity of antibodies) which makes it qualitatively distinct. These qualitatively different responses mean that with specific and sensitive autoantibody assays one can often predict future disease in asymptomatic individuals.7-9 The same quality of tests are not yet currently available for the T cell arm of the immune response. Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

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As the understanding of immune function and of autoimmunity have advanced over the past two decades, investigators and clinicians studying different autoimmune diseases are usually asking similar questions. These questions include: What “genes” determine susceptibility? What environmental factors initiate or inhibit autoimmunity? What are the target autoantigens? What are the effector molecules? What is the “natural” history of the disorder? What therapies will interrupt disease pathogenesis? With the wide diversity of autoimmune disorders, the study of different autoimmune diseases provides likely answers to many of theses questions. Our current understanding for given questions differs markedly depending upon the specific disease. It is likely that where specific answers are available they are the best guide in the search for answers to the same but unanswered question for another disease. For example, a very common autoimmune disease, celiac disease, is dependent upon the ingestion of the wheat protein gliadin.9-12 Removal of gliadin reverses a series of gastrointestinal, skin and dental manifestations and leads to a loss of autoantibodies which react with the molecule transglutaminase.13 Ubiquitous dietary proteins and peptides thus must be considered a potential “trigger” for autoimmune disorders where the “trigger” remains elusive. In addition to a common framework of questions relating to disparate autoimmune disorders for many diseases, one can divide the disorder into a series of “stages” beginning with genetic susceptibility, followed by “triggering” events, followed by the first evidence of autoimmune B or T lymphocyte responses, followed by increasing tissue damage which ultimately leads to clinically recognized disease.14 Multiple factors are likely to underlie progression through these various stages. The ability to provide stage specific prognostic information tests the relevance and accuracy of given immunologic assays. This has been particularly apparent for the field of type I diabetes, where individuals with increasing risk of type I diabetes can be identified first genetically, and then by the expression of a series of anti-islet autoantibodies, and finally with subclinical abnormalities of insulin secretion.7,14 The ability to predict type I diabetes, though it has allowed the design of diabetes prevention trials, is far from complete. An alternate view in the field of type I diabetes was that immunologic abnormalities were too variable or too often present in normal individuals to have prognostic relevance. This view initially reflected the utilization of assays with limited disease specificity, and for the most part such assays have been abandoned.15 Perhaps we will best understand the immunologic basis of a disorder when we can both predict disease and safely intervene with immunologic therapies to prevent or cure the disease. In that few “autoimmune” diseases are currently prevented or cured there is obviously much that we do not understand.

Genetics of the Immune Response At the basis of essentially all autoimmunity appears to be genetic susceptibility.16 One of the major accomplishments in basic immunology has been discovery of genes which control immune responses and the molecular and crystallographic characterization of the products of these genes.17-22 T lymphocytes as opposed to B lymphocytes recognize only fragments of antigens presented on the surface of cells. The T cell receptor reacts with peptides which are presented by what are termed class I and class II histocompatibility antigens.22-24 Figure 1.1 illustrates the crystallographic structure of a class II molecule. It resembles a hot dog bun with a cleft which binds peptides. The bound peptides are the “hot dog”. The T cell receptor reacts with amino acid residues from both the “top” of the class II molecule and the peptide.

Immunobiology of Autoimmunity

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HLA class II molecules are composed of highly polymorphic regions as well as conserved or nonpolymorphic regions. The T cell bears an antigen specific receptor that binds to the peptide in the cleft of the class II MHC molecule and a CD4 coreceptor that binds a nonpolymorphic, nonpeptide containing region of the class II molecule. The combination of receptor and coreceptor binding generates a CD4+ T cell that is class II restricted.25 In contrast, T lymphocytes which bear the CD8 molecule bear an antigen specific receptor that reacts with peptides presented in the groove of class I molecules and a CD8 coreceptor that binds a nonpolymorphic region of the class I MHC molecule. T cells cannot know when they are born whether they will be class I or class II restricted. This restriction occurs only after an antigen specific receptor is expressed. Therefore immature T lymphocytes within the thymus express both CD4 and CD8 molecules. These double positive thymocytes eventually choose to express only one coreceptor and in so doing progress to the more mature, single positive phenotype that dominates in the periphery. Genes for both class II and class I molecules are within the major histocompatibility complex which is on the short arm of chromosome 6.26 In man, there are three class II molecules termed DP, DQ and DR.27 The DQ molecule is homologous to mouse I-A, and the DR molecule is homologous to I-E. Class II molecules are made up of two chains, termed α and β. Both the α and β chains of DQ are polymorphic. The chains vary in amino acid sequence between different humans. Each unique nucleotide sequence of an α and β chain is given a unique identifying number (e.g. DQA1*0102, DQB1*0602).27 Each of the two sixth chromosomes codes for one DQα and one DQβ chain in “cis” (from the same chromosome) and additional combinations of α and β chains are often possible in “trans”. Only the β chain of DR molecules is polymorphic. Thus while it takes two numbers to define the

Fig. 1.1. Model of DQ molecule DQA1*0401, DQB1*0402 with position 56 and 57 of the DQB chain highlighted. This molecule is associated with high type 1 diabetes risk despite having aspartic acid at position 57 of DQβ chain with leucine at position 56 rather than proline suggesting one potential molecular explanation for this exception.

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amino acid sequence of a DQ molecule (α and β), it requires only one number to define a DR molecule (e.g. DRB1*1501).28 Polymorphic class II genes, and especially DQ and DR alleles are not randomly associated and show extensive linkage disequilibrium. Thus DRB1*1501 is almost always associated with DQA1*0102, DQB1*0602. There are however important exceptions, and, for example we have studied a family with three children with type I diabetes all of who expressed DRB1*1501, with DQA1*0102, DQB1*0502.29 As will be apparent from the chapters that follow, specific polymorphisms of the above histocompatibility molecules are important for disease susceptibility. For example DQA1*0102, DQB1*0602 provides dominant protection from type I diabetes.30,31 DQA1*0102, DQB1*0602, though protective for type I diabetes, is associated with risk for multiple sclerosis. Though DQ alleles often show the strongest association with autoimmune disorders, an exception is the class I molecule HLA B27 which is associated with ankylosing spondylitis.32 In addition, risk of disease can be influenced by accompanying DR molecules, and with extensive linkage disequilibrium within the major histocompatibility complex multiple genes may influence disease risk. In Figure 1.1, two amino acids of a class II molecule have been highlighted. These amino acids are aspartic acid at position 57 of the DQβ chain and leucine at position 56 of the DQB chain. The specific amino acids lining the pocket of histocompatibility molecules determine the binding of peptides and thus the ability to present peptides to T lymphocytes. These two amino acids are highlighted to illustrate the complexity of disease associations. Aspartic acid at position 57 is usually associated with DQ molecules with a low risk for type I diabetes.33-37 In the DQB chains termed DQB1*0401 and DQB1*0402 (the former common in Japan38 and the latter present in Western Caucasoid populations), there is a leucine at position 56 rather than a proline as found in all other DQB chains. DQB1*0401 and DQB1*0402 are associated with a high risk for type I diabetes despite having aspartic acid at position 57.

Antigen Presentation Peripheral T lymphocytes only recognize peptide presented within the context of class I or class II MHC molecules. The MHC restriction of T cells is accomplished in the thymus where only T cells that bear antigen specific receptors with affinity for MHC molecules presented on thymic tissue are provided with survival signals to continue in the maturation process. Mechanisms must be present to process proteins to peptides, to colocalize peptides with histocompatibility molecules intracellularly, to load peptides into the groove of histocompatibility molecules and to translocate peptide loaded class II and class I molecules to the cell surface.39 Class I molecules usually contain peptides from intracellular proteins (e.g. viral derived peptides) while class II molecules usually contain peptides derived from extracellular and membrane components. A number of genes within the major histocompatibility complex are essential for appropriate peptide processing and presentation such as the TAP genes (Transporter Associated with Antigen Processing), 40 LMP2 and LMP7 (proteosome subunits) which play a specific role in generation of peptides presented by class I histocompatibility molecules, and HLA-DM (acts as a class II molecular “chaperone” assisting in the loading of class II molecules). TAP gene polymorphisms may contribute to disease susceptibility but their effects are often obscured by much stronger disease associations of DQ polymorphisms.40 Antigen processing and provision of peptide “loaded” class I and class II molecules on the cell surface constitute only a fraction of the molecular machinery of antigen presentation. There are a series of T cell “coreceptors” (e.g. CD4, CD8) and accessory molecules on the cells on which antigens are presented which influence T cell responses. These accessory molecules are predominantly present on professional antigen presenting cells including B

Immunobiology of Autoimmunity

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lymphocytes.41 Because dendritic cells, macrophages and B lymphocytes all expressed class II molecules, the expression of class II molecules became widely held as a generic marker for professional antigen presenting cells. One hypothesis for the generation of organ specific autoimmunity posited that endocrine organs expressed class II molecules and such expression led to autoimmunity directed at the class II positive cell.42 The simplest version of this hypothesis was directly tested with the transgenic induction of class II expression by islet β-cells. Autoimmunity did not develop and class II positive β-cells were not even capable of stimulating proliferation of T lymphocytes. In retrospect it is now apparent that antigen presenting cells such as macrophages, dendritic cells, and B lymphocytes, express a series of costimulatory molecules whose presence is essential for a positive signal to T lymphocytes. In the absence of these costimulatory signals “anergy” rather than activation is likely. These accessory molecules include B7-1 and B7-2 (CD80/CD86)43 molecules binding to (CTLA4) CD28, LFA1 and ICAM, CD40 and CD40L (e.g. CD40 ligand on B lymphocytes).44 The interactions are complex and synergistic and for example CD40/CD40L interaction increases synthesis of cytokines such as IL-12 and the expression of B7 by antigen presenting cells.

Antigen Recognition All specific immunity is provided by T lymphocytes and B lymphocytes using their respective homologous receptors, T cell receptors and immunoglobulin.39,45 The major paradigm underlying specific immune responses is the “clonal” selection theory. This theory posits that a large number of clones of B lymphocytes (and as now known T lymphocytes) exist with unique receptors. Upon receptor engagement with antigen, lymphocytes proliferate to produce expanded clones of cells which underlie immunologic memory. The major objection to this theory was that it required the presence of millions to billions of different receptors (immunoglobulin molecules) and there were not enough genes in a mammal to encode such an array of receptors with the axiom of “one gene-one protein”. In retrospect, it is obvious that the axiom was in error. The remarkable diversity of the immune system is created by a combinatorial process in which variable gene segments are combined to form either the α and β chains of T cell receptors or the heavy and light chains of immunoglobulin. Immunoglobulins are made up of a heavy and light chain, each with variable and constant regions. As a precursor B lymphocyte matures the heavy chain is created by selection from an array of variable gene segments and combined with diversity (D) and joining (J) region genes to produce mature variable (V) regions of immunoglobulins. The light chain combines V region segments with J segments in a similar manner. Given the large number of variable gene segments, nucleotide additions and subtractions at the sites of joining of V, D and J segments, the presence of two chains, and the hypermutation of immunoglobulin genes of proliferating B lymphocytes (particularly in germinal centers of lymph nodes), the ability to respond to “all” antigens is readily appreciated. T cell receptor genes function in a directly analogous manner. V(D)J recombination creates the β chain and VJ recombination creates α chain. In contrast to immunoglobulin, T cell receptor genes do not undergo hypermutation of variable regions. The mutation of immunoglobulin underlies “affinity maturation” of humoral immunity, where with repeated antigenic exposure the affinity of antisera increases. T cells also differ from B cells in that their antigen specific receptors are restricted to recognizing antigen presented in the groove of an MHC molecule. Therefore T cell receptors and the cells on which they are presented are committed to the recognition of cell bound (presented on antigen presenting cells) antigen while B cell immunoglobulin can bind to soluble antigen. Within both immunoglobulin genes and T cell receptor gene segments are what are termed hypervariable regions. These regions directly interact with antigen and are present in complementarity determining regions (CDRs). The three major complementarity

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determining regions of the T cell receptor (CDR1, CDR2, CDR3), and in particular CDR3 overlie the peptide in the groove of major histocompatibility complex molecules.18,19 Given high affinity interaction of the T cell receptor with peptide and MHC, a signal through a tyrosine kinase cascade is conveyed to the T cell which can result in any one of a large number of responses depending upon the accessory signals received. A productive immune response results in the secretion of a series of lymphokines, upregulation of cell surface molecules, proliferation and activation.

Tolerance Operationally the term tolerance referred to the acceptance of a tissue graft by an immunocompetent organism which would normally undergo rejection. Thus immunosuppression with drugs such as cyclosporine A and acceptance of grafted tissue does not meet the definition of immune tolerance. It was recognized very early in the development of the field of immunology that mechanisms existed to allow the “distinction” between “self ” and “nonself ” or as recently suggested between dangerous and nondangerous “molecules” in that individuals could rapidly destroy erythrocytes from different individuals but did not destroy their own erythrocytes. Understanding the manner by which “tolerance” develops and in particular the manner by which “tolerance” is lost in autoimmune disorders is at the cutting edge of current immunologic research. T cell receptors and immunoglobulin at a molecular level cannot distinguish self molecules from nonself molecules. In addition, the great majority of peptides within major histocompatibility complex molecules are derived from self proteins. Thus the basic machinery for recognition does not make the distinction between self and nonself which is obviously essential for survival and avoidance of “horror autotoxicus”. Because the developing immune system would develop lymphocytes with receptors for antigens expressed by the organism (self), a mechanism would need to be in place to insure that self reactive lymphocytes were eliminated or negated in some way. A classic experiment by Billingham, Brent and Medawar demonstrated that the immune system learns self tolerance and that this education to distinguish self from nonself is best accomplished when the immune system is immature. Medawar was awarded the Nobel Prize for his work on “neonatal” tolerance. He and his collaborators showed that the immune system of a developing mouse could be taught to perceive cell bound antigens from genetically incompatible animals as “self” if they were presented to the animal when the immune system was immature or in this case when the cells were presented to neonatal animals. Like any good system it is best that redundancies exist to guard against the failure of any one component. The immune systems approach to self tolerance is no exception. It appears that a multiplicity of mechanisms underlie the control of damaging immune reactivity to self.

Central Deletion This is probably the major mechanism eliminating the bulk of autoreactive T cells. The term “central” refers to the thymus where the great majority of T lymphocytes differentiate and die. It is thought that within the thymus T lymphocytes whose T cell receptors fail to react with self MHC plus peptide die and those whose T cell receptors strongly interact with self MHC plus peptide are deleted. Thus the only T lymphocytes which mature and leave the thymus have intermediate reactivity with self-peptides and self MHC molecules. These T cells are poised to react strongly with foreign peptides and self MHC. In a similar manner it appears that B lymphocytes whose immunoglobulin reacts with self membrane antigens within the bone marrow are deleted. Not all molecules are expressed within the thymus and thus T lymphocytes are likely to escape from the thymus with high affinity receptors for a

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7

number of tissue specific molecules. Of note an increasing number of “tissue” specific molecules have been found to be expressed within the thymus, such as insulin.46,47 Though expressed at very low levels, the levels of insulin are apparently sufficient to induce central tolerance.

Peripheral Tolerance In a number of experimental systems engagement of T cell receptors by antigens results in a lack of T cell response rather than stimulation. In particular, activation of T lymphocytes by superantigens, infusion of large amounts of soluble antigen, inhibition of accessory molecular function of antigen presenting cells all can lead to the deletion of T cells or induction of anergy within T lymphocytes. Anergy is a state in which T lymphocytes are refractory to usual productive antigen stimulation in the absence of the addition of exogenous lymphokines.48 Anergy can result from T cell antigen recognition in the absence of costimulation. Costimulation is the subsequent signals sent to lymphocytes after engaging antigen with their antigen specific T cell receptor. A simplified way of defining antigen receptor engagement and costimulation is to ascribe to the former status as signal one and the latter as signal two. Signal one and or two are central to the issue of tolerance. In the thymus when a T cell expresses a receptor that can bind an antigen present in the thymus the nature of the antigen presenting cell, i.e. the nature of the costimulation, has a major effect on the life or death of the cell. If the antigen is not presented on a cell that can provide costimulation, signal one alone appears to be sufficient to permit the maturing thymocyte to continue in the maturation process. However if signal one is combined with signal two the thymocyte receives a death signal and dies. What becomes of the T cell that does not bind a self antigen in the thymus because the antigen is only presented in the periphery? First, this thymocyte has already been selected and therefore has matured. When it now receives signal one in the periphery it dies. Therefore T cells run a maturation gauntlet where they are only permitted to continue in maturation if they respond appropriately to antigen presented in the context of costimulation. Signal one plus costimulation leads to the deletion of self-reactive thymocytes and signal one without costimulation leads to anergy in the periphery. In addition a number of experimental forms of “tolerance” can be induced which appear to depend on active regulatory cells. Such cells can be transferred to a secondary host and limit T cell reactivity. An additional mechanism by which a tissue may escape destruction is termed immunologic “ignorance’ and occurs when T cells which can target a given antigen fail to interact with the cells expressing the antigen. This form of tissue acceptance can be abrogated in experimental models by providing additional costimulatory molecules.

Cytokines/Th1 and Th2 T Cells A large and growing series of cytokines have been cloned and characterized. These molecules underlie many of the interactions between lymphocyte subsets and between lymphocytes and the cells they regulate or target. Antibodies to lymphokines, antagonists of lymphokines and the lymphokines themselves have profound effects both in vitro and in vivo. Lymphokines such as interferon (when utilized for the therapy of viral infections) are associated with the induction of autoimmunity, particularly thyroid autoimmunity. Interleukin-2 is the major stimulatory growth factor for T lymphocytes. Molecules such as tumor necrosis factor have opposite effects upon the induction or suppression of autoimmunity depending upon the age of the animal treated.49 A series of lymphokines can down regulate immune responses and include TGF-β, IL-10 and IL-4. CD4 lymphocytes in the mouse have been divided into two major categories termed Th1 and Th2 which differ by the cytokines they produce.50,51 IL-10 and IL-4 are typically utilized as markers of Th2 cells. Th2 T cells enhance humoral immunity and IgE

8

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responses, while Th1 T cells characteristically produce IFN-γ and are associated with cellmediated immunity. Differences in T cell responses are characteristic of several strains of mice and influence the ability to resist certain parasitic infections. A dominant hypothesis is that induction of a “shift” to TH2 cells will ameliorate cell-mediated autoimmunity, while Th1 responses enhance autoimmunity. The importance of a shift to TH2 cells and the important role of costimulation in this process was recently demonstrated with transgenic NOD mice. NOD mice develop diabetes spontaneously. CD28 is a coreceptor on T cells that binds the B7 molecules on antigen presenting cells. CD28 engagement of B7 is a costimulatory signal. What would happen if diabetogenic NOD mouse T cells were denied the CD28-B7 costimulatory interaction? To address this question NOD mice were bred with CD28 knockout mice. Diabetes was exacerbated and the results interpreted to indicate that without costimulation T cells could never shift to the Th2 phenotype and consequently autoimmunity was favored. This provides experimental support for a central role of costimulation in the Th1/Th2 paradigm and its association with autoimmunity. It is likely that Th1 and Th2 rigid divisions are an oversimplification as some Th2 clones transfer autoimmune destruction. Nevertheless the paradigm is a useful starting point for characterizing a variety of immune responses. T lymphocytes can induce activation of other cells such as macrophages which secrete cytokines such as IL-1, and free radicals such as nitric oxide. Such interactions link specific and nonspecific (inflammatory) immune functions.52

Apoptosis The manner by which lymphocytes are destroyed within the thymus or following nonproductive antigen interactions as well as the mechanisms by which T lymphocytes destroy target cells is the subject of increased interest.53,54 In part this interest resulted from the characterization of two immunologically abnormal mouse strains. One strain has a mutation in the molecule Fas (lymphoproliferative lpr mice)55,56 and the other, a mutation in the molecule gld or FasL (Fas ligand). These strains both have marked lympho-accumulation and lupus-like autoimmunity. The Fas molecule is a member of the TNF (tumor necrosis factor) family of receptors and engagement of Fas by FasL results in programmed cell death (apoptosis). In that T lymphocytes express Fas and can be induced to express Fas ligand, they are subject to Fas mediated fratricide and suicide. Fas and Fas ligand are also involved in peripheral tolerance. T cells that are chronically exposed to antigen express Fas and they or their cohorts can also express Fas ligand. The Fas ligand interaction with Fas terminates these activated T cells. Fas/Fas ligand interactions ensure that the immune response is controlled. The failure of these interactions coincides with the lymphoproliferative disease observed in the lpr and gld mice. Interestingly Fas/Fas ligand interactions appear to be more important in the periphery than in the thymus. This most likely reflects the inability of thymocytes to express Fas ligand rather than an insensitivity of thymocytes to killing via Fas. A remarkable recent discovery utilizing these strains of mice concerns the concept of privileged sites, namely tissues which are able to avoid immunologic destruction.53,57-59 Both Sertoli cells of the testis and corneal cells, express FasL and the ligand is an essential feature of their ability to be transplanted across histocompatibility barriers. Sertoli cells and cornea from FasL deficient gld in contrast to these cells from normal mice are destroyed when transplanted. Normal testis transplanted into Fas deficient (lpr) mice is also destroyed. These studies led to a “stampede” to introduce FasL into islet cells to protect against graft rejection and autoimmunity. Depending upon the levels of Fas expressed by β-cells, the results varied widely. It appears that β-cells can be induced to express Fas and therefore be destroyed in

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the presence of FasL.60 Expression of FasL on myoblasts mixed with transplanted islet cells in contrast promoted islet acceptance.61 Another very important observation resulting from further studies of Fas and FasL is that many T cell clones are unable to destroy islet β-cells unless they have intact Fas. Thus these T cell clones utilize Fas to mediate destruction. Studies of thyroid autoimmunity also implicate Fas as an important effector pathway of disease.62

Unusual T Cells and T Cell Ligands The predominant circulating T lymphocytes express standard T cell receptors (α/β) and characteristically respond to antigens presented within the groove of class I or class II histocompatibility molecules. In addition there are other fascinating T cell subsets and antigens, including γ/δ T cells and NK1.1 T lymphocytes.63,64 The function of neither of these classes of T lymphocytes is currently known. NK1.1 T lymphocytes are so-called because they share cell surface molecules with natural killer cells.65,66 They are extremely unusual in that all express a TCRα chain consisting of Vα14-Jα28 with highly conserved invariant junctional sequences (the sequences between standard Vα and Jα T cell receptor chains is a position where nucleotides are usually added or deleted). NK1.1 cells produce high levels of IL-4.63 These T cells react with a nonpolymorphic class I molecule termed CD-1.63 What is particularly interesting concerning these T cells is that they can skew immune responses toward “Th2” and are deficient in several models of autoimmunity. “Superantigens” are molecules frequently produced by pathogenic bacteria which bind to class II molecules and are able to activate whole classes of T cells bearing selective T cell receptor β-chains.67-70 The hallmark of superantigen stimulation is the expansion (or deletion) of certain T cells expressing specific Vβ chains independent of α-chain expression or the junctional regions of the T cell receptor. In autoimmune disorders, “skewing” of T cell receptor β-chain utilization with heterogeneous junctional T cell receptor sequences is often utilized as indirect evidence for an infectious etiology.69,71

“Etiologic” Classification of Autoimmunity Table 1.1 lists a series of factors linked to the initiation and perpetuation of autoimmunity. Even for a single disorder such as myasthenia gravis the disease can be initiated by a drug such as penicillamine,72 by a tumor (thymoma),73-77 and most often the initiating events are unknown (idiopathic).78 It is thus clear that there is no single factor responsible for initiating all autoimmune diseases. For most common autoimmune disorders only a small subset of the disease can be ascribed to known initiating factors. In contrast, for both celiac disease10,11 and insulin autoimmune syndrome (Hirata’s disease),79,80 the disorder is ascribed to specific initiating factors (the wheat protein gliadin for celiac disease and the drug Methimizole for insulin autoimmune syndrome). Knowledge of such initiating factors can immediately suggest effective therapies (e.g. the avoidance of wheat proteins for celiac disease). Prior to the realization of the importance of diet, many children with celiac disease “starved” to death due to their intestinal lesions. The presence of a large group of idiopathic disorders suggests that their initiating factors are currently unknown. The category of oncogenic autoimmunity is one of the most interesting with its classic disease associations.81-85 For example the rare occurrence of autoimmune cerebellar degeneration should prompt a search for occult ovarian tumors.84 The link between the tumor and the autoimmune disorder appears to be the specific presence in ovarian cancers associated with cerebellar degeneration of a protein also found in neurons. It is likely that the tumor with the specific protein creates a milieu in which self proteins are presented to the immune system. Therapies directed at the tumor such as surgical removal or medical therapy

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Table 1.1. Etiologic classification of autoimmunity ETIOLOGY

DISEASE EXAMPLE

FACTOR

Food

Celiac disease

Wheat gliadin

Drug

Myasthenia gravis Insulin autoimmune syndrome Thyroiditis

Penicillamine Methimizole Amiorodirone

Cytokine

Thyroiditis

Interferon α

Infection

Rheumatic heart disease Type 1 diabetes

Streptococci Rarely congenital rubella

Tumor (oncogenic)

Myasthenia gravis Cerebellar degeneration Stiff man syndrome

Thymoma Ovarian carcinoma Breast cancer

Idiopathic

Type I diabetes Myasthenia gravis Stiff man syndrome Thyroiditis Etc.

(somatostatin analogue therapy of thymoma) may ameliorate the secondary autoimmune disorder.

“Effector” Classification of Autoimmunity The major distinction between autoimmune disorders is between those diseases predominantly mediated by autoantibodies and those predominantly mediated by T lymphocytes. A characteristic of antibody mediated disease is the ability to transfer the disease with serum antibodies and a corollary of such transfer is the frequent observation of a neonatal form of the disorder secondary to transplacental transfer of immunoglobulin. Thus neonatal hyperthyroidism of offspring of a mother with Graves’ disease as well as neonatal hypothyroidism are both associated with autoantibodies reacting with the thyrotropin (TSH) receptor.86-90 The autoantibodies of Graves’ disease stimulate the receptor while in the case of hypothyroidism the antibodies block receptor function. There are a number of classic experiments in both animals and man whereby disease transfer with immunoglobulin has been demonstrated. Perhaps the most “famous” is the induction of thrombocytopenia in the investigator himself with the infusion of serum from patients with immune mediated thrombocytopenia prurpura. Myasthenia gravis (anti-acetlycholine receptor autoantibodies) and bullous pemphigoid can also be induced with the transfer of immunoglobulin. In contrast to diseases where transplacental passage of antibodies results in a self-limited disease (neonatal heart block associated with a subset of lupus autoantibodies is permanent) most diseases thought to be T cell mediated do not have a transient neonatal disorder. For example expression of a series of anti-islet autoantibodies are characteristic of type I diabetes mellitus, and many infants of diabetic mothers are born with such autoantibodies

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(which can be present for as long as 9 months in the infant). Neonatal diabetes associated with such antibodies is not observed. In disorders where active cellular destruction is present such as type 1 diabetes and Addison’s disease, the role of autoantibodies in disease pathogenesis is poorly defined. The distinction between antibody mediated and cell mediated disorders is only an approximation in that for most autoimmune disorders both arms of the immune system may be important for initiating and maintaining autoimmunity. For example high affinity IgG autoantibodies as found in type 1 diabetes require T lymphocyte help for their generation. In addition a B lymphocyte reacting with a specific autoantigen is likely to be the most efficient antigen presenting cell, presenting peptides of the antigen to T lymphocytes.

“Natural” History of Autoimmunity The development of many autoimmune disorders, particularly those characterized by tissue destruction can be divided into a series of stages (Fig. 1.2) usually beginning with genetic susceptibility.14 Genetic susceptibility is followed by the presence of detectable immunologic abnormalities even though organ dysfunction may either not be present or may be subclinical. In type I diabetes mellitus it appears that the majority of insulin secreting cells must be destroyed before hyperglycemia develops. In patients followed to the development of diabetes, a long prodrome is present with most individuals followed to diabetes demonstrating progressive loss of the ability to secrete insulin. The extent of this loss of insulin secretion is such that many individuals fail to respond with insulin secretion to intravenous glucose stimulation for more than a year and as much as five years prior to the development of overt diabetes. In the NOD mouse model of type I diabetes there is evidence for both chronic islet beta cell destruction beginning as early as three weeks of age,

Fig. 1.2. Hypothetical stages for the development of a destructive autoimmune disorder illustrated by the development of type 1 diabetes mellitus.

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Endocrine and Organ Specific Autoimmunity

and acceleration of beta cell destruction for individual animals within weeks of the onset of their diabetes which usually varies between 15 and 52 weeks.91,92 It is likely that autoimmune islet infiltration and destruction is a “spotty” process (e.g. in man and animals in the same pancreas normal islets, islets with all beta cells destroyed and islets with infiltration and remaining beta cells can all be present at the same time). Perhaps the best example of the spottiness of autoimmunity is vitiligo with autoimmune destruction of melanocytes within the skin resulting in white skin patches. The course of progression of vitiligo is highly variable but almost always is characterized by localized regions of the skin lacking all melanocytes with adjoining normal skin. A major goal of both clinical immunologic research and basic research is to be able to predict disease and to understand the factors which either accelerate or moderate disease progression. Prediction of type I diabetes is now accurate enough to allow the design of large clinical trials for the prevention of the disease and is likely to improve with better understanding of disease pathogenesis. Often the best predictive models combine determination of immunologic abnormalities and subclinical organ dysfunction. This is true for type I diabetes (autoantibodies and loss of first phase insulin secretion), Addison’s disease (anti-adrenal autoantibodies and elevations of adrenocorticotropic hormone (ACTH)) and hypothyroidism (antithyroid autoantibodies and minor elevations of thyrotropin (TSH)). Prediction of disease in individuals with only genetic susceptibility is obviously much less secure. For most autoimmune disorders concordance for disease of identical twins ranges between 30 and 70%, with concordance for “autoimmunity” even higher. Recent studies in type 1 diabetes suggest that there may be T lymphocyte (e.g. NK1.1 T cells) differences between twins who do and who do not progress to diabetes. It is likely that when we understand the factors which distinguish identical twins who are discordant for any of the autoimmune diseases our knowledge of pathogenesis will have been greatly enhanced.

Therapy of Autoimmunity There are relatively few drugs available for the therapy of autoimmune disorders,93 but there is a wealth of novel therapies for such diseases which are effective in animal models. Thus it is possible that over the next decade therapies for a number of autoimmune disorders will be revolutionized. Table 1.2 lists a series of therapies which either are currently in use for specific autoimmune disorders or for which experimental studies (Expt) are ongoing either in man or experimental animal models. The most effective immunodulatory drug, Rhogam, is used not to treat an actual autoimmune disease but as therapy to prevent sensitization of mothers to their infants who are Rh incompatible. This therapy which consists of administration of human anti-Rh antibodies is given at the time of birth of an Rh incompatible infant. It is thought that Rhogam administration prevents sensitization of the mother by Rh antigens because the antigens are coated by immunoglobulin. Human immunoglobulin is standard therapy for Kawasaki’s disease a disease of unknown etiology. It is also used to treat individuals who have developed high titer anti-factor VIII autoantibodies. These autoantibodies block the action of factor VIII.5 One hypothesis for the effectiveness of such infusions is that the immunoglobulin preparations contain anti-idiotypic antibodies. Most therapies for autoimmunity in man rely upon drugs with immunosuppressive or anti-inflammatory properties. With these drugs serious side effects are likely and a major goal has been the development of medications with less long-term toxicity. Glucocorticoids in amounts necessary to control autoimmunity often create disorders of the same severity as the diseases they are meant to treat. Autoimmune hepatitis is one disease where early therapy with glucocorticoids can be lifesaving.94

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Table 1.2. Examples of Therapies for Autoimmunity Therapeutic Class

Example

Disease

Eliminate inciting factor

Wheat gliadin Streptococcal infection Rubella vaccination

Celiac disease Rheumatic heart disease Rare subset type I DM

Immunosuppression

Mycophenolate mofetil Cyclosporine A Expt anti-CD4 antibody

Psoriasis Psoriasis Rheumatoid arthritis

Anti-Inflammatory

Glucocorticoids

Autoimmune hepatitis

Cytokine Based

Interferon beta

Multiple sclerosis

“Autoantigen” Based

Copolymer

Multiple sclerosis

Oral “Tolerance”

Expt uveal antigens Expt insulin Expt collagen

Uveitis Type I diabetes Rheumatoid arthritis

Antigen “Vaccination”

Expt insulin Expt GAD65

Type I diabetes animals Type I diabetes animals

T Cell Receptor Vaccination

Expt Vbeta8.2

Multiple sclerosis

Altered Peptide Ligands MHC Blocking Peptides

Insulin peptides Peptide ligands

Expt type I DM animals Expt type I DM animals

Immunomodulatory

Rhogam

Rh incompatibility

Unknown

Pooled immunoglobulin

Anti-factor VIII Antibodies

Expt=Experimental

An important principal in the therapy of transplant rejection is the utilization of a combination of drugs with synergistic effects. For example, the inhibitor of guanosine synthesis, mycophenolate mofetil, when replacing azothioprine in transplantation regimens has allowed the reduction of glucocorticoid dosage to almost nontoxic levels, while preserving graft function. Both mycophenolate mofetil and cyclosporine A have dramatic effects on psoriatic lesions. Despite effectiveness of drugs such as cyclosporine A in preventing the beta cell destruction associated with type I diabetes, it is likely that long-term immunosuppression with such a potent agent will not be acceptable because of the risk of malignancy (as well as nephrotoxicity for cyclosporine A). For most autoimmune disorders development of safer and more effective therapies is a major goal. For example many experimental autoimmune disorders can be prevented or

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Endocrine and Organ Specific Autoimmunity

ameliorated with therapies directed at antigen recognition or the immunologic response to given antigens. There are many therapies under active investigation which interfere with the recognition of antigens presented in the context of class I and class II histocompatibility antigens. Thus investigators are studying altered antigenic peptides,25,95-97 antibodies to accessory molecules such as CD4, antibodies to costimulatory molecules such as CD40 ligand, peptides which bind with high affinity to specific class II molecules, and antibodies to selected T cell receptors,98 or immunization with T cell receptors themselves. Perhaps one of the most intriguing ways of preventing autoimmunity is the utilization of self antigens administered as a “vaccine”.98,99 Such vaccination may prove to be a double edge sword, but studies in animal models such as the diabetes prone NOD mouse indicate that a single injection of, for example, a peptide of insulin (amino acids B9 to B23 of insulin) can lead to prolonged protection from diabetes.

Conclusion There are a wealth of unanswered questions concerning autoimmunity, but also fortunately there is an expanding set of answers available for many of these questions for specific autoimmune diseases. It is likely that the search for a fundamental understanding of autoimmunity is at the threshold of an autocatalytic period whose end result will hopefully be the prevention or amelioration of a vexing group of human diseases.

Acknowledgments This work was supported in part by grants from the National Institutes of Health (R37 DK32083, GSE; U01 DK46639, GSE; R01 AI39213, GSE; R01 DK32493 GSE; and R01 DK48805, DB), the Juvenile Diabetes Foundation International (196009, DB) and Bayer Corporation (GSE).

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59. Cheng J, Zhou T, Liu C et al. Protection from fas-mediated apoptosis by a soluble form of the fas molecule. Science 1994; 263:1759-1762. 60. O’Brien BA, Harmon BV, Cameron DP et al. Apoptosis is the mode of β-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 1997; 46:750-757. 61. Lau HT, Yu M, Fontana A et al. Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice. Science 1996; 273:109-112. 62. Giordano C, Stassi G, De Maria R et al. Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto’s thyroiditis. Science 1997; 275:960-963. 63. Chen YH, Chiu NM, Mandal M et al. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 1997; 6:459-467. 64. Jullien D, Brossay L, Sieling PA et al. CD1: Clues on a new antigen-presenting pathway. Res Immunol 1996; 147:321-328. 65. Nishimura T, Santa K, Yahata T et al. Involvement of IL-4-producing Vbeta8.2+ CD4+ CD62L-CD45RB- T cells in nonMHC gene-controlled predisposition toward skewing into T helper type-2 immunity in BALB/c mice. J Immunol 1997; 158:5698-5706. 66. Davodeau F, Peyrat MA, Necker A et al. Close phenotypic and functional similarities between human and murine alphabeta T cells expressing invariant TCR alpha-chains. J Immunol 1997; 158:5603-5611. 67. Jardetzky TS, Brown JH, Gorga JC et al. Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen. Nature 1994; 368:711-718. 68. Swaminathan S, Furey W, Pletcher J et al. Crystal structure of staphylococcal enterotoxin B, a superantigen. Nature 1992; 359:801-805. 69. Conrad B, Weldmann E, Trucco G et al. Evidence for superantigen involvement in insulin-dependent diabetes mellitus etiology. Nature 1994; 371:351-355. 70. Kim J, Urban RG, Strominger JL et al. Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule HLA-DR1. Science 1994; 266:1870-1874. 71. Leung DYM, Travers JB, Giorno R et al. Evidence for a streptococcal superantigen-driven process in acute guttate psoriasis. J Clin Invest 1995; 96:2106-2112. 72. Garlepp MJ, Dawkins RL, Christiansen FT. HLA antigens and acetylcholine receptor antibodies in penicillamine induced myasthenia gravis. BMJ 1983; 286:338-340. 73. Mygland A, Tysnes O, Aarli JA et al. IgG subclass distribution of ryanodine receptor autoantibodies in patients with myasthenia gravis and thymoma. J Autoimmun 1993; 6:507-515. 74. Geuder KI, Marx A, Witzemann V et al. Genomic organization and lack of transcription of the nicotinic acetylcholine receptor subunit genes in myasthenia gravis-associated thymoma. Lab Invest 1992; 452:458. 75. Maggi G, Casadio C, Cavallo A et al. Thymoma: results of 241 operated cases. Ann Thorac Surg 1991; 51:152-156. 76. Piccolo G, Martino G, Moglia A et al. Autoimmune myastenia gravis with thymoma following the spontaneous remission of stiff-man syndrome. Ital J Neurol Sci 1990; 11 (2):177-80. 77. Souadjian JV, Enriquez P, Silverstein MN et al. The spectrum of diseases associated with thymoma. Arch Intern Med 1974; 134:374-379. 78. Marx A, Wilisch A, Schultz A et al. Pathogenesis of myasthenia gravis. Virchows Arch 1997; 430:355-364. 79. Uchigata Y, Kuwata S, Tsushima T et al. Patients with graves’ disease who developed insulin autoimmune syndrome (Hirata disease) possess HLA-Bw62/Cw4/DR4 carrying DRB1*0406. J Clin Endocrinol Metab 1993; 77:249-254. 80. Hirata Y. Methimazole and insulin autoimmune syndrome with hypoglycemia. Lancet 1983; 99:182-184. 81. Palmieri G, Lastoria S, Colao A et al. Successful treatment of a patient with a thymoma and pure red-cell aplasia with octreotide and prednisone. N Engl J Med 1997; 336:263-265.

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Endocrine and Organ Specific Autoimmunity

82. Sakai K, Gofuku M, Kitagawa Y et al. A hippocampal protein associated with paraneoplastic neurologic syndrome and small cell lung carcinoma. Biochem Biophys Res Commun 1994; 199:1200-1208. 83. Anhalt GJ, Kim S, Stanley JR et al. Paraneoplastic pemphigus: an autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990; 323:1729-1735. 84. Hetzel DJ, Stanhope R, O’Neill BP et al. Gynecologic cancer in patients with subacute cerebellar degeneration predicted by anti-purkinje cell antibodies and limited in metastatic volume. Mayo Clin Proc 1990; 65:1558-1563. 85. Kornguth SE. Neuronal proteins and Paraneoplastic Syndromes. N Engl J Med 1989; 321 No. 23:1607-1608. 86. Smith BR, McLachlan SM, Furmaniak J. Autoantibodies to the thyrotropin receptor. Endocr Rev 1988; 9:106-121. 87. Cho BY, Chung JH, Shong YK et al. A strong association between thyrotropin receptorblocking antibody-positive atrophic autoimmune thyroiditis and HLAL-DR8 and HLADQB1*0302 in Koreans. J Clin Endocrinol Metab 1993; 77:611-615. 88. Costagliola S, Swillens S, Niccoli P et al. Binding assay for thyrotropin receptor autoantibodies using the recombinant receptor protein. J Clin Endocrinol Metab 1992; 75:1540-1544. 89. Singer PA. Will postpartum recurrence of Graves’ hyperthyroidism become a thing of the past? J Clin Endocrinol Metab 1992; 75:6-10. 90. Takasu N, Yamada T, Takasu M et al. Disappearance of thyrotropin-blocking antibodies and spontaneous recovery from hypothyroidism in autoimmune thyroiditis. N Engl J Med 1992; 326:513-518. 91. Katz JD, Wang B, Haskins K et al. Following a diabetogenic T cell from genesis through pathogenesis. Cell 1993; 74:1089-1100. 92. Shimada A, Charlton B, Taylor-Edwards C et al. β-cell destruction may be a late consequence of the autoimmune process in nonobese diabetic mice. Diabetes 1996; 45:1063-1067. 93. Halloran PF. Immunosuppressive agents in clinical trials in transplantation. Am J Med Sci 1997; 313:283-288. 94. Czaja AJ. Autoimmune hepatitis: Evolving concepts and treatment strategies. Dig Dis Sci 1996; 40:435-456. 95. Gaur A, Boehme SA, Chalmers D et al. Amelioration of relapsing experimental autoimmune encephalomyelitis with altered myelin basic protein peptides involves different cellular mechanisms. J Neuroimmunol 1997; 1-11. 96. López-Moratalla N, Ruiz E, López-Zabalza MJ et al. A common structural motif in immunopotentiating peptides with sequences present in human autoantigens. Elicitation of a response mediated by monocytes and Th1 cells. Biochem Biophys Acta 1996; 1317:183-191. 97. Kersh GJ, Allen PM. Structural basis for T cell recognition of altered peptide ligands: A single T cell receptor can productively recognize a large continuum of related ligands. J Exp Med 1996; 184:1259-1268. 98. Howell MD, Winters ST, Olee T et al. Vaccination against experimental allergic encephalomyelitis with T cell receptor peptides. Science 1989; 246:668-670. 99. Daniel D, Wegmann DR. Protection of nonobese diabetic mice from diabetes by intranasal or subcutaneous administration of insulin peptide B-(9-23). Proc Natl Acad Sci USA 1996; 93:956-960.

Autoimmune Polyendocrine Syndrome Type I (APECED)

19

CHAPTER 2

Autoimmune Polyendocrine Syndrome Type I (APECED) Jaakko Perheentupa and Aaro Miettinen

T

his disease is known by many names, most commonly as autoimmune polyglandular syndrome type I (APS-I). We prefer the name autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED), because it reminds of the three groups of components of this disease. “Syndrome,” implying a consistent set of manifestations not necessarily uniform in etiology, is here in our opinion a misnomer, because as an autosomal recessive condition, this is a disease uniform in etiology but widely variable in manifestation. This review is mostly based on our experience with all the 78 patients documented by 1996 to have been given the diagnosis of APECED in Finland (Perheentupa unpublished).1,2

Disease Components Endocrinopathies (Table 2.1, Fig. 2.1) Hypoparathyroidism appeared in 85% of our patients, at the age of 1.6 to 43 years. In some patients its progression from latent to severe was observed over some months. Addison’s disease developed in 72%, at 4.2 to 41 years. In 11 patients a 0.5 to 5.8-year interval was documented in the appearance of overt clinical deficiencies of cortisol and aldosterone, their order being random. In some patients transition from normal secretion of cortisol to its severe deficiency occurred within a few months. In others this often took several years, with fluctuation in the secretory capacity3 between defined stages of impairment.4 In Iranian Jewish patients, Addison’s disease appears to develop later than in the Finnish patients. At the age of 20 years only 3 of 17 patients had it in contrast to 34 of 58 Finnish patients.5 Hypogonadism was present in 60% of our female patients >14 years old and in 14% of male patients >20 years old; it appeared by the ages of 33 and 38 years, respectively. In all but one patient it was primary, a male had secondary hypogonadism. Half the females with ovarian atrophy failed in pubertal development, the others had secondary amenorrhea. In a few patients we demonstrated with successive gonadorelin tests slow destruction of the ovaries.3 A 26-year-old man is on record with infertility caused by antisperm antibodies in association with normal endocrine function of the testes.6 IDDM appeared in 18% of our total of 78 patients, at the age of 4.1 to 45 years. Its highest incidences were 0.014 cases per patient year at the age of 15.0 to 20.0 years, and 0.03 at 40.0 to 50.0 years. Primary atrophic hypothyroidism appeared in 6% of our patients, at 18 to 32 years. Enlargement of the thyroid gland was never observed. None of our patients developed hyperthyroidism, but one patient with Graves’ disease is on record.7 We know of only three patients with central

Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

Endocrine and Organ Specific Autoimmunity

20

diabetes insipidus.8,9 Three of our patients have developed growth hormone deficiency. Two patients with ACTH deficiency are on record.10

Other Organ-Specific Autoimmunity (Table 2.1) Alopecia appeared in 27% of our patients, at 5 to 28 years; its highest incidence was 0.024 at 5.0 to 10.0 years. Hairless patches developed first, progressing to almost complete lack of hair, and eye and body hair. Two patients have normal hair growth after a period of partial alopecia. Vitiligo developed in 13% of our patients, by 15 years of age. Initially small unpigmented skin patches tended to grow slowly. Gastric parietal cell failure with vitamin

Table 2.1. Prevalence of Components of APECED at Different Ages in the Finnish series (Perheentupa, unpublished) Age, years

No. of patients in age group No. of females

1

2

5

10

15

20

30

40

50

60

All

78

78

78

77

70

58

41

17

7

3

78

40

40

40

39

35

28

23

9

2

1

40

percent of patients No. of endocrine components per patient 0 100 95 65 26 ≥1 0 5 35 74 ≥2 0 0 4 30 ≥3 0 0 0 3 ≥4 0 0 0 0 ≥5 0 0 0 0 Hypoparathyroidism Adrenal failure Diabetes mellitus Parietal-cell atrophy Hypothyroidism Ovarian failure1 Testicular failure2

0 0 0 0 0

5 0 0 0 0

33 4 3 0 0

Candidiasis 18 31 55 Alopecia 0 0 3 Vitiligo 1 1 1 Kerotopathy 1 4 9 Hepatitis 1 3 3 Intestinal 0 0 5 malabsorption Enamel hypoplasia3 Tympanic membrane calcification4 Nail dystrophy5

64 40 3 3 0

89 14 8 19 8 6

14 86 50 19 1 0

10 90 50 34 5 0

2 98 61 44 15 0

0 100 65 24 6 0

0 100 35 18 6 14

0 100 67 33 0 0

0 100 67 38 9 1

77 59 4 4 0 37

79 59 10 9 3 54 7

83 68 22 12 7 61 22

71 65 6 18 12 44 37

71 72 43 57 14 50 29

100 67 33 67 0 50

85 72 18 15 6 60 17

95 33 14 26 1 5

100 34 12 32 0 5

100 41 12 24 0 6

100 29 14 29 0 14

100 67 0 33 0 33

100 27 13 22 13 10

94 23 11 23 9 7

1Calculated for females ≥15 years of age. 2Calculated for males ≥20 of age. n=344, 442, 550 patients.

77 33 52

Autoimmune Polyendocrine Syndrome Type I (APECED)

21

Fig. 2.1. Incidence rates of six components of APECED in the Finnish series of 78 patients.

B12 malabsorption appeared in 15% of our patients, at 6 to 48 years. The known complications of pernicious anemia, epithelial cell dysplasia, hyperplastic polyps, and gastric cancer seem not to have been reported. Hepatitis developed in 13% of our patients at 0.7 to 17 years. It was chronic in all but two patients, who died of fulminant hepatitis and liver failure at 7 and 17 years. Periodic fat malabsorption appeared in 10% of our patients, at 2.5 to 27 years. Three of them recovered normal intestinal function. The pathogenesis is unknown, but an autoimmune lesion of the small intestinal mucosa is not excluded. Of ocular disease some may be autoimmune.8

Other Autoimmunity Dermal vasculitis was part of the presenting picture in the 0.7-year-old boy with hepatitis, and developed in another patient at the age of 1.2 years. These patients had 2 to 7-day episodes of fever recurring 4 to 5 times monthly. An urticaria-like rash appeared during the episodes. Plasma IgG was polyclonally increased to 23-42 g/l. One of the two had small amounts of circulating immunocomplexes. Rheumatoid arthritis developed in a severe rapidly progressive form in one patient in her late twenties. Two of our patients developed terminal renal failure and received kidney transplants, which functioned without undue complications. Some others have impaired glomerular filtration. The etiology of this problem is unknown. Periodic hypercalcaemia may contribute and an autoimmune component is not excluded.

Clinical Immune Defect Chronic or periodic oral candidiasis affected all our patients. It commonly appeared during the first year of life; the incidence then slowly decreasing but continuing to the third decade (Fig. 2.1). One of our patients had her first bout of (oral) candidiasis only in her twenties. The picture ranges from intermittent or continuous angular cheilosis to acute inflammation of most of the oral mucosa, hyperplastic chronic candidiasis with thick white

22

Endocrine and Organ Specific Autoimmunity

coating of the tongue, and atrophic disease with scant coatings and a scarred thin mucosa.11 Chronic mucosal candidiasis is carcinogenic. Four of our patients developed epithelial carcinoma of the oral mucosa and three died of it. Candidiasis should be carefully suppressed by good dental care and oral hygiene, with systemic and local antimycotics. Candidal esophagitis causing retrosternal pain was confirmed by esophagoscopy in four patients, one of them had a stricture. Eleven others had periods of retrosternal pain, which resolved within a few days of oral anticandidal therapy. Periodic abdominal pain, meteorism and diarrhea occurred in some patients who had strong candidal growth in fecal cultures; the symptoms promptly subsided during systemic anticandidal therapy. Nail and skin candidiasis was common. Vulvovaginal candidiasis developed at puberty. Affected nails were darkly discolored, thickened or eroded. Candidal eczema of the hands tended to develop if the hands were frequently wetted and could spread to the face. A case of generalized dermal candidiasis has been reported,12 but we are not aware of any patient with APECED having suffered from deep candidal infection. Candidiasis appears to be markedly less prevalent in the Iranian Jewish (17% of 23 patients) than in the Finnish patients (100% of 78).5 Tuberculin anergy was a feature of most of our patients.13 Whether it is associated with abnormal susceptibility to tuberculosis is unclear. Anergy to candidal antigen was also common.

Ectodermal Dystrophy Dental enamel hypoplasia affected the permanent teeth of most of our patients, but some others had faultless teeth.11,14 Deciduous teeth were never affected. Commonly, all the permanent teeth have hypoplastic enamel, either transverse hypoplastic bands alternating with zones of well-formed enamel or hypoplasia of all enamel. Enamel hypoplasia is not associated with hypoparathyroidism. Pitted nail dystrophy was another characteristic of most patients. It appeared to be unrelated to nail candidiasis: one third of patients with nail dystrophy had no ungual candidiasis. The pits were 0.5-1 mm in diameter. This minor abnormality may help to identify patients with otherwise insufficient diagnostic features. Atrophy of the tympanic membranes was common, and 33% of our patients had conspicuous calcium deposits in the membranes without a history of middle ear disease.

Ocular Disease Keratopathy developed in 22% of our patients, at 2.0 to 16 years. It was the first or part of the first manifestation in 14%. In one patient is was the sole manifestation for 20 years. If not carefully treated it may cause permanent impairment of visual acuity and even total blindness.15 The occurrence of keratopathy was not associated with hypoparathyroidism. Two patients had iritis, another cyclitis, and two others optic atrophy.

Number of Components The number of disease components, excluding the features of ectodermal dystrophy, was 0-3 (most commonly 0) at the ages of 1.0 and 2.0 years, 0-4 (1) at 5.0 years, 0-5 (3) at 10.0 years, 0-7 (3) at 15.0, 1-8 (3) at 20, 1-7 (4) at 30, 2-6 (4) at 40, 4-6 (5) at 50 and 5-6 at 60 years. The number of endocrine deficiencies increased similarly by age (Table 2.1). The triad of candidiasis, hypoparathyroidism and Addison’s disease, often regarded as pathognomonic for APECED, developed in 58% of our patients, by the age of 3.4 to 43 (median 10.2) years.

Timing and Sequences of the Components The disease developed in infancy in many patients, and most were symptomatic by the age of 5 years (Table 2.1). Five patients had first (non-endocrine) manifestations only at 10

Autoimmune Polyendocrine Syndrome Type I (APECED)

23

to 15 years, and in 5 patients the first endocrine manifestation only at 20 to 35 years. A clear nonendocrine manifestation appeared before the first endocrinopathy in 78% of the patients.1 It was oral candidiasis in 60%, intestinal malabsorption in 9%, keratopathy in 4%, and vitiligo, alopecia or hepatitis in 4%. Their interval to the first endocrinopathy was 0.1 to 33 (median 4.1) years. Of the other 22% of patients, the initial manifestation was hypoparathyroidism in 19% (with simultaneous oral candidiasis in 10%) and either hypoparathyroidism or oral candidiasis in two (sequence uncertain). Thus candidiasis was part of the first manifestation in at least 70%.1 Of the endocrine components the first appeared at 1.6 to 35 (median 6.5) years, the second at 4.1 to 45 (11.0) years, the third at 5.5 to 41 (15.4), the fourth at 15 to 35 (19), and the fifth at 22 to 44 (26) years. The first endocrinopathy was hypoparathyroidism in 69% and Addison’s disease in 23% of the 78 patients; both were diagnosed at the same time in 6%. Of the 54 patients whose first endocrinopathy was hypoparathyroidism alone, 61% later developed Addison’s disease. Of the 18 patients whose first endocrinopathy was Addison’s disease alone, 39% have developed hypoparathyroidism, after an interval of 3-16 years, the others remain euparathyroid already for 5.4-34 (median 15) years. Overall, patients with Addison’s disease as the first component other than candidiasis developed significantly fewer further components (mean 0.6) than the others (2.2).1 Steatorrhea was part of the initial manifestation in 6% of our patients. One of these patients remains euparathyroid 22 years after onset of the intestinal problem, which continued for 15 years. In patients with hypoparathyroidism and steatorrhea, watery diarrhea tended to recur whenever hypocalcemia developed; a vicious circle easily develops because fat malabsorption renders the control of hypocalcemia difficult. Other patients with the same degree of hypocalcemia maintained normal fat absorption.

What Are the Genes Determining Susceptibility? APECED is unique among the well known autoimmune diseases in being caused by one single gene pair, homozygosity for defect in the AIRE gene (autoimmune regulator) located on chromosome 21q22.3.16,17 It is rare in most populations, but has been enriched in the Finnish1 and the Iranian Jews.5 This enrichment is based on a founder effect. Study of the marker phenotypes indicates presence of one major mutation, the Finnish one being responsible for about 90 % of the cases in Finland.18 A study of the marker haplotypes in affected families from eight other European countries and Israel indicated locus homogeneity but a spectrum of haplotypes and, presumably, of mutations.18 Several observations attest to contribution of other genes (more likely than environmental factors) in determining the clinical picture, but no such genes have been identified. Firstly, some observations suggest differences between affected sibships. Our Finnish family series includes 8 sets of 2-4 affected siblings. Adrenocortical failure was present in all affected members of 6 sets, and absent in all affected members of 2. Secondly, the disease appears to differ between the two relatively large ethnic series. Candidiasis was markedly less prevalent in Iranian Jewish (17% of 23) patients5 than in Finnish patients (100% of 68).1 Also, in the Iranian Jews Addison’s disease appears to develop later than in the Finnish. At the age of 20 years only 18% of 17 patients had it5 in contrast to 59% of 78 Finnish patients (Perheentupa, unpublished). Perhaps pertinent to both family and ethnic differences, among the more than 70 Finnish patients none has diabetes insipidus, whereas in an English family both affected sons had it.8

Nature of the Immune Defect As the product of the APECED gene is unknown, the nature of the immune defect remains elusive. Both clinical and laboratory evidence suggest that the defect is in the T cells.

24

Endocrine and Organ Specific Autoimmunity

The chronic candidiasis is a hallmark of T cell defects, both congenital and acquired.19 Cutaneous anergy or weak delayed type hypersensitivity reactions to PPD13,20 and candida antigens21-23 in the patients with APECED likewise suggest T cell defect. Patients with major T cell defects typically suffer from serious infections, viral, parasitic, and intracellular. Severe measles was described in some patients with APECED, 24 but none of our numerous patients had such infections, and neither have live BCG and Vaccinia, given for immunization to most of our patients, caused generalized infections. A common feature of immunosuppression is increased incidence of malignancies. Four of our patients developed epithelial carcinoma of the oral mucosa, thought to be secondary to the chronic oral candidiasis. Interestingly, one of these patients was receiving cyclosporine A after renal transplantation. The patients apparently mount normal immune responses to protein antigens such as tetanus and diphtheria toxins, indicating normal T cell help. Thus the T cell defect is limited. No lymphocyte abnormality shared by all patients has been detected in vitro. In a family with 3 affected and 5 nonaffected children mixed leukocyte reaction revealed decreased lymphocyte responses to polyclonal T cell activators and Candida antigens in affected and nonaffected family members alike.24 A test of suppressor T cell activity showed impairment in two affected and one nonaffected sibling. Slightly elevated T cell counts, decreased B cell counts, and varying degrees of defective lymphocyte reactivity to polyclonal T cell activators or LPS, Candida antigens, PPD, and staphylococcal protein A are on record.20 In a study of lymphocyte surface markers (CD2, CD3, CD4, CD8, surface Ig) and responses to polyclonal activators in 42 of our patients, no single abnormality appeared characteristic. However, in line with previous reports, variable abnormalities were found significantly more often in the patients (52%) than in controls (14%) (Ahonen et al, unpublished). These observations speak for disordered immunoregulation.24 Studies with modern methods are needed of the functional subsets of T cells (a/b, g/d, TH1, TH2, NK 1.1), their cytokine production patterns, T and B cell receptors and adhesion receptors. No clear defect has been observed in these patients’ humoral immunity, although some of them have supranormal circulating B cell, immunoglobulin or IgE levels, or selective IgA-deficiency.20,24 Of the 42 of our patients studied some had elevated plasma levels of IgG and IgM, but only 1 patient had a subnormal IgA level, and none had supranormal levels of IgE (Ahonen et al, unpublished). Despite their cutaneous anergy to Candida antigens and inability to eradicate C. albicans from body surfaces, the patients have high levels of (protective) antibodies against the major Candida antigens.25 Also, the apparently normal vaccination responses and the high levels of various autoantibodies of IgG class against several epitopes of different protein autoantigens speak for intact antibody formation. Resistance to many intracellular bacteria and parasites is linked to induction of TH1 lymphocyte responses, in particular to the macrophage-activating cytokines INF-γ and TNF-α. For viral infections natural killer cells, cytotoxic CD8 lymphocytes and neutralizing antibodies are also needed.26 For the formation of effective neutralizing antibodies, B cells need help from TH1 cells. These TH1 functions seem to proceed normally in patients with APECED. In experimentally induced chronic infections and inflammations, chronic TH1 responses may result in damaging autoimmunity, if not restricted by TH2 cells or the cytokines normally suppressing TH1 responses.27-29 The normal unresponsiveness to self may at least partially depend on immunosuppressive cytotoxic TH2 or other regulatory T cells, while the activation of proinflammatory TH1 cells is essential for tissue destruction.26 We know nothing of the TH1/TH2 balance in APECED, but many features of this disease, including the high titres of autoantibodies, and the high levels of IgE in some patients, are compatible with the concept of regulatory imbalance. It is noteworthy that ectodermal or epithelial abnormalities are part of APECED. This suggests that the primary defect may be an ectodermal one leading to defective function of

Autoimmune Polyendocrine Syndrome Type I (APECED)

25

the surface epithelia and thymic epithelium, as is in the DiGeorge syndrome, and the nude mice and rats.19 Alternatively, the ectodermal manifestations may also be of autoimmune origin. Of note, none of the disease components have been described in utero or in neonates. This could be due to some maternal factors substituting for the missing gene product, or perhaps to the immaturity of the neonatal immune system. Alternatively, it may indicate that microbes or some other external triggers are needed for induction of the autoimmune process.

Target Autoantigens Most studies of autoimmunity in APECED have focused on occurrence of autoantibodies and, recently, identification of the autoantigens recognized by those antibodies. Studies of cellular immunity are few.

Parathyroid Glands That hypoparathyroidism is the most common component of this polyendocrine autoimmune disease makes causal the role of autoimmune destruction of the parathyroid glands extremely likely. Furthermore, the histology of affected parathyroid glands is characterized by lymphocytic infiltration and atrophy. Yet the nature of the autoantigens remains obscure. First, parathyroid specific antibodies were observed by indirect immunofluorescence (IF) in 38% of 74 patients with idiopathic hypoparathyroidism, 26% of 93 patients with idiopathic Addison’s disease, and 6% of 245 controls.30 With the same method 10 of our 34 hypoparathyroid patients and 1 of 6 euparathyroid patients, but none of 55 sibling and parents were antibody positive.31 An antibody to parathyroid oxyphil cells was reported in 1 of 9 patients with idiopathic hypoparathyroidism.32 Then the oxyphil cell reactivity was attributed to human specific mitochondrial antibodies.33 This was confirmed; these antibodies were shown to be of IgG class, and no specific parathyroid antibody was detectable in 32 patients with idiopathic hypoparathyroidism.34 Next, autoantibodies directed toward antigenic determinants on the surface of human parathyroid cells were observed in 8 of 23 adult patients with idiopathic hypoparathyroidism.35 Three of these sera (apparently from nonAPECED patients) inhibited the secretion of parathyroid hormone (PTH) in an in vitro dispersed human parathyroid cell system. Some monoclonal antibodies directed toward specialized differentiation antigens expressed on endocrine cells were reported to inhibit or stimulate PTH secretion in such system.36 In a system of long-term serum-free culture of bovine parathyroid cells, cytotoxic IgM antibodies were demonstrated by IF and by cytotoxicity utilizing 51Cr release technique in sera of all of seven hypoparathyroid patients with APECED. In addition to the parathyroid cells, the antibodies were in the presence of complement cytotoxic to bovine adrenal medullary cells.37 Later the same group showed that these antibodies are directed against proteins associated with bovine endothelial cells.38 They recognize molecules of 200 and 130 kD solubilized from the membrane fraction of bovine parathyroid endothelial cells. The reactivity of these antibodies with endothelium-related structures of human parathyroid adenomas was much less consistent: only the serum from 1 of 6 patients reacted with all of three different adenomas. The immunohistologic picture of this adenoma closely resembled the one observed with bovine parathyroid tissue: the antibodies reacted with determinants closely related to the vascular endothelium and in close apposition to the epithelial cell membrane in the region of vascular cells. The conclusion was that these antibodies are disease-specific but not organ- or species-specific. The group speculated that endothelium may serve an important local function.37 Circulating antibodies against Ca2+ receptors were searched in 61 of our patients, 51 of them hypoparathyroid. All were negative (Spiegel et al, unpublished).

Endocrine and Organ Specific Autoimmunity

26

Adrenal Cortex and Gonads In APECED all identified autoantigens in the adrenal cortex and the gonads are cytochrome P450 enzymes involved in the synthesis of steroids: steroid 21-hydroxylase (P450c21, microsomal, present only in the adrenal cortex), cholesterol side-chain cleaving enzyme (P450scc, mitochondrial, in the adrenal cortex and the steroid-producing cells of the gonad and placenta) and steroid 17α-hydroxylase (P450c17, microsomal, in the adrenal cortex and the gonads ) (Fig. 2.2, Table 2.2) .39-47 (Rorsman et al, unpublished) By a sensitive method, of sera from 49 of our patients with Addison’s disease of APECED, 92% recognized at least one of these three enzymes and 80% at least one of P450scc and P450c17 (equivalent to steroid cell antibodies) (Table 2.2). The true incidences of these autoantigens are undoubtedly higher, because antibodies disappear with time, especially adrenocortical cell antibodies3 and, presumably, P450c21 antibodies. Most of our antibody-negative patients have either a very long-standing Addison’s disease or high-dose pharmacological glucocorticoid medication. In contrast to previous claims,48 antibodies recognizing P450scc or P450c17 occur in absence of antibodies recognizing P450c21 (Rorsman et al, unpublished). This may be due to earlier disappearance of the latter. Differences in observed prevalences probably depend more on the nature of the antigen preparations used in testing45,47,49 and other method differences than true prevalence variation. Even the occurrence of antibodies to P450c21 and P450c17 in patients with APECED was previously disputed by the group that now finds them in our patients. 45 Three- β -hydroxysteroid dehydrogenase 44 and 11β-hydroxysteroid dehydrogenase41,44 were tested for in sera from small series of patients with negative results. This hardly excludes the possibility that these or other unrecognized enzyme or other autoantigens may also be involved in the destruction of the adrenal cortex and the ovaries.43,45 In contrast to previous claims,41 the pattern of occurrence of antibodies against the enzymes of steroid synthesis does not differ between the two forms of autoimmune polyendocrinopathy.47 P450scc activity was inhibited by sera from patients with APECED41 and P450c21 activity by sera from patients with Addison’s disease.50

Pancreatic βCells

Cytoplasmic islet cell antibodies (ICA)51 and antibodies against glutamic acid decarboxylase (GAD-ab)52-54 are common in patients with APECED and are often present in high titers. ICA were determined in 313 sera taken over 1.5-13.3 (mean 7.4) years from 47 of

Table 2.2. Reported prevalences in patients with APECED of circulating antibodies recognizing steroid 21-hydroxylase (21, P450c21), 17(-hydroxylase (17, P450c17) and the side-chain cleaving enzyme (SCC, P450scc). The numbers are counts of positives. Type of patients: AD+ with, AD- without Addison’s disease Reference

Number and type of patients

21

17

SCC

Any1

42

36 AD+ 14 AD5 AD+ 7 AD+ 11 AD? 49 AD+ 13 AD-

15 1 4 0 7 41 3

15 1 0 1 6 24 2

21 1 0 7 5 32 2

29 3 4 7 ? 45 5

44 45 47 Rorsman et al2

1 Any of 21, 17, or SCC; 2unpublished

Autoimmune Polyendocrine Syndrome Type I (APECED)

27

Fig. 2.2. Synthetic pathways of steroids in the adrenal cortex. In the gonads, androstenedione is further transformed to testosterone and the estrogens.

28

Endocrine and Organ Specific Autoimmunity

our patients.51 Of them 5 were diabetics; only 1 of these was ICA-positive, in several samples. Of the 42 nondiabetic patients 9 (21%) were positive during the follow-up, 6 of them persistently for 0.5-11 years, with fluctuation between positive and negative state in 2. In these patients, no change in glucose tolerance was observed in sequential intravenous glucose tolerance tests (IVGTTs). In a later study 8 of our 47 patients were diabetics.54 Six of them were positive for GAD65-Ab, 1 for GAD67-Ab and 4 for ICA; 2 were negative for all three. Sera before the clinical onset of IDDM were analyzed in 6 patients: in 4 GAD65-ab appeared 0.9-8 years before the onset, and in 2 children disappeared by 1 and 4 years after the onset. Of the 39 nondiabetic patients, 16 had GAD65-ab, 11 had GAD67-ab and 11 had ICA; 20 had at least one of the three antibodies. The levels of both GAD67-ab and ICA correlated with those of GAD65-ab, but not with each other. Except for two patients with GAD67-ab only, the reactivity with 35S-labeled GAD67 was abolished by incubation with unlabeled GAD65. Age at the time of the first GAD65-ab-positive sample or the duration of the positivity did not differ between the patients who developed IDDM and those who did not. The mean duration of the antibody positivity was 10.1 years in the nondiabetics by the time of their last sample, compared with 4.4 years in the patients followed to IDDM. Nine of the 39 nondiabetics had high GAD65-ab levels (index >10) compared with 2 of 6 of the patients who developed IDDM. Among the nondiabetics fasting serum C-peptide levels (0.5 ± 0.24 vs. 1.03 ± 0.49 nmol/L, P=0.003) and first phase insulin responses (FPIR) (75.6 ± 37.9 vs. 166 ± 113 mU/L, P=0.019) were lower in the GAD65-antibody positive group than in the negative group. Four of the antibody negative and four of the positive nondiabetic patients were tested repeatedly. FPIR decreased in all the positive patients but increased in three of the negative patients. The antibody positivity was suggested to reflect subclinical insulitis that, in the absence of genetic susceptibility to IDDM, progresses to clinical diabetes only in a minority of the patients. As another explanation, GAD65-ab in APECED and in the common IDDM have been suggested to differ in their autoantigen recognition, as only those from (five nondiabetic) patients with APECED inhibited GAD enzyme activity and bound to denatured GAD by western blotting (WB).52 Such difference may be partly explained by antibody levels, because dilution of strongly reactive sera may reveal up to 10,000-fold differences in antibody levels. Humoral autoimmunity in patients with polyendocrinopathy may not associate with visible β-cell damage, in contrast to patients with the common IDDM.55 In post mortem examination of well preserved pancreases of two elderly patients with APS type 2 and the autolytic gland of a 18-year-old girl with APECED, all nondiabetic but with ICA and GAD-antibodies, none showed evidence of increased HLA I or II, or islet infiltration by T or B cells or macrophages, islet capillary hypertrophy, or immunoglobulin deposition around the islets. None of the patients had whole islet ICA, insulin autoantibodies or antibodies against the nonGAD-derived 37k islet antigen, which appear to be more closely related to IDDM than the GAD-antibodies. Cellular immunity to GAD65, detected in vitro as proliferation response to GAD65 (stimulation index (SI) >3.0), was observed in 15 of 44 of our patients vs. 3 of 28 controls (p=0.026), and the median SI was higher in the patients (p=0.009).56 Increased IFN-γ secretion (>50 pg/ml) by GAD65-stimulated peripheral blood mononuclear cells (PBMC) was observed in 16 of 28 patients tested (57%) vs. 19% of healthy controls (p=0.009). The levels of IFN-γ secreted by the GAD-stimulated cells were higher in the patients than in the controls (p=0.006). The IFN-γ response occurred in several patients without positive proliferation response, and the two cellular responses showed no mutual correlation. Of the patients who had both tests, 68% showed at least one of the two cellular responses. Serum levels of GAD65-ab were elevated in 14 of the 44 patients. Both the SIs and the IFN-γ secretory

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responses correlated negatively with the antibody levels. The antibody and proliferative responses coincided in only four patients. This supports the concept of reciprocal regulation of humoral and cellular immunity.57 In 14 nondiabetics of the patients who underwent intravenous glucose tolerance test, no difference in insulin response was observed between patients with cellular reactivity to GAD65 and those without. Eight of the patients had IDDM (since 4.6-19.6 mean 11.2 years before the study). They did not differ from the nondiabetic patients in the two cellular responses or the antibody positivity to GAD65. Three patients developed IDDM within 12 months of the testing, which included the IFN-γ secretory response for only one of them. That one was only positive for the IFN-γ response. Of the two others both had the GAD65-ab and one the proliferation response to GAD65. When the GAD65-ab data of this study are assessed together with the results of the above-cited earlier study of the same group,54 GAD65-ab positivity associated weakly with development of IDDM within a year (8/11 positive in diabetic vs. 18/49 in nondiabetic patients).56 Two observations concerning HLA may be relevant. The cellular proliferation response to GAD65 was significantly associated with the IDDM risk allele HLA DQB1*0201 (p=0.03, Chi-Square test).56 Though the humoral immunity against GAD65 was not associated with the IDDM susceptibility HLA alleles, GAD65-ab positive nondiabetic patients had a significantly lower frequency of IDDM susceptibility HLA DQ and HLA DR alleles than the GAD65-ab negative nondiabetic patients (P 0.008-0.043 for different alleles) or controls (P 0.038-0.067). This might explain the low frequency of IDDM in the GAD65-ab positive patients.54 The role of gut-specific lymphocyte homing receptor α4β7-integrin was explored.58 Using immunomagnetic cell sorting the lymphocytes with high expression of the α4β7-integrin were depleted from peripheral blood mononuclear cell preparation from patients with the common IDDM or APECED, all selected because of cellular immunity to the β-cells. The depletion led to marked (mean 70%) decrease in the cellular response to GAD65 in 3 of 6 patients with the common IDDM and in another patient at high risk of it. A 37% decrease occurred in 1 of the 3 patients with APECED, the only one with IDDM; in the 2 others the cellular response remained unaltered. Cellular response to tetanus toxoid increased in the majority of the patients as well as in all three healthy controls. Thus a remarkable population of islet cell antigen-reactive lymphocytes express the gut specific homing receptor, which emphasizes the role of gut-specific immunity in IDDM. The immune responses to GAD, though frequent in patients with APECED, may not be pathogenic determinants of IDDM in this disease. But if they are, some special characteristics of GAD-reactive lymphocytes might be important for the induction of β cell destruction, such as epitopic recognition or homing properties.56,58 Circulating autoantibodies against aromatic L-amino-acid decarboxylase (AADC) were studied in 69 patients with APECED, 9 of them diabetic. This enzyme is present in the pancreatic β cells. The antibodies were observed in 35 of the patients, including 5 of the diabetic patients. Hence there was no indication of an association of the AADC-antibodies with IDDM.59

Thyroid Gland Antibodies directed against thyroid peroxidase (the autoantigen for anti-thyroid microsomal antibodies) and thyroglobulin are common in patients with APECED. Of our 62 patients significant titers (≥400 and ≥100, respectively) were observed against one of these autoantigens at least once in 19 and against both in 9, while only 5 have developed hypothyroidism (Miettinen and Perheentupa, unpublished).

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Liver Liver-kidney microsomal (LKM) antibodies are frequently present in sera of patients with APECED. Two cytochromes, P450 2A6 and P450 1A2, are the hepatic target antigens; antibodies against them might represent hepatic markers for APECED.60-63 Of 11 Sardinian patients LKM staining pattern was observed in 5: one with lethal liver involvement, another with chronic hepatitis, and 3 with only rare and mild elevations in serum transaminases. With WB, P450 2A6 was identified in 4 and P450 1A2 in 1 of the IF-positive patients, and absorption studies confirmed antigen specificity.63 P450 1A2 -antibodies had earlier been observed only in hydralazine-induced hepatitis,64 though they have been searched for in patients with various autoimmune and infectious hepatic diseases. The patient with antibodies against P450 1A2 had chronic active hepatitis. In her liver IF revealed centrolobular and in kidney proximal tubular staining pattern. The staining pattern was characterized by predominant staining of perivenous hepatocytes. This pattern differs from the homogeneous staining found in patients with isolated autoimmune hepatitis. P450 1A2 is expressed in the liver but not in the kidney. Absorption with recombinant P4501A2 made the IF in both the liver and the kidney disappear. The kidney IF must thus have been due to a cross-reacting antigen.63 Antibodies against both P450 1A2 and P450 1A1 were also observed in a Yugoslav boy with APECED including chronic nonspecific hepatitis.60 These observations were confirmed in a series of 64 Finnish patients.65 IF revealed in 9 of them liver staining, in 5 with staining of the kidney. In addition, two patients showed IF in the kidney only. The sera of five patients recognized P450 1A2. Of other human P450 enzymes 1A1, 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and epoxide hydroxylase, sera from 10 of the patients recognized 2A6. Serum from a patient with active hepatitis recognized four hepatic P450s: 1A1, 1A2, 2A6 and 2B6. Of the others with current or past clinical hepatitis, two tested positive for 1A2, and the third for 2A6. Only 2 of the 4 patients with hepatitis tested positive for the IF: the boy with the 4 P450 antibodies strongly in both liver and kidney, and a girl strongly in the liver. Five of the patients with positive IF of liver did not react with any of the specific P450 enzymes. This might be due either to other protein targets or the presence of conformational epitopes that are destroyed upon denaturation with SDS. Overall, the findings by IF and WB did not correlate. WB with recombinant cytochromes is more sensitive than IF.

Pituitary Gland Autoantibodies against arginine vasopressin (AVP)-secreting hypothalamic cells were searched in sera from 39 patients with central idiopathic diabetes insipidus (DI) including a 22-year-old male with APECED.9 Of them 13 had an overt autoimmune disease or associated organ-specific antibodies. Eight of these 13 were positive for the AVP-cell antibodies, including the patient with APECED. Of 81 patients with DI secondary to hypothalamic lesions only 7 of 13 patients with histiocytosis X and two others were positive. The autoantigen is unknown. Serum samples (N= 138) from 47 of our patients with APECED were tested for antipituitary autoantibodies by immunoblotting.66 Antibodies to a 49 kD cytosolic protein were detected in the sera of 6 of the 47 and to a 45 kD protein in three patients. Seroconversion was observed in two of them, at 14 and 31 years. None of 16 nonAPEDED patients had anti-pituitary antibodies.67 A 12 year-old girl with APECED developed hypophysitis with growth hormone deficiency.68 At the age of 11.9 years her height was -2.9 SD, height velocity 2.4 cm/year and bone age 8 years. Plasma IGF-I was repeatedly low before and 12 months after beginning nutritional supplementation (46 and 65 γg/L respectively). Maximum serum GH level at

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L-dopa-propranolol and clonidine tests was 0.8 and 0.3 γg/L. MRI showed a halo effect with perihypophyseal gadolinium enhancement consistent with hypophysitis.

Gastric Mucosa The most relevant target cell in the autoimmune destructive process leading to gastric fundal atrophy and pernicious anemia (PA) is the gastric parietal cell (PC). Autoantibodies against the PC or intrinsic factor (IF) (their product besides acid), which binds avidly to dietary vitamin B12, constitute the most important immunological features in PA.69 PC-antibodies are of IgG and IgA classes (IgM can also be detected) and tend to fix complement. Sera positive to PC cytoplasmic antibodies are also reactive to autoantigens on the surface of the cells in almost 100% of cases. In the presence of complement the antibodies are cytotoxic in vitro. Both the α and the β subunits of the H+, K+ ATPase, the H+ pump responsible for acid production, are major target molecules for the autoimmune process against the PC.70-75 Presumably that is also true for the PA of APECED. Against IF two distinct forms of antibodies may be detected by RIA. Type 1, IF-blocking antibodies, react with the vitamin B12 binding site of IF and inhibit the attachment of vitamin B12 to it. Type 2, binding antibodies, react with a spatially distinct epitope on vitamin B12 on the B12-IF complex. The pathogenic role of IF-antibodies is indisputable in juvenile PA, where their prevalence reaches almost 100% when searched both in serum and gastric juice. Cell-mediated immunity has been demonstrated in vitro by lymphoblastic transformation assay, using the patients’ peripheral blood lymphocytes in the presence of IF, gastric juice or homogenate of gastric mucosa.69 Our clinical experience with the significance of these autoantibodies in patients with APECED is consistent with this general information.

Intestine According to a recent case report, the fat malabsorption in APECED may also be of autoimmune origin.76 In a 15-year-old patient with APECED suffering from recurrent episodes of severe intractable diarrhea, steatorrhea and hypocalcemia, the only treatment that controlled the malabsorption was immunosuppresion with high dose i.v. administration of methylprednisolone, and oral methotrexate maintenance. With such therapy clinical remissions of the intestinal disorder were repeatedly achieved (Fig. 2.3). In the patient’s serum antibodies of all three Ig isotypes were demonstrated by indirect IF microscopy against the brush border of normal gut enterocytes.

Skin Vitiligo in association with autoimmune diseases, at least the polyendocrinopathies, is clearly an autoimmune disease with anti-melanocyte autoantibodies. These antibodies can lyse cultured melanocytes by both complement activation and antibody-dependent cellular cytotoxicity.77-79 Of 28 patients with vitiligo associated with autoimmune polyendocrine disease type 2, autoimmune thyroid disease or IDDM, 18 patients and 8 immediate family members were demonstrated to have autoantibodies for a 69 kD protein in HTB-70 melanoma cells that was not present in control cells. The autoantibody-positive sera reacted with recombinant human tyrosinase but not with recombinant tyrosinase-related protein. Not one of 31 normal controls or 8 patients with alopecia or systemic lupus erythematosus had these autoantibodies, but 12% of 42 patients with autoimmune endocrine disease without a history of vitiligo had them. Tyrosinase, an enzyme important in melanin formation, was concluded to be the principal autoantigen of autoimmune vitiligo.80 The autoantigens of melanocytes seem not to have been studied in patients with APECED.

Stool volume

Day

Day

Age 13

Age 13.3

Age 16

Fig. 2.3. Responses of watery diarrhea in a 15-year-old boy with APECED to intravenous pulse therapy with methylprednisolone (18 mg/kg/day) and oral methotrexate (15mg/m2/week). CFA = coefficient of fat absorption. From ref. 76.

CFA (%)

32 Endocrine and Organ Specific Autoimmunity

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Alopecia is frequently associated with organ-specific81 and systemic82 autoimmune diseases. It is a putative autoimmune disease in which the anagen hair follicles are the target. Infiltrates of both CD4 and CD8 lymphocytes are present around the hair follicles, and autoantibodies against various structures of the follicles have been described. These autoantibodies seem not to have been studied in patients with APECED.

Eyes

Unidentified autoantigens may be present in cornea, iris and ciliary body.8

What Activates or Inhibits Autoimmunity? Very little is known of the events triggering the individual organ specific autoimmune processes in APECED. Infections have been suspected in other autoimmune diseases to lead to autoimmunity via molecular mimicry or other mechanisms (see chapter 1). Mucocutaneous candidiasis precedes adrenal failure in most patients with APECED. In a search for molecular mimicry between the P450 steroidogenic enzymes of C. albicans and human steroid producing cells no antigenic cross-reactivity was found with APECED autoantibodies.25 In one patient hypoparathyroidism started shortly after BCG vaccination.20 In our patients no association has been observed with infections or immunizations. Increased activity of endocrine glands may alter the antigen and/or HLA expression on the cell surface of the secretory cells and could predispose to the autoimmune process.84 In one patient with early onset adrenal insufficiency, who had lost adrenal and steroid cell autoantibodies detected during the active adrenal disease, steroid cell antibodies reemerged at puberty when gonadal autoimmunity developed.3 In most patients, however, such association is not obvious. In SLE the type II HLA antigens of the patient influence the clinical manifestations and the autoantibodies formed,85 but not so in APECED. Direct evidence is missing of the pathogenic mechanisms leading to the organ specific damages in APECED. It is generally believed that autoaggressive cytotoxic T cells are needed for the destruction of endocrine glands. B cells may have a role in the presentation of autoantigens to T cells, but the role of the organ specific autoantibodies in tissue damage is less clear. Antibodies binding to the cell surface receptors may cause hyper- or hypofunction of endocrines, but such antibodies (often TH2 isotypes) have not been found in APECED. Antibodies binding to the cell surface may damage the target cells by activating the complement cascade or via antibody mediated cellular cytotoxicity. Steroid cell antibodies cytotoxic to cultured human granulosa cells or bovine parathyroid endothelial cells have been described in Addison’s disease and primary ovarian failure86 and in autoimmune hypoparathyroidism.38 Also, complement fixing antibodies against adrenocortical cells seem to be stronger predictors of overt disease than noncomplement fixing antibodies.87 As discussed above, adrenal and steroidal cell antibodies are good predictors of the development of disease in both children and adults with APECED, while even high titers of anti-GAD65, islet cell, or thyroid autoantibodies do not correlate with the development of clinical disease. However, all steroid and β-cell antigens described are intracellular enzymes and in intact cells thus out of reach of the circulating autoantibodies. Thyroid peroxidase and H+, K+-ATPase are present in the cell membrane, but usually so located that antibodies do not reach them normally. Thus the pathogenic role of autoantibodies as effector molecules is probably small in APECED. GAD65 induced mitotic activity and secretion of INF-γ in peripheral blood T-cells of patients with APECED, but the patients with IDDM were no different from those without.56 What triggers the formation of autoaggressive cytotoxic effector cells remains an open question. During the long follow-up period of the Finnish patients with APECED, autoantibody levels frequently rose and fell, sometimes disappearing with time. This may reflect activation and deactivation of the autoimmune process, but its determinants remain unknown.1

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Are There Assays Which Allow for Prediction of the Disorder or Its Components? Recognizing Persons at Risk of APECED Presumably, every homozygote for a mutation of the APECED gene rendering the gene nonfunctional will sooner or later develop at least some components of the disease. At present, homozygotes and heterozygotes for an APECED mutation can usually be identified in sibships known to be at risk because of a proband with APECED. This requires analysis of the parents and the proband for polymorphic markers in chromosome 21q22.3, and that the parental chromosomes differ in those polymorphisms. In the near future, ongoing intense work should result in identification of the APECED gene and its common mutations.17 In general, APECED should be remembered in unexplained cases of chronic mucocutaneous candidiasis or keratopathy, and isolated hypoparathyroidism or Addison’s disease, especially in children. The above described features of ectodermal dystrophy should increase suspicion. If molecular genetic diagnostics is not available, such individuals need follow-up aimed at early detection of other disease components.

Predicting Course of the Disease Determinants of the widely variable clinical course and picture of APECED, identity and number of disease components and timing and order of their development, are unknown. Because of the rarity of the disease no conclusive study has been performed of possible dissimilarity between affected sibships, beyond the concordance for Addison’s disease cited above. That concordance was associated with HLA haploidentity or identity in all sets with the exception of a single patient.88 Overall, the disease components show no clear dependence on class I or class II MHC. In a study of correlations of alleles of HLA-A, -B, -C, and -DR with the clinical picture in 45 patients from 34 families MHC, only HLA-A28 showed some weak associations with some disease components, the strongest ones with keratopathy and alopecia (P corrected for multiple correlation analysis 0.04).88 In the above-cited studies of GAD-antibodies in 47 patients,54 and GAD-antibodies and cellular immunity in 44 patients,56 no indication was observed in patients with APECED of dependence of IDDM on the IDDM susceptibility alleles HLA-DQA1, -DQB1, -DRB1.

Predictive Value of Antibodies Circulating organ-specific antibodies are quite reliable in predicting future development of failure of adrenal cortex and ovaries. Adrenal antibodies and steroid cell antibodies usually precede clinical Addison’s disease. For adrenal binding antibodies, reported sensitivity in predicting adrenal failure is 0.91, specificity 0.89 and predictive value 0.92.3 However, these values were observed in a follow-up study of 1-12 years, and in some patients the antibodies appeared a few weeks before the clinical disease, and in others more than 5 years earlier (Fig. 2.4) waxing and waning over the years and even completely disappearing for years. Not even did high antibody titers predict imminent failure. Similarly, the functional capacity of the adrenal cortex, as followed by an ACTH test response and plasma renin activity, may fluctuate and slowly decrease over several years, or be completely lost over a few weeks.4 The secretions of aldosterone and cortisol may fail simultaneously, or even many years apart. Circulating steroid cell antibodies preceded the development of ovarian failure in all patients in the same follow-up study (Fig. 2.4).3 Ovarian failure usually developed after adrenocortical failure, and circulating steroid cell antibodies may persists for years and even decades after development of adrenocortical failure. Hence the specificity (0.55) and predictive value (0.69) of these antibodies in predicting ovarian failure is lower than in predict-

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ing adrenocortical failure, though the sensitivity is high (1.0). If these antibodies disappear in a female patient without ovarian failure, their reappearance seems to indicate development of the latter.3 Presence of circulating antibodies against the enzymes of steroid synthesis, be they demonstrated by cell specific IF or the specific enzyme antibodies, calls for functional endocrine monitoring of the adrenal cortex and ovaries. There are no follow-up studies with the enzyme-specific antibodies. Presumably, they bring increased assay sensitivity. Of them, antibodies against P450c21 probably have the greatest diagnostic sensitivity, because their prevalence is highest, even higher in patients developing adrenal failure than among patients with long-standing failure. The known β-cell autoantibodies, ICA and GAD-ab, evidently have little value in the prediction of IDDM in patients with APECED. In our updated data the value of GAD65-ab in predicting IDDM is 33%, and the negative predictive value of GAD65-ab negativity 92%. The predictive value of cellular immunity against the β-cells needs to be further explored. Many patients have thyroid antibodies but most of them probably never develop the clinical disease. Presumably the reverse is true: thyroid failure may not develop without preceding high levels of antibodies. In our experience circulating antibodies to PC or blocking IF are present when vitamin B12 deficiency is developing. Laboratory tests are diagnostic: serum vitamin B12 100 V (variable) genes with one of >50 J (junctional) genes, with a highly mutated D (diversity) region and with a C (constant) chain.193,194 Clonal expansion has been shown to be present in Graves’ and Hashimoto’s diseases. Intrathyroidal T lymphocytes from Graves’ disease thyroids more easily than Hashimoto’s tissue, showed restricted use of TcR V genes, as compared with peripheral blood lymphocytes. Using RT-PCR with individual alpha gene-specific primers, an average of 5 out of 18 α genes studied were used by thyroidal lymphocytes obtained from surgical specimens, while in peripheral blood more than 17 were found.195 When samples derived from fine needle aspirations of thyroids were used, especially helpful in Graves’ disease patients who were at an earlier stage in the natural history of their disease, both TCR V α and TCR V β restrictions were found in Graves’ disease, with different V genes used preferentially in each patient.196 Selective use of TCR V α or β genes within the thyroid, retro-orbital tissue and pretibial lesions has been confirmed197 although not all studies using surgically obtained thyroids have been in agreement,186,198 the latter perhaps secondary to technical difficulties with background peripheral blood mononuclear cells (PBMC) contaminating the specimens. Further insight into the mechanism of clonal expansion of autoreactive thyroid T cells has been recently obtained with a different approach. Amplification of the complete rearranged TcR α and β genes from PBMC and from thyroid infiltrating lymphocytes using TcR V gene-specific reverse primers and radiolabeled constant region-specific forward primers yielded a number of products of many different sizes from PBMC. In contrast, intrathyroidal lymphocytes gave a limited number of bands, some of them represented by monoclonally selected TcR genes as indicated by sequence analysis, and thought to represent antigen-specific receptors (Fig. 6.6).199 Such observations confirm that the TcR V gene restrictions seen in our earlier studies do indeed represent the results of an antigen driven T cell accumulation. Ophthalmic Graves’ Disease The etiology of the retro-orbital inflammatory response in patients with Graves’ disease has many similarities to the thyroid abnormality. As mentioned earlier, TSH receptor mRNA and antigen expression have been observed in fibroblasts and adipocytes75,76,79,81 and cross-over specificity with the thyroid has become an interesting hypothesis.80,235 The presence of extrathyroidal TSH receptors in many tissues, however, suggests that tissue specific post-translational processing must be involved in any unique antibody responses. Unfortunately, the role of antibodies to retro-orbital antigens remains confusing and appears to be secondary to tissue destruction rather than primary in disease etiology.80 However, analyzing T cell receptor V gene use in retro-orbital, pretibial, and thyroidal tissues of two patients with Graves’ disease, Heufelder et al found a marked oligoclonality, suggesting that similar antigenic determinants may have contributed to T cell expansion in the thyroid and extrathyroidal tissues. 200 Such findings once again emphasize the similarity in the immunopathogenesis of both the retro-orbital and thyroid infiltrates.

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Fig. 6.6. An example of a radiolabeled RT-PCR of the Vb5 T cell receptor gene family in the intrathyroidal T cells and peripheral blood T cells (PBMC) of a patient with Graves’ disease. Note the restricted pattern of activity with the thyroid sample. Since the RT-PCR products represented the CDR3 regions of the T cell receptors present, it appeared that two clones were present in the thyroid rather than the peripheral circulation. This was confirmed for the most prominent band by the sequencing as shown with 9 of the 10 sequences being identical.199

Potential Pathogenic Mechnisms Aberrant HLA Expression by Thyroid Epithelial Cells It is now well known that thyroid epithelial cells (TEC) from patients with Graves’ disease and Hashimoto’s thyroiditis can express enhanced class I and aberrant MHC class II molecules on their surface201-202 in a similar manner to professional antigen presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been proposed that aberrant MHC class II expression by TEC could trigger autoantigen presentation and initiate thyroid autoimmunity directly, without the involvement of professional APCs.203 Such expression may also be a secondary phenomenon due to cytokine production (especially IFNγ) by the activated thyroidal lymphocytic infiltrate, enhancing rather than initiating the autoimmune response.204-206 A similar phenomenon is seen in many other, if not all, autoimmune diseases. Class II expression by TEC has also been induced by viral infections, as demonstrated by the ability of SV40, and reovirus to up-regulate MHC class II expression in human and rat thyroid cells.207,208 This would suggest that, under appropriate circumstances, MHC class II expression may indeed be a primary rather than a secondary phenomenon. Further supportive evidence for MHC class II expression by a nonprofessional APC initiating autoimmune disease has come from the recent study in which mice immunized with fibroblasts spontaneously expressing both the TSHR and MHC class II molecules,69 developed many features of Graves’ disease. In our enthusiasm for the thyroid cell acting as an APC, we sometimes forget that professional APCs such as dendritic cells, macrophages, and B cells, exist within the thyroid lymphocytic infiltrate in an intimate relationship with thyrocytes, and that they are able to most efficiently present thyroid autoantigens to the appropriate T cells.209,210 Furthermore, professional APCs express costimulatory proteins on their surface (like B7-1 and B7-2), which interact with CD28 and CTLA-4 molecules expressed on CD4+ T cells during antigen presentation.211 Expression of costimulatory molecules is critical for the initiation of an immune response, since in their absence anergy or clonal deletion of T cells will ensue. B7-1 and B7-2 molecules are only expressed on intrathyroidal APCs and not on TECs, but CD40 is expressed and may provide the necessary signal support. Alternatively, the TEC may rely

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on costimulatory molecules from the professional APCs in their vicinity.212,213 Recent studies suggest that thyrocytes may cooperate with such infiltrating APCs. Blocking B7-1 and B7-2 molecules, present only on intrathyroidal APCs and not on TEC, suppressed T cell responses to levels noted when thyrocytes were used alone.213 To complicate matters further, intrathyroidal or retrobulbar fibroblasts have been proposed as immunologically active cells capable of cytokine secretion in response to activation of their own expressed CD40 antigen by CD40 ligand on T cells (Smith, T et al (in press)).214

The Role of Infection Background Epidemiological and experimental evidence suggests that infection could play a role in the pathogenesis of AITD.20 Both seasonal and geographic variation in the incidence of Graves’ disease have been reported,215,216 and a recent infection was serologically evident in a higher percentage of Graves’ disease and Hashimoto’s thyroiditis patients than in normal controls.217 Although much has been written in this regard, surprisingly little hard data are available. Infection and the Cryptic Epitope Hypothesis T cells from both normal and Graves’ patients are able to recognize TSHR peptides (Fig. 6.5) and this observation requires explanation. Surely such cells would induce disease? Since they do not, we assume that a state of tolerance to the hTSHR must be present in normals and is likely due to the anergizing of such potential autoreactive CD4+ T cells. This would, of course, suggest that AITD was simply a failure to anergize T cells. While we and others have provided some evidence for such a problem, it is too simplistic to be the total story. Another way of looking at this question is to realize that not all T cell and B cell epitopes are necessarily seen easily by the immune system. Some may be hidden, particularly B cell epitopes, while others may be seen only in small amounts, insufficient to initiate an immune response. These so-called cryptic epitopes may, however, become more available because of an insult to the target organ. They would then be seen as new antigens and initiate a vigorous response.218 Such an hypothesis would explain the presence of normal T cells recognizing certain hTSHR, Tg, and TPO peptides since they would be intolerant of any cryptic epitopes. Furthermore, the mechanistic explanation requires a major insult to the target organ to expose large concentrations of cryptic epitope to the immune system; such an insult may be an infection. Since viral infections are known to initiate MHC class II expression on thyroid epithelial cells, this hypothesis appears very attractive. Infection and the Molecular Mimicry Hypothesis Another possible mechanism for AITD which potentially involves infection is the failure of the immune system to efficiently differentiate a foreign infectious antigen from a self antigen.219,220 It is now well know that T cells and B cells activated by foreign antigen may acquire dual specificity for both an eliciting antigen and a self antigen. Normally such an immune response is controlled by the phenomena of anergy and deletion (via apoptosis). For example, when apoptosis was prevented by immunizing mice with foreign antigen fused to bcl-2 protein, monoclonal antibodies with specificity to the immunogen and self double stranded DNA were generated. Such antibodies were deposited in the kidney, as seen in systemic autoimmune diseases.221 This phenomenon of molecular mimicry (or specificity cross-over) may well play a role in both Graves’ and Hashimoto’s diseases. Such patients have been shown to have a high prevalence of circulating antibodies against Yersinia enterocolitica.222-224 Yersinia

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antibodies interacted with thyroid structures,225,226 and some Yersinia antigens cross-reacted with thyroid autoantigens.227 Furthermore, saturable binding sites for TSH have been found in Yersinia, which also bind TSHR-Ab.228 Under low stringency conditions, a Yersinia cDNA fragment could be amplified using human TSH receptor primers.229 Rabbits or mice immunized with Yersinia proteins developed antibodies against human thyroid epithelial cells,230 and against the TSH receptor.231 However, none of these observations have shown that infection with Yersinia could lead to AITD, and the majority of patients with Yersinia infection do not develop thyroid autoimmune disease.232 Further complicating this issue has been the observation that Yersinia can act as a superantigen.233 Therefore, Yersinia infection may also trigger AITD disregarding the phenomenon of molecular mimicry, via nonspecific activation of a predisposed immune system. As mentioned briefly above, the presence of TSHR mRNA transcripts and TSH binding sites in a variety of nonthyroidal tissues, including retro-orbital and pretibial tissues has suggested that molecular mimicry (cross-over specificity) between thyroidal and extrathyroidal TSHRs may help explain Graves’ orbitopathy and dermopathy.80,234 While an attractive explanation it will be important to explain the role of such transcripts in many other tissues which are not obviously involved significantly in the disease. This would include many fibroblasts, pituitary cells and cardiac myoblasts.84 Evidence for Viral Infection Inducing AITD Viruses could trigger AITD through a variety of mechanisms, some of which we have already discussed above, including aberrant expression of HLA antigens, abnormal exposure to cryptic antigens and the involvement of molecular mimicry. Additional potential mechanisms include viral-induced alterations to thyroid autoantigens and new expression of viral proteins on the surface of thyroid epithelial cells (reviewed in 20). Although data remain sparse, reports have appeared finding retroviral (HIV-I gag protein) sequences,235 and human foamy virus antigens236,237 in the thyroid and peripheral blood of patients with Graves’ disease but not in controls. These findings, however, have not been confirmed in subsequent studies.238-240 Recently, circulating antibodies against a human intracysternal type A retroviral particle (HIAP-1) were reported in 87.5% of patients with Graves’ disease as compared to just 10-15% in patients with other thyroid diseases, other autoimmune diseases, and healthy controls.241 When 35 members of three families with a high prevalence of Graves’ disease (31.5%) were examined for antibodies to HIAP-1 and HLA susceptibility alleles, the association between anti-HIAP-1 antibody positivity, HLA susceptibility and the presence of Graves’ disease was claimed in 67%, 80% and 100% of the members of the three families (p< 0,001).242 Such data await confirmation.

Apoptosis Background Apoptosis (programmed cell death) is a primary mechanism for down-modulation of an immune response, leading to deletion of both foreign-reactive and self-reactive T cells and B cells. Apoptosis also contributes to nonimmune tissue homeostasis independent from necrosis, characterized by oligonucleosomal DNA fragmentation.243 Apoptosis can be induced by the interaction of Fas (CD95 / APO-1) with Fas-ligand,244 both membrane proteins expressed mainly on the surface of activated immune cells but also on many other types of cells, and inhibited by a variety of mechanisms including expression of the bcl-2 oncogene.245 Animal models have provided much insight into the important role of apoptosis in autoimmune disease. For example:

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A) Mice with a mutation in the Fas (APO-1) apoptosis gene (lpr mice), leading to low expression of Fas mRNA, develop lymphoproliferation and a generalized autoimmune disease resembling SLE. Early correction of this defect in T cells was sufficient to eliminate the acceleration of autoimmune disease in such mice.246 B) In the absence of costimulatory signals, peripheral T cells can be deleted or anergized when they encounter antigen. High doses of specific antigen (MBP) injected intravenously induced mature T cells to undergo apoptosis and improved the clinical evolution of experimental allergic encephalomyelitis (EAE).247 This mechanism was also seen with MBP-specific T cells derived from patients with multiple sclerosis.248 C) B cells also undergo apoptosis upon membrane bound immunoglobulin cross-linking. Strong “in vivo” cross-linking of cell surface IgGs induced apoptotic death of mature peritoneal B cells in normal mice, but not in activated B cells in bcl-2-transgenic (apoptosis resistant) mice, and in autoimmune-disease-prone New Zealand mice. B cell activation may require a second signal, such as expression of “rescue molecules”, like the bcl-2 gene product in addition to antigen binding. Resistance to B cell apoptosis may play a crucial role in autoantibody production in such mice.249 Apoptosis and AITD Several lines of evidence support the idea that apoptosis may be involved in the pathogenesis of AITD although the importance of its role is presently unclear. It has been shown that thyroid cells and retrobulbar fibroblasts from patients with AITD have an increased expression of MHC class I, MHC class II, and several adhesion molecules, which may lead to increased signaling via the T cell receptor. In particular, as compared to control fibroblasts, retrobulbar fibroblasts from Graves’ patients had an increased capacity to protect T cells from apoptosis via diminished induction of Fas (APO-1) in T lymphocytes.250 This might enable T cells to escape peripheral elimination and contribute to the perpetuation of disease. Besides ADCC and thyroid-specific T cell cytolysis, apoptosis may be an important mechanism of tissue damage in autoimmune thyroiditis. Immunohistochemical and electron microscopy studies have shown that almost all nuclei of follicular epithelial cells from atrophic thyroid follicles showed nuclear DNA fragmentation compared to only 7-21% of nuclei from intact thyroid follicles.251 A recent study reported that Fas was expressed in thyrocytes from Hashimoto’s patients but not in normal thyrocytes and that IL-1β was able to induce Fas expression and apoptotic death even in normal thyrocytes.252 The same investigators claimed that Fas-ligand was expressed on all thyroid cells. The data would imply that normal thyrocytes do not undergo apoptosis because of the absence of Fas. However, simultaneous expression of functional Fas in AITD or IL-1β stimulated thyrocytes, and Fasligand, induced apoptosis. IL-1β could be released by infiltrating lymphocytes and could theoretically interact with thyrocytes and trigger Fas-mediated apoptosis. Surprisingly, expression of FAS-ligand on infiltrating T lymphocytes was negligible compared to FAS-ligand expression on HT thyrocytes, suggesting a minor role for cytotoxic T cells and a prevailing involvement of Fas/Fas-ligand mediated tissue destruction.253 These interesting observations require further exploration and, to date, their confirmation has not been forthcoming.

Other Precipitating Factors Gender It is well recognized that women have a higher prevalence of AITD than men. An influence of sex steroids has been proposed based on the observations of the many effects of sex

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steroids on the immune system.253 Graves’ disease is uncommon before puberty and estrogens may influence the immune system, particularly the B-cell repertoire.254 Testosterone down-regulates lymphoproliferative responses to mitogens, T cell maturation and the humoral response to several antigens, while opposite effects were suggested for estrogens.255 It was also suggested that androgens protect and estrogens enhance thyroiditis in mice after thyroglobulin immunization.256

Stress Environmental influences, such as stressful life events, have been associated with the initiation of Graves’ disease in recent controlled studies.257-259 These reports suggest that stress-induced immune suppression may be followed by immune system hyperactivity and lead to a break in self-tolerance for thyroid antigens in susceptible individuals as seen in the postpartum period. Smoking has been particularly associated with ophthalmic Graves’ disease but may have its influence through an anoxic mechanism distinct from just stress.305

Iodine Epidemiological evidence has shown that increased iodine intake, such as the introduction of iodized salt, iodized oil or potassium iodine tablets for the prophylaxis of iodine deficiency, may increase the incidence of AITD.260-262 Amiodarone, an iodine containing anti-arrhythmic drug increased the incidence of thyroid autoantibodies and may lead to both hyper- and hypothyroidism.263,264 The obese strain (os) chicken develops spontaneous thyroiditis and has a defect in iodine uptake, resulting in excessive amounts of iodine entering the thyroid epithelial cells.265 Indeed, excessive amounts of iodine induce thyroiditis in genetically susceptible animal strains, while intrathyroidal depletion of iodine prevents disease. Several hypotheses by which iodine could promote experimental thyroiditis have been proposed. T and B cells may react specifically to iodinated portions of Tg, in murine induced thyroiditis.96,188 A defect in the iodine processing machinery of thyroid epithelial cells of a susceptible animal may result in elevated levels of oxygen or iodine radicals, which could damage membrane lipids or proteins (other than Tg) and which could act as autoantigens (reviewed in 266). Such a mechanism has not been described in patients with AITD, but few investigations have been performed to date.267

New Insights into Immunologic Diagnosis and Treatment Immunologic Diagnosis The measurement of TSHR-Ab by radio receptor assay remains the appropriate clinical test for most patients with AITD.115 It is cheap, precise and sensitive. While this assay is measuring IgGs able to compete for binding of labeled TSH to solubilized porcine TSHR, it does not explore the bioactivity of hTSHR-Ab (stimulatory or blocking). CHO cells transfected with recombinant full length human TSHR have become widely available for the measurement of both binding inhibition activity and TSHR-Ab bioactivity.268,269 The hTSHRCHO cell assay is also as sensitive as the porcine TSHR radio receptor assay for TSH binding inhibition evaluation, and may be more sensitive than the previously used rat thyroid cell (FRTL-5) assay for measuring TSHR-Ab bioactivity. Although such assays require cell culture and are more tedious and expensive, they have an important place in the diagnostic armamentarium available to the physician. Using chimeric LH-TSHR constructs expressed on CHO cells, it was possible to show that binding sites specific for stimulatory and blocking TSHR-Ab may be different,128,130 and that one single serum may contain a heterogeneous mixture of such TSHR-Ab.

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Although radio assays using natural human TPO are still primarily used for the measurement of TPO-Ab, the use of recombinant human TPO (rTPO) has shown promising results. Purified soluble rTPO expressed in insect cells was used to measure TPO-Ab in an ELISA assay.270,271 Most TPO-Ab containing sera from patients with AITD bound to insect cell expressed rTPO, and the ELISAs had a high sensitivity and specificity. Such binding correlated well with binding to natural human TPO and rTPO expressed on CHO cells. Another study demonstrated that the enzyme activity of rTPO expressed in insect cells was comparable to that of native TPO.272 In contrast to these results with rTPO, only a small percentage of hTSHR-Abs from patients with Graves’ disease, recognized prokaryotic or eukaryotic recombinant human TSHR-ecd273 or soluble purified murine rTSHR-ecd131 expressed in insect cells, or E. coli.132,274 Here the conformational epitopes require both correct glycosylation and folding.

The Immunosuppressive Effect of Antithyroid Drugs (ATD) Although the mechanism of stable remission of Graves’ disease after long-term treatment with ATD is not fully understood, there is evidence suggesting an immunosuppressive action of ATD. A direct suppressive effect of methimazole and prophylthiouracil on the immune system has been proposed,275 and in vitro studies have shown that carbimazole can inhibit autoantibody production by affecting antigen presenting cells.276 Studies in animals have suggested that methimazole is able to reduce the severity of experimental autoimmune thyroiditis.277 Additional evidence has come from the fact that in patients with Graves’ disease the titers of hTSHR-Ab, TPO-Ab and Tg-Ab fall during ATD therapy.278-280 Hence, besides the well known antithyroid effects (blocking iodine organification and thyroid hormone synthesis), ATD may have important immunosuppressive effects but the relevance of such immunosuppression on the clinical outcome of AITD is not known.

Oral Immunotherapy A novel approach to immunotherapy of autoimmune disease has been that of “oral tolerance”. Gastrointestinal feeding of target antigen was shown to induce anergy or deletion of autoreactive T and B lymphocytes in an antigen-specific fashion and induce a population of suppressor cells, thus inhibiting or down-regulating autoimmunity. “Oral tolerance” has been successfully applied to treating experimental allergic encephalomyelitis (EAE),281 collagen-induced arthritis, adjuvant arthritis, uveoretinitis, experimental myasthenia gravis, and diabetes, in susceptible animal models.282 Initial clinical trials for human diseases including multiple sclerosis, rheumatoid arthritis, and uveitis demonstrated initial enthusiasm with no apparent toxicity, and decreases in T cell autoreactivity283,284 but more extensive human trials underway have shown only modest results.285-287 This approach has been recently applied to murine Tg-induced EAT, which could be prevented in most mice (80%) when Tg was fed before disease induction, and also reduced in 40% of mice when Tg was fed after disease induction.288,289 EAT induced in recipients after transfer of splenocytes from Tg-immunized donor mice, was also suppressed if donor mice were fed Tg before immunization.290 These models define an experimental system with possible relevance to immunosuppression of human AITD. However, oral immunotherapy was effective only with large doses of protein and tolerance was maintained just for 8-14 weeks, which would not be practical for human autoimmune thyroiditis. On the positive side, oral tolerance could lead to bystander suppression of all immune responses at the same tissue location, and because the immune response to all three thyroid autoantigens occurs in the same tissue, it would be conceivable that feeding Tg could suppress TSHR-Ab production and could be effective in treating Graves’ disease.291 However, caution is necessary in applying oral-antigen administration, since recently diabetes was induced in mice by

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feeding oral autoantigen.292 Therefore, such an approach may, under certain conditions which need to defined, also cause induction rather than prevention of autoimmunity.293

T cell Vaccination This is a procedure in which autoimmune T cells are administered to induce specific resistance to autoimmune disease and has been applied to the treatment of EAE,294 experimental diabetes,295,296 and also human multiple sclerosis.297 In EAT, both an attenuated mouse Tg-derived thyroiditogenic T cell line and a porcine-Tg T cell hybridoma, inhibited development of thyroiditis following immunization with Tg.298,299 Although EAT can be transferred only with CD4+ T cells, it seems that both CD4+ and CD8+ specific T cells can mediate vaccination induced suppression of murine EAE.300 Because T cell vaccination could be a potential treatment option for human AITD, much effort has been devoted to the mapping of disease related T cell epitopes in AITD (see above).180 This would enable generation of specific pathogenic T cell lines, which could be used for T cell vaccination.

DNA Immunization Vaccination of mice with DNA encoding a specific pathogenic T cell receptor has been shown to protect them from EAE.301 This type of “DNA vaccination” is another approach to specific immunotherapy and is based on the presence of intramuscular DNA which will induce protein expression. For example DNA encoding mycobacterial proteins injected intramuscularly into mice, induced protection against subsequent infection because of specific cellular response to the protein.302 A similar protective cellular immunity has been shown with DNA encoding surface glycoproteins of influenza virus.303,304 Immunization of mice with hTSHR-cDNA in the attempt to induce experimental Graves’ disease has generated TSHR-Abs and may signal an approach to AITD.303 However, such antibodies were not functional and extensive future research is needed to explore the potential preventive or curative aspect of such an approach.

Conclusions The autoimmune thyroid diseases are common examples of polygenic diseases with highly variable penetrance. The variable penetrance causes uncertainty as to the degree that environmental influences contribute to their etiology. Nevertheless, circumstantial evidence suggests that stress, infection and other undetermined factors may be important. The disease phenotype is variable, with some patients exhibiting overactive thyroid glands and others rapidly progressing to total thyroid failure and unpredictable extrathyroidal manifestations. The characteristics of the immune response conform to the clinical phenotypes, with thyroid-stimulating antibodies explaining the thyroid overactivity, because they act as TSH agonists, and cytotoxic T cells and antibody-mediated cytotoxicity explaining the cell damage of autoimmune thyroiditis. The identification of the responsible susceptibility genes will lead to our next phase of understanding in these model autoimmune diseases.

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228. Heyma P, Harrison LC, Robins BR. Thyrotropin (TSH) binding sites on Yersinia enterocolitica recognized by immunoglobulins from humans with Grave’s disease. Clin Exp Immunol 1986; 64:249-254. 229. Burman KD, Lukes YG, Gemiski P. Molecular homology between the human TSH receptor and Yersinia enterocolitica (abstract). Thyroid 1991; 1:S62. 230. Wenzel BE, Heesemann J, Heufelder A et al. Enteropathogenic Yersinia enterocolitica and organ-specific autoimmune diseases in man. Contrib Microbiol Immunol 1991; 12:80-88. 231. Luo G, Fan JL, Seetharamaiah GS et al. Immunization of mice with yersinia enterocolitica leads to the induction of antithyrotropin receptor antibodies. J Immunol 1993; 151:922-928. 232. Lindholm H, Visakorpi R. Late complications after a Yersinia Enterocolitica epidemic: A follow up study. Ann Rheum Dis 1991; 50:694-696. 233. Stuart PM, Woodward JG. Yersinia Enterocolitica produces superantigenic activity. J Immunol 1992; 148:225-233. 234. Davies TF. The TSH receptors spread themselves around. J Clin Endocrinol Metabol 1995; 79:1232-1233. 235. Ciampolillo A, Mirakian R, Schulz T et al. Retrovirus-like sequences in Graves’ disease: implications for human autoimmunity. Lancet 1989; 1:1096-1099. 236. Wick G, Grubeck-Loehenstein B, Trieb K et al. Human foamy virus antigens in thyroid tissue of Graves’ disease patients. Int Arch Allergy Immunol 1992; 99:153-156. 237. Wick G, Trieb K, Aguzzi A et al. Possible role of human foamy virus in Graves’ disease. Intervirology 1993; 35:101-107. 238. Humphrey M, Baker JJ, Carr FE et al. Absence of retroviral sequences in Graves’ disease. Lancet 1991; 337:17-18. 239. Tominaga T, Katamine S, Namba H et al. Lack of evidence for the presence of human immunodeficiency virus type 1-related sequences in patients with Graves’ disease. Thyroid 1991; 1:307-314. 240. Schweizer M, Turek R, Reinhardt M et al. Absence of foamy virus DNA in Graves’ disease. AIDS Res Hum Retroviruses 1994; 10:601-605. 241. Jaspan JB, Luo H, Ahmed B et al. Evidence for a retroviral trigger in Graves’ disease. Autoimmunity 1995; 20:135-142. 242. Jaspan JB, Sullivan K, Garry RF et al. The interaction of type A retroviral particle and class II leukocyte antigen susceptibility genes in the pathogenesis of Graves’ disease. J Clin Endocrinol Metab 1996; 81:2271-2279. 243. Jacobson M, Burne JF, Raff MC. Programmed cell death and Bcl-2 protection in the absence of a nucleus. EMBO J 1994; 13:1899-1910. 244. Nagata S. Apoptosis mediated by the Fas system. Prog Mol Subcell Biol 1996; 16:87-103. 245. Mountz JD, Zhou T, Su X et al. The role of programmed cell death as an emerging new concept for the pathogenesis of autoimmune diseases. Clin Immunol Immunopathol 1996; 80:S2-S14. 246. Wu J, Zhou T, Zhang J et al. Correction of accelerated autoimmune disease by early replacement of the mutated lpr gene with the normal Fas apoptosis gene in the T cells of transgenic MRL-lpr/lpr mice. Proc Natl Acad Sci USA 1994; 91:2344-8. 247. Racke MK, Critchfield JM, Quigley L et al. Intravenous antigen administration as a therapy for autoimmune demyelinating disease. Ann Neurol 1996; 39:46-56. 248. Pelfrey CM, Tranquill LR, Boehme SA et al. Two mechanisms of antigen-specific apoptosis of myelin basic protein (MBP)-specific T lymphocytes derived from multiple sclerosis patients and normal individuals. J Immunol 1995; 154:6191-6202. 249. Tsubata T, Murakami M, Honjo T. Antigen-receptor cross-linking induces peritoneal Bcell apoptosis in normal but not autoimmunity-prone mice. Curr Biol 1994; 4:8-17. 250. Mohacsi A, Trieb K, Anderl H et al. Retrobulbar fibroblasts from patients with Graves’ ophthalmopathy induce downregulation of APO-1 in T lymphocytes and protect T cells from apoptosis during coculture. Int Arch Allergy Immunol 1996; 109:327-333. 251. Kotani T, Aratake Y, Hirai K et al. Apoptosis in thyroid tissue from patients with Hashimoto’s thyroiditis. Autoimmunity 1995; 20:231-236.

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252. Giordano C, Stassi G, De Maria R et al. Potential involvement of Fas and its ligand in the pathogenesis of Hashimoto’s thyroiditis [see comments. Science 1997; 275:960-963. 253. Ansar Ahmed S, Penhale WJ, Talal N. Sex hormones, immune response and autoimmune disease. Am J Pathol 1985; 121:531-551. 254. Kincade PW, Medina KL, Smithson G et al. Pregnancy: a clue to normal regulation of B lymphopoiesis. Immunol Today 1994; 15:539-544. 255. Grossman CJ. Regulation of the immune system by sex steroids. Endocr Rev 1984; 5:435-455. 256. Chiovato L, Lapi P, Fiore E et al. Thyroid autoimmunity and female gender. J Endocrinol Invest 1993; 1:384-391. 257. Winsa B, Adami H, Bergstrom R et al. Stressful life events and Graves’ disease. Lancet 1991; 338:1475-1479. 258. Leclere J, Germain M, Weryha G et al. Role of stressful life-events in the onset of Graves’ disease. 10th International Thyroid Conference,The Hague 1991. 259. Sonino N, Girelli ME, Boscaro M et al. Life events in the pathogenesis of Graves’ disease. A controlled study. Acta Endocrinol Copenh 1993; 128:293-296. 260. Koutras D, Karaiskos K, Evangelopoulou K et al. Thyroid autoantibodies after iodine supplementation. In: The Thyroid and Autoimmunity (Drexhage H and Wiersinga W eds.), Excerpta Medica, Amsterdam 1986. 261. Weaver D, Nishiyiami R, Burton W et al. Surgical thyroid disease: a survey before and after iodine prophylaxis. Arch Surg 1966; 92:796-801. 262. Boukis MA, Koutras DA, Souvatzoglou A et al. Thyroid hormone and immunological studies in endemic goiter. J Clin Endocrinol Metab 1983; 57:859-862. 263. Martino E, Buratti L, Bartalena L et al. High prevalence of subacute thyroiditis during summer season in Italy. J Endocrinol Invest 1987; 10:321-323. 264. Monteiro E, Galvao-Teles A, Santos M et al. Antithyroid antibodies as an early humoral marker for thyroid disease induced by amiodarone. Br Med J 1986; 292:227-228. 265. Bagchi N, Brown T, Urdanivia E et al. Induction of autoimmune thyroiditis in chickens by dietary iodine. Science 1985; 230:325-327. 266. Sundick RS, Bagchi N, Brown TR. The role of iodine in thyroid autoimmunity: from chickens to humans: a review. Autoimmunity 1992; 13:61-88. 267. Braverman L. Iodine induced thyroid disease. In: Werner’s The thyroid, 5th edition (Ingbar S. and Braverman L, eds.), 1986:735-746, Lippincott, Philadelphia. 268. Costagliola S, Swillens S, Niccoli P et al. Binding assay for thyrotropin receptor autoantibodies using the recombinant receptor protein. J Clin Endocrinol Metab 1992; 75:1540-1544. 269. Filetti S, Foti D, Costante G et al. Recombinant human TSH receptor in a radio receptor assay for the measurement of TSH receptor autoantibodies. J Clin Endocrinol Metab 1991; 72:1096-1101. 270. Kendler DL, Brennan V, Davies TF et al. Expression of human thyroid peroxidase in insect cells using recombinant baculovirus. Mol Cell Endocrinol 1993; 93:199-206. 271. Haubruck H, Mauch L, Cook NJ et al. Expression of recombinant human thyroid peroxidase by the baculovirus system and its use in ELISA screening for diagnosis of autoimmune thyroid disease. Autoimmunity 1993; 15:275-284. 272. Grennan Jones F, Wolstenholme A, Fowler S et al. High-level expression of recombinant immunoreactive thyroid peroxidase in the High Five insect cell line. J Mol Endocrinol 1996; 17:165-174. 273. Huang GC, Collison KS, McGregor AM et al. Expression of a human TSH receptor fragment in E. coli and its interaction with the hormone and autoantibodies from patients with Graves’ disease. J Mol Endocrinol 1992; 8:137-144. 274. Graves PN, Vlase H, Davies TF. Folding of the recombinant human thyrotropin (TSH) receptor extracellular domain: identification of folded monomeric and tetrameric complexes that bind TSH receptor autoantibodies. Endocrinology 1995; 136:521-527. 275. Weetman AP, McGregor AM, Hall R. Evidence for an effect of antithyroid drugs on the natural history of Graves’ disease. Clin Endocrinol 1984; 21:163-172.

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276. Weetman AP, McGregor AP, Hall R. Methimazole inhibits thyroid autoantibody production by an action on accessory cells. Clin Immunol Immunopathol 1983; 28:39-45. 277. Weiss I, Davies TF. Inhibition of immunoglobulin-secreting cells by antithyroid drugs. J Clin Endocrinol Metab 1981; 53:1223-1228. 278. Davies TF, Yeo PP, Evered DC et al. Value of thyroid-stimulating-antibody determinations in predicting short-term thyrotoxic relapse in Graves’ disease. Lancet 1977; 1:1181-1182. 279. McGregor AM, Ibbertson HK, Rees SB et al. Carbimazole and autoantibody synthesis in Hashimoto’s thyroiditis. Br Med J 1995; 281:968-969. 280. McGregor AM, Petersen MM, McLachlan SM et al. Carbimazole and the autoimmune response in Graves’ disease. N Engl J Med 1980; 303:302-304. 281. Meyer AL, Benson JM, Gienapp IE et al. Suppression of murine chronic relapsing experimental autoimmune encephalomyelitis by the oral administration of myelin basic protein. J Immunol 1996; 157:4230-4238. 282. Whitacre CC, Gienapp IE, Meyer A et al. Treatment of autoimmune disease by oral tolerance to autoantigens. Clin Immunol Immunopathol 1996; 80:S31-S39. 283. Weiner HL, Friedman A, Miller A et al. Oral tolerance: immunologic mechanisms and treatment of animal and human organ-specific autoimmune diseases by oral administration of autoantigens. Annu Rev Immunol 1994; 12:809-837. 284. Hafler DA, Weiner HL. Antigen-specific immunosuppression: oral tolerance for the treatment of autoimmune disease. Chem Immunol 1995; 60:126-149. 285. Weiner HL. Oral tolerance for the treatment of autoimmune diseases. Annu Rev Med 1997; 48:341-351. 286. Matsui M. Application of oral tolerance to the treatment of autoimmune diseases—active suppression and bystander suppression. [In Process Citation. Nippon Rinsho 1997; 55:1537-1542. 287. Kagnoff MF. Oral tolerance: mechanisms and possible role in inflammatory joint diseases. Baillieres Clin Rheumatol 1996; 10:41-54. 288. Guimaraes VC, Quintans J, Fisfalen ME et al. Suppression of development of experimental autoimmune thyroiditis by oral administration of thyroglobulin. Endocrinology 1995; 136:3353-3359. 289. Guimaraes VC, Quintans J, Fisfalen ME et al. Immunosuppression of thyroiditis [see comments. Endocrinology 1996; 137:2199-2207. 290. Peterson K, Braley-Mullen H. Suppression of murine experimental autoimmune thyroiditis by oral administration of porcine thyroglobulin. Cell Immunol 1995; 166:123-130. 291. Rapoport B, McLachlan S. Editorial: Food for thought-Is induction of oral tolerance feasible and practical in human thyroid autoimmunity? Endocrinology 1996; 137:2197-2198. 292. Blanas E, Carbone FR, Allison J et al. Induction of autoimmune diabetes by oral administration of autoantigen. Science 1996; 274:1707-1709. 293. Heath W, Miller F. Oral tolerance: Feeding autoantigens can exacerbate rather than ameliorate autoimmune diseases. J NIH Res 1997; 9:35-39. 294. Ben NA Wekerle H, Cohen I. Vaccination against autoimmune encephalomyelitis with Tlymphocite line cells reactive against myelin basic protein. Nature 1981; 292:60-61. 295. Elias D, Markovits D, Reshef T et al. Induction and therapy of autoimmune diabetes in the nonobese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc Natl Acad Sci USA 1990; 87:1576-1580. 296. Elias D, Reshef T, Birk O et al. Vaccination against autoimmune mouse diabetes with a T cell epitope of human 65-kDa heat shock protein. Proc Natl Acad Sci 1991; 88:3088-3091. 297. Medaer R, Stinissen P, Truyen L et al. Depletion of myelin-basic-protein autoreactive T cells by T cell vaccination: pilot trial in multiple sclerosis. Lancet 1995; 346:807-808. 298. Maron R, Zerubavel R, Friedman A et al. T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice. J Immunol 1983; 131:2316-2322. 299. Roubaty C, Bedin C, Charreire J. Prevention of experimental autoimmune thyroiditis through the anti-idiotypic network. Journal of Immunology 1990; 144:2167-2172.

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300. Flynn J, Kong M. In vivo evidence for CD4+ and CD8+ suppressor T cells in vaccinationinduced suppression of murine experimental autoimmune thyroiditis. Clin Immunol Immunopath 1991; 60:484-494. 301. Waisman A, Ruiz J, Hirschberg D et al. Suppressive vaccination with DNA encoding a variable region gene of the T cell receptor prevents autoimmune encephalomyelitis and activates Th-2 immunity. Nat Med 1996; 2:899-905. 302. Tascon R, Colston M, Ragno S et al. Vaccination against tuberculosis by DNA injection. Nat Med 1996; 2:888-892. 303. Fu TM, Friedman A, Ulmer JB et al. Protective cellular immunity: cytotoxic T-lymphocyte responses against dominant and recessive epitopes of influenza virus nucleoprotein induced by DNA immunization. J Virol 1997; 71:2715-2721. 304. Bot A, Bot S, Garcia-Sastre A et al. DNA immunization of newborn mice with a plasmidexpressing nucleoprotein of influenza virus. Viral Immunol 1996; 9:207-210. 305. Dooper DS. Tobacco and Graves’ disease. J Am Med Assoc 1993; 269:518-519.

CHAPTER 7

Insulin Autoimmune Syndrome (IAS, Hirata Disease) Yasuko Uchigata and Yukimasa Hirata

Introduction

A

lthough HLA and disease association has been studied for many diseases, only four diseases have been identified in which almost all patients have the same HLA antigen; B27 in 88% of ankylosing spondylitis,1 DR4 in 91% of patients with pemphigus vulgaris,2 DR2 in 100% of patients with narcolepsy,3 DRw52a in 100% of patients with primary sclerosing cholangitis.4 When the first patient with spontaneous hypoglycemia associated with the production of insulin autoantibodies, so-called insulin autoimmune syndrome (IAS), was reported in Japan by Hirata et al5 in 1970, no one could forecast that IAS was the fifth disease with such a strong HLA-association. Many questions were raised by the first case, including its differential diagnosis from factitious hypoglycemia, the causes of this syndrome, the mechanisms to produce hypoglycemia in this syndrome and so on, raised doubt whether IAS was a “disease”. Immediately after the first patient was diagnosed with IAS, several other patients with the same symptoms and findings were reported over five years.6-9 The strong association of IAS with HLA-DR410 gave IAS a citizenship of “disease”, which was named Hirata’s disease. One hundred and ninety seven Japanese IAS patients have been registered from 1970 to 199211 and a total of 226 Japanese IAS patients have been registered through the end of 1996. Besides the analysis of those reports, several studies concerning the causes of IAS and the hypoglycemia have been clarified by us.

Insulin Autoimmune Syndrome as the Third Leading Cause of Spontaneous Hypoglycemia in Japan To determine the further characteristics of IAS in Japanese, we performed two nationwide surveys for causes of spontaneous hypoglycemia. Questionnaires were sent to 2094 hospitals with more than 200 beds; the first, from 1979 to 1981, the second, from 1985 to 1987. The first and the second surveys revealed the same results.12 Cases with hypoglycemia showed three main causes for the hypoglycemic attacks: insulinoma, extrapancreatic neoplasms and IAS. IAS was found to be the third leading cause of spontaneous hypoglycemia in Japan.

Onset Age, Sex Distribution, and Duration of Hypoglycemia of 226 Japanese IAS Patients Registered in Japan from 1970 to 1996

The records of 197 patients with IAS reported from 1970 to 199211 and 29 patients from 1993 to 1996 were analyzed. The records of a total of 226 patients were obtained from

Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

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Table 7.1. Age at Onset and Sex Distribution in Japanese IAS Patients, 1970-1996

Age at Onset 0 10 20 30 40 50 60 70 80

-

IAS Patient Male(n)

9 19 29 39 49 59 69 79 89

Total

Female(n)

Total

0 1 4 10 20 25 26 23 3

1 1 14 7 19 20 28 17 7

1 2 18 17 39 45 54 40 10

112

114

226

Table 7.2. Diseases and Drug Exposure Ahead of the Development of IAS Total n=131 Drugs

Diseases

Methimazole(MTZ) α-mercaptopropionyl glycine (MPG) α-mercaptopropionyl glycine (MPG) α-mercaptopropionyl glycine (MPG) α-mercaptopropionyl glycine (MPG) Glutathione(GTT)

Graves’ disease chronic liver dysfunction cataract dermatitis rheumatoid arthritis urticaria

miscellaneous

n 42 25 6 5 2 7 44

nationwide hypoglycemia surveys, abstracts in local or national medical congresses, and personal communications to us. Age of onset and sex distribution of the 226 patients are listed in Table 7.1. The age distribution was wide at onset of IAS. The peak age of onset was 60-69 years for both sexes; there was no remarkable sex difference in the other age distribution except 20-29 year group, in which 77% were female IAS patients. It seems that the 20-29 year group had a larger number of female patients with Graves’ disease. The duration of the transient and spontaneous hypoglycemia was shown to be less than 1 month in approximately 30% of the patients, more than 1 month and less than 3 months in 40% of the patients.11 A few of the patients have continued to suffer mild hypoglycemic attacks for more than 1 year. The geographic distribution of IAS in Japan showed no characteristic pattern in the areas of residence of the patients.

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Drug Exposure Ahead of Development of IAS and Associated Diseases As Hirata already noted in 1983, patients with Graves’ disease who had received methimazole (MTZ) had a predisposition to develop IAS.13 In addition to methimazole (MTZ) for the treatment of Graves’ disease, α-mercaptopropionyl glycine (MPG) for the treatment of chronic hepatitis, dermatitis, cataract and rheumatoid arthritis, and glutathione (GTT) for urticaria, which contains the sulfhydryl (SH) group, were proposed to be related to the develop IAS.11 Approximately 38% of Japanese IAS patients had received drugs with an SH group (Table 7.2). After such drugs were discontinued, the hypoglycemic attacks subsided. We have 4 IAS patients who developed IAS at the second treatment after interruption of MTZ therapy, 1 IAS patient who developed the disease after the third challenge (after two interruptions of MTZ therapy), and 1 IAS patient at both the first and the second MTZ treatment. Another 3 patients redeveloped IAS at MPG challenge.14 Such evidence may support the breakdown of T cell immunotolerance in the circumstance described above.

Clinical Features of IAS Patients Out of Japan Although there have been 226 IAS patients reported from 1970 to 1996 in Japan, 10 IAS patients have been reported in East Asians excluding Japanese patients (Table 7.3A). Nine of 10 IAS patients have associated Graves’ disease with the treatment of MTZ. Such patients were female and developed IAS at a younger age. HLA class II in 3 of them were analyzed, which was compatible with that in Japanese IAS patients, and insulin autoantibodies were all polyclonal, as described later. So far, 26 IAS patients in Caucasians have been reported in the past 26 years (Table 7.3B). MTZ for treatment of Graves’ disease and penicillamine for treatment of rheumatoid arthritis were administered to 3 IAS patients, which all contained SH group. Insulin autoantibodies of 6 IAS patients so far examined were monoclonal as described later.

Insulin in the Sera of the Patients with IAS Insulin in the sera of IAS patients was found to be native human insulin by HPLC analysis.15 Figure 7.1 shows total extractable IRI and 125I-insulin binding of the sera of patients with IAS. The IRI levels during hypoglycemic attacks were quite enormous.8 When hypoglycemia was severe, Scatchard analysis of the insulin antibodies showed that a highaffinity (k1)/low-capacity (b1) population of the antibodies was changed to relatively low affinity with very high binding capacity compared with the same population of antibodies in insulin-treated diabetic patient.16 When the attacks were relieved, the total IRI was decreased and the high-affinity (k1)/low-capacity (b1) population of antibodies showed a higher affinity constant and a lower binding capacity than those during the attacks.16 A possible monoclonal insulin autoantibody which was of IgG1(γ) subclass and has a very low affinity constant and a large binding capacity against human insulin was found to be directed at a determinant at the asparagine site on insulin B-chain.17 One of the idiotypic antibodies against the insulin autoantibody was found to express insulin action through the insulin receptor.18

Two Groups of IAS Defined by Clonality of Insulin Autoantibodies The immunoglobulin class, the subclass and the light chain types of insulin autoantibodies were examined.19 All insulin autoantibodies belonged to the IgG group with various ratios of κ:λ light chains. Insulin autoantibodies from IAS patients were classified as polyclonal or monoclonal on the basis of affinity curves for binding to human insulin (Scatchard analysis) and presence of solitary light chain. So far, 1 Japanese, 1 Norwegian, 1 Swiss, and 3 Italian IAS patients

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Fig. 7.1. Total extractable immunoreactive insulin (IRI), and 125Iinsulin binding% of the sera of male and female patients, immediately after diagnosis of insulin autoimmune syndrome (IAS). The methods for the IRI and 125I-human insulin binding assay have been described elsewhere. At diagnosis of IAS, the peak of the hypoglycemic attacks had passed. The normal range of total IRI and 125I-insulin binding was