Endocrine and Organ Specific Autoimmunity (Molecular Biology Intelligence Unit)

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.

2

Endocrine and Organ Specific Autoimmunity

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

3

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.

4

Endocrine and Organ Specific Autoimmunity

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

5

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

6

Endocrine and Organ Specific Autoimmunity

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

Immunobiology of Autoimmunity

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

Endocrine and Organ Specific Autoimmunity

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

Immunobiology of Autoimmunity

9

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

Endocrine and Organ Specific Autoimmunity

10

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

Immunobiology of Autoimmunity

11

(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.

12

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

Immunobiology of Autoimmunity

13

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

14

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).

References 1. Eisenbarth G, Bellgrau D. Autoimmunity. Science Med 1994; 1:38-47. 2. Theofilopoulos AN. The basis of autoimmunity: Part I. Mechanisms of aberrant self-recognition. Immunol Today 1995; 16:90-98. 3. Davis MM, Bjorkman PJ. T cell antigen receptor genes and T cell recognition. Nature 1988; 334:395-402. 4. Talmage DW, Sanderson RJ. Theoretical Essays: Why doesn’t everyone develop autoimmune diabetes? In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:292-295. 5. Gilles JG, Desqueper B, Lenk H, Vermylen J, Saint-Remy J-M. Neutralizing antidiotypic antibodies to factor VIII inhibitors after desensitization in patients with hemophilia A. J Clin Invest 1996; 97:(6)1382-1388. 6. Naquet P, Ellis J, Kenshole A et al. Sulfated beef insulin treatment elicits CD8+ T cells that may abrogate immunologic insulin resistance in type I diabetes. J Clin Invest 1989; 84:1479-1487. 7. Verge CF, Gianani R, Kawasaki E et al. Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes 1996; 45:926-933. 8. Bingley PJ, Christie MR, Bonifacio E et al. Combined analysis of autoantibodies improves prediction of IDDM in islet cell antibody-positive relatives. Diabetes 1994; 43:1304-1310. 9. Not T, Horvath K, Hill ID et al. Endomysium Antibodies in blood donors predicts a high prevalence of celiac disease in the USA. Gastroenterology 1996; 110:Suppl:A351 Abstract. 10. Friis SU. Coeliac disease, pathogenesis and clinical aspects. Acta Pathol Microbiol Immunol Scand 1996; 61:5-48. 11. Halsted MD. CH. The many faces of celiac disease. N Engl J Med 1996; 334:(18)1190-1191.

Immunobiology of Autoimmunity

15

12. Brown WR, Claman HN, Strober W. Immunological diseases of the gastrointestinal tract. In: Frank MM, Austen KF, Claman HN et al. Samter’s Immunologic Diseases. Fifth ed. Boston: LIttle, Brown and Company, 1994:1151-1178. 13. Dietrich W, Ehnis T, Bauer M et al. Identification of tissue transglutaminase as the autoantigen of cleiac disease. Nature Medicine 1997; 3:797-801. 14. Eisenbarth GS. Type I diabetes mellitus: A chronic autoimmune disease. N Engl J Med 1986; 314:1360-1368. 15. Greenbaum C, Palmer JP, Kuglin B et al. Insulin autoantibodies measured by radioimmunoassay methodology are more related to insulin-dependent diabetes mellitus than those measured by enzyme-linked immunosorbent assay: Results of the fourth international workshop on the standardization of insulin autoantibody measurement. J Clin Endocrinol Metab 1992; 74:1040-1044. 16. Theofilopoulos AN. The basis of autoimmunity: Part II. Genetic predisposition. Immunol Today 1995; 16:150-158. 17. Zinkernagel RM, Doherty PC. The discovery of MHC restriction. Immunol Today 1997; 18:14-17. 18. Garboczi DN, Ghosh P, Utz U et al. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2 [comment]. Nature 1996; 384:134-141. 19. Jardetzky TS, Brown JH, Gorga JC et al. Crystallographic analysis of endogenous peptides associated with HLA- DR1 suggests a common, polyproline II-like conformation for bound peptides. Proc Natl Acad Sci USA 1996; 93:734-738. 20. Bjorkman PJ, Saper MA, Samraoui B et al. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 1987; 329:506-512. 21. Bjorkman PJ, Saper MA, Samraoui B et al. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 1987; 329:512-518. 22. Brown JH, Jardetzky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993; 364:33-39. 23. Gautam AM, Lock CB, Smilek D, E. et al. Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: implications in induction of autoimmunity. Proc Natl Acad Sci USA 1994; 91:767-771. 24. Hunt DF, Michel H, Dickinson TA et al. Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad. Science 1992; 256:1817-1820. 25. Madrenas J, Chau LA, Smith J et al. The efficiency of CD4 recruitment to ligand-engaged TCR controls the agonist/partial agonist properties of peptide-MHC molecule ligands. J Exp Med 1997; 185:219-229. 26. Noble JA, Valdes AM, Cook M et al. The role of HLA class II genes in insulin-dependent diabetes mellitus: molecular analysis of 180 Caucasian, multiplex families. Am J Hum Genet 1996; 59:1134-1148. 27. Bodmer JG, Marsh SGE, Albert ED et al. Nomenclature for factors of the HLA system, 1995. Hum Immunol 1995; 43:149-164. 28. Bodmer WF. HLA: What’s in a name? A commentary on HLA nomenclature development over the years. Tissue Antigens 1997; 49:293-296. 29. Erlich HA, Griffith RL, Bugawan TL et al. Implication of specific DQB1 alleles in genetic susceptibility and resistance by identification of IDDM siblings with novel HLA-DQB1 allele and unusual DR2 and DR1 haplotypes. Diabetes 1991; 40:478-481. 30. Baisch JM, Week T, Giles R et al. Analysis of HLA-DQ genotypes and susceptibility in insulin-dependent diabetes mellitus. N Engl J Med 1990; 322:1836-1841. 31. Pugliese A, Gianani R, Moromisato R et al. HLA-DQB1*0602 is associated with dominant protection from diabetes even among islet cell antibody-positive first degree relatives of patients with IDDM. Diabetes 1995; 44:608-613. 32. Feltkamp TEW, Khan MA, Lopez de Castro JA. The pathogenic role of HLA-B27. Immunol Today 1996; 17:5-7. 33. Trucco M. To be or not to be ASP 57, that is the question. Diabetes Care 1992; 15:705-715.

16

Endocrine and Organ Specific Autoimmunity

34. Dorman JS, LaPorte RE, Stone RA et al. Worldwide differences in the incidence of type 1 diabetes are associated with amino acid variation at position 57 of the HLA-DQb chain. Proc Natl Acad Sci U S A 1990; 87:7370-7374. 35. Morel PA, Dorman JS, Todd JA et al. Aspartic acid at position 57 of the HLA-DQ beta chain protects against Type I diabetes: A family study. Proc Natl Acad Sci USA 1988; 85:8111-8115. 36. Acha-Orbea H, McDevitt HO. The first external domain of the nonobese diabetic mouse class II I-A chain is unique. Proc Natl Acad Sci U S A 1987; 84:2435-2439. 37. McDevitt H, Singer S, Tisch R. The role of MHC class II genes in susceptibility and resistance to type I diabetes mellitus in the NOD mouse. Horm Metab Res 1996; 28:287-288. 38. Ikegami H, Ogihara T. Genetics of insulin-dependent diabetes mellitus. Endocr J 1996; 43:605-613. 39. Lanzavecchia A. Understanding the mechanisms of sustained signaling and T cell activation [comment]. J Exp Med 1997; 185:1717-1719. 40. Ronningen KS, Undlien DE, Ploski R et al. Linkage disequilibrium between TAP2 variants and HLA class II alleles: no primary association between TAP2 variants and insulin-dependent diabetes mellitus. Eur J Immunol 1993; 23:1050-1056. 41. Wright-Browne V, McClain KL, Talpaz M et al. Physiology and pathophysiology of dendritic cells. Hum Pathol 1997; 28:563-579. 42. Todd I, Bottazzo GF. On the issue of inappropriate HLA class II expression on endocrine cells: an answer to a sceptic. J Autoimmun 1995; 8:313-322. 43. Racke MK, Scott DE, Quigley L et al. Distinct roles for B7-1 (CD-80) and B7-2 (CD-86) in the initiation of experimental allergic encephalomyelitis. J Clin Invest 1995; 96:2195-2203. 44. Kishimoto T, Goyert S, Kikutani H et al. CD antigens 1996. Blood 1997; 89:3502 45. Rose DR. The generation of antibody diversity. Am J Hematol 1982; 13:91-99. 46. Pugliese A, Zeller M, Fernandez A et al. The insulin gene is transcribed in the human thymus and transcription levels correlate with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type I diabetes. Nat Genet 1997; 15:293-297. 47. Vafiadis P, Bennett ST, Todd JA et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nat Genet 1997; 15:289-292. 48. Cao Y, Yang M, Luo Z et al. Vaccination with a multi-epitopic recombinant allergen induces specific immune deviation via T-cell anergy. Immunology 1997; 90:46-51. 49. Jacob CO, Also S, Michie S et al. Prevention of diabetes in nonobese diabetic mice by tumor necrosis factor (TNF): Similarities between TNF-alpha interleukin 1. Proc Natl Acad Sci USA 1990; 87:968-972. 50. Bellgrau D, Gold DP. Theoretical essays: The new math of diabetes: when Th1 - Th2 = Th0. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York: Oxford University Press, 1996:289-291. 51. Romagnani S. Th1 and Th2 subsets of CD4+ T lymphocytes. Science Med 1994; 1:68-77. 52. Calcinaro F, Lafferty KJ, Shehadeh NN. Inflammatory mediators and development of autoimmune diabetes. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:91-117. 53. Griffith TS, Ferguson TA. The role of FasL-induced apoptosis in immune privilege. Immunol Today 1997; 18:240-244. 54. Cohen JJ, Duke RC. Apoptosis and programmed cell death in immunity. Ann Rev Immunol 1992; 10:267-293. 55. Watanabe-Fukunaga R, Brannan CI, Copeland NG et al. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediate apoptosis. Nature 1992; 356:314-317. 56. Itoh N, Yonehara S, Ishii A et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991; 66:233-243. 57. Bellgrau D, Gold D, Selawry H et al. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377:600-602. 58. Stuart PM, Griffith TS, Usui N et al. CD95 ligand (FasL)-induced apoptosis is necessary for corneal allograft survival. J Clin Invest 1997; 99:396-402.

Immunobiology of Autoimmunity

17

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.

18

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

Autoimmune Polyendocrine Syndrome Type I (APECED)

29

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).

30

Endocrine and Organ Specific Autoimmunity

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

Autoimmune Polyendocrine Syndrome Type I (APECED)

31

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

Autoimmune Polyendocrine Syndrome Type I (APECED)

33

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

34

Endocrine and Organ Specific Autoimmunity

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-

Autoimmune Polyendocrine Syndrome Type I (APECED)

35

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 <200 pg/ml, pepsinogen 1 <20 mg/ml, gastrin >100 pmol/l. The definitive diagnosis of vitamin B12

Fig. 2.4. Percentage of patients with adrenocortical failure (left panel) and of female patients with ovarian failure (right panel) by time elapsed after the first antibody-positive serum sample (+) or, for antibody-negative patients (o), by time elapsed after the first serum sample. The number of patients is given at each point. From ref. 3.

36

Endocrine and Organ Specific Autoimmunity

malabsorption is based on subnormal absorption (<5%) of vitamin B12 in the Schilling test. The value in prediction of the life-threatening component of hepatitis of the antibodies recognizing CYP1A2 and CYP2A6 is unknown. At least, they are presumably useful in following the activity of hepatitis and its response to immuno-suppressive therapy.

Therapy Our own experience with immunosuppressive therapy in patients with APECED is limited to patients with hepatitis. This potentially life-threatening disease component can usually be suppressed by glucocorticoid or azathioprine, or a combination of both. For milder cases treatment indications have not been adequately defined. Prolonged pharmacological therapy with glucocorticoids tends to add growth failure to the already overwhelming problems of a child with APECED. Hence, other kinds of immunosuppression seem preferable. With azathioprine we have had no complications. We know of no reports on experience with the use of cyclosporine in these patients. Replacement therapy for endocrine deficiencies is so simple that immunosuppressive prevention of endocrinopathies has not appeared indicated considering its risks. Against the background of fluctuation of secretory capacity and slow destruction of the adrenal cortex in many patients, and the evidence of subclinical damage to pancreatic β-cells that may never lead to IDDM, bringing a gland to a complete rest by full replacement therapy, initiated at the first sign of autoimmune aggression against it, might protect the gland.45,89,90 A patient with APECED and infertility caused by antisperm antibodies gained fertility during cyclical prednisolone medication.6 In the future specific immunosuppressive therapies may become available with the identified autoantigens.92,93 Another hope is gene therapy after cloning of the culprit gene.

References 1. Ahonen P, Myllärniemi S, Sipilä I et al. Clinical variation of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. New Engl J Med 1990; 322:1829-1836. 2. Perheentupa J. Autoimmune endocrinopathy-candidiasis-ectodermal dystrophy (APECED). Horm Metab Res 1996; 28:353-356. 3. Ahonen P, Miettinen A, Perheentupa J. Adrenal and steroidal cell antibodies in patients with autoimmune polyglandular disease type I and risk of adrenocortical and ovarian failure. J Clin Endocrinol Metab 1987; 64:494-500. 4. Leisti S, Ahonen P, Perheentupa J. The diagnosis and staging of hypocortisolism in progressing autoimmune adrenalitis. Pediatr Res 1983; 17:861-867. 5. Zlotogora J, Shapiro MS. Polyglandular autoimmune syndrome type I among Iranian Jews. J Med Genet 1992; 29:824-826. 6. Tsatsoulis A, Shalet M. Antisperm antibodies in the polyglandular autoimmune (PGA) syndrome type I: Response to cyclical steroid therapy. Clin Endocrinol 1991; 35:299-303. 7. Betterle C, Caretto A, Zeviani M et al. Demonstration and characterization of anti-human mitochondria auto-antibodies in idiopathic hypoparathyroidism and in other conditions. Clin Exp Immunol 1985; 62:353-360. 8. Hung SO, Patterson A. Ectodermal dysplasia associated with autoimmune disease. British J Ophthalmol 1984; 68:367-369. 9. Scherbaum WA, Wass JAH, Besser GM et al. Autoimmune cranial diabetes insipidus: its association with other endocrine diseases and with histiocytosis X. Clin Endorinol 1986; 25:411-420. 10. Arvanitakis C, Knouss RF. Selective hypopituitarism. Impaired cell-mediated immunity and chronic mucocutaneous candidiasis. J Am Med Assoc 1973; 225:1492-1495.

Autoimmune Polyendocrine Syndrome Type I (APECED)

37

11. Myllärniemi S, Perheentupa J. Oral findings in the autoimmune polyendocrinopathycandidosis syndrome and other forms of hypoparathyroidism. Oral Surg 1978; 45:721-729. 12. Greenberg MS, Brightman VJ, Lynch MA et al. Idiopathic hypoparathyroidism, chronic candidiasis, and dental hypoplasia. Oral Surg 1969; 28:42-53. 13. Perheentupa J. Autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED). In: Eriksson AW, Forsius HR, Nevanlinna H et al. (eds.): Population Structure and Genetic Disorders. London: Academic Press Inc Ltd 1980:583-587. 14. Lukinmaa P-L, Waltimo J, Pirinen S. Microanatomy of the dental enamel in autoimmune polyendocrinopathy-candidiasisis-ectodermal dystrophy: Report of three cases. J Craniofac Genet Dev Biol 1996; 16:174-181. 15. Tarkkanen A, Merenmies L, Perheentupa J. Ocular changes in autoimmune-polyendocrinopathy-candidosis-ectodermal dystrophy syndrome. R Soc Med Int Congr Symp Ser 1981; 50:677-681. 16. Aaltonen J, Björses P, Sandkuijl L et al. An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21. Nature Gen 1994; 8:83-87. 17. Aaltonen J, Horelli-Kuitunen N, Bin-Fan J et al. High resolution physical and transcriptional mapping of the autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) locus on chromosome 21q22.3 by FISH. Genome Res 1997; 7:820-829. 18. Björses P, Aaltonen J, Vikman A et al. Genetic homogeneity of autoimmune polyglandular disease Type I. Am J Hum Genet 1996; 59:879-886. 19. Buckley RH: Primary immunodeficiency diseases. In: Paul WE (ed): Fundamental Immunity, 3rd Ed. New York: Raven Press Ltd. 1993:1353-1374. 20. Wirfält A: Genetic heterogeneity in autoimmune polyglandular failure. Acta Med Scand 1981; 210:7-13. 21. Chilgren RA, Quie PG, Meuwissen HJ et al. Chronic mucocutaneous candidiasis, deficiency of delayed hypersensitivity, and adrenocorticotrophic hormone deficiency. Lancet 1967; ii:688-693. 22. Castelles C, Fikrig S, Inamdar S et al. Familial moniliasis, defective delayed hypersensitivity, and adrenocorticotrophic hormone deficiency. J Pediatr 1971; 79:72-79. 23. Tomar RH, Rao RJ, Lawrence A et al. Moniliasis and anergy in hypoparathyroidism: treatment with transfer factor. Ann Allergy 1979; 42:241-45. 24. Arulanantham K, Dwyer JM, Genel M. Evidence for defective immunoregulation in the syndrome of familial candidiasis endocrinopathy. N Engl J Med 1979; 300:164-168. 25. Peterson P, Perheentupa J, Krohn KJ. Detection of candidal antigens in autoimmune polyglandular syndrome type I. Clin Diagn Lab Immunol 1996; 3:290-294. 26. Abbas AK, Murphy KM, Sher A. Functional diversity of helper T lymphocytes. Nature 1996; 383:787-793. 27. Liblau RS, Singer SM, McDevitt HO. TH1 and TH2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995; 16:34-38. 28. André I, Gonzalez A, Wang B et al. Checkpoints in the progression of autoimmune disease: Lessons from diabetes models. Proc Natl Acad Sci USA 1996; 93:2260-2263. 29. Segal BM, Klinman DM, Shevach EM. Microbial products induce autoimmune disease by an IL-12 dependent pathway. J Immunol 1997; 158:5087-5090. 30. Blizzard RM, Chee D, Davis W. The incidence of parathyroid and other antibodies in sera of patients with idiopathic hypoparathyroidism. Clin Exp Immunol 1966; 1:119-128. 31. Miettinen A, Ahonen P, Perheentupa J. Parathyroid and other auto-antibodies (AAb) in patients with autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED) (abstract). Ped Res 1982; 16:889. 32. Irvine WJ, Scarth L. Antibody to oxyphil cells of the human parathyroid in idiopathic hypoparathyroidism. Clin Exp Immunol 1969; 4:505-510. 33. Doniach D, Bottazzo GF. Polyendocrine auto-immunity. In: Franklin EC (ed): Clinical Immunology Update. Reviews for Physicians. Edinburgh: Churchill Livingstone 1981:95-121.

38

Endocrine and Organ Specific Autoimmunity

34. Betterle C, Caretto A, Zeviani M et al. Demonstration and characterization of anti-human mitochondria auto-antibodies in idiopathic hypoparathyroidism and in other conditions. Clin Exp Immunol 1985; 62:353-360. 35. Posillico JT, Wortsman J, Srikanta SS et al. Parathyroid cell surface auto-antibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells. J Bone Mineral Res 1986; 1:475-483. 36. Posillico JT, Wilson RE, Srikanta SS et al. Monoclonal antibody-mediated modulation of parathyroid hormone secretion by dispersed parathyroid cells. Arch Surg 1987; 122:436-442. 37. Brandi M-L, Aurbach GD, Fattorossi A et al. Antibodies cytotoxic to bovine parathyroid cells in idiopathic hypoparathyroidism. Proc Natl Acad Sci USA 1986; 83:8366-8369. 38. Fattorossi A, Aurbach GD, Sakaguchi K et al. Anti-endothelial cell antibodies: Detection and characterization in sera from patients with autoimmune hypoparathyroidism. Proc Natl Acad Sci USA 1988; 85:4015-4019. 39. Winqvist O, Karlsson FA, Kämpe O. 21-hydroxylase, a major auto-antigen in idiopathic Addison’s disease. Lancet 1992; 339:1559-1562. 40. Krohn K, Uibo R, Aavik A et al. Identification by molecular cloning of an auto-antigen associated with Addison’s disease as steroid 17a-hydroxylase. Lancet 1992; 339:770-773. 41. Winqvist O, Gustafsson J, Rorsman F et al. Two different cytochrome P450 enzymes are the adrenal antigens in autoimmune polyendcrine syndrome type I and Addison’s disease. J Clin Invest 1993; 92:2377-2385. 42. Uibo R, Aavik E, Peterson P et al. Antibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison’s disease. J Clin Endocrinol Metab 1994; 78:323-328. 43. Uibo R, Perheentupa J, Ovod V et al. Characterization of adrenal auto-antigens recognized by sera from patients with autoimmune polyglandular syndrome (APS) type I. J Autoimm 1994; 7:399-411. 44. Song Y-H, Connor EL, Muir A et al. Auto-antibody epitope mapping of the 21-hydroxylase in autoimmune Addison’s disease. J Clin Endocrinol Metab 1994; 78:1108-1112. 45. Winqvist O, Gebre-Medhin G, Gustafsson J et al. Identification of the main gonadal autoantigens in patients with adrenal insufficiency and associated ovarian failure. J Clin Endocrinol Metab 1995; 80:1717-1723. 46. Colls J, Betterle C, Volpato M et al. Immunoprecipitation assay for auto-antibodies to steroid 21-hydroxylase in autoimmune adrenal disease. Clin Chem 1995; 41:375-380. 47. Chen S, Sawickka J, Betterle C et al. Auto-antibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison’s disease, and premature ovarian failure. J Clin Endocrinol Metab 1996; 81:1871-1876. 48. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocrine Rev 1997; 18:107-134. 49. Asawa T, Wedlock N, Baumann-Antczak A et al. Naturally occurring mutations in human steroid 21-hydroxylase influence adrenal autoantibody binding. J Clin Endocrinol Metabolism 1994; 79:372-376. 50. Furmaniak J, Kominami S, Asawa T et al. Autoimmune Addison’s disease—evidence for a role of steroid 21-hydroxylase auto-antibodies in adrenal insufficiency. J Clin Endocrinol Metab 1994; 79:1517-1521. 51. Ahonen P, Miettinen A, Perheentupa J. Cytoplasmic antibodies in patients with autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy. Acta Endocrinol 1985; 109:suppl 270:78. 52. Björk E, Velloso LA, Kämpe O et al. GAD auto-antibodies in IDDM, stiff-man syndrome and autoimmune polyendocrine syndrome type 1 recognize different epitopes. Diabetes 1993; 43:161-165. 53. Velloso LA, Winqvist O, Gustafsson J et al. Auto-antibodies against a novel 51 kDa islet antigen and glutamate decarboxylase isoforms in autoimmune polyendocrine syndrome type I. Diabetologia 1994; 37:61-69.

Autoimmune Polyendocrine Syndrome Type I (APECED)

39

54. Tuomi T, Björses P, Falorni A et al. Antibodies to glutamic acid decarboxylase and insulin-dependent diabetes in patients with autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 1996; 81:1488-1494. 55. Wagner R, McNally JM, Bonifazio E et al. Lack of immunohistochemical changes in the islet of nondiabetic, autoimmune, polyendocrine patients with β selective GAD-specific islet cell antibodies. Diabetes 1994; 43:851-856. 56. Klemetti P, Björses P, Tuomi T et al. T cell proliferation and interferon-g response to glutamate decarboxylase in patients with autoimmune polyendocrinopathy -candidosis— ectodermal dystrophy. Diabetologia (in print) 57. Harrison LC, Honeyman MC, DeAizpura HJ et al. Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes. Lancet 1993; 341:1365-1369. 58. Paronen J, Klemetti P, Kantele JM et al. Glutamate decarboxylase-reactive peripheral blood lymphocytes from patients with IDDM express gut-specific homing receptor a4b7-integrin. Diabetes 1997; 46:583-588. 59. Husebye ES, Gebre-Medhin G, Tuomi T et al. Auto-antibodies against aromatic L-amino-acid decarboxylase in autoimmune polyendocrine syndrome Type I. J Clin Endocrinol Metab 1997; 82:147-150. 60. Manns MP, Griffin KJ, Quattrochi L et al. Identification of cytochrome P450IA2 as a human auto-antigen. Arch Biochem Biophys 1990; 280:229-232. 61. Sacher M, Blümel P, Thaler H et al. Chronic active hepatitis with vitiligo, nail dystrophy, alopecia and a new variant of LKM antibodies. J Hepatol 1990; 10:364-369. 62. Clemente MG, Meloni A, Obermayer-Straub P et al. Cytochrome P450 2A6: a new auto-antigen in liver disease. Hepatology 1996; 24:233A. 63. Clemente MG, Obermayer-Straub P, Meloni A et al. Cytochrome P450 1A2 is a hepatic auto-antigen in autoimmune polyglandular syndrome type 1. J Clin Endocrinol Metab 1997; 82:1353-1361. 64. Bourdi M, Larrey D, Nataf J et al. Anti-liver endoplasmic reticulum auto-antibodies directed against human cytochrome P-450IA2. Specific marker off hydralazine-induced hepatitis. J Clin Invest 1990; 85:1967-1973. 65. Obermayer-Straub P, Braun S, Grams B et al. Different liver cytochromes P450 are auto-antigens in hepatitis patients with autoimmune hepatitis and with autoimmune polyglandular syndrome type 1. Hepatology 1996; 24:234A. 66. Crock P, Salvi M, Miller A et al. Detection of anti-pituitary autoantibodies by immunoblotting. J Immunol Methods 1993; 162:31-40. 67. Dwyer DO, Perheentupa J, Crock P. Antipituitary auto-antibodies associated with autoimmune polyendocrinopathy (APECED) (abstract), Horm Res 1997; 48(suppl 2):187. 68. Ward L, Seidman E, Huot C, Alvarez F, Crock P, Delvin E, Deal C. Exocrine pancreatic insufficiency and autoimmune hypophysitis in a child with autoimmune polyendocrine syndrome type I. (abstract), Horm Res 1997; 48(suppl 2):186. 69. Mirakian R, Bottazzo GF. The autoimmune pathogenesis of chronic gastritis and pernicious anemia. In: Bhatt HR, James VHT, Besser GM, Bottazzo GF, Keen H (eds) Advances in Thomas Addison’s Diseases. Bristol: Journal of Endocrinology Ltd 1994:193-203. 70. Burman P, Mardh S, Norberg L et al. Parietal cell antibodies in pernicious anemia inhibit H+,K+ -adenosine triphosphatase, the proton pump of the stomach. Gastroenterology 1989; 96:1434-1438. 71. Goldkorn I, Gleeson PA, Tob B-H. Gastric parietal cell antigens of 60-90, 92 and 100-120 kDa associated with autoimmune gastritis and pernicious anemia. J Biol Chem 1989; 264:18769-18774. 72. Hall K, Perez G, Anderson D et al. Location of the carbohydrates present in the H+ ATPase vesicles isolated from the hog gastric mucosa. Biochemistry 1990; 29:801-806. 73. Reuben MA, Lasater LS, Sachs G. Characterization of a b subunit of the gastric H+/K+ transporting ATPase. Proc Soc Nat Acad Sci USA 1990; 87:6767-6771. 74. Shull GE. cDNA cloning of the b subunit of the rat gastric H+, K+-ATPase. J Biol Chem 1990; 265:12123-12126.

40

Endocrine and Organ Specific Autoimmunity

75. Goldkorn I, Gleeson PA, Tob B-H. Reverse immunoaffinity chromatography: Application to purification of the 60-90 kDa gastric parietal cell auto-antigen associated with autoimmune gastritis. Anal Biochem 1991; 194:433-438. 76. Padeh S, Theodor R, Jonas A et al. Severe malabsorption in autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy syndrome successfully treated with immunosuppression. Arch Dis Childh 1997; 76:532-534. 77. Brostoff J, Bor S, Feiwel M. Auto-antibodies in patients with vitiligo. Lancet 1969; 2:177-178. 78. Hertz KC, Gazze LA, Kirkpatrick CH et al. Autoimmune vitiligo: detection of autoantibodies to melanin-producing cells. N Engl J Med 1977; 297:246-251. 79. Naughton GK, Eisinger M, Bystryn JC. Antibodies to normal human melanocytes in vitiligo. J Exp Med 1983; 158:246-251. 80. Song Y-H, Conner E, Li Y et al. The role of tyrosinase in autoimmune vitiligo. Lancet 1994b; 344:1049-1052. 81. Montazeri A, Serre G, Kanitakis J. Alopecia areata. Eur J Dermatol 1996; 471-478. 82. Vitali C, Doria A, Tincani A et al. International survey on the management of patients with SLE. 1. General data on the participating centers and the results of a questionnaire regarding mucocutaneous involvement. Clin Exp Rheumatol 1996; 14 (suppl 16):S17-S22. 83. Tobin DJ, Hann SK, Song MS et al. Hair follicle structures targeted by antibodies in patients with alopecia areata. Arch Dermatol 1997; 133:57-61. 84. Weetman AP. Antigen presentation in the pathogenesis of autoimmune endocrine disease. J Autoimm 1995; 8:305-312. 85. Harley JB, Scofield RH. Systemic lupus erythematosus: RNA-protein auto-antigens, models of disease heterogeneity, and theories of etiology. J Clin Immunol 1991; 11:297-316. 86. McNatty KP, Short RV, Barnes EW et al. The cytotoxic effect of serum from patients with Addison’s disease and autoimmune ovarian failure on human granulosa cells in culture. Clin Exp Immunol 1975; 22:378-384. 87. Betterle C, Zanchetta R, Trevisan A et al. Complement-fixing adrenal auto-antibodies as a marker for predicting onset of idiopathic Addison’s disease. Lancet 1983; 1:1238-1241. 88. Ahonen P, Koskimies S, Lokki M-L et al. Expression of autoimmune polyglandular disease type I appears associated with several HLA-A antigens but not with HLA-DR. J Clin Endocrinol Metab 1988; 66:1152-1157. 89. Jansson R, Karlsson A, Dahlberg PA. Thyroxine, methimazole, and thyroid microsomal auto-antibody titres in hypothyroid Hashimoto’s thyroiditis. Br Med J [Clin Res] 1985; 290:11-12. 90. Kämpe O, Andersson A, Björk E et al. High-glucose stimulation of 64,000-Mr islet cell auto-antigen expression. Diabetes 1989; 38:1326-1328. 91. Buschard K. The functional state of the beta cells in the pathogenesis of insulin-dependent diabetes mellitus. Auto-immunity 1991; 10:65-69. 92. Adorini L, Sinigaglia F. Pathogenesis and immunotherapy of autoimmune diseases. Immunol Today 1997; 18:209-211. 93. Weiner HL. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997; 18:335-43.

CHAPTER 3

Autoimmune Polyendocrine Syndrome Type II Maria J. Redondo and George S. Eisenbarth

Introduction

T

he two major autoimmune polyendocrine syndromes are termed autoimmune polyendocrine syndrome type I (APS-I) and APS-II.1-4 These syndromes are of particular interest in that they have led to the identification of a series of diseases of autoimmune etiology5-8 and they have provided theoretical links between various autoimmune diseases. The two syndromes are very distinct in terms of their genetic etiology and have different, though somewhat overlapping disease associations (Table 3.1). The APS-I syndrome is reviewed in detail in chapter 2 by Miettine and Perheentupa.

Autoimmune Polyendocrine Syndrome Type II Syndrome Definition APS-II is characterized by more than one of the following disorders: Addison’s disease, autoimmune thyroid disease (Graves’ disease and hypothyroidism), type I diabetes, celiac disease, hypogonadism, vitiligo, alopecia, pernicious anemia, myasthenia gravis and a series of less common disorders (Table 3.2). It fundamentally differs from APS-I which is characterized by hypoparathyroidism and mucocutaneous candidiasis. At present, one cannot distinguish in a fundamental way (e.g. genetic, additional disease associations) individuals with Addison’s disease plus Graves’ disease from individuals with, for example, autoimmune type 1 diabetes plus Graves’ disease. The association of autoimmune thyroid disease with an autoimmune disorder other than Addison’s disease and hypoparathyroidism has been called APS-III.2,9 Finally association of other diseases such as immune type I diabetes and myasthenia gravis, but not Addison’s disease, hypoparathyroidism or autoimmune thyroid disease, has been termed APS-IV. We believe that the features of APS-III and APS-IV are not distinctive enough from APS-II to constitute unique syndromes and we distinguish only APS-I and APS-II, where APS-II includes APS-II, APS-III and APS-IV. The APS-II syndrome is far more common than APS-I. It is three times more frequent in females than in males and the usual age of onset is adulthood, in the second decade or later. APS-I characteristically presents during childhood but it may appear between 19 months and 35 years of age and affects males and females in the same proportion. The most characteristic disorder of APS-II is Addison’s disease. Addison’s disease results from destruction of the adrenal cortex, as opposed to secondary or tertiary adrenal insufficiency, in which there is a lack of secretion of stimulating Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

42

Endocrine and Organ Specific Autoimmunity

DQB1*0302 DQB1*0201 Fig. 3.1. Family with four members with Addison’s disease in two generations. All four family members with Addison’s disease have what we believe is the major genotype of the disorder namely DRB1*0404, DQ8 with DRB1*0301, DQ2 (Indicated B/C). Of note the DR3 haplotype (DRB1*0301) of the patients with Addison’s disease came from three different haplotypes (c, d, and e) of two parents suggesting that the “common” DR3 haplotype carries Addison’s disease susceptibility. In contrast the DR4 (DRB1*0404) haplotype (B) was introduced only once into the family.

hormones by the hypothalamus or pituitary. Addison’s disease is most frequently of autoimmune origin in developed countries, although in the past it was often tuberculosisrelated. 5,6 There are additional rare causes of primary adrenal insufficiency such as adrenoleukodystrophy.10 The clinical manifestations of primary adrenal insufficiency are caused mainly by cortisol, aldosterone and adrenal sex hormone deficiency. Nausea, vomiting and weight loss are frequently presenting symptoms. Cortisol deficiency causes fatigue, muscle weakness, anorexia, weight loss, hypoglycemia, normocytic anemia, lymphocytosis and eosinophilia. Aldosterone deficiency causes orthostasis, salt craving, hyponatremia (also due to cortisol deficiency), hypotension, hyperkaliemia and metabolic acidosis. Darkening of the skin is secondary to the decreased cortisol feed back, which stimulates the overproduction of ACTH and related peptides, among them, melanocyte stimulating hormone. Loss of axillary and pubic hair is seen in women as a consequence of the lack of adrenal androgenic steroids.

Autoimmune Polyendocrine Syndrome Type II

43

Table 3.1. Comparison of APS-I and APS-II APS-I

APS-II

Inheritance

Autosomal recessive1-4 chromosome 21

Autosomal dominant5,30,52 polygenic/oligogenic

HLA association

None

DQ and DR Alleles5

Immunodeficiency defined

Mucocutaneous Candidiasis125 Asplenism126

Not well defined

Usual age of onset

Infancy3

Adulthood31

Mucocutaneous candidiasis

73-100% 4

Not associated

Hypoparathyroidism

80%-89%4,127

Not associated

Addison’s disease

60-72%4,127

70%

Type 1 diabetes

4-15%46,127,128

52%46

Autoimmune thyroid disease

10-40%4,46

70%46,11

Gonadal failure

38-60% of females, 7-14% of males 4,80,127

3.5-10%46 5-50% 127

Vitiligo

4-9% 46,127

4.5% 46

Hepatitis

10-15%4,127

Rare

Pernicious anemia

12-15%4,127

< 1% 46,127

Malabsorption

18%4

with celiac disease

Autoimmune thyroid disease is a constellation of disorders including autoimmune thyroiditis (which has a goitrous form known as Hashimoto’s disease and an atrophic form), Graves’ disease, painless thyroiditis and postpartum thyroiditis. The latter is more common in postpartum women with type I diabetes. Graves’ disease is characterized by hyperthyroidism, diffuse goiter, ophthalmopathy and dermopathy. Any of these signs, even the goiter, may be absent in a given patient. Autoimmune thyroid disease is more common (70%)11 than type I diabetes in APS-II patients and can present at any age. 12 It is reviewed in chapter 5. Immune mediated type I diabetes is present in approximately half of patients with Addison’s disease and APS-II. It is reviewed in chapter 8. Celiac disease is a disorder characterized by gluten-induced malabsorption. It often appears soon after onset of type I diabetes.13,14 Celiac disease is reviewed in chapter 5.

Endocrine and Organ Specific Autoimmunity

44

Table 3.2. Diseases Associations Reported for Addison’s Disease and Type 1 Diabetes Mellitus Disease

Prevalence with Addison’s

Type 1 diabetes mellitus

50-10%

Addison’s disease

N/A

0.5-1%130

Celiac disease

1.2%131,132

2.4-6%133

Hypothyroidism

20-70%129,131

4%130

Graves’ disease

6-11%129,131

0.02-2 %130,134

Pernicious anemia

0.4-1.1%130

3.9-11%96

Premature ovarian failure

7.3-20%129,131,135,136

6.7 %137

Hypophysitis

Not reported

Occasional reports138,139

Vitiligo

9.6 %131

10-18%

Myasthenia gravis

Occasional reports140,141

Not available

Hypoparathyroidism

1.2%131

Rare reports142

Mucocutaneous candidiasis

with APS-I

Rare

Serositis

Case report

Not available

Sjogren’s disease

2.4%131

Not available

129

Prevalence with type 1 diabetes N/A

Myasthenia gravis is an autoimmune disease characterized by weakness that worsens during repeated muscular contractions.15 The disease is caused by anti-acetylcholine receptor autoantibodies and is reviewed in chapter 9. The thymus probably plays an important role in the pathophysiology of this disease since 10-15% of the patients with myasthenia gravis harbor a thymoma and up to 65-80% have thymic hyperplasia. Thymectomy improves myasthenia gravis in up to 85% of patients. The clinical features may be modified by association with Graves’ disease. Thymic disease seems to be less frequent than in other subsets of patients and the proportion of ocular myasthenia is higher in Graves’ disease patients.16 Over 10% of women under 40 years of age with APS-II develop hypergonadotropic hypogonadism. It can present as primary or secondary amenorrhea. Primary amenorrhea, or absence of menarchia, results from ovarian failure before the age of the first menstruation. Secondary amenorrhea is the cessation of menstruations in a women before the expected age of physiological menopause.

Autoimmune Polyendocrine Syndrome Type II

45

Involvement of the pituitary region is seen in a subset of patients with APS-II. The more common disorder is hypophysitis with isolated failure of secretion of ACTH, TSH, FSH or LH.17-19 Empty sella syndrome20 and an FSH-producing adenoma21 have also been reported as has isolated gonadotropin deficiency resulting in hypogonadotropic hypogonadism associated with APS-II.22 A variety of other conditions have been reported in association with APS-II, such as primary biliary cirrhosis,23 achalasia24 and sarcoidosis.9,25 A sensory-autonomic polyneuropathy has been described with a potential autoimmune inflammatory contribution.26 Interstitial myositis with a perifascicular denervation has also been reported.27 Ulcerative colitis has been occasionally associated with Addison’s disease.28 In patients with APS-II the number of associated disorders which will develop and their age of appearance are clinically unpredictable and thus long-term follow-up is necessary. A variety of autoantibodies aid in disease diagnosis or prediction and, in some cases, such as celiac disease, Addison’s disease and type 1 diabetes, presence of autoantibodies confers a high risk of developing disease. It is advisable to screen patients with Addison’s for a series of disorders (e.g., TSH determination, serum B12, anti-endomysial autoantibodies, anti-islet autoantibodies). Patients with type 1 diabetes should be screened for autoimmune thyroid disease (TSH measurements), celiac disease (anti-endomysial autoantibodies) and Addison’s disease (21-hydroxylase autoantibodies). There is no specific therapy for APS-II though there are a number of special situations where clinical diagnosis or management is specific for certain disease combinations. The clinical management of APS-II patients includes early diagnosis of associated disorders. Associated disorders are then treated as they are diagnosed. Therapy for hypothyroidism may trigger an Addisonian crisis in a patient with unsuspected Addison’s disease. An unexpected decrease in insulin needs or the occurrence of increasing hypoglycemic episodes in a patient with diabetes should prompt evaluation for Addison’s disease. Steroid therapy for sarcoidosis might mask the presence of Addison’s disease. Steroid therapy for Addison’s disease may trigger a hypocalcemic crisis in a patient with hypoparathyroidism. Addisonian hypercalcemia may mask hypocalcemia due to hypoparathyroidism and delay its diagnosis.29 Hypocalcemia in a patient with APS-I is more likely to be due to celiac disease rather than the rare (for APS-II) hypoparathyroidism. In a patient with type 1 diabetes, the onset of hyperthyroidism may result in increased insulin needs whereas the appearance of hypothyroidism may cause hypoglycemic events.

Genetics The genetic susceptibility to APS-II is mainly conferred by genes in the human leukocyte antigen (HLA) region (major histocompatibility complex, MHC), on the short arm of chromosome 6 (e.g. DQA1*0501, DQB1*0201).3,30-33 Many but not all of the diseases of APS-II are associated with DR3 haplotypes. Type 1 diabetes and Addison’s disease are both strongly HLA associated.34,35 Only a weak association between HLA and thyroiditis has been documented.36 There are approximately 100 genes within the major histocompatibility complex.37 Many of these genes are intimately involved in the regulation of immune function.38 In addition, alleles of these genes are nonrandomly associated with each other and evidence “linkage dysequilibrium” (i.e., the distribution of alleles are not random and have not reached evolutionary equilibrium).39,40 Such groups of alleles nonrandomly associated are termed “extended haplotypes”. Disease susceptibility and resistance can be associated with specific DQ alleles and then secondarily to specific DR molecules (Table 3.3). Both the α and β chains of DQ are polymorphic. DQα and DQβ chain alleles can form a complete DQ molecule either in cis (DQ α and DQ β coded on the same chromosome) or in trans (DQ α from one

Endocrine and Organ Specific Autoimmunity

46

chromosome and DQβ from the other chromosome). The ability to form trans DQ heterodimers is of particular importance for celiac disease (see chapter on celiac disease) and potentially for Addison’s disease. APS-II is classically associated with the extended HLA haplotype HLA A1, B8, DR3, DQA1*0501, DQB1*0201.30,41 Initial studies associated the class I HLA alleles A1 and B8 with APS-II prior to the discovery of the class II MHC genes.30 A1 and B8 are associated with Addison’s disease, type 1 diabetes mellitus and celiac disease because they are in linkage dysequilibrium with DR3 and DQA1*0501, DQB1*0201 (DQ2).39 In addition to its association with DQA1*0501, DQB1*0201 Addison’s disease is strongly associated with the class II genotype consisting of the DR3 haplotype and a DR4 haplotype with DQA1*0301, DQB1*0302 (DQ8). In our studies approximately 50% of patients with Addison’s disease have this genotype in comparison to 2.3% of the general population. What is particularly interesting concerning this haplotype is that it is also a high risk genotype for type 1 diabetes mellitus (approximately 40% of type 1 diabetes patients are DQ8/DQ2 heterozygotes). In addition we find that more than 80% of patients with Addison’s disease and this genotype have a specific DR4 DRB1 allele, DRB1*0404. (Figure 3.1) The genotype DRB1*0404,

Table 3.3. HLA Association of APS-II Disorders DISEASE

HLA ASSOCIATION DRB DQA/DQB

COMMENT

Addison’s disease

0404 0301 05

0301/0302 0501/0201 0501/0301

DR “3/4” with DRB1*0404 is predominant genotype

Type 1 diabetes mellitus

0401, 0402 and 0404 0301

0301/0302 0501/0201

DR “3/4” with DRB1*0401, 0402, or 0404 is predominant genotype

Celiac disease

0501/0201-2 DR “3” in cis DR “5” and “7” in trans

Graves’ disease

0301

0501/0201

Myasthenia gravis

0301 DR1

0501/0201

Atrophic thyroiditis Goitrous thyroiditis

B8,Dw3-DR3 DR546

0501/0201

Pernicious anemia

DR546

Sjogren syndrome

B8,Dw3,DR346 0501/0201

Myasthenia gravis

B88,Dw3,DR346 0501/0201

Spontaneous Penicillamine induced

When associated with other endocrinopathies may be B8,Dw3,DR3-associated46

In young females46

Autoimmune Polyendocrine Syndrome Type II

47

DQA1*0301, DQB1*0302 with DRB1*0301, DQA1*0501, DQB1*0201 occurs in 1/200 newborns in Denver, Colorado, and in more than 50% of patients with Addison’s disease. There is one report that Addison’s disease in the absence of type 1 diabetes mellitus is only associated with DR3, DQA1*0501, DQB1*0201 and is not associated with DR4.35 In that many patients with Addison’s disease have type 1 diabetes mellitus, and the DR3/4 genotype is enriched in type 1 diabetes mellitus, exclusion of patients with diabetes may have reduced the ability to detect the association with DR4. We have recently found that Addison’s disease and type 1 diabetes mellitus share a major “risk” genotype (DR3/DR4) with DRB1*0404. Addison’s disease is associated with only one of three DRB1 alleles (DRB1*0404) commonly found in patients with type 1 diabetes mellitus. This association is likely to account for a significant portion of the association between Addison’s disease and type 1 diabetes mellitus. We estimate that approximately 1/20 patients with type 1 diabetes mellitus with the DR3/4 genotype will have anti-adrenal autoantibodies.42 Another high risk haplotype for Addison’s disease consists of a DR5 haplotype with DQA1*0501 with DQB1*0301. The DQ molecule formed by DQA1*0501, DQB1*0301 is very similar to the molecule formed in trans in DR3/4 individuals DQA1*0501,DQB1*0302 suggesting that these heterodimers may underlie a major portion of Addison’s disease susceptibility. DR2-DQA1*0102/DQB1*0602 is present in less than 1% of children with type 1 diabetes but greater than 20% of the general populations.33 The protection conferred by this haplotype is dominant since when present with high risk DQ alleles, the risk of type 1 diabetes remains low. Of importance, approximately 7% of relatives of patients with type 1 diabetes mellitus who express anti-islet autoantibodies have DQA1*0102, DQB1*0602, and the great majority of such individuals do not progress to diabetes.43 Thus DQA1*0102, DQB1*0602 appears to provide protection even in the presence of anti-islet autoantibodies. In a similar manner, patients expressing 21-hydroxylase autoantibodies with DRB1*0401 or DRB1*0402 appear to less often progress to Addison’s disease. This has led to the hypothesis that DRB1 typing may identify a subgroup of 21-hydroxylase autoantibody positive individuals with a low risk of progression to Addison’s disease. Celiac disease is associated with both DR3 and DR4 molecules, with 78% and 56% of the celiac patients being positive for them, respectively.13 Celiac disease is most strongly associated with HLA-DQ2 (alpha*0501, beta*0201). DR3 haplotypes almost always (>99%) have DQA1*0501 and DQB1*0201 (genes in cis). In addition an individual with DR5 (DQA1*0501, DQB1*0301) and DR7 (DQA1*0201, DQB1*0202) can form a similar 0501/ 0202 heterodimer in trans. A pathogenic role for this DQ2 molecule has been posited as a restriction element for intestinal gliadin-specific T cells.44 Other DR3 associated diseases are Graves’ disease (65% of the patients have DR3DQA1*0501, DQB1*0201), myasthenia gravis15 and other autoimmune diseases that are not classically associated with APS-II such as lupus eythematosus. The goitrous form of chronic autoimmune thyroiditis is DR5-associated. In Down’s syndrome, chronic autoimmune thyroiditis is associated with HLA-DQA1*0301.45 Clinical heterogeneity of myasthenia gravis patients correlates with its genetic heterogeneity. DR3 alleles are associated with myasthenia gravis in patients without a thymoma and with high titers of anti-acetylcholine-receptor antibodies.46 Penicillamine-induced myasthenia gravis has a different HLA association, with an increase in DR1.47-49 Specific amino acids of DQ molecules have been associated with disease. A valine, serine or alanine at position 57 in DQB (instead of an aspartic acid) is associated with higher risk for type 1 diabetes in Caucasoid populations.50,51 There are other polymorphisms, such as an arginine at position 52 of DQA1, which are also associated with type 1 diabetes mellitus (94%), Graves disease (80%) and Addison’s disease (89%) compared to controls (66%).33 Such single amino acid associations are only generalizations. For example the DQ alleles

48

Endocrine and Organ Specific Autoimmunity

DQB1*0402 in Causcasoid populations and DQB1*0401 in Japanese patients, both have aspartic acid at position 57, but confer high risk for type 1 diabetes mellitus. It is likely that HLA alleles such as the DR”3/4” genotype account for a portion but not all of the disease associations of the APS-II syndrome. It is likely that there are genes outside of the HLA region which influence immune function and contribute to the multiple organs to which “tolerance” is lost in this syndrome. At present the identity of these additional loci is largely unknown despite genome wide studies of a number of selected diseases such as type 1 diabetes mellitus, celiac disease and Graves’ disease. Recently it has been suggested that an alanine at codon 17 in the sequence of the cytotoxic T lymphocyte antigen 4 (CTLA 4 located on chromosome 2q33) is associated with susceptibility to Graves’ disease and type 1 diabetes mellitus.52 The association is relatively weak and for type 1 diabetes has not been replicated in a number of populations.

“Triggering Factors” For the component disorders of the APS-II syndrome less than 70% of identical twin pairs are concordant. This suggests a role for nonMendelian genetic factors (e.g. mutations or the influence of “random” joining of T cell receptor or immunoglobulin gene segments), “chaotic” events, or environmental factors in disease etiology. Epidemiological studies in man and experiments in animal models indicate that environmental factors can initiate or suppress the development of a number of autoimmune disorders. Only a few environmental factors have been identified. For instance, in celiac disease, ingestion of gluten induces disease53 and congenital rubella infection is associated with thyroiditis and type 1 diabetes mellitus.54 A number of drugs are known to be able to trigger autoimmune disease. Treatment with procainamide may be followed by APS diseases as well as by lupus.55 Anti-thyroglobulin or anti-microsomal autoantibodies appear in up to 20% of interferon alpha-treated patients and clinical hypothyroidism occurs in 5%.56-58 This drug may also cause seroconversion or increase in the titer of antimitochondrial autoantibodies, parietal cell autoantibodies and smooth muscle cell autoantibodies. Appearance of anti-microsomal and anti-thyroglobulin antibodies along with the development of hypothyroidism can also follow treatment with interleukin-2 and lymphokine-activated killer cells.59 There is a report on the development of hypothyroidism after treatment with granulocyte-macrophage colony-stimulating factor.60 Penicillamine, used in rheumatoid arthritis patients, can induce myasthenia gravis and anti-insulin antibodies. There are several ways in which environmental factors may be involved in disease pathogenesis. In a T cell mediated autoimmune disease such as type I diabetes or autoimmune thyroiditis, molecular “mimicry” with an environmental antigen might trigger activation of T cells and subsequent clonal expansion of the activated T cells reactive with a self-protein. Viral infections may also reactivate autoimmunity through direct tissue destruction. A number of viruses have been documented to trigger diabetes in animal models. In man, autoantibodies to Coxsackie viruses are in some reports more frequent in patients than controls. The only infection convincingly associated to type 1 diabetes is congenital rubella infection, which is followed by diabetes in up to 33% of the affected children. Thyroiditis is also very common following congenital rubella infection.61,62 An intriguing hypothesis for celiac disease is that gluten which is greatly enriched in glutamine is coupled to transglutaminase (transglutaminase is a major intestinal autoantigen) and creates a “neoantigen” recognized by the immune system.

Autoimmune Polyendocrine Syndrome Type II

49

Humoral Autoimmunity Multiple organ-specific autoantibodies have been identified and even when their pathogenic significance is not clear, many of these autoantibodies are useful for diagnosis or disease prediction. Although autoantibodies usually precede the clinical onset of the disease, in many cases it is not known whether their presence is a cause or a consequence of cellular damage. In some disorders the pathogenic role played by autoantibodies is well established. Type 1 diabetes is believed to be a T cell mediated autoimmune disease and the associated autoantibodies are not probably “causative” but a consequence of tissue damage. The observation that the passage of anti-islet antibodies from a diabetic pregnant woman to the fetus does not cause neonatal diabetes, as opposed to what may happen in Graves’ disease and myasthenia gravis, supports the hypothesis that the antibodies of type 1 diabetes are not pathogenic. “Causative Autoantibodies” Graves’ disease and myasthenia gravis are autoantibody-mediated autoimmune diseases, in which the pathogenic role of autoantibodies is established. In Graves’ hyperthyroidism, autoantibodies specifically bind to the thyroid stimulating hormone (TSH) receptor and act as agonists. Increased thyroid hormone levels result in a feed-back inhibition of TSH secretion. Other components of Graves’s disease such as ophtalmopathy and pretibial dermopathy are not clearly linked to TSH autoantibodies and T cell mediated immunity is probably implicated in their pathogenesis. The above mentioned antibodies are termed stimulating TSH receptor autoantibodies (s-TSHR) as opposed to antibodies in chronic autoimmune thyroiditis, which bind to TSHR but do not act as agonists but rather as antagonists. Differences in epitope recognition could account for differences in clinical presentation of Graves’ disease: certain epitopes within a 64 Kda-antigen are more frequently associated with ophthalmopathy.63 Myasthenia gravis is another disease in which autoantibodies play a causative role. Antibodies directed to the alpha chain of the acetylcholine receptor can act as antagonists. In addition, these antibodies cause receptor internalization and degradation. Infants born to myasthenic mothers may develop neonatal myasthenia gravis due to the transplacental exchange of antibodies. Thyroid autoimmunity, defined by the presence of autoantibodies against TSHR, thyroid peroxidase or thyroglobulin, is much more common than symptomatic thyroid disease. Using monoclonal antibodies, two major conformational epitopes on the thyroglobulin molecule have been recognized and anti-thyroglobulin antibodies in autoimmune thyroid disease patients seem to be directed against a different epitope than in healthy controls.64 The discordance between thyroid autoimmunity and disease is probably also due to the large number of subclinical patients, which may only manifest disease after a further challenge. For instance, treatment with amiodarone and lithium can induce hypothyroidism secondary to inhibition of thyroid hormone synthesis. This adverse effect is more frequent in subjects with thyroid autoimmunity. Sera from some patients with APS inhibit the secretion of anterior pituitary hormones in vitro,18 suggesting a causative role for these antibodies in the development of anterior pituitary failure. Inhibitory activity in vivo remains to be demonstrated. A pathogenic role for 21-hydroxylase autoantibodies in Addison’s disease has been suggested since a subset of antibodies inhibit the target enzyme in vitro.65 However, hormonal studies in vivo do not support this hypothesis.66 Steroid cell autoantibodies are cytotoxic for cultured granulosa cells in the presence or complement but a causative action in vivo has not been shown yet.

50

Endocrine and Organ Specific Autoimmunity

“Associated” Autoantibodies Steroid Cell Autoantibodies There are a series of autoantibodies whose direct involvement in disease pathogenesis is not apparent. Adrenal cortex autoantibodies (ACA) are markers of autoimmune Addison’s disease. The indirect immunofluorescence assay for such antibodies uses sections of simian, bovine or human adrenal cortex. Measured by this method, ACA have been documented in 43-84%67,68 of patients with idiopathic adrenal insufficiency. ACA are useful markers of progression to overt Addison’s disease. Betterle and co-workers69 found that 9 out of 10 ACA-positive children developed overt Addison’s disease and the remaining child had subclinical adrenal insufficiency after 3 to 121 months of follow-up. However, in adults the progression rate in ACA-positive is not as high.70 In one study,70 25% of ACA-positive adults were diagnosed with subclinical Addison’s disease when initially assessed and a further 37% developed clinical or subclinical disease on follow-up. In the adult study, there was a correlation between progression to Addison’s disease and the titers of ACA. On the other hand, none of the ACA-negative subjects, children or adults, showed impairment of the adrenal function after a mean follow-up of 2.6 years. ACA have been also found in 1-2% of individuals with autoimmune disease other than adrenal insufficiency, such as type 1 diabetes.67,69 The frequency is higher among adults with premature ovarian failure (8.9%) and in children with hypoparathyroidism (48%).69 The major component of ACA measured by immunofluorescence is directed against the steroidogenic enzyme P450c21 (21-hydroxylase). This enzyme converts 17-alpha-progesterone and progesterone into 11-deoxy-cortisol and deoxycorticosterone. Between 80 and 100% of patients positive for ACA are positive for 21-hydroxylase autoantibodies. Simple assays using radiolabelled recombinant human 21-hydroxylase are far less cumbersome to perform than the immunofluorescence method for ACA. Immunoprecipitations assays with 35S or 125I labeled antigen71,72 are available. The majority of 21-hydroxylase autoantibodies bind to the central and C-terminal parts of the molecule,73,74 which form a major conformational epitope. Autoantibodies against 21-hydroxylase have been documented in 90-100% patients with APS-II and Addison’s disease and 92% of patients with APS-I and Addison’s disease.71,75 These antibodies are reported to be absent in patients with APS-I without Addison’s disease.75 Approximately 1.5% of patients with type 1 diabetes are positive for these autoantibodies and one study found that 86% of 21-hydroxylase-positive IDDM patients had DQB1*0201.76 Similarly, 1.5% of patients with autoimmune thyroid disease are positive for 21-hydroxylase autoantibodies. Autoantibodies to 21-hydroxylase have not been found in nonautoimmune adrenal insufficiency, type 2 diabetes, myasthenia gravis, isolated premature ovarian failure, lupus erythematosus or rheumatoid arthritis.71,77 Between 0% and 2.5%71,72,77of healthy controls are reported to have antibodies reacting with 21-hydroxylase and in our own studies with a sensitivity for Addison’s of greater than 90%, none of 240 controls were positive for 21-hydroxylase autoantibodies using a recombinant antigen radioassay format. The presence of 21-hydroxylase autoantibodies correlates inversely with illness duration: 100% of patients with Addison’s disease with a duration of less than 20 years had autoantibodies, while the percentage decreased to 67% in patients with a longer duration.77 21-hydroxylase antibodies correlate with progression to adrenal insufficiency in a similar way to ACA. In adults but not in children an association between HLA-DR3 and high titers of ACA and 21-hydroxylase has been reported.70 Seroconversion to ACA or 21-hydroxylase autoantibodies usually precedes the clinical onset of Addison’s disease. It is recommended that patients with organ-specific autoimmunity should be screened for the presence of these autoantibodies. A yearly hormonal evalua-

Autoimmune Polyendocrine Syndrome Type II

51

tion of adrenal function in ACA or P450c21-positive patients should allow early diagnosis and treatment. Given that children progress to overt disease at a higher rate, it has been suggested to schedule the hormone tests on a six monthly-basis in this population.69 There are two types of adrenal cell antibodies (ACA). One ACA only reacts with the adrenal cortex while the other also reacts with steroid producing cells from the ovary, testis and placenta and are termed steroid cell autoantibodies (SCA).78 SCA have a high sensitivity for ovarian autoimmunity. These autoantibodies are present in almost 100% of the patients with primary amenorrhea and Addison’s disease and 60% of the patients with secondary amenorrhea and Addison’s disease.79 On the other hand, SCA are not detected in isolated premature ovarian failure, that is, ovarian failure not associated with adrenal autoimmunity and, of note, not even in the “idiopathic” form of ovarian failure (the autoimmune etiology of this latter condition is not clearly defined).80 The presence in serum of SCA confer an increased risk (approximately 40%) for ovarian failure in Addisonian female patients. In males, these autoantibodies do not seem to increase the risk of hypergonadotropic hypogonadism.79 Possible targets of SCA are other cytochrome P450 steroidogenic enzymes, namely 17alpha-hydroxylase (P450c17) and cholesterol side-chain cleavage enzyme (P450scc). P450c17 is expressed in Leydig cells of the testis and theca interna cells of the ovary and the adrenal gland, (excluding the zona glomerulosa). P450scc is present in adrenal, Leydig cells of the testis, theca internal cells of the ovary and placental trophoblasts. Autoantibodies against both P450c17 and P450scc are more frequent in APS, especially when adrenal insufficiency is part of the syndrome,81 than in patients with isolated Addison’s disease.75,82 Most patients with APS-I or APS-II with Addison’s disease have autoantibodies to one or more of P450c21, P450scc or P450c17 while only 23% of the patients with an APS without Addison’s disease are positive for one of these three antibodies.83 There are discrepancies in reported frequencies of autoantibodies against P450c21, P450c17 and P450scc. One study did not find antibodies to P450c21 and P450c17 in APS-I patients in contrast to other reports.84 Technical differences in the autoantibody assays employed may contribute to these differences85 but it is also likely that expression of this autoantibody is related to the presence of ovarian autoimmunity. Recently, 3-beta-hydroxysteroid dehydrogenase has been identified as part of SCA reactivity. Antibodies against this enzyme are present in 23% of the patients with premature ovarian failure, a higher percentage than that found for SCA measured by immunofluorescence.86 There are also reports of a 51 kDa protein which is targeted by autoantibodies from APS-I patients’ sera. This protein is expressed in islets, granulosa cells and placenta and is apparently L-aminodecarboxylase.87,88 Anti-Islet Autoantibodies Non-diabetic APS patients are often positive for anti-islet autoantibodies including antibodies reacting with sections of human pancreas (cytoplasmic ICA) and glutamic acid decarboxylase autoantibodies (GAD-Ab). Amongst APS patients progression to diabetes of such autoantibody positive individuals appears to be less in comparison to similarly autoantibody positive relatives of patients with type 1 diabetes mellitus.89 The cytoplasmic ICA of patients with APS-II with limited progression to diabetes are often what have been termed “restricted” or “selective” ICA and are exclusively high titer anti-GAD autoantibodies.43,90 Thus for these patients, despite the presence of high titer cytoplasmic ICA, they express only a single autoantibody. It has been suggested that first phase insulin secretion with intravenous glucose tolerance testing is not a good predictor of progression to diabetes in APS patients.89 The actual data in the manuscript indicates that 3/6 polyendocrine autoimmune patients expressing cytoplasmic ICA and with first phase

52

Endocrine and Organ Specific Autoimmunity

secretion less than the first percentile of normal progressed to diabetes versus 3/32 with insulin secretion above the first percentile. Autoantibodies reactive with a novel islet 37K antigen was a better predictor of progression to type 1 diabetes in APS patients compared to cytoplasmic ICA.91 The 37Kd molecule and related 40Kd molecules are now sequenced and are ICA512/IA-2 and phogrin/IA-2β. Autoantibodies to these molecules are associated with expression of multiple anti-islet autoantibodies. Similar to relatives of patients with type 1 diabetes mellitus the best predictor of progression to diabetes appears to be the expression of multiple autoantibodies. Five of six APS patients progressing to diabetes expressed ≥ 2 of GAD65, insulin or ICA512 autoantibodies.89 Celiac Disease Autoantibodies Celiac disease is preceded by seropositivity for autoantibodies against endomysium, reticulin, gliadin and transglutaminase.13,14,92 Among patients with type 1 diabetes, 6% are positive for antiendomysial autoantibodies. A biopsy concordant with celiac disease can be documented in most of patients with the endomysial autoantibodies even though most patients are asymptomatic. One study found that 2.4% among 776 new onset type 1 diabetes patients had celiac disease.13 Anti-Parathyroid Autoantibodies/Anti-Gastric Autoantibodies The extracellular domain of the calcium sensing receptor has recently been identified as the target for autoantibodies associated with hypoparathyroidism.93,94 Patients with this condition associated to APS-I or to hypothyroidism had these autoantibodies, whereas it was absent in patients with other autoimmune diseases and in healthy controls. Patients with autoimmune gastritis and pernicious anemia have autoantibodies reacting with HK-ATPase, the proton pump of gastric parietal cells.95,96

Cell Mediated Autoimmunity The study of T cell-mediated immunity is more difficult than the study of humoral autoimmunity. Specifically activated T cells are often located mainly in the target organ, which in many conditions, such as Addison’s disease or type I diabetes, is not biopsied. The percentage of activated T cells from peripheral blood is very small, less than 1% in normal subjects and over four-fold higher in patients with an autoimmune disease. T cell reactivity to specific antigens utilizing lymphocytes from peripheral blood of patients with type I diabetes is difficult to conclusively demonstrate as evidenced by the disappointing results of the recent Canberra Immunology of Diabetes T cell workshop and recent publications.97,98 In the Canberra workshop a series of islet antigens and peptides were sent in a “blinded” manner to multiple laboratories. No laboratory with any of the antigens could distinguish the T cell proliferative response of controls from that of patients with recent onset diabetes. An intensive effort is underway to develop specific and sensitive T cell assays.

Other Endocrine Syndromes Anti-Insulin Receptor Autoantibodies Type B insulin resistance is a rare condition secondary to circulating anti-insulin receptor antibodies. Anti-insulin receptor antibodies can block insulin action and cause insulin resistance, in a degree that varies from very mild forms to severe insulin resistance. Paradoxically the antibodies can also cause hypoglycemia99 and this may occur at any time in the clinical course. The hypoglycemic action apparently results from agonist action of the autoantibodies. A fall in antibody levels may result in remission of the disease.100

Autoimmune Polyendocrine Syndrome Type II

53

Among the associated autoimmune disorders of these patients are systemic lupus erthematosus, Sjogren’s syndrome, alopecia, vitiligo, arthritis, glomerulonephritis (membranous and prolipherative), tubulointerstitial nephiritis and lupus nephritis. Sera from asymptomatic patients frequently have antinuclear antibodies, hypergammaglobulinemia, anti-DNA antibodies and anti-thyroid antibodies.

Kearns-Sayre Syndrome This rare syndrome is also known as oculocraniosomatic neuromuscular disease. It is a mitochondrial myopathy with progressive external ophthalmoplegia and further involvement of somatic nerves. Cardiac conduction defects, sensorineural and retinal pigmentary degeneration may be present. There is a defect in enzymatic complexes involved in oxidative phosphorylation and crystalline mitochondrial inclusions in muscle and cerebellum cells can be seen. Mitochondrial DNA mutations and deletions have been described.101,102 Associated endocrinological disorders include hypoparathyroidism, hypergonadotropic hypogonadism, insulin-dependent diabetes mellitus and hypopituitarism. The link between the neuromuscular and endocrine disturbances has not been clarified.

Poems Syndrome Poems syndrome is characterized by the presence of Polyneuropathy, Organomegaly, Endocrinopathy, M protein and Skin changes. It is also called Crow-Fukase syndrome, Takatuki syndrome and PEP syndrome (pigmentation, edema and plasma cell dyscrasia). Sensory-motor polyneuropathy is progressive and disabling. Cranial nerves are not usually involved but reported phrenic nerve palsy has been reported.103 Hepatomegaly, splenomegaly and lymph node enlargement are seen. Proliferation of hepatocytes is induced in vitro when hepatocytes are cultured in the serum of patients with POEMS, suggesting the presence of humoral factors responsible for in vivo hepatocyte proliferation.104 Endocrinopathies in this syndrome include gynecomastia, testicular atrophy, amenorrhea, chronic autoimmune thyroiditis (present in 50% of the cases),11 and diabetes mellitus. The M protein is a monoclonal IgG or IgA. Myeloma is very common. Vincristine and other agents used to treat myeloma may severely exacerbate the neuropathy and should be avoided in patients with POEMS syndrome.105 Skin changes include hyperpigmentation, thickening, Raynaud phenomenon103 and hypertrichosis. Pulmonary hypertension has been also reported.106 The etiology of this syndrome is unknown but increased circulating levels of proinflamatory cytokines (IL-6, TNF-α, IL-1 β) suggest activation of the monocyte/macrophage system.107 Endothelial cell abnormalities and chronic intravascular coagulation are also reported.108 Fibrinogen, fibrinopeptide A, thrombin-antithrombin complexes and circulating vascular endothelial growth factor109 are increased in sera of POEMS patients.

Thymoma Over 40% of patients with a thymoma have an associated disorder.110 The commonest associated disorders are myasthenia gravis,110,111 pure red cell aplasia112 and hypogammaglobulinemia. Fifteen percent of the patients with myasthenia gravis have a thymoma.113 Other diseases are less common and include Addison’s disease, autoimmune thyroid disease114 and stiff-man syndrome.115 Thymoma cells overexpress neurofilaments that contain epitopes of a striated muscle protein, titin. This protein is the target of autoantibodies detected in patients with thymoma-associated myasthenia gravis but anti-titin autoantibodies are not found with nonparaneoplastic myasthenia gravis.116 Over 50% of patients with thymoma and myasthenia

Endocrine and Organ Specific Autoimmunity

54

gravis have autoantibodies to the ryanodine receptor, a ligand-gated calcium-release channel in the sarcoplasmic reticulum of striated muscle and these may interfere with the excitation-contraction mechanism in striated muscle.117 These antibodies are more frequent in patients with a severe form of the disease.118 Actinin is another target antigen for antistriational antibodies.

Turner’s Syndrome/Trisomy 21 In Turner’s syndrome, a missing X chromosome (cariotype 45X0 or 45X0/46XX) results in a gonadal dysgenesis, with impaired development of the secondary sexual characters, primary amenorrhea and a series of somatic features such as short stature, webbing of the neck, low hairline and pectus excavatum. These patients also present kidney abnormalities, coarctation of the aorta, neurosensorial hearing loss and cataracts. There is an increased susceptibility to autoimmune diseases, particularly to chronic autoimmune thyroid disease, present in 50% of the patients,11 and diabetes mellitus.119 Trisomy 21, also called Down’s syndrome, is associated with mental retardation and congenital cardiac abnormalities. There is an increased susceptibility to autoimmune disease. Thyroid disease is detected in more than 60% of the patients.120 Autoimmune thyroid disease in patients with Down’s syndrome is associated with HLA-DQA*0301.45 Celiac disease and type 1 diabetes are other autoimmune disorders frequently present in these patients.9,121,122 There is an increased susceptibility to hepatitis B among patients with trisomy 21, probably secondary to cellular immunity alterations and it has been found that chronic thyroid autoimmune disease is more frequent among Down’s individuals with serological markers of chronic hepatitis B.123 An increased sensitivity of cells to interferon has been suggested to be the reason for increased frequency of transient leukemoid reaction, myelofibrosis and leukemia among these patients.124

Conclusion The APS-II syndrome is remarkable for its diversity of associated diseases. The major genetic determinants of disease are within the HLA region of chromosome 6. For patients with Addison’s disease and type 1 diabetes a common genotype is DRB1*0404, DQ8 with DRB1*0301, DQ2. It is likely that genes which influence immune regulation in addition to HLA class II alleles contribute to disease susceptibility. The eventual characterization of these genes should provide a better understanding of APS-II. At present, one can identify individuals at high genetic risk for the syndrome and with autoantibody screening identify individuals with a high risk of having or developing a given disorder. This knowledge aids both basic research concerning these associated disorders and the clinical care of patients with APS-II and their relatives.

Acknowledgments This work was in part supported by a grant from F.I.S. (97/5094) and grants from the Children’s Diabetes Foundation, Juvenile Diabetes Foundation, American Diabetes Association and the National Institutes of Health (DK32083). We thank research nurse Terry Smith of the Joslin Diabetes Center for evaluating families and Sherman Gates for referring family illustrated in Figure 3.1.

References 1. Verge C, Eisenbarth GS. Autoimmune polyendocrine syndromes. In: Wilson JD, Foster DW, ed(s). Williams Textbook of Endocrinology. 9th ed. Philadelphia: W.B. Saunders, 1997.

Autoimmune Polyendocrine Syndrome Type II

55

2. Muir A, Schatz DA, Maclaren NK. Polyglandular failure syndromes. In: DeGroot LJ, Besser M, Burger HG et al, Endocrinology. Philadelphia: W.B. Saunders, 1995:3013-3024. 3. Neufeld M, Maclaren NK, Blizzard RM. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 1981; 60:355-362. 4. Ahonen P, Myllarniemi S, Sipila I et al. Clinical variation of autoimmune poly-endocrinopathy-candidiasis-ectodermal dystrophy (APECED) in a series of 68 patients. N Engl J Med 1990; 322:1830-1836. 5. Nerup J. Addison’s disease-clinical studies: A report of 108 cases. Acta Endocrinol 1974; 76:127-141. 6. Irvine WJ, Stewart AG, Scarth L. A clinical and immunological study of adrenocortical insufficiency (Addison’s Disease). Clin Exp Immunol 1967; 2:31-69. 7. Irvine WJ. Autoimmunity in endocrine disease. Recent Prog Horm Res 1980; 36:509-527. 8. Bottazzo GF, Florin-Christensen A, Doniach D. Islet cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974; 2:1279-1283. 9. Papadopoulos KI, Hornblad Y, Hallengren B. The occurrence of polyglandular autoimmune syndrome type III associated with coeliac disease in patients with sarcoidosis. J Intern Med 1994; 236:661-663. 10. Dumic M, Gubarev N, Sikic N et al. Sparse hair and multiple endocrine disorders in two women heterozygous for adrenoleukodystrophy. Am J Med Genet 1992; 43:829-832. 11. Dayan CM, Daniels GH. Chronic autoimmune thyroiditis. N Engl J Med 1996; 335:99-107. 12. Papadopoulos KI, Hallengren B. Polyglandular autoimmune syndrome type II in patients with idiopathic Addison’s disease. Acta Endocrinol (Copenh) 1990; 122:472-478. 13. Saukkonen T, Savilahti E, Reijonen H et al. Coeliac disease: frequent occurrence after clinical onset of insulin-dependent diabetes mellitus. Diabetic Med 1995; 13:464-470. 14. Mäki M, Huupponen T, Holm K et al. Seroconversion of reticulin autoantibodies predicts coeliac disease in insulin dependent diabetes mellitus. Gut 1995; 36:239-242. 15. Drachman DB. Myasthenia gravis. N Engl J Med 1995; 330:1797-1810. 16. Marino M, Ricciardi R, Pinchera A et al. Mild clinical expression of myasthenia gravis associated with autoimmune thyroid diseases. J Clin Endocrinol Metab 1997; 82:438-443. 17. Powrie JK, Powell M, Ayers AB et al. Lymphocytic adenohypophysitis: magnetic resonance imaging features of two new cases and a review of the literature. [Review]. Clin Endocrinol (Oxf) 1995; 42:315-322. 18. Kurokawa H, Numata Y, Nishioka T et al. Secretion of anterior pituitary hormones in patients with insulin- dependent diabetes mellitus and polyglandular autoimmune syndrome. Intern Med 1996; 35:686-692. 19. Hashimoto K, Kurokawa H, Nishioka T et al. Four patients with polyendocrinopathy with associated pituitary hormone deficiency. Endocr J 1994; 41:613-621. 20. Tanaka T, Watanabe K, Furukawa Y et al. Comprehensive adrenocortical steroid measurements in two cases of Schmidt’s syndrome. Endocr J 1994; 41:379-386. 21. Otsuka F, Ogura T, Hayakawa N et al. A case of Schmidt’s syndrome accompanied by a pituitary adenoma. Endocr J 1996; 43:495-502. 22. Barkan AL, Kelch RP, Marshall JC. Isolated gonadotrope failure in the polyglandular autoimmune syndrome. N Engl J Med 1985; 312:1535-1540. 23. Ko GT, Szeto CC, Yeung VT et al. Autoimmune polyglandular syndrome and primary biliary cirrhosis. Br J Clin Pract 1996; 50:344-346. 24. Fritzen R, Bornstein SR, Scherbaum WA. Megaoesophagus in a patient with autoimmune polyglandular syndrome type II. Clin Endocrinol (Oxf) 1996; 45:493-498. 25. Watson JP, Lewis RA. Schmidt’s syndrome associated with sarcoidosis. Postgrad Med J 1996; 72:435-436. 26. Watkins PJ, Gayle C, Alsanjari N et al. Severe sensory-autonomic neuropathy and endocrinopathy in insulin- dependent diabetes. QJM 1995; 88:795-804. 27. Heuss D, Engelhardt A, Gobel H et al. Myopathological findings in interstitial myositis in type II polyendocrine autoimmune syndrome (Schmidt’s syndrome). Neurol Res 1995; 17:233-237.

56

Endocrine and Organ Specific Autoimmunity

28. Govindarajan R, Galpin OP. Coexistence of Addison’s disease, ulcerative colitis, hypothyroidism and pernicious anemia. J Clin Gastroenterol 1992; 15:82-83. 29. Inaba M, Morii H. Polyglandular autoimmune syndrome. Nippon Rinsho 1995; 53:974-978. 30. Eisenbarth GS, Wilson P, Ward F et al. HLA type and disease occurrence in familial polyglandular failure. N Engl J Med 1978; 298:92-94. 31. Eisenbarth GS, Wilson P, Ward F et al. The polyglandular failure syndrome: disease inheritance, HLA-type and immune function. Ann Intern Med 1979; 91:528-533. 32. Partanen J, Peterson P, Westman P et al. Major Histocompatibility Complex class I and II in Addison’s Disease. Hum Immunol 1994; 41:135-140. 33. Badenhoop K, Walfish PG, Rau H et al. Susceptibility and resistance alleles of human leukocyte antigen (HLA) DQA1 and HLA DQB1 are shared in endocrine autoimmune disease. J Clin Endocrinol Metab 1995; 80:2112-2117. 34. Nepom GT, Erlich H. MHC class-II molecules and autoimmunity. Ann Rev Immunol 1991; 9:493-525. 35. Huang W, Connor E, Dela Rosa T et al. Although DR3-DQB1*0201 may be associated with multiple component diseases of the autoimmune polyglandular syndromes, the human leukocyte antigen DR4-DQB1*0302 haplotype is implicated only in b-cell autoimmunity. J Clin Endocrinol Metab 1996; 81:2559-2563. 36. McLachlan SM. Editorial: The genetic basis of autoimmune thyroid disease: Time to focus on chromosomal loci other than the major histocompatibility complex (HLA in man). J Clin Endocrinol Metab 1995; 77:605A-605C. 37. Bodmer WF. HLA: What’s in a name? A commentary on HLA nomenclature development over the years. Tissue Antigens 1997; 49:293-296. 38. Eisenbarth GS. Primer: Immunology/autoimmunity. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:3-17. 39. Raum D, Awdeh Z, Yunis EJ et al. Extended major histocompatibility complex haplotypes in type I diabetes mellitus. J Clin Invest 1996; 74:449-454. 40. Pugliese A, Eisenbarth GS. Human type I diabetes mellitus: genetic susceptibility and resistance. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:134-152. 41. Garg SK, Klingensmith GJ, Eisenbarth GS. Autoimmune Polyendocrine Syndromes. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:153-171. 42. Brewer K, Parziale VS, Eisenbarth GS. Screening patients with insulin-dependent diabetes mellitus for adrenal insufficiency. N Engl J Med 1997; 337:202 43. Gianani R, Pugliese A, Bonner-Weir S et al. Prognostically significant heterogeneity of cytoplasmic islet cell antibodies in relatives of patients with Type I Diabetes. Diabetes 1992; 41:347-353. 44. van de Wal Y, Kooy YMC, Drijfhout JW et al. Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule. Immunogenetics 1996; 44:246-253. 45. Nicholson LB, Wong FS, Ewins DL et al. Susceptibility to autoimmune thyroiditis in Down’s syndrome is associated with the major histocompatibility complex class II DQA*0301 allele. Clin Endocrinol 1994; 41:381-383. 46. Trence DL, Morley JE. Polyglandular autoimmune syndromes. Am J Med 1984; 77:107-116. 47. Garlepp MJ, Dawkins RL, Christiansen FT. HLA antigens and acetylcholine receptor antibodies in penicillamine induced myasthenia gravis. BMJ 1983; 286:338-340. 48. Drosos AA, Christou L, Galanopoulou V et al. D-penicillamine induced myasthenia gravis: Clinical, serological and genetic findings. Clin Exp Rheumatol 1993; 11:387-391. 49. O’Keefe M, Morley KD, Haining WM et al. Penicillamine-induced ocular myasthenia gravis. Am J Ophthalmol 1985; 99:66-67. 50. Boehm BO, Manfras B, Seidl S et al. The HLA-DQbeta nonAsp-57 allele: a predictor of future insulin-dependent diabetes mellitus in patients with autoimmune Addison’s disease. Tissue Antigens 1991; 37:130-132.

Autoimmune Polyendocrine Syndrome Type II

57

51. Morel PA, Dorman JS, Todd JA et al. Aspartic acid at position 57 of the HLA-DQ beta chain protects against Type I diabetes: A family study. Proc Natl Acad Sci U S A 1988; 85:8111-8115. 52. Donner H, Rau H, Walfish PG et al. CTLA4 alanine-17 confers genetic susceptibility to Graves’ disease and to type-1 diabetes mellitus*. J Clin Endocrinol Metab 1997; 82:143-146. 53. Friis SU. Coeliac disease, pathogenesis and clinical aspects. Acta Pathol Microbiol Immunol Scand 1996; 61:5-48. 54. Rubenstein P. The HLA system in congenital rubella patients with and without diabetes. Diabetes 1982; 31:1088-1091. 55. Finger DR, Bernet VJ, Doyle JJ. Polyglandular autoimmune endocrinopathy following procainamide induced lupus. J Rheumatol 1995; 22:574-575. 56. Imagawa A, Itoh N, Hanafusa T et al. Autoimmune endocrine disease induced by recombinant interferon-a therapy for chronic active type C hepatitis. J Clin Endocrinol Metab 1995; 80:922-926. 57. Burman P, Totterman TH, Oberg K et al. Thyroid autoimmunity in patients on long term therapy with leukocyte- derived interferon. J Clin Endocrinol Metab 1986; 63:1086-1090. 58. Gisslinger H, Gilly B, Woloszczuk W et al. Thyroid autoimmunity and hypothyroidism during long-term treatment with recombinant interferon-alpha. Clin Exp Immunol 1992; 90:363-367. 59. Atkins MB, Mier JW, Parkinson DR et al. Hypothyroidism after treatment with interleukin2 and lymphokine-activated killer cells. N Engl J Med 1988; 318:1557-1563. 60. Hoekman K, von Blomberg-van der Flier BM, Wagstaff J et al. Reversible thyroid dysfunction during treatment with GM-CSF [see comments]. Lancet 1991; 338:541-542. 61. Ginsberg-Fellner F, Witt ME, Yagihashi S et al. Congenital rubella syndrome as a model for Type I (insulin-dependent) diabetes mellitus: Increased prevalence of islet cell surface antibodies. Diabetologia 1984; 27:87-89. 62. Clarke W, Shaver K, Bright GA et al. Autoimmunity in congenital rubella syndrome. J Pediatr 1984; 104:370-373. 63. Zhang Z, Wall JR, Bernard NF. Identification of antigenic epitopes of 1D antigen recognized by antibodies in the serum of patients with thyroid-associated ophthalmopathy. Clin Immunol Immunopathol 1995; 77:193-200. 64. Prentice L, Kiso Y, Fukuma N et al. Monoclonal thyroglobulin autoantibodies: variable region analysis and epitope recognition. J Clin Endocrinol Metab 1995; 80:977-986. 65. Furmaniak J, Kominami S, Asawa T et al. Autoimmune Addison’s disease-evidence for a role of steroid 21-hydroxylase autoantibodies in adrenal insufficiency. J Clin Endocrinol Metab 1994; 79:1517-1521. 66. Boscaro M, Betterle C, Volpato M et al. Hormonal responses during various phases of autoimmune adrenal failure: no evidence for 21-hydroxylase enzyme activity inhibition in vivo. J Clin Endocrinol Metab 1996; 81:2801-2804. 67. Falorni A, Laureti S, Nikoshkov A et al. 21-hydroxylase autoantibodies in adult patients with endocrine autoimmune diseases are highly specific for Addison’s disease. Clin Exp Immunol 1997; 107:341-346. 68. Soderbergh A, Winqvist O, Norheim I et al. Adrenal autoantibodies and organ-specific autoimmunity in patients with Addison’s disease. Clin Endocrinol (Oxf) 1996; 45:453-460. 69. Betterle C, Volpato M, Smith BR et al. II. Adrenal cortex and steroid 21-hydroxylase autoantibodies in children with organ-specific autoimmune diseases: markers of high progression to clinical Addison’s disease. J Clin Endocrinol Metab 1997; 82:939-942. 70. Betterle C, Volpato M, Rees Smith B et al. I. Adrenal cortex and steroid 21-hydroxylase autoantibodies in adult patients with organ-specific autoimmune diseases: Markers of low progression to clinical Addison’s disease. J Clin Endocrinol Metab 1997; 82:932-938. 71. Tanaka H, Perez MS, Powell M et al. Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipitation assay. J Clin Endocrinol Metab 1997; 82:1440-1446. 72. Colls J, Betterle C, Volpato M et al. Immunoprecipitation assay for autoantibodies to steroid 21- hydroxylase in autoimmune adrenal diseases. Clin Chem 1995; 41:375-380.

58

Endocrine and Organ Specific Autoimmunity

73. Wedlock N, Asawa T, Baumann-Antczak A et al. Autoimmune Addison’s disease: analysis of autoantibody binding sites on human steroid 21-hydroxylase. FEBS Lett 1993; 332:123-126. 74. Song YH, Connor EL, Muir A et al. Autoantibody epitope mapping of the 21-hydroxylase antigen in autoimmune Addison’s disease. J Clin Endocrinol Metab 1994; 78:1108-1112. 75. Chen S, Sawicka J, Betterle C et al. Autoantibodies to steroidogenic enzymes in autoimmune polyglandular syndrome, Addison’s disease, and premature ovarian failure. J Clin Endocrinol Metab 1996; 81:1871-1876. 76. Peterson P, Samni H, Hyoty H et al. Steroid 21-Hydorxylase Autoantibodies in InsulinDependent Diabetes Mellitus. Clin Immunol Immunopathol 1997; 82:37-42. 77. Falorni A, Nikoshkov A, Laureti S et al. High diagnostic accuracy for idiopathic Addison’s disease with a sensitive radiobinding assay for autoantibodies against recombinant human 21-hydroxylase. J Clin Endocrinol Metab 1995; 80:2752-2755. 78. McNatty KP, Short RV, Barnes EW et al. The cytotoxic effect of serum from patients with Addison’s disease and autoimmune ovarian failure on human granulosa cells in culture. Clin Exp Immunol 1975; 22:378-384. 79. Betterle C, Rossi A, Dalla Pria S et al. Premature ovarian failure: autoimmunity and natural history. Clin Endocrinol 1993; 39:35-43. 80. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocr Rev 1997; 18:107-134. 81. Peterson P, Uibo R, Peranen J et al. Immunoprecipitation of steroidogenic enzyme autoantigens with autoimmune polyglandular syndrome type I (APS I) sera; further evidence for independent humoral immunity to P450c17 and P450c21. Clin Exp Immunol 1997; 107:335-340. 82. Krohn K, Uibo R, Aavik E et al. Identification by molecular cloning of an autoantigen associated with Addison’s disease as steroid 17alpha-hydroxylase. Lancet 1992; 339:770-773. 83. Uibo R, Aavik E, Peterson P et al. Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison’s Disease. J Clin Endocrinol Metab 1994; 78:323-328. 84. Winqvist O, Gustafsson J, Rorsman F et al. Two different cytochrome P450 enzymes are the adrenal antigens in autoimmune polyendocrine syndrome type I and addison’s disease. J Clin Invest 1993; 92:2377-2385. 85. Smith BR, Furmaniak J. Editorial: Adrenal and gonadal autoimmune diseases. J Clin Endocrinol Metab 1995; 80:1502-1505. 86. Arif S, Vallian S, Farzaneh F et al. Identification of 3β-hydroxysteroid dehydrogenase as a novel target of steroid cell autoantibodies: association of autoantibodies with endocrine autoimmune disease*. J Clin Endocrinol Metab 1996; 81:4439-4445. 87. Velloso LA, Winqvist O, Gustafsson J et al. Autoantibodies against a novel 51 kDa islet antigen and glutamate decarboxylase isoforms in autoimmune polyendocrine syndrome type I. Diabetologia 1994; 37:61-69. 88. Husebye ES, Gebre-Medhin G, Tuomi T et al. Autoantibodies against aromatic L-amino acid decarboxylase in autoimmune polyendocrine syndrome type I*. J Clin Endocrinol Metab 1997; 82:147-150. 89. Wagner R, Genovese S, Bosi E et al. Slow metabolic deterioration towards diabetes in islet cell antibody positive patients with autoimmune polyendocrine disease. Diabetologia 1994; 37:365-371. 90. Genovese S, Bonifacio E, McNally JM et al. Distinct cytoplasmic islet cell antibodies with different risks for type I (insulin-dependent) diabetes mellitus. Diabetologia 1992; 35:385-388. 91. Christie MR, Genovese S, Cassidy D et al. Antibodies to islet 37k antigen, but not to glutamate decarboxylase, discriminate rapid progression to IDDM in endocrine autoimmunity. Diabetes 1994; 43:1254-1259. 92. Dietrich W, Ehnis T, Bauer M et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nature Medicine 1997; 3:797-801.

Autoimmune Polyendocrine Syndrome Type II

59

93. Li Y, Song Y, Rais N et al. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. J Clin Invest 1996; 97:910-914. 94. Posillico JT, Wortsman J, Srikanta S et al. Parathyroid cell surface autoantibodies that inhibit parathyroid hormone secretion from dispersed human parathyroid cells. Bone Miner Res 1986; 5:475-485. 95. Karlsson FA, Burman P, Loof L et al. Major parietal cell antigen in autoimmune gastritis and pernicious anemia is the acid producing HK-ATPase of the stomach. J Clin Invest 1988; 81:475-479. 96. Davis RE, VcCann VJ, Stanton KG. Type 1 diabetes and latent pernicious anemia. Med J Aust 1992; 156:160-162. 97. Schloot NC, Roep BO, Wegmann DR, Yu L, Wang TB, Eisenbarth GS. T cell reactivity to GAD65 peptide sequences shared with Coxsackie virus protein in IDDM patients and controls. Diabetologia 1997; In press. 98. Schloot N, Roep BO, Wegmann D et al. Altered immune response to insulin in newly diagnosed compared insulin-treated diabetic patients and healthy control subjects. Diabetologia 1997; 40:564-572. 99. Howell W, Leung ST, Jones DB et al. HLA-DRB, -DQA, and -DQB polymorphism in celiac disease and entropathy-associated T cell lymphoma. Common features and additional risk factors for malignancy. Hum Immunol 1995; 43:29-37. 100. Flier JS, Bar RS, Muggeo M et al. The evolving clinical course of patients with insulin receptor autoantibodies: Spontaneous remission or receptor proliferation with hypoglycemia. J Clin Endocrinol Metab 1978; 47:985-995. 101. Collombet JM, Faure-Vigny H, Mandon G et al. Expression of oxidative phosphorylation genes in muscle cell cultures from patients with mitochondrial myopathies. Mol Cell Biochem 1997; 168:73-85. 102. Akaike M, Kawai H, Yokoi K et al. Cardiac dysfunction in patients with chronic progressive external ophthalmoplegia. Clin Cardiol 1997; 20:239-243. 103. de la Pena A, Subtil JC, Rodriguez-Rosado R et al. The POEMS syndrome, apropos of 2 cases and review of the literature. An Med Interna 1996; 13:291-294. 104. Fushimi T, Inoue A, Koh CS et al. A study on the pathogenesis of hepatomegaly in patients with Crow- Fukase syndrome. Rinsho Shinkeigaku 1996; 36:534-539. 105. Schey S. Osteosclerotic myeloma and ‘POEMS’ syndrome. Blood Rev 1996; 10:75-80. 106. Ribadeau-Dumas S, Tillie-Leblond I, Rose C et al. Pulmonary hypertension associated with POEMS syndrome. Eur Respir J 1996; 9:1760-1762. 107. Gherardi RK, Authier FJ, Belec L. Pro-inflammatory cytokines: a pathogenic key of POEMS syndrome. Rev Neurol (Paris) 1996; 152:409-412. 108. Saida K, Kawakami H, Ohta M et al. Coagulation and vascular abnormalities in CrowFukase syndrome. Muscle Nerve 1997; 20:486-492. 109. Soubrier M, Dubost JJ, Serre AF et al. Growth factors in POEMS syndrome: evidence for a marked increase in circulating vascular endothelial growth factor. Arthritis Rheum 1997; 40:786-787. 110. Maggi G, Casadio C, Cavallo A et al. Thymoma: results of 241 operated cases.. Ann Thorac Surg 1991; 51:152-156. 111. Marx A, Wilisch A, Schultz A et al. Pathogenesis of myasthenia gravis. Virchows Arch 1997; 430:355-364. 112. 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. 113. Rosenow III EC, Hurley BT. Disorders of the Thymus. Arch Intern Med 1984; 144:763-770. 114. Combs RM. Malignant thymoma, hyperthyroidism and immune disorder. South Med J 1968; 61:337-341. 115. Piccolo G, Martino G, Moglia A et al. Autoimmune myasthenia gravis with thymoma following the spontaneous remission of stiff-man syndrome. Ital J Neurol Sci 1990; 11 (2):177-80.

60

Endocrine and Organ Specific Autoimmunity

116. Wilisch A, Schultz A, Greiner A et al. Molecular mimicry between neurofilaments and titin as the basis for autoimmunity towards skeletal muscle in paraneoplastic myasthenia gravis. Verh Dtsch Ges Pathol 1996; 80:261-266. 117. 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. 118. Mygland A, Aarli JA, Matre R et al. Ryanodine receptor antibodies related to severity of thymoma associated myasthenia gravis. J Neurol Neurosurg Psychiatr 1995; 57:843-846. 119. Larizza D, Bianchi MM, Lorini R et al. Autoimmunity, HLA, Gm and Km polymorphism in Turner’s syndrome. Autoimmunity 1989; 4:69-78. 120. Zori RT, Schatz DA, Ostrer H et al. Relationship of autoimmunity to thyroid dysfunction in children and adults with Down syndrome. Am J Med Genet 1990; 7 (suppl):238-241. 121. Rabinowe SL, Rubin L, George KL et al. Trisomy 21 (Down’s Syndrome): autoimmunity, aging and monoclonal antibody defined T cell abnormalities. J Autoimmun 1989; 2:25-30. 122. Bonamico M, Rasore-Quartino A, Mariani P et al. Down syndrome and celiac disease: Usefulness of antigliadin and antiendomysium antibodies. Acta Paediatr 1996; 85:1503-1505. 123. May P, Kawanishi H. Chronic hepatitis B infection and autoimmune thyroiditis in Down syndrome. J Clin Gastroenterol 1996; 23:181-184. 124. Zihni L. Down’s syndrome, interferon sensitivity and the development of leukemia. Leukemia Res 1994; 18:1-6. 125. Ahonen P, Myllarniemi S, Kahanpaa A et al. Ketoconazole is effective against the chronic mucocutaneous candidosis of autoimmune polyendocrinopathy-candidosis-ectodermal dystrophy (APECED). Acta Med Scand 1986; 220:333-339. 126. Friedman TC, Thomas PM, Fleisher TA et al. Frequent occurrence of asplenism and cholelithiasis in patients with autoimmune polyglandular disease type I. Am J Med 1991; 91:625-630. 127. Leshin M. Southwestern Internal Medicine Conference: Polyglandular autoimmune syndromes. Am J Med Sci 1985; 290:77-88. 128. Tuomi T, Björses P, Falorni A et al. Antibodies to glutamic acid decarboxylase and insulin-dependent diabetes in patients with autoimmune polyendocrine syndrome type I. J Clin Endocrinol Metab 1996; 81:1488-1494. 129. Kong M-F, Jeffcoate W. Eighty-six cases of Addison’s disease. Clin Endocrinol 1994; 41:757-761. 130. Betterle C, Eisenbarth GS, Pedini B et al. Clinical and subclinical organ-specific autoimmune manifestations in type 1 (insulin-dependent) diabetic patients and their first-degree relatives. Diabetologia 1984; 26:431-436. 131. Zelissen PM, Bast EJEG, Croughs RJM. Associated autoimmunity in Addison’s disease. J Autoimmun 1995; 8:121-130. 132. Viskorpi JK. Diabetes and coeliac disease. Lancet 1969; ii:1192. 133. Barera G, Bianchi C, Calisti L et al. Screening of diabetic children for coeliac disease with antigliadin antibodies and HLA typing. Arch Dis Child 1991; 66:491-494. 134. Burek CL, Rose NR, Guire KE et al. Thyroid autoantibodies in black and in white children and adolescents with type 1 diabetes mellitus and their first degree relatives. Autoimmunity 1990; 7:157-167. 135. Winqvist O, Gebre-Medhin G, Gustafsson J et al. Identification of the main gonadal autoantigens in patients with adrenal insufficiency and associated ovarian failure. J Clin Endocrinol Metab 1995; 80:1717-1723. 136. Karlsson FA, Kampe O, Winqvist O et al. Autoimmune disease of the adrenal cortex, pituitary, parathyroid glands and gastric mucosa. J Intern Med 1993; 234:379-386. 137. Kjaer K, Hagen C, Sando SH et al. Epidemiology of menarche and menstrual disturbances in an unselected group of women with insulin-dependent diabetes mellitus compared to controls. J Clin Endocrinol Metab 1992; 75:524-529. 138. Pholsena M, Young J, Couzinet B et al. Primary adrenal and thyroid insufficiencies associated with hypopituitarism: a diagnostic challenge. Clin Endocrinol (Oxf) 1994; 40:693-695.

Autoimmune Polyendocrine Syndrome Type II

61

139. Mirakian R, Cudworth AG, Bottazzo GF et al. Autoimmunity to anterior pituitary cells and the pathogenesis of IDDM. Lancet 1982; I:755-759. 140. Okada T, Kawamura T, Tamura T et al. Myasthenia gravis associated with Addison’s disease. Intern Med 1994; 33:686-688. 141. Dumas P, Archambeaud-Mouveroux F, Vallat JM et al. Myasthenia Gravis associated with adrenocortical insufficiency. Report of two cases. J Neurol 1985; 232:354-356. 142. Yoshioka K, Ohsawa A, Yoshida T et al. Insulin-dependent diabetes mellitus associated with Graves’ disease and idiopathic hypoparathyroidism. J Endocrinol Invest 1993; 16:643-646.

CHAPTER 4

Oncogenic Autoimmunity Robert P. Friday and Massimo Pietropaolo

Introduction

T

oward the end of the nineteenth century Louis Pasteur demonstrated that widespread immune-mediated self-destruction of neural tissue occurs in animals exposed to crude extracts of central nervous system (CNS) antigens. Based on these observations, Paul Ehrlich hypothesized that the natural mechanisms involved in host defense can turn against self, promoting the development of a disease state which he designated horror autotoxicus.1 From these early experiments has developed the concept of autoimmunity, simply defined as an immune response to antigens of the host’s own tissues.2,3 An autoimmune response can be demonstrated by the occurrence of either circulating autoantibodies or T lymphocytes in peripheral blood which are reactive with self antigens. Importantly, the majority of autoimmune responses against self antigens do not result in the development of a disease process. Only when sustained immune responses cause tissue damage is the consequence of autoimmunity identified as autoimmune disease.4 The mechanism of immune stimulation which leads to autoimmune pathology is all too frequently unknown (hence, the familiar “idiopathic” autoimmune disease). However, immunologic autoreactivity which develops in association with neoplasia, termed oncogenic autoimmunity, likely signifies a cause and effect relationship between the presence of cancer and the observed autoimmune phenomena. It has long been known that malignancies can be associated with systemic and organ-specific disease symptoms seemingly unrelated to the location or nature of the primary malignancy. These cancer-associated phenomena were collectively termed paraneoplasia, defined in literal terms by Waldenström (1978) as “something that occurs beside the tumor and is somewhat ambiguous.”5 A common theme of paraneoplastic disease is its relation to ectopic protein production by tumor cells. The proteins produced may be peptide hormones or hormone-producing enzymes in cases of endocrine paraneoplasia, or in the case of oncogenic autoimmunity, antigenic proteins which are normally limited in their expression to immunologically privileged sites such as the CNS. Often the demonstration of antibodies against molecular targets localized within the neoplastic milieu may aid in the establishment of a diagnosis of occult malignancy, even preceding the appearance of clinically detectable cancer in some patients. A 1965 published symposium entitled The Remote Effects of Cancer on the Nervous System details the discussions of perplexing, cancer-associated neurological problems, many of which at the time had been only recently described in the medical literature.6,7 A number of syndromes could not be attributed to the direct effects of patients’ tumors (i.e., local invasion or metastasis) nor to endocrine or metabolic imbalances induced by humors manufactured by malignant cells. Among these disorders were included what we today identify as Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

Endocrine and Organ Specific Autoimmunity

64

LEMS MG NMT Thymoma

Small Cell Lung Carcinoma

PP

LE

PEM/SN

SMS

PCD

Lymphoma

CAR OMS-A

XER

Gynecologic Tumors OMS-C

DM Neuroblastoma

ABBREVIATIONS CAR DM LE LEMS MG NMT OMS-A

Cancer-associated retinopathy Dermatomyositis Limbic encephalitis Lambert-Eaton myasthenic syndrome Myasthenia gravis Neuromyotonia Opsoclonus-myoclonus (adult)

OMS-C PCD PEM/SN PP SMS XER

Opsoclonus-myoclonus (child) Paraneoplastic cerebellar degeneration Paraneoplastic encephalomyelitis/ sensory neuropathy Paraneoplastic pemphigus Stiff man syndrome Paraneoplastic autoimmune xerostomia

Fig. 4.1. Venn diagram showing a significant overlap in the occurrence of the most common paraneoplastic autoimmune syndromes associated with malignancies. Thymoma is characteristically associated with myasthenia gravis, occurring in approximately 10% of myasthenia gravis. The incidence of myasthenia gravis in patients with a thymic tumor may be as great as 80%. Thymoma is found also in about 20% of patients with neuromyotonia, some of whom also have myasthenia gravis. Small cell lung cancer is found in approximately 60% of Lambert-Eaton myasthenic syndrome patients. A small number of SMS or opsoclonus-myoclonus patients have also cancer of the breast, ovary or uterus. Childhood opsoclonus-myoclonus is diagnosed in approximately 10% of neuroblastomas. Paraneoplastic pemphigus is associated more often with non-Hodgkin’s lymphoma and thymoma, whereas dermatomyositis is more frequently related with ovarian cancer.

myasthenia gravis, Lambert-Eaton myasthenic syndrome, and paraneoplastic cerebellar degeneration. The named syndromes are thought to be due to autoimmunity which is in some way induced by the presence of specific malignancies. Numerous additional associations between specific neoplastic processes and autoimmune syndromes have been described since the publication of the Remote Effects symposium, adding more complexity to the unfolding mystery of oncogenic autoimmunity. It should be noted, however, that this group of disorders is generally limited to association with relatively few tumor types, as illustrated in the Venn diagram of Figure 4.1 and summarized in Table 4.1. It is evident that there is significant overlap in the occurrence of some autoimmune neurological syndromes with

Oncogenic Autoimmunity

65

Table 4.1. Tumor-Paraneoplastic Autoimmune Syndrome Associations* Type of Tumor

Associated Autoimmune Syndrome

Gynecologic cancers breast fallopian ovarian uterine Ganglioneuroma/-neuroblastoma Lymphoproliferative Neuroblastoma Small-cell lung carcinoma Thymoma

PCD, SMS, OMS OMS DM, PCD DM Idiopathic hypothalamic dysfunction DM, PP, XER PCD, OMS CAR, DM, LEMS, PCD, PEM/SN, OMS Limbic encephalitis, MG, NMT, PP

* Tumor syndrome pairings for which there is ample evidence to suggest a true correlation are highlighted in bold. Associations for the remaining disorders are less grounded and are subject to question until further immunologic and/or molecular evidence is established. Abbreviations: PCD, paraneoplastic cerebellar degeneration; SMS, stiff-man syndrome; OMS, opsoclonus-myochlonus; DM, dermatomyositis; PP, paraneoplastic pemphigus; XER, paraneoplastic autoimmune xerostomia; CAR, cancer-associated retinopathy; LEMS, LambertEaton myasthenic syndrome; PEM/SN, paraneoplastic encephalomyelitis/sensory neuropathy; MG, myasthenia gravis; NMT, neuromyotonia.

different tumor types,8 suggesting that the parent cell types from which these tumors arise may share a common embryological derivation. The recognition of a paraneoplastic process as the result of oncogenic autoimmunity is currently based on the ability to detect antibody reactivity toward molecularly-defined target antigens in the sera of affected individuals. Ideally, expression of the autoantigen is limited to the organ or tissue which is the target of immune aggression, as this finding implies a specificity of the immune response. Catalogued in Table 4.2 are molecularly characterized autoantigens which have been thus far reported in the literature. The grouping of autoantigens according to tissue and cellular localization and/or function—nerve-terminal and vesicle-associated, neuron-specific RNA-binding, putative neuronal signaling, and neuromuscular junction proteins—follows the reclassification of “onconeural antigens” by Darnell.9 Inquiry into the importance of these and other yet undiscovered antigens to the generation of oncogenic autoimmunity will likely produce vital insights into the physiological regulation and dysregulation of autoimmunity as well as the biology of anti-tumor immunity in general. To this end, we will attempt to address in the following pages the many difficult questions which have arisen regarding the possible mechanisms by which transformed cells coax the immune system into attacking that which it is meant to protect.

Paraneoplastic Autoimmune Disorders of the Peripheral Nervous System Myasthenia Gravis The most widely recognized example of oncogenic autoimmunity is the occurrence of myasthenia gravis in association with thymoma and thymic carcinoma. Numerous patient series report that approximately 10% of myasthenia gravis patients have an associated

Acetylcholine binding subunit of ion channel receptor Repolarization of neurons

Voltage-gated K+ channel

cGMP-gated signal transductionn

Recoverin

Acetylcholine receptor alpha-subunit

Cytoplasmic protein; N-terminal amphipathic helix-leucine zipper

Putative neruronal signalling proteins cdr2 (Yo)

Signal transduction for neurotransmitter release

RNA-binding protein motifs RNA-binding protein motifs

Neuron-specific RNA-binding proteins Nova family (≥2 members) Hu family (≥ 4 members)

NMJ and peripheral nerve proteins Presynaptic Ca2+ channel

?

AP2 interaction; Ca2+ sensor

Synaptotagmin

Anti-VGKC

Anti-AChR

Anti-VGCC

Anti-CAR

Anti-Yo (APCA-1)

Anti-Ri (ANNA-2) Anti-Hu (ANNA-1)

Anti-128kDa

ANTIBODY

Dynamin and AP2 interaction

FUNCTION

Nerve-terminal/vesicle associated proteins Amphiphysin

For syndrome abbreviations, see Table 4.1

PNS

CNS

ANTIGEN

Table 4.2. Molecularly Characterized Onconeural Antigens

NMT

MG

LEMS

Retinopathy (blindness)

PCD

OMS PEM/SN (multifocal)

LEMS

SMS

SYNDROME

66 Endocrine and Organ Specific Autoimmunity

Oncogenic Autoimmunity

67

thymoma or thymic carcinoma, whilst the incidence of myasthenia gravis in patients with a thymic tumor may be as great as 80%.10,11 Although the majority of non-thymoma-associated myasthenia gravis patients show pathological evidence of thymic epithelial cell hyperplasia (70% of all myasthenia gravis patients),12 the mechanism of autoimmune sensitization is believed to differ in these two subsets of myasthenia gravis patients.11-13 Irrespective of the thymic pathology associated with a diagnosis of myasthenia gravis, the demonstration of anti-acetylcholine receptor (anti-AChR) antibodies in serum is possible in virtually all cases. Early animal experiments involving passive transfer of antibodies and active immunization with purified AChR have produced excellent correlative models for the study of human disease,14-16 whilst more recently a system employing adoptive transfer of human autoantibody-producing lymphocytes to severe-combined immunodeficiency (SCID) mice has also been reported.17 These animal models have led to the observations that anti-AChR antibodies target epitopes on the extracellular domains of the AChR α-subunit resulting in the disruption of ion channel function, accelerated turnover of receptors at the neuromuscular junction, and the activation of complement with subsequent muscle membrane damage.18,19 Additionally, autoantibodies against other functional components of skeletal muscle have been detected in a significant proportion of myasthenia gravis patients, including those directed against neurofilament proteins (titin epitopes)20-22 and the ryanodine receptor (RyR),23 a Ca2+-release channel located in the sarcoplasmic reticulum membrane which plays an important role in striated muscle excitation-contraction coupling. The logical conclusion from these observations is that the impairment of neuromuscular transmission characteristic of myasthenia gravis results from autoantibody inhibition and/or accelerated destruction of target molecules within the muscle membrane. Taken together, these data support the belief that humoral autoimmunity plays a key role in the pathogenesis of myasthenia gravis. The reclassification of thymoma pathology by Marino and Müller-Hermelink24 has provided possible clues regarding the pathogenesis of myasthenia gravis. Their revised classification system relates tumor features to the normal organization of thymic microenvironments, yielding five categories: medullary thymoma, mixed thymoma, predominantly cortical thymoma, cortical thymoma, and well-differentiated thymic carcinoma. The utility of this reclassification has been well demonstrated by data from Müller-Hermelink and colleagues regarding the degree of tumor aggressiveness and the occurrence of myasthenia gravis symptoms in patients with different histologic types.11 In general, myasthenia gravis is increasingly associated with thymomas showing a greater degree of cortical epithelial cell derivation, with far fewer cases of myasthenia gravis diagnosed in patients found to have medullary thymomas. Additional studies in other patient populations have confirmed these data.25,26 Based on the excellent correlation between thymic pathology and the occurrence of myasthenia gravis in patients with thymoma, it has been proposed that some aberration of T lymphocyte positive or negative selection is operating in the neoplastic thymic environment which promotes the maturation of self-reactive T cells.11 It is often observed in cortical-type thymoma tissue that immature CD1+ thymocytes (T cells) are intimately associated with neoplastic epithelial cells, the same cell type normally responsible for positive selection of T cells via TCR-peptide antigen-MHC interactions.11,27 Consistent with this “aberrant selection” hypothesis is the finding that cultured thymoma epithelial cells have the ability to present synthetic AChR peptides to AChR-specific CD4+ T cell responders in vitro, though somewhat less efficiently as compared to presentation of the same antigens by peripheral blood mononuclear cells.28 It is still unknown why the abnormally selected T cells of this disease model orchestrate an humoral immune response preferentially targeted to skeletal muscle proteins, although expression of AChR mRNA29 and neurofilament epitopes

68

Endocrine and Organ Specific Autoimmunity

by thymic tumor cells has been demonstrated, supporting a role for abnormal and/or premature T cell maturation in the pathogenesis of myasthenia gravis.

Lambert-Eaton Myasthenic Syndrome Lambert-Eaton myasthenic syndrome is a second widely recognized PNS autoimmune paraneoplastic syndrome which complicates a small number of small cell lung cancer cases. Approximately 60% of diagnosed Lambert-Eaton myasthenic syndrome patients have an associated tumor. Clinically, myasthenia gravis and Lambert-Eaton myasthenic syndrome may present with quite similar symptoms, but the two disorders can be readily distinguished on the basis of electromyography studies and the presence [myasthenia gravis] or absence [Lambert-Eaton myasthenic syndrome] of anti-AChR antibodies.30 Electromyographic differentiation of Lambert-Eaton myasthenic syndrome from myasthenia gravis is possible because the site of neuromuscular junction pathology is slightly different in each case. In myasthenia gravis, autoantibody inhibition of neurotransmitter receptor (AChR) function at the postsynaptic muscle membrane results in myasthenia, whereas in Lambert-Eaton myasthenic syndrome the majority of patients have circulating antibodies to a voltage-gated Ca2+ channel located in the membrane of the presynaptic terminal at the motor endplate. Since voltage-gated Ca2+ channel activation with subsequent Ca2+ influx to the presynaptic terminal is the electrochemical signal which facilitates acetylcholine release at the neuromuscular junction synapse, autoantibody-mediated functional blockade of voltage-gated Ca2+ channels results in lowered quantal release of neurotransmitter packets and impaired muscle contraction. As with myasthenia gravis, the likely importance of anti-voltage-gated Ca2+ channel (VGCC) antibodies to the pathogenesis of Lambert-Eaton myasthenic syndrome is supported by animal models of passive disease transfer using autoantibodies purified from Lambert-Eaton myasthenic syndrome patients.31-33 Small-cell tumors of the lung are believed to arise from the neuroectodermally-derived Kulchitsky cell, so it is not surprising that mRNA transcripts representing a number of neuronal Ca2+ channel subtypes can be found in small cell lung cancer cell lines.34 A strong anti-tumor immune response to malignant cells expressing functional Ca2+ channels is presumed to initiate oncogenic autoimmunity, the consequence of which is antibody-mediated blockade of VGCC function. This role for anti-voltage-gated Ca2+ channel antibodies is supported by in vitro findings of reduced45 Ca2+ flux in response to K+-stimulation of cultured tumor cell lines pretreated with Lambert-Eaton myasthenic syndrome antisera.35 Antibodies to a synaptic vesicle protein, synaptotagmin, have also been reported in association with Lambert-Eaton myasthenic syndrome,36 but the importance of these antibodies to disease pathogenesis is not entirely clear.

Acquired Neuromyotonia Acquired neuromyotonia (NMT, or Isaac’s syndrome), is a rare peripheral neurological syndrome with symptoms involving continuous, involuntary muscle contraction and myokymia which in some cases may be the result of oncogenic autoimmunity. Approximately 20% of neuromyotonia cases are associated with thymoma or small cell lung cancer, the former often associating with anti-AChR antibodies but no evidence of myasthenia gravis.37 In one study of six patients with classic acquired neuromyotonia, antibodies directed against voltage-gated K+ channel were demonstrated in three individuals, although none of the six patients had an associated malignancy.38 Nevertheless, the possibility of autoimmune mechanisms acting in paraneoplastic neuromyotonia exists, with impairment of muscular relaxation and, therefore, symptomatic disease being the likely consequences of autoantibody inhibition of voltage-gated K+ channels function in peripheral nerves.

Oncogenic Autoimmunity

69

Paraneoplastic Autoimmune Disorders of the Central Nervous System Cancer-Associated Retinopathy The classification of cancer-associated retinopathy as a disorder of the CNS is based on knowledge of the existence of a blood-retinal barrier, much like the blood-brain barrier which envelopes most brain structures.39 Such barriers are the anatomic and physiologic elements which confer ‘immune privilege’ to these sites. However, in some patients with small cell lung cancer or extrapulmonary small-cell carcinomas (implying a similar neuroendocrine derivation), there is some compromise of the blood-retinal barrier which allows the immune system to direct an attack on retinal photoreceptor cells.39 Humoral immune sensitivity to a 23-kDa cancer-associated retinopathy antigen, recoverin, can be assayed in the serum of cancer patients suffering from retinal degeneration.40,41 One recent study of anti-recoverin antibodies purified from patient sera has demonstrated apoptotic cell death in a rat retinal cell line presumably due to the presence of antibody,42 but the results of these experiments await confirmation by other investigators. The gene for recoverin has been assigned to a region of chromosome 17 (17p13.1) which contains a number of cancer-related loci, including the tumor suppresser gene p53. This finding raises the possibility that ectopic expression of recoverin in human cancers is related to a single mutational event which inactivates a functional cell-cycle control gene (p53) at the same time it activates the transcription of recoverin mRNA and subsequent protein production.39,43 The end result of this molecular cascade is enhanced growth of malignant cells which are capable of sensitizing the immune system to a self antigen normally sequestered from immune surveillance by the blood-retinal barrier. Expression of recoverin mRNA and protein has in fact been demonstrated in small cell lung cancer tumors and tumor cell lines,44,45 suggesting a likely mechanism whereby auto-immunization occurs.

Paraneoplastic Cerebellar Degeneration The symptomatic hallmark of the paraneoplastic cerebellar degeneration syndrome is progressive global cerebellar dysfunction manifesting as symmetric ataxia, dysarthria, and nystagmus. The incidence of paraneoplastic cerebellar degeneration is substantially higher than that of other CNS paraneoplastic syndromes, with clinical onset generally preceding or coinciding with the diagnosis of malignancy.30 It is most commonly reported in association with ovarian cancer or small cell lung cancer but is seen in breast cancer and Hodgkin’s disease patients as well.46 Pathologically, the disease is characterized by a selective loss of Purkinje cells from the cerebellum in the absence of local inflammation, although other neural structures may also be involved.47 The detection of anti-Purkinje cell antibodies was first reported in 1983, followed four years later by the cloning of a 34 kDa autoantigen, CDR (cerebellar-degeneration-related) 34.46 Antigenically related to this molecule, and now considered to be the major paraneoplastic cerebellar degeneration-related autoantigen, is a 62 kDa protein (CDR 62) known as cdr2, the so-called “Yo” antigen.48 It has been observed that cdr2 mRNA is widely expressed, although translation into protein appears to be regulated by a post-transcriptional mechanism which limits protein production to the immune privileged tissues of testis and brain.49 This limited expression pattern combined with the confirmation of cdr2 protein detection in paraneoplastic cerebellar degeneration-associated tumors48 is consistent with the hypothesis that Purkinje cell destruction is mediated by a cross-reactive immunologic response. However, in mouse experiments, passive transfer of human anti-Yo antibodies, induction of anti-Yo antibodies by immunization with recombinant Yo protein, and passive transfer of Yo protein-activated murine mononuclear cells have all failed to induce symptomatic

70

Endocrine and Organ Specific Autoimmunity

cerebellar disease or Purkinje cell loss in mice.50,51 These data serve to underscore our limited understanding of the interplay between humoral and cellular immune effector mechanisms in the generation of CNS oncogenic autoimmunity and paraneoplastic autoimmune disease, and they suggest that as yet undetermined factors likely play a role in the pathogenesis of paraneoplastic cerebellar degeneration.

Stiff Man Syndrome Stiff-man syndrome (SMS) is a rare CNS autoimmune disease characteristically presenting as chronic body musculature rigidity with periodic painful spasms. Sixty percent of patients are found to have circulating autoantibodies to glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing the inhibitory neurotransmitter gammaaminobutyric acid (GABA).52 The decrement of inhibitory neuronal input from GABA-ergic neurons which likely accounts for the clinical manifestations of SMS is believed to result from GAD-directed autoimmunity against the presynaptic (GABA-ergic) terminal. High titer GAD autoantibodies have been detected in the cerebrospinal fluid (CSF).53 However, a transitory remission of the muscular rigidity in patients with SMS has been documented following treatment with plasmapheresis or intravenous immunoglobulins,54 without a significant decline in GAD autoantibody levels. A small number of SMS patients are also afflicted with breast cancer, (paraneoplastic SMS).55-57 Interestingly, whilst GAD autoantibodies are not found in patients affected by paraneoplastic SMS, these patients display a high titer of autoantibodies against amphiphysin in the serum and cerebrospinal fluid.55,56,58,59 Amphiphysin is a 128 kDa synaptic vesicle-associated protein also found in the presynaptic terminal. Recent observations indicate that amphiphysin is involved in endocytosis of synaptic vesicles.60 It is hypothesized that the neurotransmitter release mechanism requiring docking of synaptic vesicles with the presynaptic neuronal membrane is impaired by anti-amphiphysin autoimmunity, in contrast to the antibody-mediated functional impairment of GABA synthesis believed to occur in GAD-antibody positive patients. A potential pathogenic role for anti-amphiphysin antibodies in breast cancer-associated SMS is supported by the observed reversal of symptomatic neurological disease in some patients following tumor removal and steroid therapy.55,56,59 Therefore, the detection of amphiphysin antibodies represents a tool for the diagnosis of SMS and is a strong indication to search for an occult breast tumor. Stiff man syndrome patients often develop insulin-dependent diabetes (IDDM), and the majority of these patients are positive for islet cell antibodies.60-62 Nevertheless, with regard to the relationship between IDDM and SMS, a number of questions are still without answer.60 In patients with coexistent IDDM and stiff-man syndrome (SMS), an HLA association with DQ*0201, an allele which is found on the DQ2-DR3 haplotype, has been described.54 In both disorders, GAD antibodies are preferentially directed against the Mr 65,000 isoform. However they differ in several respects.60,63 Their levels are 10- to 100-fold higher in SMS sera than in IDDM sera. Secondly, GAD65 antibodies, unlike the majority of those from IDDM patients, usually react with GAD65 on Western blotting. Thirdly, GAD65 autoantibodies present in SMS patients react mainly with two epitopes at the N-terminus and C-terminus of GAD65, whilst IDDM GAD65 autoantibodies recognize epitopes in the middle and C-terminus of the protein.64 Immunoblot analyses using deletion mutants of GAD65 suggest that the epitope at the C-terminus recognized by specific antibodies from patients with SMS is conformational. This possibility is supported by the evidence that sera from SMS patients can inhibit the enzymatic activity of glutamic acid decarboxylase.60,65 From a clinical perspective, it is of interest that antibodies against the IDDM-related autoantigen ICA512 (IA-2),66,67 unlike GAD65 antibodies, are exclusively detected in SMS patients who

Oncogenic Autoimmunity

71

have concurrent IDDM,68 raising the concept that they may be used as predictive markers in SMS-affected individuals who have a significant risk of developing IDDM.

Paraneoplastic Encephalomyelitis, Sensory Neuropathy and Limbic Encephalitis The development of peripheral neuropathies and generalized neurological disturbances is relatively common in cancer patients, although these symptoms are usually attributed to nutritional deficiencies, metabolic disturbances, or toxic effects of treatment. However, within the context of certain tumors, namely small cell lung cancer and neuroblastoma, oncogenic autoimmunity generated by an immune response to ectopic protein expression by tumor cells appears to be capable of initiating damage to various areas of the CNS. The result is a constellation of syndromes recognized as paraneoplastic encephalomyelitis/sensory neuropathy and limbic encephalitis. The unifying element of these disorders is evidence of a humoral immune response to the CNS nucleoprotein known as Hu antigen.69-72 An immunohistochemical study of Hu antigen expression in small cell lung cancer and neuroblastoma tissues revealed that 26/26 small cell lung cancer and 39/50 neuroblastoma tumors expressed the protein, some at very high levels. This antigen expression did not, however, correlate with the presence of anti-Hu antibodies in serum, as only 15/26 small cell lung cancer patients and 4/50 neuroblastoma patients showed evidence of Hu antigen reactivity by Western blot analysis. In the same report, expression of MHC class I molecules was also analyzed by immunohistochemistry. The correlation of Hu antigen and MHC class I co-expression with evidence of an anti-Hu immune response led the authors to conclude that a significant T cell-mediated anti-tumor response against MHC-expressing tumor cells is involved in the disease.71 However, the importance of anti-Hu antibodies to the primary autoimmune pathology of paraneoplastic encephalonmyelitis/sensory neuropathy and limbic encephalitis has yet to be determined. Likewise, the occurrence of anti-amphiphysin antibodies in three patients with paraneoplastic encephalonmyelitis/sensory neuropathy has also been reported,73 although the significance of these observations with respect to disease pathogenesis is not yet known.

Opsoclonus-Myoclonus Syndrome Opsoclonus-myoclonus syndrome, the “dancing eyes, dancing feet” syndrome, has been reported to occur in association with childhood neuroblastoma, small cell lung cancer, breast cancer, and other gynecologic malignancies. Among pediatric patients diagnosed with opsoclonus-myoclonus, approximately 10% have an underlying neuroblastoma, whilst <20% of adult patients have an associated malignancy.8 In the paraneoplastic form associated with breast cancer, an autoantibody against the 55 kDa Ri antigen has been demonstrated,74-76 but similar autoantibodies have not been detected in children affected with opsoclonusmyoclonus. As noted in Table 4.1, Ri antigen belongs to the Nova gene family, the products of which are believed to function as RNA binding proteins specifically expressed within the nuclei of CNS neurons.77 This limited distribution of Nova gene expression to brain tissue is consistent with tumor-induced peripheral immune sensitization to CNS antigens normally protected from immune surveillance.

Other Paraneoplastic Disorders Associated with Oncogenic Autoimmunity Paraneoplastic syndromes may occur in the setting of malignancies distinct from tumors of the nervous system. Although more than one syndrome may arise with a given tumor, certain clinical manifestations are often associated with particular types of neoplasms.

72

Endocrine and Organ Specific Autoimmunity

Paraneoplastic pemphigus, a devastating disease with high mortality, has been established as a distinct entity from pemphigus vulgaris.78-80 Paraneoplastic pemphigus is more frequently associated with non-Hodgkin’s lymphoma, thymoma,81 chronic lymphocytic leukemia and poorly differentiated spindle cell sarcoma.82 Autoantibodies reactive with desmoplakin I and II, the 230 kD bullous pemphigoid antigen and an as yet uncharacterized 190 kDa antigen are present in the serum of some paraneoplastic pemphigus patients. These antibodies appear to be distinct from those found in pemphigus vulgaris. The mechanism(s) by which the tumor cells in this disorder drive oncogenic autoimmunity is so far unknown. Early diagnosis and intervention with combined prednisone-cyclosporine therapy have provided some control of the disease process. Dermatomyositis or polymyositis are inflammatory myopathies characterized by proximal symmetrical muscle weakness, elevated serum muscle enzymes and electromyographic abnormalities. Dermatomyositis may be the consequence of humorally mediated capillary necrosis, whereas polymyositis is hypothesized to arise through T cell-mediated inflammation that ultimately causes destruction of muscle fibers. Whilst the association of malignancy with dermatomyositis/polymyositis is still controversial,83 dermatomyositis sine myositis, known also as amyopathic dermatomyositis,84 seems to be associated with a number of tumors, particularly with ovarian cancer.85,86 It is commonly accepted that dermatomyositis/polymyositis and dermatomyositis sine myositis all represent paraneoplastic syndromes. A number of rare paraneoplastic syndromes possibly associated with oncogenic autoimmunity have been reported in the literature.7,87 Of interest is a recent example of paraneoplastic autoimmune xerostomia, occurring in association with lymphoblastic lymphoma. In this case report, after excluding a clinical diagnosis of Sjögren syndrome, it was hypothesized that autoantibodies may act to inhibit salivary secretion.37 Circulating autoantibodies against ductal cells of monkey salivary glands were detected at each relapse of xerostomia and during the second remission. The molecular target(s) of this novel paraneoplastic autoimmune disorder is still unknown. Another disease which may involve oncogenic autoimmunity is Bazex’s syndrome, a dermatological condition associated with squamous cell carcinoma of the head and neck.87 Bazex’s syndrome might develop consequent to an autoimmune reaction against a target antigen shared by both tumor and cutaneous tissue.

Theoretical Aspects and Basics Questions on Oncogenic Autoimmunity The Role of Antigen Expression There has been considerable speculation concerning factors responsible for the initiation of oncogenic autoimmunity. As introduced above, a number of onconeural antigens which are localized in normal brain and in paraneoplastic autoimmune-associated tumor tissues have now been cloned and partially characterized (Table 4.2). The occurrence of antibodies to onconeural antigens often correlates with the presence of a paraneoplastic syndrome, and their detection may help in predicting the diagnosis of a specific underlying malignancy. It is commonly accepted that tumor antigens are either identical to or closely resemble endogenous molecules of a normal cell.88 One model of paraneoplastic neurological disease indicates that the physiological expression of onconeural autoantigens is restricted to immunologically privileged tissues such as the CNS although they may be more widely expressed during fetal development (Fig. 4.2). As a consequence, these antigens are recognized as non-self when ectopically expressed in tumors arising from cells of neural crest origin. The transformation of an ancient normal cell to neoplastic tissue may be accompanied by significant overexpression of otherwise normal proteins.88 If this hypothesis holds

Oncogenic Autoimmunity

73

Fig. 4.2. Model for the pathogenesis of neurological oncogenic autoimmunity. Oncogenic autoimmunity may originate when solid tumors outside the nervous system express molecules that are restricted within the immunologically privileged state of neurons. If neuronal proteins are overexpressed in tumor cells, the immune system recognizes them as foreign and gives rise to a cascade of events culminating in autoimmune-mediated disruption of the blood-brain barrier and ultimately damage of bystander neuronal cells expressing the “oncogenic” autoantigen. Depending on the nature and the function of the target onconeural antigen, an autoimmune attack may lead to irreversible neurological injury and symptomatic clinical disease (Reprinted with permission from Darnell RB. Proc Natl Acad Sci USA 1996; 93:4529-4536. (1996 National Academy of Sciences, U.S.A.).

true, peripheral T lymphocytes, that recognize certain self peptides as a result of thymic maturation, will settle in the periphery in an anergic state, “tolerant” to their TCR-specific peptides. Those T cells which can potentially recognize “oncofetal” antigens may have escaped deletion in the thymus, having progressed to the periphery because of low “oncofetal” antigen expression in the appropriate thymic environment. Various mechanisms, including a subthreshold availability of the antigenic peptide(s) in peripheral tissues and/or the lack of a costimulatory helper signal necessary for T cell activation,89 are considered to be responsible for maintaining this state of tolerance.89,90 If tumor cells express quantitatively abnormal but qualitatively normal peptides this may lead to local activation of T lymphocytes with a certain TCR which is specific for the processed self antigen presented in the appropriate MHC context, thereby priming oncogenic autoimmunity.91,92 In autoimmune paraneoplastic disorders involving the PNS, the target antigens of autoantibodies are generally membrane proteins having extracellular domains where antibodies can bind. In contrast, autoantigens identified in the CNS are generally intracellular proteins thought to be inaccessible to circulating antibodies. However, it has been postulated that binding of antineuronal antibodies to their molecular target(s) may lead to either

74

Endocrine and Organ Specific Autoimmunity

significant neuronal death (i.e., cancer-associated retinopathy), or to reversible tissue damage with a potential for complete resolution of the neurological symptoms (i.e., opsoclonus-myoclonus). Some of these proteins are involved in essential intracellular functions, so that upon gaining access to the intracellular compartment, specific autoantibodies may inhibit these functions when reacting with their target antigen. As a result, cessation of essential cellular activities in the neuron may ensue, ultimately leading to cell death. Paraneoplasia-associated autoantibodies are, in fact, believed to target functional protein domains.93 For instance, the cdr2 (Yo antigen) epitope is the leucine zipper dimerization domain of the protein,46,49 the Hu epitope includes two RNA binding domains,70 and the Nova-1 epitope is the third KH RNA-binding domain of the Ri antigen.77 Cessation of normal cell function may therefore explain why irreversible neuronal damage is present in some paraneoplastic syndromes such as paraneoplastic cerebellar degeneration or cancer-associated retinopathy. On the contrary, when the oncogenic autoimmune process is limited to onconeural antigens that are involved in nonessential cell functions, the autoimmune process may be reversible and does not progress to permanent neuronal injury. Studies in animal models of melanoma paradoxically support the hypothesis that oncogenic autoimmunity may be beneficial when it occurs in the context of concomitant tumor rejection.94 These models involve immunization of mice bearing human melanoma tumors with altered sources of the oncogenic antigen gp75 (i.e., expressed in insect cells) rather than the native murine gp75 antigen. In animals receiving altered gp75, anti-gp75 antibodies were elicited, resulting in both rejection of melanoma and the development of skin lesions similar to those of human vitiligo. The induction of coexisting tumor rejection and autoimmune disease is reminiscent of clinical data suggesting a favorable prognosis in patients with metastatic melanoma who develop vitiligo.95 Antigen mimicry, whereby an immunologic response develops to different self proteins with similar epitopes, has been proposed as a potential mechanism underlying organ-specific human autoimmune diseases96-99 and in the origins of T cell autoimmunity in thymoma-associated myasthenia gravis.23 However, there is no convincing evidence that shared epitopes between tumor and target normal tissues contributes to the generation of oncogenic autoimmunity.100

Are Antibodies Involved in Oncogenic Autoimmunity? The evidence for direct involvement of humoral autoimmunity in the pathogenesis of paraneoplastic disorders lies in the success of passive transfer experiments using myasthenia gravis and Lambert-Eaton myasthenic syndrome antisera in animal models of disease9,14,18,31,32,101 and in the efficacy of plasmapheresis and B cell immunosuppressive treatments in ameliorating disease symptoms.9,102,103 Even though most CNS paraneoplastic disorders do not respond to plasmapheresis or other immunomodulatory treatments, both adult and childhood forms of opsoclonus respond to immunotherapy, plasmapheresis or Protein A adsorption, resulting in a decline of autoantibody levels.104 These clinical observations support a role for circulating autoantibodies in the pathogenesis of paraneoplastic autoimmune diseases of the PNS and at least some paraneoplastic disorders of the CNS. The fact that there is a higher antibody titer in paraneoplastic autoimmune disease patients in the CSF relative to the serum (index > 1),105 suggests that an active B-cell inflammatory response is present within the CSF compartment. Additional evidence of antibody-mediated inhibition of protein synthesis within neurons, is that antibodies, unlike cytotoxic T lymphocytes (CTLs) recognizing the same antigens via MHC class I molecules, may mediate clearance of latent infections with alphavirus.106 If intracellular antigens or viral products potentially involved in the generation of oncogenic autoimmunity are expressed at the surface of neurons, an antibody-mediated signal may be able to inhibit

Oncogenic Autoimmunity

75

antigen expression and potentially disrupt neuronal function or induce the eradication of a latent viral infection capable of sustaining oncogenic autoimmunity. Additional evidence for the pathogenic potential of autoantibodies in paraneoplastic disorders is given by the finding of autoantibodies against glutamate receptor (GluR) subunits GluR1, GluR2, and/or GluR5/6 in serum from seven patients with paraneoplastic autoimmune disease (three paraneoplastic cerebellar degeneration, two PEM and two opsoclonus-myoclonus).107 Electrophysiological analysis of cultured cortical neurons demonstrated that 5 out of 7 GluR5 antibody positive patients enhanced glutamate-elicited currents, suggesting that some paraneoplastic neurological neuronal loss or dysfunction may result from glutamate-mediated excitotoxicity. Similar antibodies, directed predominantly against GluR3, were observed in Rasmussen’s encephalitis, a childhood disorder characterized by an inflammatory insult of the brain associated with severe epileptic seizures.108 Although a conspicuous body of data suggests that autoantibodies play a central role in the pathogenesis of some human oncogenic autoimmune diseases, results of active and passive immunization in some animal models of paraneoplastic disorders, particularly of CNS paraneoplastic disorders, have been rather disappointing.8 Perhaps the whole story of oncogenic autoimmunity cannot be explained by the humoral immune response; nevertheless, autoantibodies in paraneoplastic autoimmune disease, as for other autoimmune diseases, have been important tools for the molecular cloning, identification and characterization of novel onconeural antigens. They have the practical advantage to be used as markers of occult malignancy that can precede the clinical diagnosis of certain tumors.

Are T Cells Involved in Oncogenic Autoimmunity? The fact that there is induction of IgG antibodies in several paraneoplastic disorders suggests that helper T cell responses are involved in the generation of these antibodies. Dependence upon helper T cell signaling is thought to be crucial to the generation of antibodies that target AChR in myasthenia gravis associated with thymoma.109 AChR autoantibodies are mainly IgG1 and IgG3 isotypes,110 a pattern of expression considered to be the hallmark of T cell dependent antibody responses.111 It has been demonstrated that thymoma epithelial cells have the capacity to stimulate T cells and likely to autosensitize against AChR in vivo.28 Interestingly, T cell responses against epitopes corresponding to amino acid (aa) residues 257-269 of the AChR α-subunit are apparently specific for myasthenia gravis, particularly thymoma-associated myasthenia gravis.113 It is noteworthy that there are sequence similarities between aa residues 231-301 of the AChR α-subunit and aa residues 4628-4861 of the RyR molecule.114,115 It has been postulated that developing T cells in the thymoma are initially sensitized against epitopes shared by the RyR and AChR molecules, and then will stimulate B cells to produce antibody responses.23 Unlike what has been demonstrated in myasthenia gravis, the role of T cells in generating oncogenic autoimmunity in the context of CNS paraneoplastic disorders is not quite clear.9,116 The lack of readily detectable MHC expression on neurons, weakens the hypotheses that T cells play a pathogenic role in autoimmune paraneoplasia of the CNS, although some studies have described the induction of MHC molecule expression on neurons in vitro.117 Transgenic expression of a murine class I MHC molecule (Db) in neurons using the neuron-specific enolase promoter did not appear to cause neuronal lysis in vivo following infection with lymphocytic choriomeningitis virus and adoptive transfer of virus-reactive CTLs.118 Class I MHC expressed in the context of Hu antigen-bearing tumors may play a role in the development of anti-Hu autoimmunity associated with the paraneoplastic syndromes, paraneoplastic encephalonmyelitis/sensory neuropathy.70 The strong association (χ2: ρ < 0.0001) found between expression of MHC class I glycoproteins in neuroendocrine-related

76

Endocrine and Organ Specific Autoimmunity

tumors such as small cell lung cancer and neuroblastoma and an anti-Hu antibody response, suggests that T cell-mediated events may be responsible for the development of oncogenic autoimmunity in these conditions.71 Whether the Hu antigens may serve as targets for cytotoxic T cell or antibody responses is still unknown.70 It was postulated that enhanced expression of MHC class II proteins following transduction of melanoma cells with IFN genes is responsible for presentation of self antigens to tumor infiltrating CD4+ cells.119 This raised the concept that in autoimmune diseases self antigens can be presented by MHC class II bearing cells and promote autoimmunity.120 Enhanced or abnormal expression of MHC class II proteins has also been described for carcinoma of the gastrointestinal tract and small cell lung cancer,71,121 but this may simply be the consequence of either endogenous release of anti-tumor factors or secretion of cytokines from activated tumor-infiltrating lymphocytes.

Is Apoptotic Cell Death Induced in Oncogenic Autoimmunity? The manner in which target organs are destroyed by immunologically-mediated responses remains an issue of great interest and discussion. To date, the only paraneoplastic syndrome in which immune-mediated apoptotic cell death was clearly demonstrated in vitro is cancer-associated retinopathy.42 Anti-recoverin antibodies obtained from sera of patients with cancer-associated retinopathy and animals immunized with the antigen cause target cells expressing recoverin to undergo programmed cell death. Also, the fact that anti-Hu antibodies, which are present in small cell lung cancer, can be found in the nucleus of living target cells,122,123 prompted speculation that autoantibodies, after penetration into living cells, may alter their function and ultimately cause apoptosis.124 It has been shown that inappropriate expression or excessive Fas (CD95/Apo-1) activity results in apoptotic-induced tissue damage.125-128 Whether Fas induction is an important effector pathway of disease as a result of oncogenic autoimmunity, similar to what has been recently demonstrated in studies of autoimmune thyroid disease129 and autoimmune diabetes,130,131 remains to be established.

Anti-Tumor Therapy in Autoimmune Paraneoplastic Disorders There are relatively few interventions used for the treatment of paraneoplastic disorders associated with oncogenic autoimmunity. A number of early reports suggested that removal of the underlying neoplasm may improve the paraneoplastic disease, but currently it is widely accepted that surgery, chemotherapy, radiation, immunosuppression, or a combination of these interventions may be beneficial in some patients.132-136 Thymectomy for myasthenia gravis, long considered treatment of choice for this condition, shows greatest benefit in patients with moderate to severe myasthenia.134 Review of a decade of therapeutic results132 from 258 patients suffering from various paraneoplastic disorders (Table 4.3), showed encouraging results following specific treatment only in Lambert-Eaton myasthenic syndrome.132,137 Even though nearly half of the cases of Lambert-Eaton myasthenic syndrome are not associated with cancer, an autoimmune-mediated attack on the target tissue in this disease is present in all cases.138 Specific treatment of an associated small cell lung cancer may induce remission.137 Drugs like 3,4-diaminopyridine seem to produce significant improvement in muscle strength and autonomic function.138 The majority of patients appear to benefit from 3,4-diaminopyridine in combination with anti-tumor therapy and immunosuppression such as steroids or azathioprine.138 Plasmapheresis and intravenous immunoglobulins,139 together with peripherally acting drugs such as pyridostigmine may potentiate the positive effects. In the majority of other syndromes described in this review, therapeutic results have been rather disappointing. Overall, only 12.8% of all patients showed amelioration after

Oncogenic Autoimmunity

77

Table 4.3. Pooled Results of Therapy for Paraneoplastic Neurological Disorders132 DISEASE

TREATMENT STRATEGY

n OF SUCCESSFUL THERAPIES

Paraneoplastic encephalomyelitis/ Limbic encephalitis Opsoclonus myoclonus Spinal cord myelitis Paraneoplastic sensory neuropathy Paraneoplastic cerebellar degeneration Lambert-Eaton myasthenic syndrome137 Stiff man syndrome

CT

8/72

CT, steroids* CT, PL, steroids* PL CT, SG, Symp* ** steroids, Symp*†

7/11 3/5 3/22 7/134 11/16 3/4

n = number of patients; CT = chemotherapy; PL = plasmapharesis; SG = surgery; Symp = symptomatic. * Some patients responded to a combination of these therapies. ** For treatment strategies in LEMS, see text. † Anti-tumor therapy (breast cancer) was also employed, but methods used were unspecified.

specific treatment (Table 4.3). Among those with PEM/SSN, limbic encephalitis, or paraneoplastic cerebellar degeneration, only 18 of 228 patients had a favorable outcome. These data suggest that in autoimmune paraneoplastic neurological disorders, combined therapies such as chemotherapy and immunosuppression, seem more effective than monotherapy.132,133 In summary, results of therapeutic trials performed in paraneoplastic neurological disorder patients have been unsuccessful with the exception of Lambert-Eaton myasthenic syndrome and myasthenia gravis. A possible explanation for the poor responses to treatment is that if severe neurological damage has been caused by oncogenic autoimmunity, it is very unlikely that any therapeutic procedure can reverse the neurological injury. Theoretically, the success of treatment depends upon early detection of signs and markers of oncogenic autoimmunity (i.e., anti-Ri antibodies etc.), followed by a careful search for occult cancer, prompt removal of tumor and initiation of a specific treatment consisting of combined therapies.

Concluding Remarks An important unsolved issue in the generation of oncogenic autoimmunity is the ability to understand whether oncogenes may act as potent immunogens or how tumor antigens and autoimmune antigens are related, since specific tumor cell types consistently express a specific oncogenic antigen. Certainly the ability to clone, identify and classify target oncogenic antigens has aided in the improvement of the classification and differentiation of paraneoplastic disorders otherwise considered to be similar in their clinical aspects and in their pathology. The wide heterogeneity of autoimmune responses present in paraneoplastic disorders provides evidence that both antibodies and T cells are involved in these disorders. Nevertheless, the existence of MHC-independent pathways for T cell activation may have substantial implications for many aspects of cell-mediated autoimmunity in the context of paraneoplastic diseases (Fig. 4.3). In addition to the usual T cells and T cell ligands (TCR α/β) which respond to self antigens (including oncogenic antigens) classically presented by MHC class I and class II molecules,140 there are non-classical T cell types, including CD4-/CD8phenotype T cells141,142 and natural killer (NK) cells. The latter are capable of recognizing tumoral cells that do not express MHC class I molecules. 143-145 Exploration of the

Endocrine and Organ Specific Autoimmunity

78 MHC molecule + Oncogenic antigen

CD1 molecule + Oncogenic antigen

B7

TCR

CD4 CD28

α β ε δ

γ δ ε δ

γ ε ζ ζ

γ ε ζ ζ

Lck

Fyn CYTOPLASM

ZAP70 2nd messenger signal generation Ca++

DAG

PKC

Calcineurin

NUCLEUS

p21ras

Alternative pathways of T cell activation

Nuclear signaling events with activation of gene transcription

Fig. 4.3. Hypothetical pathways of antigen presentation and T cell activation in oncogenic autoimmunity. T cells that have encountered the potential “oncogenic” peptide during thymic development escape deletion and assume a state of T cell unresponsiveness in the periphery. Upon encountering the same “oncogenic” peptide overexpressed by neoplastic cells, CD4+or CD8+ T cells can be activated through classical MHC-TCR α/β or γ/δ interactions. Alternatively, CD4-/ CD8- phenotype T cells could be stimulated by nonclassical oncogenic antigen presentation in the context of CD1 molecules expressed on the surface of tumor cells. Conventional costimulatory signals or other factors may play a role in reversing the anergic state upon stimulation by either of these mechanisms.

mechanisms operating in the presentation of unusual antigens (i.e., oncogenic antigens presented by isoforms of CD1 molecules) to unusual T lymphocytes expressing either α/β or γ/δ TCRs, will provide a more comprehensive and integrated view of the mechanisms underlying the generation of oncogenic autoimmunity.

References 1. Ehrlich P, Morgenroth J. On hemolysins, fifth communication, Berl Klin Wochenschr. In: The collected papers of Paul Ehrlich. London: Pergamon Press 1901; 1957:246. 2. Eisenbarth GS, Bellgrau D. Autoimmunity. Science Med 1994; 16:90-98. 3. Theofhilopoulos AN. The basis of autoimmunity: Part I. Mechanisms of aberrant self-recognition. Immunol Today 1995; 16(2):90-98. 4. Rose NR. Immunologic diagnosis of autoimmunity. In: Leffel MS, Donnenberg AD eds. Handbook of Immunology. New York: CRC Press, 1997:111-123.

Oncogenic Autoimmunity

79

5. Waldenström JG ed. Paraneoplasia: Biological Signals in the Diagnosis of Cancer. New York: John Wiley & Sons, 1978. 6. Brain WR, Norris F eds. The Remote Effects of Cancer on the Nervous System. New York: Grune & Stratton, 1965. 7. Agarwala SS. Paraneoplastic syndromes. Med Clin N Am 1996; 80:173-184. 8. Lang B, Vincent A. Autoimmunity to ion-channels and other proteins in paraneoplastic disorders. Current Opinin Immunol 1996; 8:865-871. 9. Darnell RB. Onconeural antigens and the paraneoplastic neurologic disorders: At the intersection of cancer, immunity, and the brain. Proc Natl Acad Sci USA 1996; 93:4529-4536. 10. Souadjian JV, Enriquez P, Silverstein MN et al. The spectrum of diseases associated with thymoma. Arch Intern Med 1974; 134:374-379. 11. Muller-Hermelink HK, Marx A, Geuder K et al. The pathological basis of thymoma-associated myasthenia gravis. Ann NY Acad Sci 1992; 56-65. 12. Hohlfield R, Wekerle H. The thymus in myasthenia gravis. Neurologic Clini N Am 1994; 12(2):331-342. 13. Compston AX, Vincent A, Newsom-Davis J et al. Clinical, pathological, HLA-antigen and immunological evidence for disease heterogeneity in myasthenia gravis. Brain 1980; 103:597-601. 14. Toyka KV, Brachman DB, Pestronk A et al. Myasthenia gravis: Passive transfer from man to mouse. Science 1975; 190:397-399. 15. Tarrab-Hazdai R, Aharonov A, Silman I et al. Experimental autoimmune myasthenia induced in monkeys by purified acetylcholine receptor. Nature 1975; 256:128-130. 16. Lennon VA, Lindstrom JM, Seybold ME. Experimental autoimmune myasthenia: a model of myasthenia gravis in rats and guinea pigs. J Exp Med 1975; 141:1365-1375. 17. Martino G, DuPont BL, Wollmann RL et al. The human-severe combined immunodeficiency myasthenic mouse model: A new approach for the study of myasthenia gravis. Ann Neurol 1993; 34:48-56. 18. Tzartos SJ, Lindstrom JM. Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits. Proc Natl Acad Sci USA 1980; 77(2):755-759. 19. Engel AG, Lambert EH, Howard FM. Immune complexes (IgG and C3) at the motor end-plate in myasthenia gravis: Ultrastructure and light microscopic localization and electrophysiological correlations. Mayo Clin Proc 1977; 52:267-280. 20. Marx A, Kirchner T, Greiner A et al. Neurofilament epitopes in thymoma and antiaxonal autoantibodies in myasthenia gravis. Lancet 1992; 339:707-708. 21. Marx A, Kirchner T, Greiner A et al. Myasthenia gravis-associated thymic epithelial tumors express neurofilaments and are associated with antiaxonal autoimmunity. Ann NY Acad Sci 1993; 681:107-109. 22. Marx A, Wilisch A, Schultz A et al. Expression of neurofilaments and of a titin epitope in thymic epithelial tumors. Am J Pathol 1996; 148(6):1839-1850. 23. Mygland A, Tysnes O-B, Aarli JA et al. IgG subclass distribution of ryanodine receptor autoantibodies in patients with myasthenia gravis and thymoma. J Autoimmun 1993; 6:507-515. 24. Marino M, Muller-Hermelink HK. Thymoma and thymic carcinoma: Relation of thymoma epithelial cells to the cortical and medullary differentiation of thymus. Virchows Arch A 1985; 407:119-149. 25. Ho FCS, Fu KH, Lam SY et al. Evaluation of a histogenetic classification for thymic epithelial tumors. Histopathology 1994; 25:21-29. 26. Close PM, Kirchner T, Uys CJ et al. Reproducibility of a histogenetic classification of thymic epithelial tumors. Histopathology 1995; 26:339-343. 27. Chilosi M, Iannucci A, Menestrina F et al. Immunohistochemical evidence of active thymocyte proliferation in thymoma. Its possible role in the pathogenesis of autoimmune diseases. Am J Pathol 1987; 128 (3):464-70.

80

Endocrine and Organ Specific Autoimmunity

28. Gilhus NE, Willcox N, Harcourt G et al. Antigen presentation by thymoma epithelial cells from myasthenia gravis patients to potentially pathogenic T cells. Journal of Neuroimmunol 1995; 56:65-76. 29. Gattenlohner S, Brabletz T, Schultz A et al. Cloning of a cDNA coding for the acetylcholine receptor alpha-subunit from a thymoma associated with myasthenia gravis. Thymus 1994; 23(2):103-113. 30. Meehan KR, Ernstoff MS. Systemic secretions of cancer cells and their effects. Paraneoplastic syndromes. In: Bennett JC, Plum F, eds. Cecil Textbook of Medicine. 20th ed. Philadelphia: W.B. Saunders Company, 1996:1017-1021. 31. Lang B, Newsom-Davis J, Wray D et al. Autoimmune aetiology for myasthenic (Eaton-Lambert) syndrome. Lancet 1981; 2:224-226. 32. Fukunaga H, Engel AG, Lang B et al. Passive transfer of Lambert-Eaton myasthenic syndrome with IgG from man to mouse depletes the presynaptic membrane active zones. Proc Natl Acad Sci USA 1983; 80(24):7636-7640. 33. Lang B, Molenaar PC, Newsom-Davis J et al. Passive transfer of Lambert-Eaton myasthenic syndrome in mice: decreased rates of resting and evoked release of acetylcholine from skeletal muscle. J Neurochem 1984; 42(3):658-662. 34. Oguro-Okano M, Griesmann GE, Wieben ED et al. Molecular diversity of neural-type calcium channels identified in small cell lung carcinoma. Mayo Clin Proc 1992; 67(12):1150-1159. 35. Roberts A, Perera S, Lang B et al. Paraneoplastic myastenic syndrome IgG inhibits 45Ca2+ flux in a human small cell carcinoma line. Nature 1985; 317:737-739. 36. Takamori M, Takahshi M, Yasukawa Y et al. Antibodies to recombinant syaptotagmin and calcium channel subtypes in Lambert-Eaton myasthenic syndrome. J Neurol Sci 1995; 133:95-101. 37. Folli F, Ponzoni M, Vicari AM. Paraneoplastic autoimmune xerostomia. Ann Intern Med 1997; 127(2):167-168. 38. Shillito P, Molenaar PC, Vincent A et al. Acquired neuromyotonia: Evidence for autoantibodies directed against K+ channels of peripheral nerves. Ann Neurol 1995; 38(5):714-722. 39. Thirkill CE. Cancer-induced retinal hypersensitivity. BritJ Biomed Sci 1996; 53:227-234. 40. Thirkill CE, Keltner JL, Tyler NK et al. Antibody reactions with retina and cancer-associated antigens in 10 patients with cancer-associated retinopathy. Arch Ophthalmol 1993; III:931-937. 41. Adamus G, Guy J, Schmied JL et al. Role of anti-recoverin autoantibodies in cancer-associated retinopathy. Invest Ophth Vis Sci 1993; 34(9):2627-2633. 42. Adamus G, Machnicki M, Seigel GM. Apoptotic retinal cell death induced by antirecoverin autoantibodies of cancer-associated retinopathy. Invest Ophth Vis Sci 1997; 38(2):283-291. 43. McGinnis JF, Austin B, Klisak I et al. Chromosomal assignment of the human gene for the cancer-associated retinopathy protein (Recoverin) to chromosome 17p13.1. J Neurosci Res 1995; 40:165-168. 44. Polans AS, Witkowska D, Haley TL et al. Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy. Proc Natl Acad Sci USA 1995; 92:9176-9180. 45. Matsubara S, Yamaji Y, Sato M et al. Expression of a photoreceptor protein, recoverin, as a cancer-associated retinopathy autoantigen in human lung cancer cell lines. Brit J Cancer 1996; 74(9):1419-1422. 46. Dropcho EJ, Chen Y-T, Posner JB et al. Cloning of a brain protein identified by autoantibodies froma patient with paraneoplastic cerebellar degeneration. Proc Natl Acad Sci USA 1987; 84:4552-4556. 47. Verschuuren J, Chuang L, Rosenblum MK et al. Inflammatory infiltrates and complete absence of Purkinje cells in anti-Yo-associated paraneoplastic cerebellar degeneration. Acta Neuropathol 1996; 91(5):519-525. 48. Furneaux HM, Rosenblum MK, Dalmau J et al. Selective expression of Purkinje-cell antigens in tumor tissue from patients with paraneoplastic cerebellar degeneration. N Engl J Med 1990; 322(26):1844-1851.

Oncogenic Autoimmunity

81

49. Corradi JP, Yang C, Darnell JC et al. A post-transcriptional regulatory mechanism restricts expression of the paraneoplastic cerebellar degeneration antigen cdr2 to immune privileged tissues. J Neurosci 1997; 17(4):1406-1415. 50. Tanaka M, Tanaka K, Onodera O et al. Trial to establish an animal model of paraneoplastic cerebellar degeneration with anti-Yo antibody. 1. Mouse strains bearing different MHC molecules produce antibodies on immunization with recombinant Yo protein, but do not cause Purkinje cell loss. Clin Neurol Neurosur 1995; 97:95-100. 51. Tanaka K, Tanaka M, Igarashi S et al. Trial to establish an animal model of paraneoplastic cerebellar degeneration with anti-Yo antibody. Passive transfer of murine mononuclear cells activated with recombinant Yo protein to paraneoplastic cerebellar degeneration lymphocytes in severe combined immunodeficiency mice. Clin Neurol Neurosur 1995; 97:101-105. 52. Solimena M, Folli F, Denis-Donini S et al. Autoantibodies to glutamic acid decarboxylase in a patient with Stiff-man syndrome, epilepsy, and Type I diabetes mellitus. N Engl J Med 1988; 318:1012-1020. 53. Solimena M, Folli F, Aparisi R et al. Autoantibodies to GABA-ergic neurons and pancreatic beta cells in Stiff-Man syndrome. N Engl J Med 1990; 322:1555-1560. 54. Solimena M, Butler MH, De Camilli P. GAD, diabetes, and stiff man syndrome: some progress and more questions. J Endocrinol Invest 1994; 17:509-520. 55. Folli F, Solimena M, Cofiell R et al. Autoantibodies to a 128-kD synaptic protein in three women with the Stiff-Man syndrome and breast cancer. N Engl J Med 1993; 328(8):546-551. 56. De Camilli P, Thomas A, Cofiell R et al. The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of stiff-man syndrome with breast cancer. J Exp Med 1993; 178:2219-2223. 57. Piccolo G, Cosi V. Stiff-Man syndrome dysimmune disorder and cancer. Ann Neurol 1989; 26:105-105. 58. Yamamoto R, Li X, Winter S et al. Primary structure of human amphiphysin, the dominant autoantigen of paraneoplastic Stiff-Man syndrome, and mapping of its gene (AMPH) to chromosome 7p13-p14. Hum Mol Genet 1995; 4(2):265-268. 59. David C, Solimena M, De Camilli P. Autoimmunity in Stiff-Man syndrome with breast cancer is targeted to the C-terminal region of human amphiphysin, a protein similar to the yeast proteins, Rvs167 and Rvs161. FEBS Lett 1994; 351:73-79. 60. Lernmark A. Glutamic acid decarboxylase—gene to antigen to disease. J Intern Med 1996; 240:259-277. 61. Baekkeskov S, Aanstoot H, Christgau S et al. Identification of the 64K autoantigen in insulin dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990; 347:151-156. 62. Reetz A, Solimena M, Matteoli M et al. GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion. EMBO J 1991; 10:1275-1284. 63. Li L, Hagopian W, Brashear HR et al. Identification of autoantibody epitopes of glutamic acid decarboxylase in stiff man syndrome patients. J Immunol 1994; 152:930-934. 64. Daw K, Powers AC. Two distinct glutamic acid decarboxylase autoantibody specificities in IDDM target different epitopes. Diabetes 1995; 44:216-220. 65. Bjork E, Velloso LA, Kampe O et al. GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type I recognize different epitopes. Diabetes 1994; 43:161-165. 66. Rabin DU, Pleasic SM, Shapiro JA et al. Islet cell antigen 512 is a diabetes-specific islet autoantigen related to protein tyrosine phosphatases. J Immunol 1994; 152:3183-3188. 67. Lan MS, Lu J, Goto Y et al. Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 1994; 13:505-514. 68. Martino GV, Grimaldi LME, Bazzigaluppi E et al. The insulin-dependent diabetes mellitus-associated ICA 105 autoantigen in stiff man syndrome patients. J Neuroimmunol 1996; 69:129-134. 69. Graus F, Elkon KB, Cordon-Cardo C et al. Sensory Neuronopathy and Small Cell Lung Cancer. Antineuronal Antibody That Also Reacts with the Tumor. Am J Med 1986; 80:45-52.

82

Endocrine and Organ Specific Autoimmunity

70. Manley GT, Smitt PS, Dalmau J et al. Hu antigens: reactivity with Hu antibodies, tumor expression, and major immunogenic sites. Ann Neurol 1995; 38:102-110. 71. Dalmau J, Graus F, Cheung NV et al. Major histocompatibility proteins, anti-Hu antibodies, and paraneoplastic encephalomyelitis in neuroblastoma and small cell lung cancer. Cancer 1995; 75(1):99-109. 72. Sakai K, Gofuku M, Kitagawa Y et al. A hippocampal protein associated with paraneoplastic neurologic syndrome and small cell lung carcinoma. Biochem Bioph Res Co 1994; 199(3):1200-1208. 73. Dropcho EJ. Antiamphiphysin antibodies with small-cell lung carcinoma and paraneoplastic encephalomyelitis. Ann Neurol 1996; 39(5):659-667. 74. Budde-Steffen C, Anderson NE, Rosenblum MK et al. An antineuronal autoantibody in paraneoplastic opsoclonus. Ann Neurol 1988; 23(5):528-531. 75. Luque FA, Furneaux HM, Ferziger R et al. Anti-Ri: An antibody associated with paraneoplastic opsoclonus and breast cancer. Ann Neurol 1991; 29(3):241-251. 76. Escudero D, Barnadas A, Codina M et al. Anti-Ri-associated paraneoplastic neurologic disorder without opsoclonus in a patient with breast cancer. Neurology 1993; 43:1605-1606. 77. Buckanovich RJ, Posner JB, Darnell RB. Nova, the paraneoplastic Ri antigen, is homologous to an RNA-binding protein and is specifically expressed in the developing motor system. Neuron 1993; 11:657-672. 78. Anhalt GJ, SooChan K, Stanley JR et al. Paraneoplastic Pemphigus: An autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990; 323:1729-35. 79. Helm TN, Camisa C, Valenzuela R et al. Paraneoplastic pemphigus: a distinct autoimmune vesiculobullous disorder associated with neoplasia. Oral Surg Oral Med Oral Pathol 1993; 75(2):209-213. 80. Thivolet J. Pemphigus: past, present and future. Dermatology 1994; 189(suppl 2):26-29. 81. Patten SF, Dijkstra JWE. Association of pemphigus and autoimmune disease with malignancy or thymoma. Int J Dermatol 1994; 33(12):836-842. 82. Anhalt GJ. Paraneoplastic pemphigus. Adv Dermatol 1997; 12:77-96. 83. Callen JP. Dermatomyositis and malignancy. Clin Dermatol 1993; 11:61-65. 84. Kagan LJ. Amyopathic dermatomyositis. Arch Dermatol 1995; 131:1458-1459. 85. Whitmore SE, Watson R, Rosenshein NB et al. Dermatomyositis sine myositis: association with malignancy. J Rheumatol 1996; 23:101-105. 86. Whitmore SE, Rosenshein NB, Provost TT. Ovarian cancer in patients with dermatomyositis. Medicine 1994; 73(3):153-159. 87. Minotti AM, Kountakis SE, Stiernberg CM. Paraneoplastic syndromes in patients with head and neck cancer. Am J Otolaryng 1994; 15(5):336-343. 88. Parmiani G. Tumor immunity as autoimmunity: tumor antigens include normal self proteins which stimulate anergic peripheral T cells. Immunol Today 1993; 14(11):536-538. 89. Mueller DL, Jenkins MK. Molecular mechanisms underlying functional T-cell unresponsiveness. Curr Opin Immunol 1995; 7:375-381. 90. Lanzavecchia A. Understanding the mechanisms of sustained signaling and T cell activation. J Exp Med 1997; 185:1717-1719. 91. Guerder S, Matzinger P. A fail-safe mechanism for maintaining self tolerance. J Exp Med 1992; 176:553-564. 92. Kroemer G, Martinez AC. Mechanisms of self tolerance. Immunol Today 1992; 13:401-404. 93. Tan EM. Autoantibodies in Pathology and Cell Biology. Cell 1991; 67:841-842. 94. Naftzger C, Takechi Y, Kohda H et al. Immune response to a differentiation antigen induced by altered antigen: A study of tumor rejection and autoimmunity. Proc Natl Acad Sci USA 1996; 93:14809-14814. 95. Duhra P, Ilchyshyn A. Prolonged survival in metastatic malignant melanoma associated with vitiligo. Clin Exp Dermatol 1997; 16:303-305. 96. Luo A-M, Garza KM, Hunt D et al. Antigen mimicry in autoimmune disease. Sharing of amino acid residues critical for pathogenic T cell activation. J Clin Invest 1993; 92:2117-2123.

Oncogenic Autoimmunity

83

97. Luppi P, Rossiello MR, Faas S et al. Genetic background and environment contribute synergistically to the onset of autoimmune diseases. J Mol Med 1995; 73:381-393. 98. Barnaba V, Sinigaglia F. Molecular mimicry and T cell-mediated autoimmune disease. J Exp Med 1997; 185:1529-1531. 99. Gautman AM, Lock CB, Smilek DE et al. Minimum structural requirements for peptide presentation by major histocompatibility compless class II molecules: implications in induction of autoimmunity. Proc Natl Acad Sci USA 1994; 91(2):767-761. 100. Jaeckle KA. Autoimmune mechanisms in the pathogenesis of paraneoplastic nervous system disease. Clin Neurol and Neurosur 1995; 97:82-88. 101. Pohl KRE, Pritchard J, Wilson J. Neurological sequelae of the dancing eye syndrome. Eur J Pediatr 1996; 155:237-244. 102. Newsom-Davis J, Vincent A, Wilson SG et al. Plasmapheresis for myasthenia gravis. N Engl J Med 1978; 298(8):456-457. 103. Newsom-Davis J, Murray NMF. Plasma exchange and immunosuppressive drug treatment in the Lambert-Eaton myasthenic syndrome. Neurology 1984; 34(4):480-485. 104. Cher LM, Hochberg FH, Teruya J et al. Therapy for paraneoplastic neurologic syndromes in six patients with protein A column immunoadsorption. Cancer 1995; 75:1678-1683. 105. Borges LF, Elliott PJ, Gill R et al. Selective extraction of small and large molecules from the cerebrospinal fluid by Purkinje neurons. Science 1985; 228:346-348. 106. Levine B, Hardwick JM, Trapp BD et al. Antibody-mediated clearance of alphavirus infection from neurons. Science 1991; 254:856-860. 107. Gahring LC, Twyman RE, Greenlee JE et al. Autoantibodies to neuronal glutamate receptors in patients with paraneoplastic neurodegenerative syndrome enhance receptor activation. Mol Med 1995; 1(3):245-253. 108. Rogers SW, Andrews PI, Gahring LC et al. Autoantibodies to glutamate receptor GluR3 in Rasmussen’s encephalitis. Science 1994; 265:648-649. 109. Willcox N, Baggi F, Batocchi A-P et al. Approaches for studying the pathogenic T cells in autoimmune patients. Ann NY Acad Sci 1993; 681:219-237. 110. Rodgaard A, Nielsen FC, Djurup R et al. Acetylcholine receptor antibody in myasthenia gravis: predominance of IgG subclasses 1 and 3. Clin Exp Immunol 1987; 67:82-88. 111. Schur PH. IgG subclasses — a review. Ann Allergy 1987; 58:89-97. 112. Harcourt GC, Sommer N, Rothbard J et al. A juxta-membrane epitope on the human acetylcholine receptor recognized by T cells in myasthenia gravis. J Clin Invest 1988; 82:1295-1300. 113. Sommer N, Willcox N, Harcourt GC et al. Myasthenic thymus and thymoma are selectively enriched in acetylcholine receptor-reactive T cells. Ann Neurol 1990; 28:312-319. 114. Zorzato F, Fujii J, Otsu K et al. Molecular cloning of cDNA encoding human and rabbit forms of the Ca2+ release channel (ryanodine receptor) of skeletal muscle sarcoplasmic reticulum. J Biol Chem 1990; 265:2244-2256. 115. Takeshima H, Nishimura S, Matsumoto T et al. Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor. Nature 1989; 339:439-445. 116. Panegyres PK, Reading MC, Esiri MM. The inflammatory reaction of paraneoplastic ganglionitis and encephalitis: an immunohistochemical study. J Neurol 1993; 240:93-97. 117. Neumann H, Cavalie A, Jenne DE et al. Induction of MHC class I genes in neurons. Science 1995; 269:549-552. 118. Rall GF, Mucke L, Oldstone MBA. Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo. J Exp Med 1995; 182:1201-1212. 119. Ogasawara M, Rosenberg SA. Enhanced expression of HLA molecules and stimulation of autologous human tumor infiltrating lymphocytes following transduction of melanoma cells with γ-interferon genes. Cancer Research 1993; 53:3561-3568. 120. Bottazzo GF, Dean BM, McNally JM et al. In situ characterization of autoimmune phenomena and expression of HLA molecules in the pancreas in diabetic insulitis. N Engl J Med 1985; 313:353-360.

84

Endocrine and Organ Specific Autoimmunity

121. Ghosh AK, Moore M, Street AJ et al. Expression of HLA-DR sub-region products on human colorectal carcinoma. Int J Cancer 1986; 38:459-464. 122. Hormigo A, Lieberman F. Nuclear localization of anti-Hu antibody is not associated with in vitro cytotoxicity. J Neuroimmunol 1994; 55:205-212. 123. Fabian RH. Uptake of plasma IgG by CNS motoneurons: comparison of antineural and normal IgG. Neurology 1988; 38:1775-1780. 124. Alarcon-Segovia D, Ruiz-Arguelles A, Llorente L. Broken dogma: penetration of autoantibodies into living cells. Immunol Today 1995; 17(4):163-164. 125. Nagata S, Golstein P. The Fas death factor. Science 1995; 267:1449-1456. 126. Krammer PH. The CD95 (APO-1/Fas) receptor/ligand system: death and disease. Cell Death Differ 1996; 3:159-160. 127. Abbas AK. Die and let live: eliminating dangerous lymphocytes. Cell 1996; 84:655-657. 128. Bellgrau D, Gold D, Selawry H et al. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377:630-632. 129. 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. 130. Chervonsky AV, Wang Y, Wong I et al. The role of Fas in autoimmune diabetes. Cell 1997; 89:17-24. 131. Stassi G, De Maria R, Trucco G et al. Nitric oxide primes pancreatic beta cell destruction for Fas-mediated destruction in IDDM. J Exp Med 1997; In Press. 132. Grisold W, Drlicek M, Liszka-Setinek U et al. Anti-tumor therapy in paraneoplastic neurological disease. Clin Neurol Neurosur 1995; 97:106-111. 133. Graus F, Delattre JY. Immune modulation of paraneoplastic neurologic disorders Clin Neurol Neurosur 1995; 97:112-116. 134. Busch C, Machens A, Pichlmeier U et al. Long-term outcome and quality of life after thymectomy for myasthenia gravis. Ann Surg 1996; 224(2):225-232. 135. Maggi G, Casadio C, Cavallo A et al. Thymoma: results of 241 operated cases. Ann Thorac Surg 1991; 51:152-156. 136. Bach J-F. Immunosuppressive therapy in autoimmune diseases. Immunol Today 1993; 14(6):322-326. 137. Chalk CH, Murray NMF, Newsome-Davis J et al. Response of the Lambert-Eaton myasthenic syndrome to treatment of associated small-cell lung carcinoma. Neurology 1990; 40:1552-1556. 138. McEvoy KM, Windebank AJ, Daube JR et al. 3,4-Diaminopyridine in the treatment of the Lambert-Eaton myasthenic syndrome. N Engl J Med 1989; 321(23):1567-1571. 139. Takano H, Tanaka M, Koike R et al. Effect of intravenous immunoglobulin in LambertEaton myasthenic syndrome with small-cell lung cancer: correlation with the titer of anti-voltage-gated calcium channel antibody. J Muscle Nerve 1994; 17:1073-1075. 140. Zinkernagel RM, Doherty PC. The discovery of MHC restriction. Immunol Today 1997; 18(1):14-18. 141. Porcelli SA, Moldin RL. CD1 and the expanding universe of T cell antigens. J Immunol 1995; 155:3709-3710. 142. Bendelac A. CD1: presenting unusual antigens to unusual T lymphocytes. Science 1995; 269:185-186. 143. Kaneko T, Fukuda J, Yoshihara T et al. Nasal natural killer (NK) cell lymphoma: report of a case with activated NK cells containing Epstein-Barr virus and expressing CD21 antigen, and comparative studies of their phenotype and cytotoxicity with normal NK cells. Brit J Haematol 1995; 91:355-361. 144. Gumpez JE, Parham P. The enigma of the natural killer cell. Nature 1995; 378:245-248. 145. Mori N, Yatabe Y, Oka K et al. Expression of perforin in nasal lymphoma. Additional evidence of its natural killer derivation. Am J Pathol 1996; 149(2):699-705.

CHAPTER 5

Celiac Disease Fei Bao, Marian Rewers, Fraser Scott and George S. Eisenbarth

Introduction

C

eliac disease is a common, often asymptomatic immune-mediated disorder with a prevalence of approximately 1/200 in Western populations. The disorder is typically associated with intestinal lesions leading to diarrhea and weight loss. In the most severely diseased patients death occurs in the absence of dietary therapy. Celiac disease is a multisystem disorder with mucocutaneous, neurological and dental abnormalities in addition to intestinal lesions.1 Amongst common autoimmune disorders celiac disease is remarkable in that the disease is dependent upon the ingestion of wheat proteins (rye, also barley and possibly oats) and in particular wheat gliadins. Removal of these triggering factors from the diet results in resolution not only of the intestinal lesions but also the rapid disappearance of the autoantibodies characteristic of the disorder. However, the condition appears to be life long and the lesions re-appear following subsequent exposure to these cereal proteins. The major target autoantigen is a transglutaminase and the inducing dietary factor gliadin contains as many as 40% glutamines as its predominant amino acid.2

Pathology The symptoms and signs of celiac disease only imperfectly correlate with intestinal histopathology and it is likely that the majority of individuals with celiac disease are asymptomatic.3 The current approach to diagnosis of celiac disease includes initial serologic screening for anti-endomysial autoantibodies followed by endoscopic biopsy of the duodenal mucosa.4 In 1970, The European Society for Pediatric Gastroenterology and Nutrition (ESPGAN) proposed diagnostic criteria for celiac disease which include the demonstration of a flat jejunal mucosa in small intestinal mucosa biopsies at the time of presentation, complete restoration of the mucosal structure during gluten-free diet, and relapse after a period of gluten-challenge.5 However, these criteria have been difficult to comply within individual practice, and have been subsequently modified6 to include, among others, serologic testing. The typical pathologic changes of celiac disease are most pronounced in the duodenum and upper jejunum. The mucosa in the ileum is usually histologically normal. Grossly, the mucosa of the small bowel from a patient with celiac disease is pale and thin without prominent redundant folds. There is an almost total absence of villi with so-called subtotal villous atrophy. At times the mucosa is not completely flat but displays villi fused into ridges, convolutions, waves, or ripples, which is characteristic of partial villous atrophy.7 The major alterations of the small bowel mucosa in order of appearance in celiac disease are: 1) expansion of the lamina propria with lymphocytic infiltration, 2) crypt hyperplasia and 3) absence or blunting of villi (i.e., total or subtotal villous atrophy). Dramatic Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

86

Endocrine and Organ Specific Autoimmunity

increased size of the intraepithelial lymphocyte pool8 resulting from local differentiation of T lymphocytes, high mitotic rates with local expansion, and migration to the lamina propria of T cells may be the only histological finding. Progression through these stages takes probably weeks to months upon primary exposure to gliadin but only a few hours upon reexposure of completely normal mucosa of a patient on gluten free diet. The mucosal lesion in gluten sensitivity essentially involves the lamina propria and submucosa. It is likely that the lamina propria is the major site of gluten-driven immunologic abnormalities. Upon gluten challenge, swelling and early neutrophil infiltration of the lamina propria occur before marked changes in villous morphology. The first changes occur in the subepithelial layer of the mucosa with infiltration by both plasma cells and lymphocytes which appear within a few hours of gluten challenge. Apparent damage to epithelial cells is delayed for several days and such damage does not occur in normal individuals. Other immune responses are also observed in the early gliadin-induced reaction, including an accumulation of neutrophils, mast cells, and basophils and activation of eosinophils and neutrophils, degranulation of mast cells,9 prostaglandin secretion,10 and complement activation. There is little relation between the degree of atrophy of the mucosa and the age of the patient, the duration of disease, or its severity. Patients with mild symptoms may show a mucosa as flat as for patients with severe diarrhea. Older persons with long-standing disease may have mucosal lesions that are less likely to be reversible with therapy than younger patients. Frequent clinical complications of celiac disease include anemia caused by folate or iron deficiency, osteopenia (due to vitamin D deficiency) and slight aminotransferasemia. There are several reports of an association between long-standing celiac disease and intestinal lymphoma and carcinoma.11,12 At least one study suggested that a gluten free diet protects against these malignancies in patients with celiac disease.13 Celiac disease can progress from an inflammatory to a malignant process and it has been suggested that celiac disease may be a low-grade lymphoma of intraepithelial lymphocytes.11,12 This transformation appears to be associated with the HLA-DR3/4 genotype.14 Dermatitis herpetiformis(DH) is characterized by itchy papulovesicular skin eruptions and granular depositions of IgA in the papullary dermis. Dermatitis herpetiformis may be due to cross reactivity between dietary glutenin and dermal elastin.15,16 About two-thirds of patients with dermatitis herpetiformis have the flat, small intestinal muscosal lesion of celiac disease. The lesions respond over a prolonged period to a gluten-free diet. Patients with dermatitis herpetiformis often have no symptoms from their spruelike intestinal lesions.

What Genes Determine Susceptibility? Twins/Relatives/Associated Disorders There is an increased incidence of celiac disease among nonsymptomatic relatives of patients with celiac disease, with a prevalence of 10-12% in first degree relatives,17-19 and a high rate of concordance (71%) in monozygotic twins.20 Most patients with celiac disease are Caucasian with ancestors from northwestern Europe. Celiac disease is rare amongst individuals of Asian or African derivation. Dermatitis herpetiformis (65%),7 insulin-dependent diabetes mellitus (3-6%),21-27 Sjogren’s syndrome (3.3%),21 IgA nephropathy,4 IgA deficiency and Down’s syndrome are associated with celiac disease.28 Patients with IgA deficiency have at least a tenfold risk of celiac disease compared with the general population.29

HLA Alleles The major histocompatibility complex (MHC) class I allele B8, MHC class II allele DR3 and MHC DQ alleles DQA1*0501, DQB1*0201 are in linkage disequilibrium. This

Celiac Disease

87

Table 5.1. Familial Risk of Celiac Disease Relationship to Proband

Region

Prevalence of Celiac Disease

General Population

England,Wales,Scotland30 London31 Edinburgh32 Toronto33 West of Ireland34 U.S.A.35

Monozygotic Twin

Spain20

71%

Parent

Italy,Finland,Spain19 Birmingham,England17

5% 6.6%

Sibling

Seattle,U.S.A.36 Italy,Finland,Spain19 Birmingham,England17

13% 8.4% 4.8%

Offspring

Seattle,U.S.A.36 Italy,Finland,Spain19

19% 10%

1/8000-1/4000 1/6000-1/2000 1/1850 1/1000 1/387-1/303 1/250

haplotype (A1, B8, DR3, DQA1*0501, DQB1*0201) is associated with type 1 diabetes,24 dermatitis herpetiformis,37 selective IgA deficiency,29 Graves’ disease, and Addison’s disease.38 It was found that approximately 90% of patients with celiac disease have HLA-B8, whereas 20% of the general population have the same allele.5 Susceptibility to celiac disease is primarily associated with DQA1*0501 and DQB1*0201. This particular DQ α/β heterodimer can be encoded in either cis or trans (DR3 haplotypes in cis; in trans with DR7 (DQB1*0201) plus DR5 (DQA1*0501).39-41 Homozygosity for DQA1*0501, DQB1*0201 alleles may predispose to an earlier disease onset and to more severe disease manifestations.42 DR3 is significantly increased in patients with celiac disease in all ethnic groups studied. The DR7 association with celiac disease has been more often reported from southern Europe, due to an increased frequency of the DR5/7 genotype in these populations. The DQα chain encoded by DR3-DQ2 haplotypes (DQA1*0501) is identical to that encoded for by DR5-DQ7 haplotypes. The DQβ chain of the DR3-DQ2 haplotype (DQB1*0201) is identical to that of the DR7-DQ2 haplotype except for a single amino-acid difference (DQB1*0202, Asp135 versus Gly135 in the second domain of DQ β ). 43-45 The few DQA1*0501/DQB1*0201 negative patients with celiac disease usually have DQ8 (DQA1*0301, DQB1*0302).41 A number of studies suggest that polymorphisms of additional genes in the major histocompatibility complex including DPB1, DMB, and TAP polymorphisms contribute to celiac disease susceptibility. Other studies indicate that these associations result merely from linkage disequilibrium with high risk class II alleles.40,46-49 In a similar manner, two highly polymorphic microsatellite loci, TNFα and β in the class III region of the MHC have been associated with celiac disease. These results suggest that the TNF2 gene may have a role in the pathogenesis of celiac disease.50

88

Endocrine and Organ Specific Autoimmunity

NonHLA Genes The HLA-linked locus is a major determinant of the familial aggregation of celiac disease. Genes unlinked to HLA may also contribute to disease susceptibility. The disease was associated with an HLA-unlinked marker identified as a B-cell alloantigen.51 The non-HLA loci T cell receptor (TCR) α,β,γ chain were examined for linkage to celiac disease and no linkage was shown with the TCRα and γ chain loci, but a weak linkage was demonstrated with the TCRβ locus.52 Highly polymorphic microsatellite markers at the TCRD locus on chromosome 14q11.2 was analyzed with no significant difference in allele frequencies between celiac disease patients and controls.53 A study of Irish patients with celiac disease identified a locus on chromosome 6p about 30 cM telomeric from HLA, with a multipoint maximum lod score of 4.66 (compared with 4.44 for HLA-DQ) and a recessive mode of inheritance. This study requires replication and at present no nonHLA genes contributing to celiac disease have been identified.

What Environmental Factors Initiate or Inhibit Development of Celiac Disease? The incidence of celiac disease varies widely among different countries. The disease is reportedly decreasing in frequency, though such a decrease may only be for symptomatic disease. The existence of identical twins discordant for celiac disease implies that environmental factors play a role and the major environmental factor associated with celiac disease is gliadin. Patients with celiac disease develop lesions with gluten in the diet in excess of 5 to 20 g/ day in a time-related and dose-dependent fashion. Gluten severely affects the morphology of the small intestine and induces a characteristic flat mucosa with villous atrophy and crypt hyperplasia. Patients challenged with as little as 1.00 mg/d of gliadin show significant increases in the mean intraepithelial lymphocyte count, and a decrease in the villous height/ crypt depth ratio, while the intestinal permeability test showed an increase in the mean urinary cellobiose/mannitol ratio.54 A significant increase (p<0.05) in prostaglandin E2 secretion was noted in CD patients with gliadin administration.10 There was a progressive increase in p62c/myc (c-myc oncogene product) staining intensity in the villous enterocytes of celiac small intestinal mucosa after a 10 g oral gluten challenge. It is probable that the insult produced by gluten results in a number of oncogene products being over expressed and this may contribute to the association between long-standing celiac disease and intestinal malignancy. 55 Gluten is the water-insoluble fraction of wheat flour, which is mostly made up of two groups of proteins: ethanol insoluble glutenins and ethanol soluble gliadins. Gliadin contains large amounts of the amino acids proline and glutamine. It is known that only gliadin is toxic to celiac patients. The equivalent prolamin fractions of rye, barley, and oats are also deleterious.5 Frazer et al56 digested gluten with pepsin and trypsin in vitro. Digested gluten was not less toxic than gluten. Following these studies, this digest is referred to as Frazer fraction III. It comprises a complex mixture of peptides with a wide range of molecular weights and is toxic to the mucosa of patients with celiac disease and dermatitis herpetiformis (by feeding or in organ culture57) and it has been widely used in gluten challenge experiments in vivo and in vitro. It was initially claimed that only α-gliadin was toxic. A-gliadin, a substantial fraction of α-gliadin in some wheat varieties, is the most thoroughly studied fraction. This fraction can activate celiac disease.5 One group of investigators has synthesized three peptides corresponding to amino-acid 3-21, 31-49, and 202-220 of A-gliadin. Patients with celiac disease were challenged by intraduodenal infusion of 1 g of gliadin or 200 mg of the synthetic peptides. The oligopeptide corresponding to amino acids 31-49 of A-gliadin is toxic in vivo,

Celiac Disease

89

Table 5.2. Amino Acid Sequence of “Toxic” Gluten Peptides Peptide A58 B58 C59

Sequence

Gliadin Homology

LGQQQPFPPQQPYPQPQPF QQYPLGQGSFRPSQQNPQA (Q)IVFPSGQVQ(W)PQQ(Q)QPFP

α-gliadin 31-49 α-gliadin 202-220 Frazer III-2-VIII 2-20 (γ-gliadin)

and is recognized by HLA DQ2-restricted T cells. There was no evidence of toxicity of residues 3-21, and the C-terminal peptide 202-220 may contain an epitope to which patients display variable sensitivity.58 Another study tested the toxicity of various subfractions of Frazer fraction III in vitro (organ culture), and compared it with α-gliadin using duodenal biopsies from patients and nonceliac controls. One dominating fraction, designated Frazer III-2-VIII, was markedly toxic to duodenal explants from patients with active CD, and the in vitro toxicity of this fraction was comparable with the toxicity of higher concentrations of α-gliadin. Its N-terminal amino acid sequence was not homologous to previously reported sequences of toxic gluten peptides, and it was concluded that the so called peptide Frazer III-2-VIII is part of the γ-gliadin fraction.59 High molecular weight glutenin (HMW-g) has been shown to have structural similarities to human elastin. Dermatitis herpetiformis patients had significantly lower levels of IgA antibodies to HMW-g and to elastin than both CD patients and healthy controls with a further reduction in the amount of IgA antibodies to elastin after gluten-free diet. A significant correlation was observed between IgA antibodies to HMW-g and elastin in healthy controls and CD patients, which suggested that human serum may contain antibodies which cross-react with HMW-g and elastin.15 It is postulated that although T cell responses to gliadin have been implicated as the major pathogenic mechanism in the gut, immune response to other components of gluten may be responsible for the skin lesions. Gluten may elicit the production of T cell dependent antibodies within the intestine itself. Both IgA and IgM antibodies are increased in celiac patients. That gliadin induces immune system activation is supported by the presence of gliadin specific T cells in the mucosa of celiac disease patients and the detection of serum antibodies including antigliadin, antireticulin and antiendomysial antibodies.60 IgA-endomysial antibodies are the most reliable autoantibody marker of celiac disease. It has been established that all three antibodies are gluten-dependent and they appear with a gluten-containing diet and decrease after gluten withdrawal. Anti-endomysial autoantibodies correlate best with gliadin ingestion and with intestinal pathology.61 Between the 15th and 35th day of gluten challenge in celiac disease patients, IgA anti-gliadin antibodies were observed.62 After a gluten-free diet (GFD), IgA-AGA disappeared first.63 In organ culture, antibodies to endomysium are produced by mucosal biopsy specimens from untreated celiac disease patients as well as by specimens from patients who have been on a gluten-free diet within hours after gliadin challenge. The hypothesis is that the endomysial self-antigen (probably tissue transglutaminase) is recognized only after ingestion of gluten.64

Potential Viral or Other Environmental Factors Initiating Disease Prospective studies demonstrate the acquisition of celiac autoantibodies (e.g. antiendomysial) both in young infants and older children months to years after the introduction of dietary gliadin.25,26,65 Autoantibodies can be detected at less than 1 year of age65 and

90

Endocrine and Organ Specific Autoimmunity

anti-gliadin antibodies may precede those of endomysial autoantibodies. Classical presentation of celiac disease in the past was a 6-9 month old infant with severe diarrhea and failure to thrive due to introduction of dietary wheat in the initial 3 months of life. The search for environmental factors which may trigger celiac disease independently of gliadin has been largely unrewarding. Sequence homology between a fragment of gliadin and adenovirus has led to the hypothesis that adenovirus infection sensitizes the host to gliadin.66-68 In addition milk intolerance or allergy to soy protein may be associated with injury to the small bowel.

What Are the Target Autoantigens? Celiac disease is associated with the presence of antibodies reacting with the wheat protein gliadin65 and with the presence of autoantibodies. Autoantibody assays are both more specific and more sensitive for the diagnosis of celiac disease than the determination of anti-gliadin antibodies.60 The major autoantigens of celiac disease are distributed in multiple tissues. In terms of disease specificity and sensitivity, determination of endomysial or reticulin autoantibodies are central to the diagnosis of celiac disease.69,70 Autoantibody endomysial assays utilize primate esophagus and human placental cords.69 A recent study has demonstrated that a major autoantigen of anti-endomysial autoantibodies is the molecule tissue transglutaminase (glutamine gamma-glutamyltransferase).2 Tissue transglutaminase is an enzyme of approximately 85 kDa that catalyzes the crosslinking of proteins through glutamyl-lysine bonds (epsilon gamma-glutamyl-lysine isopeptide bonds). The evidence that transglutaminase “is” the endomysial autoantigen includes 1) immunoprecipitation of transglutaminase by anti-endomysial autoantibody positive sera, 2) the blocking of endomysial staining of monkey esophagus by preincubation of positive sera with purified transglutaminase and 3) the development of an autoantibody ELISA assay utilizing purified transglutaminase as substrate. The results of the ELISA assay paralleled that of the standard endomysial assay including the loss of the tranglutaminase autoantibodies upon institution of a gluten free diet.2 The high expression of glutamine in gliadins led to suggestions that transglutaminase might interact with gliadin and thus be involved in the pathogenesis of celiac disease.71 Gliadin can serve as a substrate of transglutaminase and one hypothesis is that gliadin may become cross-linked to transglutaminase and thereby create a “neoantigen” which induces an immune response to the self-protein transglutaminase. This novel hypothesis would explain the exquisite sensitivity of disease induction for the presence of gliadin in the diet even though a self-autoantigen is also targeted. Several forms of transglutaminase are in many tissues and in particular are present in keratinocytes. This may have relevance to the dermatitis herpetiformis associated with celiac disease which also responds to removal of gliadin from the diet.

What Are the Effector Molecules? T cells in Intestine and Skin The pathologic similarities between the intestinal damage of celiac disease and intestinal damage of graft-host disease, allograft rejection of transplanted intestine and T cell activation in fetal human small intestine support the concept that T cell mediated mechanisms may be central to celiac disease.72 Most of the intraepithelial lymphocytes of celiac disease are T cells.73 There is evidence that the mucosal lesions in gluten sensitivity within the lamina propria are associated with antigen presentation by activated macrophages that present antigen to CD4 lymphocytes. These CD4 lymphocytes acquire interleukin 2 receptors upon gliadin challenge.74-76 T cells specific for cryptic epitopes may escape thymic de-

Celiac Disease

91

letion and become activated and autoaggressive when the epitopes are presented at high concentrations.77 Further, some studies have successfully isolated gliadin-specific T cell clones from the intestinal mucosa of celiac disease patients. These T cell clones are predominantly restricted by the celiac disease associated DQ heterodimer (A1*0501,B1*0201).78-81 In one study, the minimal synthetic α-gliadin peptide recognized by T cell clones corresponded to residues 31-47 of α-gliadin and this peptide was presented by DQ2.82 Overall T cell recognition of gliadin peptides presented by DQ2 in the intestinal mucosa appear central to the immunopathogenesis of celiac disease. There are also studies that question the role of gliadin peptides in the T cell-mediated immune response of celiac disease. Overlapping gliadin peptides were found to bind with weak or intermediate affinity to DQ2 and they did not bind to DR3.83 Another study found peripheral T cell responses to α-gliadin to be DR-restricted and not DQ-restricted. This is in contrast to the strong association of disease susceptibility with DQ molecules.84 T cell receptors(TcRs) of gluten-responsive clones derived from patients with celiac disease have been sequenced. A wide diversity of T cell receptor sequences were found. The results of this study suggest that the gluten-directed CD4 response is polyclonal and does not represent a defect in a T cell using a single T cell receptor subset.85 The proportion of γδ T cell receptor-bearing lymphocytes was significantly higher both in the peripheral blood and the jejunal mucosa. A significant correlation was found between the percentage of peripheral γδ T cells and the density of γδ TcR+ cells in the lamina propria.86 Epithelial cells overexpress DR molecules within 1 hour after gliadin challenge. T lymphocytes migrating in the upper layers of the lamina propria are mainly CD4+ and show markers of activation.87 Within celiac disease lesions, a high proportion of TcR αβ+ CD8+ and TcR γδ+ (not CD4+) intraepithelial lymphocytes did not express the p55 IL-2 receptor (CD25-), but stained for a nuclear proliferation marker (Ki-67+), whereas CD4+ T lymphocytes in the lamina propria were often CD25+, Ki-67-.88 These results suggest that gluten in celiac patients induces a nonproliferative T cell activation in the lamina propria, in contrast to the proliferative activation of both TcR subsets in the epithelium. The same authors demonstrated that gluten stimulation of celiac mucosa in vitro induced CD25 expression but not proliferation of lamina propria CD4+ T cells.89 These findings were also confirmed by another study which suggested that gluten-specific T cells present in the blood and intestine of normal and dermatitis herpertiformis individuals are activated, but do not proliferate in response to specific antigen unless exogenous IL-2 or IL-4 is added.57 Recent observations of T cell responses following TcR interaction with altered peptide ligands have indicated that depending on the ligand, T cell receptors are able to selectively trigger effector functions.90 Thus it is possible that gluten preferentially stimulates cytokine production by T cells rather than proliferation.91 One study investigated the presence of mRNA coding for interferon gamma (IFN γ), TNF α, and interleukin 2 (IL-2) and interleukin 6 (IL-6) in the mucosa of celiac disease patients. Celiac disease was associated with increased expression of cytokines within the mucosa.92 A comparison of gluten-induced cytokine production by normal and dermatitis herpetiformis/celiac T cells may help to elucidate the pathogenic mechanism involved in gluten-sensitive enteropathy.

How Does Avoidance of Dietary Gluten Interrupt Disease Pathogenesis? The optimal treatment of celiac disease is a gluten-free diet (GFD). The earliest histologic changes are the reversion of the surface epithelium to normal. The epithelial mitotic count decreases to normal and later, as edema and infiltration decrease, villi begin to reappear. Histochemically, the enzymes of the surface mucosa return to normal. The complete remission of the small intestinal mucosa may take 6-12 months.5 A gluten free diet may also

Endocrine and Organ Specific Autoimmunity

92

ameliorate or prevent associated diseases. The results from one study suggest that prolonged and strict gluten-free diet provides protection against the development of malignant disorders in celiac disease patients.13

Conclusion The autoimmune hypothesis of celiac disease pathogenesis is based on identification of dietary-peptides that trigger the production of anti-endomysial autoantibodies93and intestinal lesions. According to this hypothesis in a patient with celiac disease, all of the crucial elements are present for autoimmunity to occur: the trigger (gliadin), the susceptible MHC class II alleles (DQA*0501 and DQB*0201), and the autoantigen transglutaminase. It is likely that studies of celiac disease will have a major impact on our understanding of autoimmune disorders with so many of the pieces of the “puzzle” that is celiac disease now available for detailed analysis.

Acknowledgments This work was supported by NIH grants (R37DK32083 GSE) and (R01DK50979 MR).

References 1. Branski D, Ashkenazi A, Freier S, Lerner A, Dinari G, Faber J et al. Extraintestinal manifestations and associated disorders of celiac disease. Front Gastrointest Res 1992; 19:164-175. 2. Dietrich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO et al. Identification of tissue transglutaminase as the autoantigen of cleiac disease. Nature Medicine 1997; 3:797-801. 3. Rossi TM, Kumar V, Lerner A, Heitlinger LA, Tucker N, Fisher J. Relationship of endomysial antibodies to jejunal mucosal pathology: specificity towards both symptomatic and asymptomatic celiacs. J Pediatr Gastroenterol Nutr 1988; 7:858-863. 4. Halsted M.D. CH. The many faces of celiac disease. N Engl J Med 1996; 334:(18)1190-1191. 5. Friis SU. Coeliac disease, pathogenesis and clinical aspects. Acta Pathol Microbiol Immunol Scand 1996; 61:5-48. 6. Walker-Smith JA, Guandalini S, Schmitz J, Shmerling J, Visakorpi JK. Revised criteria for diagnosis of coeliac disease. Arch Dis Child 1990; 65:909-911. 7. Howard MS, Colin EA, Kenneth WB, Fred G, Joyce DG, Cyrus RK et al. Celiac Sprue (Nontropical Sprue). In: Anonymous Clinical gastroenterology. 4th ed. New York: McGrawHill, 1993:441-461. 8. Marsh MN. Gluten, Major Histocompatibility Complex, and the small intestine: a molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). Gastroenterology 1992; 102:330-354. 9. Lavo B, Knutson L, Loof L, Odlind B, Venge P, Hallgren R. Challenge with gliadin induces eosinophil and mast cell activation in the jejunum of patients with celiac disease. Am J Med 1989; 87:655-660. 10. Lavo B, Knutson L, Loof L, Hallgren R. Gliadin challenge-induced jejunal prostaglandin E2 secretion in celiac disease. Gastroenterology 1990; 99:703-707. 11. Epstein A. Case records of the Massachusetts General Hospital, case 15-1996. N Engl J Med 1996; 334:1316-1322. 12. Egan LJ, Stevens FM, McCarthy CF. Celiac disease and T cell lymphoma. N Engl J Med 1996; 335:1611-1612. 13. Holmes GKT, Prior P, Lane MR, Pope D, Allan RN. Malignancy in coeliac disease—effect of a gluten free diet. Gut 1989; 30:333-338. 14. Howell W, Leung ST, Jones DB, Nakshabendi I, Hall A, Lanchbury JS, et al. HLA-DRB, -DQA, and -DQB polymorphism in celiac disease and entropathy-associated T cell lymphoma. Common features and additional risk factors for malignancy. Hum Immunol 1995; 43:29-37.

Celiac Disease

93

15. Bödvarsson S, Jonsdottir I, Freysdottir J, Leonard JN, Fry L, Valdimarsson H. Dermatitis herpetiformis—an autoimmune disease due to cross-reaction between dietary glutenin and dermal elastin? Scand J Immunol 1993; 38:546-550. 16. Setterfield J, Bhogal B, Black MM, McGibbon DH. Dermatitis herpetiformis and bullous pemphigoid: A developing association confirmed by immunoelectronmicroscopy. Br J Dermatol 1997; 136:253-256. 17. Rolles CJ, Myint TO, Sin WK, Anderson M. Proceedings: Family study of coeliac disease. Gut 1974; 15:827. 18. Stenhammar L, Brandt A, Wagermark J. A family study of coeliac disease. Acta Pediatr Scand 1982; 71:625-628. 19. Auricchio S, Mazzacca G, Tosi R, Visakorpi J, Mäki M, Polanco I. Coeliac disease as a familial condition: identification of asymptomatic coeliac patients within family groups. Gastroenterology 1988; 1:25-31. 20. Polanco I, Biemond I, Van Leeuwen A, Schreuder I, Meera-Khan P, Guerrero J et al. Gluten-sensitive enteropathy in Spain: genetic and environmental factors. In: McConnell RB, editor. Genetics of Coeliac Disease, Proceedings of International Symposium. Lancaster: MTP press, 1981:211-231. 21. Collin P, Reunala T, Pukkala E, Laippala P, Keyrilainen O, Pasternack A. Coeliac diseaseassociated disorders and survival. Gut 1994; 35:1215-1218. 22. Savilahti E, Simell O, Koskimies S, Rilva A, Akerblom AK. Celiac disease in insulin dependent diabetes mellitus. J Pediatr 1986; 108 (1):690-693. 23. Maki M, Hällström O, Huupponen T, Vesikari T, Visakorpi JK. Increased prevalence of coeliac disease in diabetes. Arch Dis Child 1984; 59:739-742. 24. Lorini R, Scotta MS, Cortona L, Avanzini MA, Vitali L, De Giacomo C et al. Celiac disease and type I (insulin-dependent) diabetes mellitus in childhood: Follow-up study. J Diabetes Complications 1996; 10:154-159. 25. Saukkonen T, Savilahti E, Reijonen H, Ilonen J, Tuomilehto-Wolf E, Åkerblom HK et al. Coeliac disease: Frequent occurrence after clinical onset of insulin-dependent diabetes mellitus. Diabetic Med 1995; 13:464-470. 26. Mäki M, Huupponen T, Holm K, Hällström O. Seroconversion of reticulin autoantibodies predicts coeliac disease in insulin dependent diabetes mellitus. Gut 1995; 36:239-242. 27. Lorini R, Scaramuzza A, Vitali L, d’Annunzio G, Avanzini MA, De Giacomo C et al. Clinical aspects of coeliac disease in children with insulin-dependent diabetes mellitus. J Pediatr Endocrinol Metab 1996; 9 Suppl 1:101-111. 28. Failla P, Ruberto C, Pagano MC, Lombardo M, Bottaro G, Perichon B et al. Celiac disease in Down’s syndrome with HLA serological and molecular studies. J Pediatr Gastroenterol Nutr 1996; 23:303-306. 29. Collin P, Mäki M, Keyrilainen O, Hällström O, Reunala T, Pasternack A. Selective IgA deficiency and coeliac disease. Scand J Gastroenterol 1992; 27:367-371. 30. Davidson LSP, Fountain JR. Incidence of the sprue syndrome with some observation on the natural history. BMJ 1950; 1:1157-1161. 31. Carter C, Sheldon W, Walker C. The inheritance of coeliac disease. Ann Hum Genet 1959; 23:266-278. 32. McCrae WM. The inheritance of coeliac disease. In: Booth CC, Dowling RH, editors. Coeliac Disease. Edinburgh: Livingstone, 1970:55-62. 33. Thompson MW. Heredity maternal age and birth order in the etiology of celiac disease. Am J Hum Genet 1951; 3:159-166. 34. Mylotte M, Egan-Mitchell B, McCarthy CF, McNicholl B. Incidence of coeliac disease in the West of Ireland. BMJ 1973; 1:703-705. 35. Not T, Horvath K, Hill ID, Fasano A, Hammed A, Magazzu G. Endomysium Antibodies in blood donors predicts a high prevalence of celiac disease in the USA. Gastroenterology 1996; 110:Suppl:A351 Abstract. 36. MacDonald WC, Dobbins WO, Rubin CE. Studies of the familial nature of coeliac sprue using biopsy of the small intestine. N Engl J Med 1965; 272:448-456.

94

Endocrine and Organ Specific Autoimmunity

37. Reijonen H, Ilonen J, Knip M, Reunala T. Insulin-dependent diabetes mellitus associated with dermatitis herpetiformis: evidence for heterogeneity of HLA-associated genes. Tissue Antigens 1991; 37:94-96. 38. Reunala T, Salmi J, Karvonen J. Dermatitis herpetiformis and celiac disease associated with Addison’s disease. Arch Dermatol 1987; 123:930-932. 39. Petronzelli F, Multari G, Ferrante P, Bonamico M, Rabuffo G, Campea L et al. Different dose effect of HLA-DQab heterodimers in insulin-dependent diabetes mellitus and celiac disease susceptibility. Hum Immunol 1993; 36:156-162. 40. Spurkland A, Sollid LM, Ronningen KS, Bosnes V, Ek J, Vartdal F et al. Susceptibility to develop celiac disease is primarily associated with HLA-DQ alleles. Hum Immunol 1990; 29:157-165. 41. Sollid LM, Thorsby E. HLA susceptibility genes in celiac disease: Genetic mapping and role in pathogenesis [published erratum appears in Gastroenterology 1994 Apr;106(4):1133]. Gastroenterology 1993; 105:910-922. 42. Congia M, Cucca F, Frau F, Lampis R, Melis L, Clemente MG et al. A gene dosage effect of the DQA1*0501/DQB1*0201 allelic combination influences the clinical heterogeneity of celiac disease. Hum Immunol 1994; 40:138-142. 43. Hall MA, Lanchbury JS, Lee JS, Welsh KI, Ciclitira PJ. HLA-DQB second-domain polymorphisms in susceptibility to coeliac disease. Eleventh International Histocompatibility Workshop 1991; 18:530-533. 44. Sollid LM, Thorsby E. The primary association of celiac disease to a given HLA-DQ alpha/ beta heterodimer explains the divergent HLA-DR associations observed in various Caucasian populations. Tissue Antigens 1990; 36:136-137. 45. Yasunaga S, Kimura A, Hamaguchi K, Ronningen KS, Sasazuki T. Different contribution of HLA-DR and -DQ genes in susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). Tissue Antigens 1996; 47:37-48. 46. Polvi A, Mki M, Partanen J. Celiac patients predominantly inherit HLA-DPB1*0101 positive haplotype from HLA-DQ2 homozygous parent. Hum Immunol 1997; 53:156-158. 47. Djilali-Saiah I, Benini V, Schmitz J, Timsit J, Assan R, Boitard C et al. Absence of primary association between DM gene polymorphism and insulin-dependent diabetes mellitus or celiac disease. Hum Immunol 1996; 49:22-27. 48. Polvi A, Eland C, Koskimies S, Maki M, Partanen J. HLA DQ and DP in Finnish families with celiac disease. Eur J Immunogenet 1996; 23:221-234. 49. Meddeb-Garnaoui A, Zeliszewski D, Mougenot JF, Djilali-Saiah I, Caillat-Zucman S, Dormoy A et al. Reevaluation of the relative risk for susceptibility to celiac disease of HLADRB1, -DQA1, -DQB1, -DPB1, and -TAP2 alleles in a French population. Hum Immunol 1995; 43:190-199. 50. Manus RM, Wilson AG, Mansfield J, Weir DG, Duff GW, Kelleher D. TNF2, a polymorphism of the tumor necrosis-alpha gene promoter, is a component of the celiac disease major histocompatibility complex haplotype. Eur J Immunol 1996; 26:2113-2118. 51. Mann DL, Katz SI, Nelson DL, Abelson LD. Specific B cell antigens associated with glutensensitive enteropathy and dermatitis herpetiformis. Lancet 1976; 1:110-111. 52. Hall MA, Mazzilli MC, Satz ML, Barboni F, Bartova A, Brunnler G et al. Coeliac disease study. Eleventh International Histocompatibility Workshop 1991; 6.5:722-729. 53. Roschmann E, Wienker TF, Volk BA. Role of T cell receptor delta gene in susceptibility to celiac disease. J Mol Med 1996; 74:93-98. 54. Catassi C, Rossini M, Ratsch IM, Bearzi I, Santinelli A, Castagnani R et al. Dose dependent effects of protracted ingestion of small amounts of gliadin in coeliac disease children: a clinical and jejunal morphometric study. Gut 1993; 34:1515-1519. 55. Ciclitira PJ, Stewart J, Evan G, Wight DG, Sikora K. Expression of c-myc oncogene in coeliac disease. J Clin Pathol 1987; 40:307-311. 56. Frazer AC, Fletcher RF, Ross CA, Shaw B, Sammons HG, Schneider R. Gluten-induced enteropathy: The effect of partially digested gluten. Lancet 1959; 2:252-255.

Celiac Disease

95

57. Baker BS, Garioch JJ, Bokth S, Thomas H, Walker MM, Leonard JN et al. Lack of proliferative response by gluten-specific T cells in the blood and gut of patients with dermatitis herpetiformis. J Autoimmun 1995; 8:561-574. 58. Sturgess R, Day P, Ellis HJ, Lundin KEA, Gjertsen HA, Kontakou M et al. Wheat peptide challenge in coeliac disease. Lancet 1994; 343:758-761. 59. Fluge O, Sletten K, Fluge G, Aksnes L, Elsayed S. In vitro toxicity of purified gluten peptides tested by organ culture. J Pediatr Gastroenterol Nutr 1994; 18:186-192. 60. Ferreira M, Davies SL, Butler M, Scott D, Clark M, Kumar P. Endomysial antibody: is it the best screening test for coeliac disease? Gut 1992; 33:1633-1637. 61. Lerner A, Kumar V, Iancu TC. Immunological diagnosis of childhood coeliac disease: comparison between antigliadin, antireticulin and antiendomysial antibodies. Clin Exp Immunol 1994; 95:78-82. 62. Valletta EA, Trevisiol D, Mastella G. IgA anti-gliadin antibodies in the monitoring of gluten challenge in celiac disease. J Pediatr Gastroenterol Nutr 1990; 10:169-173. 63. Cataldo F, Ventura A, Lazzari R, Balli F, Nassimbeni G, Marino V. Antiendomysium antibodies and coeliac disease: solved and unsolved questions. An Italian multicenter study. Acta Paediatr 1995; 84:1125-1131. 64. Picarelli A, Maiuri L, Frate A, Greco M, Auricchio S, Londei M. Production of antiendomysial antibodies after in vitro gliadin challenge of small intestine biopsy samples from patients with coeliac disease. Lancet 1996; 348:1065-1067. 65. Berger R, Schmidt G. Evaluation of six anti-gliadin antibody assays. J Immunol Methods 1996; 191:77-86. 66. Kagnoff MF, Austin RK, Hubert JJ, Bernardin JE, Kasarda DD. Possible role for a human adenovirus in the pathogenesis of celiac disease. J Exp Med 1984; 160:1544-1557. 67. Kagnoff MF, Paterson YJ, Kumar PJ, Kasarda DD, Carbone FR, Unsworth DJ et al. Evidence for the role of a human intestinal adenovirus in the pathogenesis of coeliac disease. Gut 1987; 28:995-1001. 68. Mantzaris GJ, Karagiannis JA, Priddle JD, Jewell DP. Cellular hypersensitivity to a synthetic dodecapeptide derived from human adenovirus 12 which resembles a sequence of A-gliadin in patients with coeliac disease. Gut 1990; 31:668-673. 69. Chan KN, Phillips AD, Mirakian R, Walker-Smith JA. Endomysial antibody screening in children. J Pediatr Gastroenterol Nutr 1994; 18:316-320. 70. Karská K, Tucková L, Steiner L, Tlaskalová-Hogenová H, Michalak M. Calreticulin—the potential autoantigen in celiac disease. Biochem Biophys Res Commun 1995; 209:597-605. 71. Bruce SE, Bjarnason I, Peters TJ. Human jejunal transglutaminase: demonstration of activity, enzyme kinetics and substrate specificity with special relation to gliadin and coeliac disease. Clin Sci 1985; 68:573-579. 72. MacDonald TT. T cell mediated intestinal injury. In: Marsh MN, ed. Coeliac Disease. Oxford: Blackwell Scientific Publications, 1992:283-304. 73. Selby WS, Janossy G, Bofill M, Jewell DP. Lymphocyte subpopulations in the human small intestine. The findings in normal mucosa and in the mucosa of patients with adult coeliac disease. Clin Exp Immunol 1983; 52:219-228. 74. Pentilla IA, Gibson CE, Forrest BD, Cummins AG, LaBrooy JT. Lymphocyte activation as measured by interleukin 2 receptor expression to gluten fraction III in coeliac disease. Clin Exp Immunol 1990; 68:155-160. 75. Crabtree JE, Heatley RV, Juby LD, Howdle PD, Losowsky MS. Serum interleukin-2 receptor in coeliac disease: Response to treatment and gluten challenge. Clin Exp Immunol 1989; 77:345-348. 76. Griffiths C, Barrison I, Leonard J, Cann K, Valdimarrson M, Fry L. Preferential activation of CD4 T lymphocytes within the lamina propria of gluten-sensitive enteropathy. Clin Exp Immunol 1988; 72:280-283. 77. Maki M. Coeliac disease and autoimmunity due to unmasking of Cryptic epitopes. Lancet 1996; 348:1046-1047.

96

Endocrine and Organ Specific Autoimmunity

78. Molberg O, Kett K, Scott H, Thorsby E, Sollid LM, Lundin KEA. Gliadin specific, HLA DQ2-restricted T cells are commonly found in small intestinal biopsies from coeliac disease patients, but not from controls. Scand J Immunol 1997; 46:103-108. 79. Lundin KE, Sollid LM, Anthonsen D, Noren O, Molberg O, Thorsby E et al. Heterogeneous reactivity patterns of HLA-DQ-restricted, small intestinal mucosa of coeliac disease patients. Gastroenterology 1997; 112:752-759. 80. van de Wal Y, Kooy YMC, Drijfhout JW, Amons R, Koning F. Peptide binding characteristics of the coeliac disease-associated DQ(a1*0501, b1*0201) molecule. Immunogenetics 1996; 44:246-253. 81. Lundin KEA, Scott H, Hansen T, Paulsen G, Halstensen TS, Fausa O et al. Gliadin-specific, HLA-DQ(a1*0501,b1*0201) restricted T cells isolated from the small intestinal mucosa of celiac disease patients. J Exp Med 1993; 178:187-196. 82. Gjertsen HA, Lundin KE, Sollid LM, Eriksen JA, Thorsby E. T cells recognize a peptide derived from alpha-gliadin presented by the celiac disease-associated HLA-DQ (alpha 1*0501, beta 1*0201) heterodimer. Hum Immunol 1994; 39:243-252. 83. Johansen BH, Gjertsen HA, Vartdal F, Buus S, Thorsby E, Lundin KEA et al. Binding of peptides from the N-terminal region of a-gliadin to the celiac disease-associated HLA-DQ2 molecule assessed in biochemical and T cell assays. Clin Immunol Immunopathol 1996; 79:288-293. 84. Franco A, Appella E, Kagnoff MF, Chowers Y, Sakaguchi K, Grey HM, et al. Peripheral T cell response to A-gliadin in celiac disease: differential processing and presentation capacities of Epstein-Barr-transformed B cells and fibroblasts. Clin Immunol Immunopathol 1994; 71:75-81. 85. Lundin K, Scott H, Hansen T, Paulsen G, Halstensen T, Fausa O et al. Activated T cells and the genetic restriction in celiac disease. J Pediatr Gastroenterol Nutr 1994; 19:250-254. 86. Klemola T, Tarkkanen J, Örmälä T, Saxen H, Savilahti E. Peripheral gamma-d T cell receptor-bearing lymphocytes are increased in children with celiac disease. J Pediatr Gastroenterol Nutr 1994; 18:435-439. 87. Maiuri L, Picarelli A, Boirivant M, Coletta S, Mazzilli MC, De Vincenzi M et al. Definition of the initial immunologic modifications upon in vitro gliadin challenge in the small intestine of celiac patients. Gastroenterology 1996; 110:1368-1378. 88. Halstensen TS, Brandtzaeg P. Activated T lymphocytes in the celiac lesion: Nonproliferative activation (CD25) of CD4+ alpha/beta cells in the lamina propria but proliferation (Ki-67) of alpha/beta and gamma/delta cells in the epithelium. Eur J Immunol 1993; 23:505-510. 89. Halstensen TS, Scott H, Fausa SO, Brandtzaeg P. Gluten stimulation of coeliac mucosa in vitro induces activation (CD25) of lamina propria CD4+ T cells and macrophages but no crypT cell hyperplasia. Scand J Immunol 1993; 38:581-590. 90. Evavold BD, Sloan-Lancaster J, Allen PM. Tickling the TCR: Selective T cell functions stimulated by altered peptide ligands. Immunol Today 1993; 14:602-609. 91. Nilsen EM, Gjertsen HA, Jensen K, Brandzaeg P, Lundin KE. Gluten activation of peripheral blood T cells induces a Th0-like cytokine pattern in both coeliac patients and controls. Clin Exp Immunol 1996; 103:295-303. 92. Kontakou M, Przemioslo RT, Sturgess RP, Limb GA, Ellis HJ, Day P et al. Cytokine mRNA expression in the mucosa of treated coeliac patients after wheat peptide challenge. Gut 1995; 37:52-57. 93. Jalava T, Maki M, Marttinen A, Partanen J, Koskimies S. The in vitro response to human fibroplast-derived extracellular matrix proteins is restricted by specific HLA class II genes. Relevance for Coeliac disease. Hum Immunol 1996; 49:106-112.

CHAPTER 6

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases Horia Vlase and Terry F. Davies

Introduction The Autoimmune Thyroid Diseases (AITDs)

T

he human AITDs1 include hyperthyroid Graves’ disease (classical Graves’ disease with thyrotoxicosis), euthyroid Graves’ disease (without thyrotoxicosis), both with or without Graves’ orbitopathy, and autoimmune thyroiditis (Hashimoto’s disease), including the goitrous form (classical Hashimoto’s thyroiditis) and the nongoitrous form (atrophic thyroiditis). Postpartum thyroiditis and silent thyroiditis are well characterized transient forms of autoimmune thyroiditis.2 Recently, we proposed a new nomenclature that clarifies this collection of autoimmune diatheses (Table 6.1).3

The Natural History of AITD Graves’ disease Often, this disease may assume a chronic course with unremitting thyrotoxicosis. In other cases the course may be characterized by cycles of remissions and relapses that can extend over many years.4 Other patients may experience a single episode of thyrotoxicosis followed by early spontaneous remission. In 10-15% of cases an evolution from hyperthyroidism to hypothyroidism has been observed.5 Ophthalmic Graves’ Disease The onset of Graves’ orbitopathy may be coincident with thyroid overactivity (33%), may develop before any thyroid dysfunction (33%) or may develop later.6 Although some cases of Graves’ orbitopathy have the natural tendency to stabilize, others can demonstrate a severe deterioration to “malignant ophthalmopathy” which may compromise visual function. Hashimoto’s Disease This may be a lifelong gradual loss of thyroid function. This has been well documented to produce thyroid failure in the susceptible population at the rate of 5% per year for patients

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

98

Endocrine and Organ Specific Autoimmunity

Table 6.1. Classification of Autoimmune Thyoiditis (reproduced from (Davies and Amino, 1993)) Type 1 Autoimmune Thyroiditis: (Hashimoto’s disease Type 1) 1AGoitrous 1BNon-goitrous Status: Euthyroid with normal TSH level. Autoantibodies to hTg and hTPO usually present. Type 2 Autoimmune Thyroiditis: (Hashimoto’s disease Type 2) 2AGoitrous (classical Hashimoto’s disease) 2BNon-goitrous (primary myxedema, atrophic thyroiditis) Status: Persistent hypothyroidism with increased TSH levels. Autoantibodies to hTg and hTPO usually present. Some of Type 2B are associated with blocking type TSH receptor autoantibodies. 2CTransient aggravation of thyroiditis Status: May start as transient destructive thyrotoxicosis (increased serum thyroid hormones with low thyroidal radioactive iodine uptake). This is often followed by transient hypothyroidism. However, some patients show transient hypothyroidism without the preceeding destructive thyrotoxicosis. Autoantibodies to hTg and hTPO present. Example, postpartum thyroiditis. Type 3 Autoimmune Thyroiditis: (Graves’ disease) 3AHyperthyroid Graves’ disease 3BEuthyroid Graves’ disease Status: Hyperthyroid or euthyroid with suppressed TSH. Diagnostic autoantibodies to the TSH receptor of the stimulating variety are present. Autoantibodies to hTg and hTPO are also usually detected. 3CHypothyroid Graves’ disease Status: Orbitopathy with hypothyroidism. Diagnostic autoantibodies to the TSH receptor of the blocking or stimulating variety may be detected. Autoantibodies to hTg and hTPO are usually present.

with incipient failure (slightly raised TSH levels and positive thyroid autoantibodies).7 If left untreated, severe hypothyroidism (myxedema) may develop leading to coma and death. Postpartum, or Silent Thyroiditis These have an initial transient course of hyperthyroidism followed by hypothyroidism but a significant number (10-20%) develop permanent hypothyroidism later in life.8

Overview of Immunological Characteristics of AITD For many years, thyroid autoantibodies have been a hallmark of human AITD because they are simple to measure. Autoantibodies to three major thyroid autoantigens, the thyrotropin receptor (TSHR-Ab), thyroid peroxidase (TPO-Ab) and thyroglobulin (Tg-Ab)

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

99

have been well characterized9,10 and no doubt the recently identified iodine transporter will also prove to be a target.11,12 Patients with Graves’ disease have functional serum TSHR-Abs which act as TSH agonists and induce excessive thyroid hormone secretion.13 Such patients may also have TPO-Ab and Tg-Ab reflecting a concurrent thyroiditis and why we have proposed that autoimmune thyroiditis is an all embracing term.3 Patients with primary autoimmune thyroiditis (Hashimoto’s disease) usually have very high titers of TPO-Ab and Tg-Ab which may be secondary to thyroid gland destruction.14 High titers of Tg-Ab and TPO-Ab are also found in patients with postpartum and silent thyroiditis. In some cases of Hashimoto’s disease, TSHR blocking autoantibodies are found.15 Such antibodies block TSHmediated receptor stimulation and may be partly responsible for the thyroid failure (review in 9). Circulating and intrathyroidal T cells, with thyroid antigen specificity, are present in all AITDs. In Graves’ disease, such T cells are mainly of the helper phenotype (CD4+)16 and many exhibit Th2 characteristics favoring antibody secretion.17 Interestingly, orbital tissue derived T cells, also mainly CD4+, recently described in Graves’ ophthalmopathy,18 are predominantly Th1 type cells favoring cytotoxicity and cell death. This type of T cell is also seen as the dominant type in Hashimoto’s disease.19 Nevertheless, the data, to date, indicate a role for both Th1 and Th2 in both types of disease.

Genetics Overview The AITDs have a multifactorial etiology and result from an interplay of a specific genetic background with certain undefined environmental factors. A particular genetic heritage may render an individual more susceptible to environmental factors (such as infectious organisms), which can precipitate the clinical expression of disease.20 AITD may be caused by a number of interacting genes, each contributing to the susceptibility status of the carrier. Given the complexity of this polygenic hypothesis, isolating the genes involved has proved difficult but new technologies are beginning to make new evidence available (Table 6.2).

Evidence for a Genetic Predisposition Familial clustering is frequently observed in relatives of patients with Graves’ and Hashimoto’s diseases. Besides this clinical observation, extensive epidemiologic evidence has supported the notion of a genetic basis for AITD. Twin studies have shown a higher than an expected concordance for Graves’ disease (30-60%) in monozygotic twins with a much lower concordance rate (11%) in dizygotic twins.21,22 The finding of a higher concordance rate in monozygotic rather than in dizygotic twins suggested the importance of a genetic predisposition in the pathogenesis of AITD and indicated the likely disease penetrance. Studies in families have shown a 22-60% prevalence for AITD in first degree relatives of patients.23,24 Moreover, multiple immunological and functional thyroid abnormalities

Table 6.2. Genes Proven to be Involved in the Autoimmune Thyroid Diseases Associated: HLA-DR CTLA-4

Linked Loci GD-1 chromosome 14 GD-2 chromosome 20 GD-3 X chromosome

100

Endocrine and Organ Specific Autoimmunity

were described in relatives of patients with AITD.25,26 The most common abnormality, found in up to 50% of relatives, was the presence of thyroid autoantibodies,25,27,28 suggesting a dominant form of inheritance. Indirect evidence of a genetic susceptibility for AITD comes from the common coexistence of other autoimmune diseases unrelated to thyroid disease in patients with AITD,29 and the presence of such autoimmune diseases in relatives of patients.

Understanding Association and Linkage Two approaches used to study susceptibility genes of AITD are association and linkage analyses. Association studies are used to identify alleles that increase the risk of developing a disease, but may not be “necessary” for the development of the disease. This is accomplished by comparing the prevalence of a marker in the diseased and nondiseased population. Association analyses are very sensitive but may detect genes with only a minor influence on disease susceptibility. Linkage studies are performed by measuring how a marker moves with disease within families. In contrast to association, linkage studies are difficult and insensitive. However, linkage detects genes that are directly involved in the pathogenesis of the disease, i.e, genes required for the development of the disease.30 This is an important concept because only linkage studies identify such “necessary genes.”

HLA Genes Because of their prominent role in the regulation of the immune system the genes of the HLA complex have been much studied as potentially involved in the pathogenesis of AITD since the 1970s. Graves’ disease is associated with HLA-DR331,32 and HLA-DQA1*0501 in Caucasians,33-35 with HLA-B35 in the Japanese population, HLA-Bw46 in the Chinese, and DQ3 in African-Americans.36 However, in all these studies the degree of association has remained relatively weak with a relative risk (RR) between 3.0-5.0. Less definitive HLA associations have been reported in Hashimoto’s disease. In Caucasians Hashimoto’s disease has been associated with HLA-DR4, 37 HLA-DR3, 38 and HLA-DR5.39,40 Recently, an association was reported with HLA-DQw7 (DQB1*0301),41 and with HLA-DRw53 in Japanese, and HLA-DR9 in Chinese (reviewed in 42). As with Graves’ disease, all these associations were weak (RR = 4.0-5.0). As opposed to association analyses, there have been few studies of HLA in AITD using linkage analysis. While one study suggested linkage of Graves’ disease to the HLA region,43 the data analyzed were highly selected and in our view inadequate in number for the type of analysis performed. Indeed, we and others, have failed to confirm these results with classical linkage analysis.44-46 Although certain HLA genes confer an increased risk of developing AITD, as evidenced by their association with AITD, such genes may not be “necessary” for disease development. Indeed, the concordance rate for Graves’ disease between HLA-identical siblings is less than 10%,47 far lower than that generally found in monozygotic twins (50-70%), clearly suggesting a role for genes located outside the HLA complex.

Non-HLA Genes Since non-HLA genes must be involved in the pathogenesis of AITD, a variety of candidate genes have begun to be investigated. In particular, immunoregulatory genes outside the HLA region have been investigated as possible candidates. A polymorphism of the T cell receptor (TcR), V beta gene, has been associated with Graves’ disease48 and Hashimoto’s disease.49 But this could not be confirmed for the TCR V α gene,32 and we have found no association. Some studies have examined the association of Graves’ disease with the IgG heavy chain (Gm) genes, showing an association with certain allotypes of the gene50 in a Japanese population. Other investigators, however, have failed to confirm this association,51 and there was no linkage to this locus in Caucasian populations.46,52 CTLA-4 is a strong

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

101

candidate gene for T cell-mediated autoimmune disease because it encodes a molecule responsible for inducing tolerance. It appears to down-regulate the secondary signal to T cells provided by the interactions between CD28 and B7.53 Recently, a microsatellite polymorphism of the CTLA-4 gene has been shown to be associated with Graves’ disease54 and IDDM.55 Thyroid specific autoantigens have also been studied as candidate genes. Despite the important role that thyroid peroxidase (TPO) and thyroglobulin (Tg) play as thyroid autoantigens, no association between AITD and a microsatellite marker inside the TPO and Tg genes has been found.56,57 The TSHR is the main antigen in Graves’ disease and, therefore, has been studied as a potential candidate gene. A polymorphism in codon 52 of the TSH receptor (TSHR) was recently described58 and associated with Graves’ disease in females and patients with orbitopathy and dermopathy.59-61 Interestingly, this polymorphism increased cAMP production to a greater extent than the wild-type TSHR receptor.62 However, recent studies could not find a significant association between this polymorphism and Graves’ disease in a different population. 63,64 Another recent study failed to show cosegregation or linkage of a microsatellite marker close to the TSHR gene with Graves’ disease,65 even after correction for the HLA-DR status. However, using a set of 19 multiplex families with AITD, we have recently described linkage to a microsatellite marker on chromosome 14q31.57 This is evidence for the identification of a susceptibility gene ~25 centimorgans from the TSH receptor gene making it unlikely that the TSHR is involved. However, this marker is found in an area where positive linkage has also been reported for type I diabetes mellitus (IDDM).66 These data suggest that genes determining a genetic predisposition to organ-specific autoimmunity do exist and further loci are now being described in AITD. No positive linkage was found for CTLA-4 or IgH genes studied, despite the previous reports of associations between these genes and AITD, suggesting that the contribution of these genes is small and that they are not “necessary” for AITD development.

Immunopathogenesis Defining the Target Autoantigens The TSH Receptor Our current understanding of the pathogenic mechanisms involved in Graves’ disease has been much enhanced by the cloning of the thyrotropin receptor (TSHR) gene on chromosome 14q.67,68 The TSHR is the major antigen in Graves’ disease and also involved in some cases of autoimmune thyroiditis. Clarification of this role for the TSHR in Graves’ disease has been shown by immunization of mice with fibroblasts expressing both the TSHR and MHC class II antigen which induced stimulatory antibodies (see below) and hyperthyroidism.69 Recently we have confirmed this experimental approach and were able to induce hyperthyroidism and stimulatory TSHR-Ab in mice by immunization with a similar TSHR and MHC class II expressing fibroblast (Kita et al) (in press) Endocrinology 1999. The deduced 764 amino acid sequence of the TSHR showed that it is a G-protein coupled receptor. The first 415 amino acids define a large extracellular domain (ecd), encoded by ten exons. The remaining 349 amino acids constitute the 7-transmembrane domain and intracytoplasmic tail (Fig. 6.1). An alternatively spliced variant (consisting of the first nine exons), has also been cloned and encodes most of the ecd, and has been suggested as a potential soluble form of the receptor.70 Regarding the binding sites for TSH on the TSHR, investigators using site-directed mutagenesis of functional TSHRs expressed on CHO cells, and synthetic peptides of the

102

Endocrine and Organ Specific Autoimmunity

Fig. 6.1. A model of the TSH receptor showing the ectodomain, the transmembrane region and the cytoplasmic tail. The proposed cleavage site is illustrated.

TSHR, have shown that such binding sites are multiple rather than a simple dominant region.71,72 In agreement with these reports, studies using epitope-mapped antibodies to the hTSHR-ecd able to block TSH binding to native TSHR have identified multiple TSH binding sites to the C-terminus of the TSHR-ecd (amino acids 322-415).73,74 Extrathyroidal TSH receptors There is also evidence that links the TSHR as a shared thyroidal, retrobulbar and pretibial dermal antigen in Graves’ patients with associated Graves’ orbitopathy (GO) and pretibial dermopathy (PTD). Using RT-PCR, TSHR mRNA was found in fibroblasts,75 adipose tissue,76 muscle of the retrobulbar space,77,78 and also in pretibial dermal fibroblasts.79 While it was unclear if the protein was translated in these extrathyroidal tissues,80 recent reports using TSHR monoclonal and polyclonal antibodies, suggest the presence of TSHR specific immunoreactivity in fibroblasts and adipose tissue from the orbital and pretibial space of patients with GO and PTD,79,81 while binding sites in adipose tissue have been long characterized.82,83 Unfortunately, the situation is more complex than this appears since TSHR mRNA has also been reported in the pituitary gland, heart muscle84 and lymphocytes themselves,85,86 with binding sites for TSH also described in the latter. Whether some of these mRNAs are false transcripts and which of a number of splicing variants are translated is unclear. In addition, the role of post-translational processing may vary from tissue to tissue70 and remains to be clarified. Thyroglobulin Thyroglobulin (Tg) is the principal constituent of the follicular colloid, produced by the thyroid epithelial cells and stored in the gland. Thyroid peroxidase (TPO) (see below) is responsible for the iodination of specific tyrosine residues on Tg to monoiodotyrosine and diiodotyrosine. When stimulated by TSH, the thyroid cell phagocytoses Tg which is then proteolyzed to yield thyroxine (T4) and triiodothyronine (T3) which are released into the

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

103

extracellular fluid. Tg is detectable in the normal circulation and serum levels are increased in all thyroid diseases. It is possible to induce experimental autoimmune thyroiditis (EAT) in mice using Tg as an immunizing antigen.87-89 The EAT model demonstrates the autoantigenic nature of Tg and has greatly contributed to our understanding of the pathogenesis of AITD. Genomic DNA encoding Tg is a 200 kb sequence located on chromosome 8 which transcribes both 9.0 Kb and 0.9 Kb mRNAs (termed Tg1 and Tg2).90 The transcription of Tg mRNAs is increased by TSH, which acts by increasing intracellular cyclic AMP, and influences a series of thyroid-specific transactivating factors (TTF1 and TTF2). 91 TSH-stimulated Tg gene transcription is also regulated by a variety of thyroid cell secreted growth factors (such as IGF1) and cytokines. Tg mRNAs are enhanced by low-dose IL-1β and suppressed by high dose IL-1β,92 while gamma interferon (γIF) inhibits TSH-stimulated Tg gene transcription.93 The Tg molecule is a large, compact, water-soluble glycoprotein dimer, each subunit being approximately 330 Kd. Although about 45 specific tyrosine residues out of a total of 72 in the molecule are accessible to iodination by TPO,94 only a few act as primary iodine acceptors and donor sites and these are located in defined polypeptide sequences near the amino and carboxyl terminals of the molecule.95 The degree of Tg iodination and posttranslational modifications are thought to be important in immunogenicity.96,97 Thyroid Peroxidase Much information has been clarified about thyroid peroxidase (TPO) with the cloning of the TPO gene on chromosome 2 (reviewed in 98,99), though its geography has not yet been fully elucidated. As mentioned earlier, TPO is a key enzyme in thyroid physiology, situated on the luminal surface of the microvilli of the thyroid epithelial cell in the appropriate location to catalyze intrafollicular reactions on stored Tg. TPO is the major “microsomal” antigen of autoimmune thyroid disease.100-102 Immunization of mice with TPO induces EAT indistinguishable from the Tg immunized model103 but with differing MHC requirements. TPO transcription and translation are closely regulated by TSH and are cyclic AMP dependent.104,105 Two different human TPO cDNA clones differing by 171 bases have been isolated and proposed as the reason for the 100 and 107 kD protein doublet often seen on Western blotting. There is also evidence for alternative splicing of TPO with at least two distinct mRNAs of 3.0 and 3.2 kB. It has been reported that Graves’ disease thyroid contains up to 50% of this second form of TPO mRNA106 raising the question of altered immunogenicity. TPO polymorphisms have also been reported.99 TPO induction by TSH is inhibited by IL-1,107 IL-6,108 and γIF.109 The deduced amino acid sequence of human TPO is 933 amino acids.110 It is a 107 Kd glycoprotein with 10% glycosylation and a membrane-spanning region close to the carboxyl-terminus (Fig. 6.2). TPO serves to iodinate tyrosine residues on Tg and to couple iodinated tyrosines to form T3 and T4. Thiourylene type antithyroid drugs, such as methimazole or propylthiouracil, act by competing for substrate with TPO. The Sodium-Iodine Symporter (NIS) Transport of iodide into the thyroid is the first step in thyroid hormone synthesis. This is a TSH mediated, active process, catalyzed by the Na+/I- symporter (NIS), an intrinsic membrane protein on thyroid epithelial cells. With the cloning of this important molecule,12 new opportunities have been opened for a better understanding of the physiological role of the NIS and its potential role as a thyroid autoantigen. While much of the physiologic function has been defined,111,112 some recent reports suggest that the NIS could be involved as an

104

Endocrine and Organ Specific Autoimmunity

Fig. 6.2. A model of thyroid peroxidase based on the known structure of myeloperoxidase. Note that it is thought to be a dimer.185

antigen in AITD. Initially, one serum from a patient with Hashimoto’s thyroiditis and one monoclonal antibody to human thyroid membranes displayed significant inhibition of TSH induced 125I-uptake in cultured thyrocytes.113 Subsequently, using recombinant NIS in Western blot assays, antibodies to this protein were identified in the sera of 85% of Graves’ disease and 15% of Hashimoto thyroiditis patients tested.11 Further studies have shown that IgG fractions from some sera of patients with Hashimoto’s thyroiditis inhibited up to 62% of TSH mediated iodide accumulation in CHO cells expressing functional NIS.114 However, these preliminary studies await further confirmation, and the potential role of NIS in thyroid autoimmunity remains to be elucidated.

Autoantibodies Characteristics of Pathogenic Human TSHR-Ab Stimulating autoantibodies to the TSHR (TSHR-Abs), found in the serum of patients with hyperthyroid Graves’ disease, act as TSH agonists, activate the TSHR, and lead to excess thyroid hormone production. The fact that such antibodies compete with TSH for binding to the receptor and stimulate cyclic AMP production has enabled investigators to

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

105

Fig. 6.3. TSH receptor antibody induced extracellular cyclic AMP generation in human fetal thyroid cells. Normal human IgG (TSH receptor antibody-TRAb-titer <15%) and Graves’ IgG (TRAb >15%) are shown. Bovine TSH served as control (I Unit/L). 187

measure their presence in Graves’ sera, as a guide to disease activity, using many different techniques. Most common have been radio receptor assay in competition with radio labeled TSH,115 or with thyroid cells in culture,116 or using mammalian cells transfected with the hTSHR117 and cyclic AMP generation as the read out (Fig. 6.3). TSHR-Ab are detectable only in patients with AITD, and are, therefore, disease specific, in great contrast to the high prevalence of Tg-Ab and TPO-Ab in normal subjects13 (see below). A direct pathogenic relationship between TSHR-Abs and Graves’ disease has been evident since long acting thyroid stimulator (LATS) activity was discovered in patient sera by Adams and Purves four decades ago.118 Most TSHR-Abs are of immunoglobulin subclass 1 (IgG1) and may show light chain restriction, suggesting that these autoantibodies are oligoclonal.119 TSHR-Ab variable region genes have not yet been thoroughly analyzed, and it remains unclear how restricted the B cell immune response is in Graves’ disease. However, the titer of TSHR-Abs are low and often a 10-fold dilution of serum will make their detection most difficult. Therefore, TSHR-Abs must be of high affinity or produced locally at their site of action. Indeed there is evidence for intrathyroidal B cells being a major source of their secretion.120 TSHR-Abs are not all TSHR-stimulating, triggering an abnormal thyroid hormone release secondary to an increase of cyclic AMP synthesis. They may also be TSHR-blocking (reviewed in 13), inhibiting the action of TSH and reducing thyroid hormone secretion. Further complicating this picture are the presence of potential neutral TSHR-Abs, which are neither stimulating nor blocking,121,122 and may coexist with stimulating and blocking TSHR-Ab in the same serum. Eighty to 100% of untreated patients with hyperthyroid Graves’ disease have detectable TSHR-Abs in their serum that are biologically stimulating. The original self infusion of such a serum from a patient with Graves’ hyperthyroidism by Adams and colleagues and the resulting thyroid stimulation,123 was the first direct evidence for the role of TSHR-Ab in the induction of hyperthyroidism in humans. Another early confirmation of this phenomenon

106

Endocrine and Organ Specific Autoimmunity

Fig. 6.4. The presence of blocking TSHR antibodies revealed by dilution of IgG from Graves’ patients. Both a highly positive (1) and a borderline sample (2) are shown. Note the initial increase in cyclic AMP generation in human thyroid cells by diluting IgG #1.187

was the observation of neonatal hyperthyroidism and hypothyroidism due to transplacental passage of maternal stimulatory TSHR-Ab.124-126 We now know that a mixture of stimulating, blocking and neutral antibodies may be present in most patients with Graves’ disease, the final effect of the mixture being determined by the concentration and affinity of each antibody species. Under such circumstances, dilution of the serum may increase its stimulating activity as the blocking antibodies are diluted out below their threshold of activity (Fig. 6.4). Since the relative concentration of TSHR-Abs in serum may fluctuate, the evolution of some Graves’ disease patients to hypothyroidism may be partly explained by the late predominance of blocking TSHR-Ab.5,127 Indeed, the existence of TSH inhibitory activity by blocking TSHR-Ab is widely recognized as a cause of hypothyroidism in some cases of Hashimoto’s thyroiditis.15 With respect to TSHR-Ab epitopes, mutant or chimeric receptor assays have shown that binding sites for stimulating TSHR-Abs (from Graves’ disease patients), are located mainly at the NH2-terminus, while binding sites for blocking TSHR-Abs (from patients with Hashimoto’s thyroiditis) are mainly at the C-terminus of the extracellular domain. Such sites are different to those seen with TSH ligand.128-130 Indeed, when the TSHR-ecd was used as immunogen, it resulted in polyclonal and monoclonal antibodies able to compete for TSH binding, and which recognized multiple epitopes in the NH2-terminus and C-terminus of the receptor.74,131 Subsequent studies using folded and unfolded recombinant human and murine TSHR-ecd’s,131,132 have also shown that folding of the TSHR is critical for TSH and TSHR-Abs binding in addition to glycosylation status. While the folded structure was preferred by most TSHR-Ab from Graves’ disease patients, underlining the importance of conformational epitopes, linear epitopes were also recognized by some Graves’ TSHR-Abs, though with lower affinities.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

107

Experimental TSHR-Ab With the availability of purified recombinant TSHR-ecd, many groups have induced experimental TSHR-Ab in mice and rabbits in attempts to mimic the pathogenic properties of TSHR-Ab and to generate an animal model of Graves’ disease. For a useful animal model, experimental TSHR-Abs should have the following characteristics: 1. They should be the equivalent of human IgG1 subclass like their functional human serum counterparts. 2. They should be of high binding affinity ( Kd > 10-10-10-9 range) and active at ng/ml concentrations. 3. They should be specific to the TSHR and be adsorbed by TSHR antigen but not control antigen. 4. They should have intrinsic biological activity, either TSHR-stimulating or TSHR-blocking. Using insect cell expressed, nonglycosylated, hTSHR-ecd, one group induced hTSHR-ecd antibodies in BALB/c mice.133,134 However, despite an inexplicable hyperthyroxinemia noticed in these mice, the antibodies showed no TSH agonist activity. Another group using prokaryotic hTSHR-ecd, also induced TSHR-Ab of IgG class in BALB/c mice. Such antibodies were blocking rather than stimulating, and thyroid function remained unchanged.135 A mild thyroiditis was also observed in these mice, but to date has not been confirmed in any other study using recombinant TSHR. In a similar way, but using glycosylated hTSHRecd and murine TSHR-ecd, we were able to induce high titers of TSHR-Abs of IgG class in BALB/c mice, which were able to block the native TSHR. However, no changes in thyroid histology were noted.74 Using a synthetic peptide approach, we were able to map the TSHRAb epitopes and found that multiple sites were sequentially recognized, especially at the NH2-terminus and C-terminus of the receptor. The reason why stimulatory TSHR-Ab and features of Graves’ disease have not been readily reproduced by immunization in mice remains unclear. The use of nonglycosylated or high mannose glycosylated recombinant TSHR extracellular domains and the unavailability of correctly glycosylated, full length, properly folded recombinant TSHR, were likely responsible. Recently, Shimojo et al, used a fibroblast line expressing both the full length hTSHR and aberrant MHC class II in an extensive immunization regimen. They found induction of stimulatory TSHR-Ab, hyperthyroidism and thyroid histological features of Graves’ disease in mice and we have recently confirmed this model.69 Hence, it seems that correctly folded, membrane bound TSHR, and an enhanced MHC class II mediated presentation to the immune system, may be key facts in reproducing pathogenic TSHR-Ab and the features of Graves’ disease in mice. Another useful tool for the study of AITD, would be the production of human TSHR monoclonal antibodies. To achieve this goal, human IgGs have been obtained from EpsteinBarr virus immortalized B cells of patients with AITD, and IgG secreting B cell lines further generated.136,137 Although specific for the TSHR, such antibodies were of low affinity, and displayed marginal stimulatory or blocking bioactivities, and only after tenfold concentration of the culture supernatants. Furthermore, none of these antibodies were capable of competing for TSH binding to native TSHR. Characteristics of Human Tg-Ab and TPO-Ab Both Tg-Ab and TPO-Ab are mainly IgGs and are hallmarks of AITD. They are present in high titer in Hashimoto’s thyroiditis, compared to lower titers in patients with Graves’ disease. Both Tg-Ab and TPO-Ab titers correlate with the extent of the thyroidal lymphocytic infiltration,138 suggesting that they are produced by intrathyroidal lymphocytes, and therefore, may have a pathogenic role. In the general population up to 20% of subjects have

108

Endocrine and Organ Specific Autoimmunity

circulating Tg-Ab, and up to 25% TPO-Ab depending upon the sensitivity of the assays used. However, such “naturally occurring” antibodies may be of low affinity and uncertain pathological significance (reviewed in 139). However, it is also likely that such natural autoantibodies may mature into high affinity antibodies in response to antigenic stimulation. Thyroglobulin Antibodies Analysis of the antibody epitopes in immune sera from Tg immunized animals has shown linear epitopes on the Tg molecule in the central and terminal regions,144,145 although heterogeniety in responsiveness does occur.146 Findings suggest that autoepitopes on Tg associated with AITD are mostly conformational.148 Some studies have demonstrated a restricted epitope recognition pattern by Tg-Ab in patients with AITD, in contrast to a polyclonal reactivity observed with Tg-Ab from healthy subjects.140,141 These findings may imply that in contrast to the oligoclonal nature of AITD-associated Tg-Ab, “natural” Tg-Ab occurring in the healthy population are not restricted in their epitope recognition and most probably are produced by polyclonal activation of B cells reactive to Tg (reviewed in 142). Tg-Ab typically do not fix complement, and are not often cytotoxic.143 Thyroid Peroxidase Antibodies TPO-Ab are predominantly of the IgG class and are frequently complement fixing, raising the possibility of a pathogenic role in follicular cell destruction. Exposure of thyroid cell cultures in vitro to TPO-Ab in the presence of complement leads to cell lysis.149 Patient sera containing TPO-Ab has been shown to have an “inhibitory effect” on the catalytic activity of TPO, implying the presence of epitopes close to the active site of the enzyme.150 With respect to TPO, immunological studies using sera from patients with AITD in Western blot assays have shown that the autoimmune response to TPO is heterogeneous and polyclonal in nature (reviewed in 151). Recent studies employing human Fabs of monoclonal anti-TPO antibodies, constructed by combinatorial libraries and representing more than 80% of the human TPO-Ab repertoire, have identified four immunodominant antigenic regions on human TPO, most of which are conformational in nature.152 The precise site of these epitopes awaits the crystal structure of the TPO molecule. Human Monoclonal Antibodies to Tg and TPO Using a phage display combinatorial library approach, investigators have generated human Tg monoclonal antibodies of IgG k and IgG l type, derived from cervical lymph nodes of a patient with Hashimoto’s thyroiditis. Sequence analysis of the clones showed a highly restricted heavy chain usage and less restricted light chain usage. The purified Fabs reacted only with conformationally intact Tg and inhibited Tg-Ab, from AITD serum, binding to human Tg.153 Using a similar approach, monoclonal human Fabs to TPO have been generated from intrathyroidal plasma cells and used to define an immunodominant region on TPO consisting of the four overlapping epitopes referred to above.154 Interestingly, these TPO-specific Fabs also showed restricted IgG variable gene usage and preferentially recognized conformationally intact TPO rather than denatured TPO.152 However, some TPO-Ab, can recognize linear epitopes in denatured TPO.155,156 Recently we have described a similar phenomenon for hTSHR-Ab from patients with Graves’ disease. While folded forms of recombinant TSHRs were clearly preferred by hTSHR-Abs, linearized (unfolded) forms were recognized with lower affinities.131,132 Two important conclusions can be drawn from these studies: 1) Small linear epitopes may be part of a larger conformational epitope and 2) pathogenic thyroid autoantibodies interact with high affinity with complex conformational epitopes on the target autoantigen.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

109

The Primary Role of T cells The Infiltrate The intrathyroidal lymphocytic infiltrate consists of a majority of T cells in AITD, and all patients with AITD have more T cells which are specific for thyroid antigens than found in the normal population. Such iniltrates have a CD4+/CD8+ ratio which was increased when compared to the peripheral blood population.157-160 When studied in detail, the phenotype of the CD4+ population was predominantly helper-inducer memory cells.157,161,162 Such infiltrating T cells were often activated, as shown by the level of HLA class II antigen expression.163 However, in recent years it has become clear that such T cell phenotypes, as studied by means of membrane markers, do not always reflect the functional activity of cell subtypes. Indeed, it is a combination of the pattern of membrane protein expression as well as of cytokine production that determines the role of the T lymphocyte in driving the immune response164,165 In particular, it has been shown that the CD4+ helper cells can be subdivided into two categories: the Th1 subset, mainly involved in delayed-type hypersensitivity reactions and cytotoxic activities, and which produce TNF-beta, IFN-gamma and IL-2, and the Th2 subset, prominently stimulating antibody production and producing IL-4, IL-5, IL-6 and IL-13. Another set of helper cells, without commitment and with mixed cytokine production has been named Th0. To date there have been only a few studies available with regard to the Th1/Th2 subtypes in Graves’ disease and Hashimoto’s disease. However, in general, the available data seem to confirm a predominantly Th1 type response in both Graves’ disease and Hashimoto’s disease within the thyroid.17,159 This finding is at first sight unexpected for Graves’ disease which is characterized by the effect of circulating antibodies. It must be remembered, however, that Th1 cells are also able to induce antibody production via IL-10 secretion.165 Moreover, TSH receptor antibodies are more often of the IgG1 subclass,119 which is probably selectively induced by Th1 cells. T Cell Clones Further analyses of thyroid antigen specific T cells have been performed by cloning the intrathyroidal T cells.159,166-170 Most of the cloned cells and lines were also of the memory (CD4+CD29+) subtype and responded with significant growth and/or cytokine production to exposure to autologous thyroid follicular cells or thyroid autoantigens. However, such clones have not yet been adequately characterized as Th1 or Th2. In summary, data are surprisingly limited at present, as far as the new definitions of T-lymphocyte functional activity are concerned, and the literature is relatively unhelpful as far as phenotypic insights into disease etiology.

Characterization of T Cell Epitopes on Thyroid Antigens TSHR Antigen T Cell Epitopes Although a solubilized, crude hTSHR preparation was shown in 1978 to induce T cell activation as measured by lymphocyte transformation,171 there have only recently been studies of the hTSHR T cell epitopes in human autoimmune thyroid disease and in animals immunized with hTSHR protein. The availability of recombinant hTSHR-ecd and the full protein sequence made such studies feasible, using both intact receptor and receptor peptides. T cell epitope recognition has been reported to be highly variable in patients with autoimmune thyroid disease and normals have also been shown to react to hTSHR-ecd peptides.172-179 We have identified 4 hTSHR peptides which were recognized by T cells from a majority of patients with Graves’ disease. Those peptides were amino acid 247-266, 202-221, 52-71 and 142-161180 (Fig. 6.5). Other studies have addressed the problem of major epitopes in a

110

Endocrine and Organ Specific Autoimmunity

variety of ways, usually starting with an identification of presumed epitopes within a given patient based on ad hoc criteria. As a result the reported immunodominant TSHR peptides have varied widely in different studies and there have been many claims of major epitopes in Graves’ disease.172-179 It was also surprising to find that two of the same epitopes we have described were also major epitopes in an inbred strain of mice (Balb/c) immunized with mouse TSHR-ecd (Kita et al, manuscript in preparation), further supporting the concept of nonMHC related T cell epitopes. The fact, that certain peptides are predominant despite probable heterogeneity in HLA-types illustrates the potential of certain peptides to override —to a degree—known HLA-restriction. Epitope 202-221 has previously been identified as a major B-cell epitope181 in Graves’ disease. This epitope is hydrophilic, a property predisposing them to exposure to the immune system182 and possibly to the T cell receptor (TcR)183 and shared with immunodominant regions such as that of proteolipid protein.184 Tg Antigen T Cell Epitopes As expected, T cells from animals immunized with Tg react to small linear Tg fragments. Some data suggest that they react preferentially to hormone-containing regions of the molecule or to Tg with higher iodine content,96,187,188 while other studies have not found this.189 However, to date, the epitopes operative in human Tg antigen-specific T cells have not been fully explored because the size of the molecule has precluded the synthetic peptide approach. TPO antigen T cell epitopes TPO-specific human T cell clones have been well characterized.190 Data defining human TPO T cell epitopes have been obtained using a series of synthesized TPO peptides encompassing the entire molecule.191 These studies have defined multiple short linear

Fig. 6.5. Mean T cell proliferative responses to TSHR-ectodomain peptides in normals (B) and Graves’ disease patients (A). Four peptide responses differed significantly in the patients and as illustrated by * with only one peptide in the normals just reaching significance.180

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

111

epitopes within the TPO molecule as T cell epitopes which vary from patient to patient There have also been reports of shared T cell epitopes between TPO and Tg.192 Amino acid data bank analysis showed greater homology of TPO to Tg than any other human protein; there is a common 8 amino acid sequence with 6 identical and 2 conserved amino acids,127 which may represent a linear T cell epitope. Studies of the T cell Receptor Reveal Intrathyroidal T cell Clonal Expansion The basis for an organ specific autoimmune process is the interaction of antigen-specific T cells with the target tissue itself in a way that leads to selection and clonal expansion of autoreactive cells. Specificity for this interaction is given by the fact that each T cell receptor chain derives from random somatic rearrangements of one out of >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.

112

Endocrine and Organ Specific Autoimmunity

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

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

113

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

114

Endocrine and Organ Specific Autoimmunity

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:

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

115

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

116

Endocrine and Organ Specific Autoimmunity

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.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

117

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

118

Endocrine and Organ Specific Autoimmunity

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.

References 1. Davies T. Human autoimmune thyroid disease-Newer views on a common breakdown in T cell tolerance. Israel J Med Sci 1994; 30:2-11. 2. Stagnaro GA, Roman SH, Cobin RH et al. A prospective study of lymphocyte-initiated immunosuppression in normal pregnancy: Evidence of a T cell etiology for postpartum thyroid dysfunction. J Clin Endocrinol Metab 1992; 74:645-653. 3. Davies TF, Amino N. A new classification for human autoimmune thyroid disease. Thyroid 1993; 3:331-333.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

119

4. Alexander WD, Harden RM, Shimmins J. Thyroidal suppression by triiodothyronine as a guide to duration of treatment of thyrotoxicosis with antithyroid drugs. Lancet 1966; 2:1041-1044. 5. Wood LC, Ingbar SH. Hypothyroidism as a late sequel in patients with Graves’ disease treated with antithyroid agents. J Clin Invest 1979; 64:1429-1436. 6. Gorman CA. Temporal relationship between onset of Graves’ ophthalmopathy and diagnosis of thyrotoxicosis. Mayo Clin Proc 1983; 58:515-519. 7. Vanderpump MP, Tunbridge WM, French JM et al. The incidence of thyroid disorders in the community: A twenty-year follow-up of the Whickham Survey. Clin Endocrinol (Oxf) 1995; 43:55-68. 8. Amino N. Postpartum thyroid disease. In: Advances in perinatal thyroidology. In: Bercu BB Shulman DI eds. New York: Plenum Press 1991:167. 9. Davies T, De Bernardo E. Thyroid autoantibodies and disease: an overview. In: Autoimmune endocrine disease. Davies TF, ed. New York: John Wiley & Sons 1983:127-138. 10. Kendler DL, Davies TF. Immunological mechanisms in Graves’ disease. New York: Harwood Academic, 1993:511-539. 11. Endo T, Kogai T, Nakazato M et al. Autoantibody against Na+/I- symporter in the sera of patients with autoimmune thyroid disease. Biochem Biophys Res Commun 1996; 224:92-95. 12. Dai G, Levy O, Carrasco N. Cloning and characterization of the thyroid iodide transporter. Nature 1996; 379:458-60. 13. Davies TF. The Pathogenesis of Graves’ disease. In: Braverman LE and Utiger EB eds. The Thyroid: A Fundamental Text, Philadelphia: Lippincot 1996:525-536. 14. Levine SN. Current concepts of thyroiditis. Arch Intern Med 1983; 143:1952-1956. 15. Kraiem Z, Lahat N, Glaser B et al. Thyrotropin receptor blocking antibodies: Incidence, characterization and in-vitro synthesis. Clinical Endocrinology 1987; 27:409-421. 16. Martin A, Schwartz AE, Friedman EW et al. Successful production of intrathyroidal human T cell hybridomas: Evidence for intact helper T cell function in Graves’ disease. J Clin Endocrinol Metab 1989; 69:1104-1108. 17. Watson PF, Pickerill AP, Davies R et al. Analysis of cytokine gene expression in Graves’ disease and multinodular goiter. J Clin Endocrinol Metab 1994; 79:355-360. 18. Otto E, Ochs K, Hansen C et al. Orbital tissue-derived T lymphocytes from patients with Graves’ ophthalmopathy recognize autologous orbital antigens. J Clin Endocrinol Metab 1996; 81:3045-3050. 19. Ajjan RA, Watson PF, McIntosh RS et al. Intrathyroidal cytokine gene expression in Hashimoto’s thyroiditis. Clin Exp Immunol 1996; 105:523-528. 20. Tomer Y, Davies TF. Infections and autoimmune endocrine disease. Bailliere’s Clin Endocrinol Metab 1995; 9:47-70. 21. Harvald B, Hauge M. A catamnestic investigation of Danish twins. Danish Med Bull 1956; 3:150-158. 22. Stenszky VK, Vozma L, Balazs et al. The role of HLA antigens in the manifestations and the course of Graves’ disease. Mol Biol Med 1986; 3:53-62. 23. Bartels ED. Twin Examinations: Heredity in Graves’ disease (Munksgaad, Copenhagen), 1941:32-36. 24. Martin L. The heredity and familial aspects of exophathalmic goitre and nodular goitre. Q J Med 1945; 14:207-219. 25. Chopra IJ, Solomon DH, Chopra U et al. Abnormalities in thyroid function in relatives of patients with Graves’ disease and Hashimoto’s thyroiditis: Lack of correlation with inheritance of HLA-B8. J Clin Endocrinol Metab 1977; 45:45-54. 26. Tamai H, Uno H, Hirota Y et al. Immunogenetics of Hashimoto’s and Graves’ diseases. Journal of Clinical Endocrinology and Metabolism 1985; 60:62-66. 27. Hall R, Stanbury JB. Familial studies of autoimmune thyroiditis. Clin Exp Immunol 1967; 2:719-725. 28. Burek CL, Hoffman WH, Rose NR. The presence of thyroid autoantibodies in children and adolescents with AITD and in their siblings and parents. Clin Immunol Immunopathol 1982; 25:395-404.

120

Endocrine and Organ Specific Autoimmunity

29. Farid NR, Stone E, Johnson G. Graves’ disease and HLA: Clinical and epidemiologic associations. Clin Endocrinol Oxf 1980; 13:535-44. 30. Greenberg DA. Linkage analysis of “necessary” loci versus “susceptibility” loci. Am J Hum Genet 1993; 52:135-143. 31. Farid NR, Sampson L, Noel EP et al. A study of human D locus related antigens in Graves’ disease. J Clin Invest 1979; 63:108-113. 32. Mangklabruks A, Cox N, DeGroot LJ. Genetic factors in autoimmune thyroid disease analyzed by restriction fragment length polymorphisms of candidate genes. J Clin Endocrinol Metab 1991; 73:236-244. 33. Barlow A, Wheatcroft N, Watson P et al. Association of HLA-DQA1*0501 with Graves’ disease in English caucasian men and women. Clin Endocrinol 1996; 44:73-77. 34. Ratanachaiyavong S, Lloyd L, Darke C et al. MHC-extended haplotypes in families of patients with Graves’ disease. Hum Immunol 1993; 36:99-111. 35. Yanagawa T, Mangklabruks A, Chang YB et al. Human histocompatibility leukocyte antigen-DQA1*0501 allele associated with genetic susceptibility to Graves’ disease in a caucasian population. J Clin Endocrinol Metab 1993; 76:1569-1574. 36. Ofosu MH, Dunston G, Henry L et al. HLA-DQ3 is associated with Graves’ disease in African-Americans. Immunol Invest 1996; 25:103-110. 37. Farid NR. Immunogenetics of autoimmune thyroid disorders. Endocrinol Metab Clin North Am 1987; 16:229-245. 38. Tandon N, Zhang L, Weetman AP. HLA associations with Hashimoto’s thyroiditis. Clin Endocrinol (Oxf) 1991; 34:383-386. 39. Farid NR, Sampson L, Moens H et al. The association of goitrous autoimmune thyroiditis with HLA-DR5. Tissue Antigens 1981; 17:265-268. 40. Weissel M, Hofer R, Zasmeta H et al. HLA-DR and Hashimoto’s thyroiditis. Tissue Antigens 1980; 16:256-259. 41. Wu Z, Stephens H, Sachs JA et al. Molecular analysis of HLA-DQ and -DP genes in caucasoid patients with Hashimoto’s thyroiditis. Tissue Antigens 1994; 43:116-119. 42. Weetman AP. Autoimmune Endocrine Disease. Cambridge University Press, Cambridge 1991. 43. Payami H, Joe S, Farid N et al. Relative predispositional effects of marker alleles with disease: HLA-DR alleles and Graves’ disease. Am J Hum Genet 1989; 45:541-546. 44. O’Connor G, Neufeld DS, Greenberg DA et al. Lack of disease associated HLA-DQ restriction fragment length polymorphisms in families with autoimmune thyroid disease. Autoimmunity 1993; 14:237-241. 45. Roman SH, Greenberg D, Rubinstein P et al. Genetics of autoimmune thyroid disease: lack of evidence for linkage to HLA within families. Clin Endocrinol Metab 1992; 74:496503. 46. Shields DC, Ratanachaiyavong S, McGregor AM et al. Combined segregation and linkage analysis of Graves’ disease with a thyroid autoantibody diathesis. Amer Hum Genet 1994; 55:540-554. 47. Stenszky V, Kozma L, Balazs C et al. The genetics of Graves’ disease: HLA and disease susceptibility. Clin Endocrinol Metab 1985; 61:735-740. 48. Demaine A, Welsh KI, Hawe SB et al. Polymorphism of the T Cell Receptor b-Chain in Graves’ disease. J Clin Endocrinol Metab 1987; 65:643-646. 49. Ito M, Tanimoto M, Kamura H et al. Association of HLA antigen and restriction fragment length polymorphism of T cell receptor beta-chain gene with Graves’ disease and Hashimoto’s thyroiditis. J Clin Endocrinol Metab 1989; 69:100-104. 50. Nakao Y, Matsumoto H, Miyazaki T et al. IgG heavy chain allotypes (Gm) in atrophic and goitrous thyroiditis. Clin Exp Immunol 1980; 42:20-26. 51. Adams DD, Adams YJ, Knight JG et al. On the nature of genes influencing the prevalence of Graves’ disease. Life Sci 1983; 31:3-13. 52. Roman SH, Hubbard M, Rubinstein P. Failure to confirm standard HLA and Gm immunogenetic typing as a predictor of familial autoimmune thyroid disease. Annual Meeting of the Endocrine Society, Seattle, WA 1989.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

121

53. Lenshow D, Bluestone J. T cell costimulation and in vitro tolerance. Curr Opin Immunol 1993; 5:747-752. 54. Yanagawa T, Hidaka Y, Guimaraes V et al. CTLA-4 gene polymorphism associated with Graves’ disease in a caucasian population. J Clin Endocrinol Metab 1995; 80:41-45. 55. Nistico L, Buzzetti R, Todd JA. The CTLA-4 gene region is linked to, and associated with, type 1 diabetes. Hum Mol Genet 1996; 5:1075-1080. 56. Pirro MT, De FV, Di CA et al. Thyroperoxidase microsatellite polymorphism in thyroid disease. Thyroid 1995; 5:461-464. 57. Tomer Y, Barbesino G, Keddache M et al. Mapping of the major susceptibility locus for Graves’ disease (GD-1) to chromosome 14q31. J Clin Endocrinol Metab 1997; 82:1645-1648. 58. Bohr U. A heritable point mutation in an extracellular domain of the TSH receptor involved in the interaction with Graves’ disease. Biochem Biophys Acta 1993; 1216:504-508. 59. Cuddihy RM, Dutton CM, Bahn RS. A polymorphism in the extracellular domain of the thyrotropin receptor is highly associated with autoimmune thyroid disease in females. Thyroid 1995; 5:89-95. 60. Bahn RS, Dutton CM, Heufelder AE et al. A genomic point mutation in the extracellular domain of the TSH receptor in patients with Graves’ ophthalmopathy. J Clin Endocrinol Metab 1994; 78:256-260. 61. Cuddihi RM, Schaid DS, Bahn RS. Multivariate analysis of HLA loci in conjunction with a thyroropin receptor codon 52 polymorphism in conferring risk to Graves’ disease. Thyroid 1996; 6:261-265. 62. Loos U, Hagner S, Bohr UR et al. Enhanced cAMP accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain. Eur J Biochem 1995; 232:62-65. 63. Watson PF, French A, Pickerill AP et al. Lack of association between a polymorphism in the coding region of the thyrotropin receptor gene and Graves’ disease. J Clin Endocrinol Metab 1995; 80:1032-1035. 64. Kotsa KD, Watson P, Weetman AP. No association between a thyrotropin receptor gene polymorphism and Graves’ disease in the female population. Thyroid 1997; 7:31-32. 65. De Roux N, Shields DC, Misrahi M et al. Analysis of the thyrotropin receptor as a candidate gene in familial Graves’ disease. J Clin Endocrinol Metab 1996; 81:3483-3486. 66. Field LL, Tobias R, Thomson G et al. Susceptibility to IDDM maps to a locus (IDDM11) on human chromosome 14q24.3-q31. Genomics 1996; 33:1-8. 67. Libert F, Lefort A, Gerard C et al. Cloning, sequencing and expression of the human TSH receptor: evidence for binding of autoantibodies. Biochem Biophys Res Comm 1989; 165:1250-1255. 68. Misrahi M, Loosfelt H, Atger M et al. Cloning, sequencing, and expression of human TSH receptor. Biochem Biophys Res Comm 1990; 166:394-403. 69. Shimojo N, Kohno Y, Yagamuchi K et al. Induction of Graves-like disease in mice by immunization with fibroblasts transfected with the thyrotropin receptor and a class II molecule. Proc Natl Acad Sci USA 1996; 93:11074-11079. 70. Graves PN, Tomer Y, Davies TF. Cloning and sequencing of a 1.3 kb variant of human thyrotropin receptor mRNA lacking the transmembrane domain. Biochem Biophys Res Commun 1992; 187:1135-1143. 71. Nagayama Y, Rapoport B. The thyrotropin receptor 25 years after its discovery: New insight after its molecular cloning. Molecular Endocrinology 1992; 6:145-156. 72. Morris JC, Berger ER, McCormick DJ. Structure-function studies of the human thyrotropin receptor. Journal of Biological Chemistry 1993; 268:10900-10905. 73. Dallas JS, Desai RK, Cunningham SJ et al. TSH interacts with multiple discrete regions of the TSH receptor: polyclonal rabbit antibodies to one or more of these regions can inhibit TSH binding and function. Endocrinology 1994; 134:1437-1445. 74. Vlase H, Nakashima M, Graves PN et al. Defining the major antibody epitopes on the human thyrotropin receptor in immunized mice: evidence for intramolecular epitope spreading. Endocrinology 1995; 136:4415-4423.

122

Endocrine and Organ Specific Autoimmunity

75. Heufelder AE, Dutton CM, Sarkar G et al. Detection of RNA encoding the TSH receptor RNA in cultured fibroblasts from patients with Graves’ ophthalmopathy and pretibila myxedema. Thyroid 1993; 3:297-300. 76. Feliciello A, Porcellini A, Ciullo I et al. Expression of thyrotropin-receptor mRNA in healthy and Graves’ disease retro-orbital tissue. Lancet 1993; 342:337-338. 77. Nakashima M, Kendler DL, Rootman J et al. Human TSH receptor variant 1.3 mRNA in human extraocular muscles. Thyroid 1994; 4:19-25. 78. Paschke R, Metcalfe A, Alcalde L et al. Presence of nonfunctional TSH receptor variant transcripts in retro-orbital and other tissues. J Clin Endocrinol Metab 1994; 79:1234-1238. 79. Stadlmayr W, Spitzweg C, Bichlmair AM et al. TSH receptor transcripts and TSH receptor-like immunoreactivity in orbital and pretibial fibroblasts of patients with Graves’ ophthalmopathy and pretibial myxedema. Thyroid 1997; 7:3-12. 80. Weetman AP. Eyeing up Graves’ ophthalmopathy. Mol Cell Endocrinol 1997; 126:113-116. 81. Burch HB, Sellitti D, Barnes SG et al. TSH receptor antisera for the detection of immunoreactive protein species in retro-ocular fibroblasts obtained from patients with Graves’ ophthalmopathy. J Clin Endocrinol Metab 1994; 78:1384-1391. 82. Davies TF, Smith BR, Hall R. Binding of thyroid stimulators to guinea-pig testis and thyroid ‘proceedings. J Endocrinol 1977; 75:39P. 83. Endo T, Ohta K, Haraguchi K et al. Cloning and functional expression of a thyrotropin receptor cDNA from rat fat cells. J Biol Chem 1995; 270:10833-10837. 84. Drvota V, Janson A, Norman C et al. Evidence for the presence of functional thyrotropin receptor in cardiac muscle. Biochem Biophys Res Commun 1995; 211:426-431. 85. Davies TF, Teng CS, McLachlan SM et al. Thyrotropin receptors in adipose tissue, retroorbital tissue and lymphocytes. Mol Cell Endocrinol 1978; 9:303-310. 86. Francis T, Burch HB, Cai W et al. Lymphocytes express thyrotropin receptor-specific mRNA as detected by the PCR technique. Thyroid 1991; 1:223-227. 87. Vladutiu AO, Rose NR. Autoimmune murine thyroiditis: relation to histocompatibility (H2) type. Science 1971; 174:1137-1139. 88. Kong YM, Bagnasco M, Canonica GW. How do T cells mediate autoimmune thyroiditis? Immunology Today 1986; 7:337-339. 89. Matsuoka N, Unger P, Ben NA et al. Thyroglobulin-induced murine thyroiditis assessed by intrathyroidal T cell receptor sequencing. J Immunol 1994; 152:2562-2568. 90. Graves PN, Davies TF. A second thyroglobulin messenger RNA species (rTg-2) in rat thyrocytes. Mol Endocrinol 1990; 4:155-161. 91. Saiardi A, Falasca P, Civitareale D. Synergistic transcriptional activation of the thyrotropin receptor promoter by cyclic AMP-responsive-element-binding protein and thyroid transcription factor 1. Biochem 1995; J 310:491-496. 92. Kung AW, Lau KS. Interleukin-1 beta modulates thyrotropin-induced thyroglobulin mRNA transcription through 3',5'-cyclic adenosine monophosphate. Endocrinology 1990; 127:1369-1374. 93. Kung AW, Lau KS. Interferon-gamma inhibits thyrotropin-induced thyroglobulin gene transcription in cultured human thyrocytes. J Clin Endocrinol Metab 1990; 70:1512-1517. 94. Turner CD, Chernoff SB, Taurog A et al. Differences in iodinated peptides and thyroid hormone formation after chemical and thyroid peroxidase-catalyzed iodination of human thyroglobulin. Arch Biochem Biophys 1983; 222:245-258. 95. Dunn JT, Anderson PC, Fox JW et al. The sites of thyroid hormone formation in rabbit thyroglobulin. J Biol Chem 1987; 262:16948-16952. 96. Champion BR, Rayner DC, Byfield P et al. Critical role of iodination for T cell recognition of thyroglobulin in experimental murine thyroid autoimmunity. J Immunol 1987; 139:3665-3670. 97. Gardas A. The influence of iodine on the immunological properties of thyroglobulin and its immunological complexes. Autoimmunity 1991; 9:331-336. 98. Ludgate M, Vassart G. The molecular genetics of three thyroid autoantigens: thyroglobulin, thyroid peroxidase and the thyrotropin receptor. Autoimmunity 1990; 7:201-211.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

123

99. Kimura SK, Kotani T, McBride OW et al. Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs. Proc Natl Acad Sci USA 1987; 84:5555-5559. 100. Libert F, Ruel J, Ludgate M et al. Thyroperoxidase, an auto-antigen with a mosaic structure made of nuclear and mitochondrial gene modules. EMBO J 1987; 6:4193-4196. 101. Seto P, Hirayu H, Magnusson RP et al. Isolation of a complementary DNA clone for thyroid microsomal antigen - homology with the gene for thyroid peroxidase. Journal of Clinical Investigation 1987; 80:1205-1208. 102. Pinchera A, Mariotti S, Chiovato L et al. Cellular localization of the microsomal antigen and the thyroid peroxidase antigen. Acta Endocrinol Suppl (Copenh) 1987; 281:57-62. 103. Kotani T, Umeki K, Hirai K et al. Experimental murine thyroiditis induced by porcine thyroid peroxidase and its transfer by antigen-specific T cell lines. Isr 1990; J Med Sci 80:11-18. 104. Mariotti S, Chiovato L, Vitti P et al. Recent advances in the understanding of humoral and cellular mechanisms implicated in thyroid autoimmune disorders. Clin Immunol Immunopathol 1989; 50:S73-S78. 105. Collison KS, Banga JP, Barnett PS et al. Activation of the thyroid peroxidase gene in human thyroid cells: effect of thyrotrophin, forskolin and phorbol ester. J Mol Endocrinol 1989; 3:1-5. 106. Zanelli E, Henry M, Charvet B et al. Evidence for an alternate splicing in the thyroperoxidase messenger from patients with Graves’ disease. Biochem Biophys Res Commun 1990; 170:735-741. 107. Ashizawa K, Yamashita S, Tobinaga T et al. Inhibition of human thyroid peroxidase gene expression by interleukin 1. Acta Endocrinol (Copenh) 1989; 121:465-469. 108. Krogh RA, Kayser L, Bech K et al. Influence of interleukin 6 on the function of secondary cultures of human thyrocytes. Acta Endocrinol (Copenh) 1991; 124:577-582. 109. Ashizawa K, Yamashita S, Nagayama Y et al. Interferon-gamma inhibits thyrotropin-induced thyroidal peroxidase gene expression in cultured human thyrocytes. J Clin Endocrinol Metab 1989; 69:475-477. 110. Nakajima Y, Howells RD, Pegg C et al. Structure-activity analysis of microsomal antigen/ thyroid peroxidase. Molecular and Cellular Endocrinology 1987; 53:15-23. 111. Carrasco N. Iodide transport in the thyroid gland. Biochem Biophys Acta 1993; 1154:65-82. 112. Kaminsky SM, Levy O, Salvador C et al. Na(+)-I- symport activity is present in membrane vesicles from thyrotropin-deprived non-I(-)-transporting cultured thyroid cells. Proc Natl Acad Sci USA 1994; 91:3789-3793. 113. Raspe E, Costagliola S, Ruf J et al. Identification of the thyroid Na+/I- cotransporter as a potential autoantigen in thyroid autoimmune disease [see comments. Eur J Endocrinol 1995; 132:399-405. 114. Endo T, Kaneshige M, Nakazato M et al. Autoantibody against thyroid iodide transporter in the sera from patients with Hashimoto’s thyroiditis possesses iodide transport inhibitory activity. Biochem Biophys Res Commun 1996; 228:199-202. 115. Shewring G, Rees SB. An improved radio receptor assay for TSH receptor antibodies. Clin Endocrinol 1982; 17:409-411. 116. Huber GK, Safirstein R, Neufeld D et al. Thyrotropin receptor autoantibodies induce human thyroid cell growth and c-fos activation. J Clin Endocrinol Metab 1991; 72:1142-1147. 117. Ludgate M, Perret J, Paramentier M et al. Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSH-R antibodies. Mol Cell Endocrinol 1990; 73:R13-R18. 118. Adams DD, Purves HD. Abnormal responses in the assay of thyrotropin. Proc Univ Otago Med School 1956; 34:11-12. 119. Weetman AP, Yateman ME, Ealey PA et al. Thyroid-stimulating antibody activity between different immunoglobulin G subclasses. J Clin Invest 1990; 86:723-727. 120. Ueki Y, Eguchi K, Otsubo T et al. Abnormal B lymphocyte function in thyroid glands from patients with Graves’ disease. J Clin Endocrinol Metab 1989; 69:939-945.

124

Endocrine and Organ Specific Autoimmunity

121. McLachlan SM, Rapoport B. Editorial: Monoclonal, human autoantibodies to the TSH receptor—the holy grail and why are we looking for it? J Clin Endocrinol Metab 1996; 81:3152-3155. 122. Davies TF, Bobovnikova Y, Weiss M et al. Characterization of the murine immune response to murine TSH receptor ectodomain: 2-Development of monoclonal antibodies which inhibit TSH binding and block TSH stimulation. Clin Exp Immunol 1997;(submitted). 123. Adams DD, Fastier FN, Howie JB et al. Stimulation of the human thyroid by infusions of plasma containing LATS protector. J Clin Endocrinol Metab 1974; 39:826-832. 124. Zakarija M, McKenzie JM. Pregnancy-associated changes in thyroid-stimulating antibody of Graves’ disease and the relationship to neonatal hyperthyroidism. J Clin Endocrinol Metab 1983; 57:1036-1040. 125. Matsuura N, Konishi J, Fujieda K et al. TSH-receptor antibodies in mothers with Graves’ disease and outcome in their offspring. Lancet 1988; 1:14-17. 126. Zakarija M, McKenzie JM, Eidson MS. Transient neonatal hypothyroidism: characterization of maternal antibodies to the thyrotropin receptor. J Clin Endocrinol Metab 1990; 70:1239-1246. 127. Tamai H, Hirota Y, Kasagi K et al. The mechanism of spontaneous hypothyroidism in patients with Graves’ disease after antithyroid drug treatment. J Clin Endocrinol Metab 1987; 64:718-722. 128. Tahara K, Toshiaki B, Minegishi T et al. Immunoglobulins from Graves’ disease patients interact with different sites on TSH receptor/LH-CR receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients. Biochem Biophys Res Commun 1991; 179:70-77. 129. Kosugi S, Ban T, Kohn LD. Identification of thyroid stimulating antibody-specific interaction sites in the N-terminal region of the TSH receptor. Mol Endocrinol 1993; 7:114-130. 130. Nagayama Y, Wadsworth HL, Russo D et al. Binding domains of stimulatory and inhibitory TSH receptor autoantibodies determined with chimeric TSH-LH/CG receptors. J Clin Invest 1991; 88:336-340. 131. Vlase H, Matsuoka N, Graves PN et al. Folding-dependent binding of thyrotropin (TSH) and TSH receptor autoantibodies to the murine TSH receptor ectodomain. Endocrinology 1997; 138:1658-1666. 132. Vlase H, Graves PN, Magnusson R et al. Human autoantibodies to the TSH receptor: recognition of linear, folded and glycosylated recombinant extracellular domain. J Clin Endocrinol Metab 1995; 80:46-53. 133. Wagle NM, Patibandla SA, Dallas JS et al. Thyrotropin receptor-specific antibodies in BALB/ cJ mice with experimental hyperthyroxinemia show a restricted binding specificity and belong to the IgG1 subclass. Endocrinology 1995; 136:3461-3469. 134. Wagle N, Dallas JS, Seetharamajah et al. Induction of hyperthyroxinemia in BALB/c but not in several other strains of mice. Autoimmunity 1994; 18:103-112. 135. Costagliola S, Many MC, Stalmans FM et al. Recombinant thyrotropin receptor and the induction of autoimmune thyroid disease in BALB/c mice: a new animal model. Endocrinology 1994; 135:2150-2159. 136. Morgenthaler NG, Kim MR Tremble J et al. Human immunoglobulin G autoantibodies to the thyrotropin receptor from Epstein-Barr virus transformed B lymphocytes: Characterization by immunoprecipitation with recombinant antigen and biological activity. J Clin Endocrinol Metab 1996; 81:3155-3161. 137. Akamizu T, Matsuda F, Okuda J et al. Molecular analysis of stimulatory anti-thyrotropin receptor antibodies (TSAbs) involved in Graves’ disease. Isolation and reconstruction of antibody genes, and production of monoclonal TSAbs. J Immunol 1996; 157:3148-3152. 138. Yoshida H, Amino N, Yagawa K et al. Association of serum antithyroid antibodies with lymphocytic infiltration of the thyroid gland: Studies of seventy autopsied cases. J Clinl Endocrinol Metab 1978; 46:859-859. 139. Davies TF D.B.E, ed. Thyroid autoantibodies and disease (Autoimmune endocrine disease), 1993:127-138, John Willey & Sons, New York.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

125

140. Dietrich G, Piechaczyk M, Pau B et al. Evidence for a restricted idiotypic and epitopic specificity of anti-thyroglobulin autoantibodies in patients with autoimmune thyroiditis. Eur J Immunol 1991; 21:811-814. 141. Caturegli P, Mariotti S, Kuppers RC et al. Epitopes on thyroglobulin: a study of patients with thyroid disease. Autoimmunity 1994; 18:41-49. 142. Tomer Y. Anti-thyroglobulin autoantibodies in autoimmune thyroid diseases: cross-reactive or pathogenic? Clin Immunol Immunopathol 1997; 82:3-11. 143. Bogner U, Hegedus L, Hansen JM et al. Thyroid cytotoxic antibodies in atrophic and goitrous autoimmune thyroiditis. Eur J Endocrinol 1995; 132:69- 73. 144. Prentice L, Kiso Y, Fukuma N et al. Rees Smith B. Monoclonal thyroglobulin autoantibodies: Variable region analysis and epitope recognition. J Clin Endocrinol Metab 1995; 80:977-986. 145. Henry M, Zanelli E, Piechaczyk M et al. A major human thyroglobulin epitope defined with monoclonal antibodies is mainly recognized by human autoantibodies. Eur J Immunol 1992; 22:315-319. 146. Henry M, Malthiery Y, Zanelli E et al. Epitope mapping of human thyroglobulin. Heterogeneous recognition by thyroid pathologic sera. J Immunol 1990; 145:3692-3698. 147. Kuppers RC, Bresler HS, Lynne Burek C et al. Immunodominant determinants of thyroglobulin associated with autoimmune thyroiditis. In: Molecular Immunobiology of SelfReactivity (Bona CA, Kausnick AK, eds.) 1992:247-284, Dekker, New York. 148. Dong Q, Ludgate M, Vassart G. Towards an antigenic map of human thyroglobulin: identification of ten epitope-bearing sequences within the primary structure of thyroglobulin. J Endocrinol 1989; 122:169-176. 149. Khoury EL, Hammond L, Bottazo GF et al. Presence of organic specific “microsomal” autoantigen on the surface of human thyroid cells in culture: Its involvement with complement mediated cytotoxicity. Exp Immunol 1981; 45:316-328. 150. Okhamoto Y, Hamada N, Saito H et al. Thyroid peroxidase activity inhibiting immunoglobulins in patients with autoimmune thyroid disease. J Clin Endocrinol Metabol 1989; 68:534-541. 151. Banga JP, Barnett BS, McGregor AM. Immunological and molecular characteristics of the thyroid peroxidase autoantigen. Autoimmunity 1991; 8:335-343. 152. Chazenbalk DG, Constante G, Portoloano S et al. The immunodominant region on human thyroid peroxidase recognized by autoantibodies does not contain the monoclonal antibody 47/c21 linear epitope. J Clin Endocrinol Metab 1993; 77:1715-1718. 153. Hexham J Furmaniak W, Pegg C et al. Cloning of a human autoimmune response: preparation and signaling of a human anti-Tg autoantibody using a combinatorial approach. Autoimmunity 1992; 12:135-141. 154. Portolano S, Chazenbalk GD, Seto P et al. Recognition of recombinant autoimmune thyroid disease-derived Fab fragments of a dominant conformational epitope on thyroid peroxidase. J Clin Invest 1992; 90:720-726. 155. Hamada N, Jaeduck N, Portmann L et al. Antibodies against denatured and reduced thyroid microsomal antigen in autoimmune thyroid disease. J Clin Endocrinol Metab 1987; 64:230-238. 156. Zanelli E, Henry M, Malthiery Y. Use of recombinant epitopes to study the heterogeneous nature of the autoantibodies against thyroid peroxidase in autoimmune thyroid disease. Clin Exp Immunol 1992; 87:80-86. 157. Martin A, Goldsmith NK, Friedman EW et al. Intrathyroidal accumulation of T cell phenotypes in autoimmune thyroid disease. Autoimmunity 1990; 6:269-281. 158. McLachlan SM, Pegg CA, Atherton MC et al. Subpopulations of thyroid autoantibody secreting lymphocytes in Graves’ and Hashimoto thyroid glands. Clin Exp Immunol 1986; 65:319-328. 159. Mariotti S, del PG, Mastromauro C et al. The autoimmune infiltrate of Basedow’s disease: analysis of clonal level and comparison with Hashimoto’s thyroiditis. Exp Clin Endocrinol 1991; 97:139-146.

126

Endocrine and Organ Specific Autoimmunity

160. Aozasa M, Amino N, Iwatani Y et al. Separation and analysis of mononuclear cells infiltrating the thyroid of patients with Graves’ disease. Clin Immunol Immunopathol 1987; 43:343-353. 161. Ishikawa N, Eguchi K, Otsubo T et al. Reduction in the suppressor-inducer T cell subset and increase in the helper T cell subset in thyroid tissue from patients with Graves’ disease. J Clin Endocrinol Metab 1987; 65:17-23. 162. Ueki Y, Eguchi K, Otsubo T et al. Phenotypic analyses and concanavalin-A-induced suppressor cell dysfunction of intrathyroidal lymphocytes from patients with Graves’ disease. J Clin Endocrinol Metab 1988; 67:1018-1024. 163. Matsunuga MK, Eguchi K, Fakuda T et al. Class II major histocompatibility complex antigen expression and cellular interactions in thyroid glands of Graves’ disease. J Clin Endocrinol Metab 1986; 62:723-728. 164. Romagnani S. Human TH1 and TH2 subsets: Doubt no more. Immunol Today 1991; 12:256-257. 165. Abbas AK, Murphy KM, Sher A. Functional diversity of T lymphocytes. Nature 1996; 383:787-793. 166. Mackenzie WA, Davies TF. An intrathyroidal T cell clone specifically cytotoxic for human thyroid cells. Immunology 1987; 61:101-103. 167. Mackenzie WA, Schwartz AE, Friedman EW et al. Intrathyroidal T cell clones from patients with autoimmune thyroid disease. J Clin Endocrinol Metab 1987; 64:818-824. 168. Londei M, Bottazzo GF, Feldmann M. Human T cell clones from autoimmune thyroid glands: specific recognition of autologous thyroid cells. Science 1985; 228:85-89. 169. Fisfalen ME, DeGroot LJ, Quintans J et al. Microsomal antigen-reactive lymphocyte lines and clones derived from thyroid tissue of patients with Graves’ disease. J Clin Endocrinol Metab 1988; 66:776-784. 170. Dayan CM, Londei M, Corcoran AE et al. Autoantigen recognition by thyroid-infiltrating T cells in Graves disease. Proc Natl Acad Sci U S A 1991; 88:7415-7419. 171. Makinen,TG Wagar G, Apter L et al. Evidence that the TSH receptor acts as a mitogenic antigen in Graves’ disease. Nature 1978; 275:314-315. 172. Tandon N, Freeman MA, Weetman AP. T cell response to synthetic TSH receptor peptides in Graves’ disease. Clin Exp Immunol 1992; 89:468-473. 173. Fan J-L, Desai RK, Dallas JS et al. Heterogenity in cellular and antibody responses against thyrotropin receptor in patients with Graves’ disease detected using synthetic peptides. J Autoimmun 1993; 6:799-808. 174. Sakata SS, Tanaka S, Okuda K et al. Autoimmune T cell recognition sites of human TSH receptor in Graves’ disease. Mol Cell Endocrinol 1993; 92:77-82. 175. Soliman M, Kaplan E, Fisfalen M-E et al. T cell reactivity to recombinant human thyrotropin receptor extracellular domain and thyroglobulin in patients with autoimmune and nonautoimmune thyroid diseases. J Clin Endocrinol Metab 1995; 80:206-213. 176. Soliman M, Kaplan E, Yanagawa T et al. J Clin Endocrinol Metab 1995; 80:905-914. 177. Nagy E, Morris JC, Burch HB et al. Thyrotropin receptor T cell epitopes in autoimmune thyroid disease. Clin Immunol Immunopath 1995; 75:117-124. 178. Kellermann SA, McCormick DJ, Freeman SL et al. TSH receptor sequences recognized by CD4+ T cells in Graves’ disease and healthy controls. J Autoimmun 1996; 8:695-698. 179. Soliman M, Kaplan E, Guimaraes T et al. T cell recognition of residue 158-176 in thyrotropin receptor confers risk for development of thyroid autoimmunity in siblings in a family with Graves’ disease. Thyroid 1996; 6:545-551. 180. Martin A, Nakashima M, Zhou A et al. Detection of major T cell epitopes on the human TSH receptor by overriding immune heterogeneity in patients with Graves’ disease. J Clin Endocrinol Metab 1997; 82:3361-3366. 181. Morris JC, Gibson JL, Haas EJ et al. Identification of epitopes and affinity purification of thyroid stimulating autoantibodies using synthetic human TSH receptor peptides. Autoimmunity 1994; 17:287-299. 182. Vlase H, Nakashima M, Graves PN et al. Defining the major antibody epitopes on the human TSH receptor in immunized mice. Endocrinology 1995; 136:4415-4423.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

127

183. Martin A, Magnusson RP, Kendler DL et al. Endogenous antigen presentation by autoantigen-transfected E-B virus lymphoblastoid cells. J Clin Invest 1993; 91:1567-1574. 184. Elliott EA, McFarland HI, Nye SH et al. Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid. J Clin Invest 1996; 98:1602-1612. 185. Nishikawa T, Rapoport B, McLachlan SM. Exclusion of two major areas on thyroid peroxidase from the immunodominant region containing the conformational epitopes recognized by human autoantibodies. J Clin Endocrinol Metab 1994; 79:1648-1654. 186. Davies TF, Platzer M, Schwartz AE et al. Functionality of thyroid-stimulating antibodies assessed by cryopreserved human thyroid cell bioassay. J Clin Endocrinol Metab 1983; 57:1021-1028. 187. Huber GK, Safirstein R, Neufeld D et al. TSH receptor autoantibodies induce human thyroid cell growth and c-fos activation. J Clin Endocrinol Metab 1991; 72:1142-1147. 188. Champion BR, Page KR, Parish N et al. Identification of a thyroxine-containing self-epitope of thyroglobulin which triggers thyroid autoreactive T cells. J Exp Med 1991; 174:363-370. 189. Kong YC, McCormick DJ, Wan Q et al. Primary hormonogenic sites as conserved autoepitopes on thyroglobulin in murine autoimmune thyroiditis. Secondary role of iodination. J Immunol 1995; 155:5847-5854. 190. Dayan CM, Londei M, Corcoran et al. Autoantigen recognition by thyroid-infiltrating T cells in Graves disease. Proc Natl Acad Sci USA 1991; 88:7415-7419. 191. Kawakami Y, Fisfalen ME, DeGroot LJ. Proliferative responses of peripheral blood mononuclear cells from patients with autoimmune thyroid disease to synthetic peptide epitopes of human thyroid peroxidase. Autoimmunity 1992; 13:17-26. 192. Kohno Y, Nakayima H, Tarutani O. Interspecies cross-reactive determinants of thyroglobulin recognized by autoantibodies. Clin Exp Immunol 1985; 61:44-48. 193. Weiss A. Structure and function of the T cell antigen receptor. J Clin Invest 1990; 86:1015-1022. 194. Davis MM, Bjorkman PJ. T cell antigen receptor genes and T cell recognition [published erratum appears in Nature 1988 Oct 20 335(6192):744. Nature 1988; 334:395-402. 195. Davies TF, Martin A, Concepcion ES et al. Evidence of limited variability of antigen receptors on intrathyoidal T cells in autoimmune thyroid disease. N Engl J Med 1991; 325:238-244. 196. Davies T, Concepcion E, Ben NA et al. T cell receptor V gene usage in autoimmune thyroid disease: Direct assessment by thyroid aspiration. J Clin Endocrinol Metab 1993; 76:660-666. 197. Heufelder AE, Wenzel BE, Scriba PC. Antigen receptor variable region repertoires expressed by T cells infiltrating thyroid, retroorbital, and pretibial tissue in Graves’ disease. J Clin Endocrinol Metab 1996; 81:3733-3739. 198. Caso-Pelaez E, McGregor AM, Banga JP. A polyclonal T cell repertoire of V-alpha and Vbeta t cell receptor gene families in intrathyroidal T lymphocytes of Graves’ disease patients. Scandinavian J Immunol 1995; 41:141-147. 199. Nakashima M, Martin A, Davies TF. Intrathyroidal T cell accumulation in Graves’ disease: delineation of mechanisms based on in situ T cell receptor analysis. J Clin Endocrinol Metab 1996; 81:3346-3351. 200. Heufelder AE, Herterich S, Ernst G et al. Analysis of retroorbital T cell antigen receptor variable region gene usage in patients with Graves’ opthalmopathy. Eur J Endocrinol 1995; 132:266-277. 201. Bottazzo GF, Pujol BR, Hanafusa T et al. Role of aberrant HLA-DR expression and antigen presentation in induction of endocrine autoimmunity. Lancet 1983; 2:1115-1119. 202. Davies TF. The role of human thyroid cell Ia (DR) antigen in thyroid autoimmunity. In: Autoimmunity and the Thyroid (Walfish, P, Wall J.R, and Volpe, R. Eds) Academic Press, New York 1985. 203. Hanafusa T, Pujol BR, Chiovato L et al. Aberrant expression of HLA-DR antigen on thyrocytes in Graves’ disease: Relevance for autoimmunity. Lancet 1983; 2:1111-1115.

128

Endocrine and Organ Specific Autoimmunity

204. Migita K, Eguchi K, Otsubo T et al. Cytokine regulation of HLA on thyroid epithelial cells. Clin Exp Immunol 1990; 82:548-552. 205. Neufeld DS, Davies TF. Strain-specific determination of the degree of thyroid cell MHC class II antigen expression: evaluation of established Wistar and Fisher rat thyroid cell lines. Endocrinology 1990; 127:1254-1259. 206. Platzer M, Neufeld DS, Piccinini LA et al. Induction of rat thyroid cell MHC class II antigen by thyrotropin and gamma-interferon. Endocrinology 1987; 121:2087-2092. 207. Belfiore A, Mauerhoff T, Pujol BR et al. De novo HLA class II and enhanced HLA class I molecule expression in SV40 transfected human thyroid epithelial cells. J Autoimmun 1991; 4:397-414. 208. Neufeld DS, Platzer M, Davies TF. Reovirus induction of MHC class II antigen in rat thyroid cells. Endocrinology 1989; 124:543-545. 209. Kabel PJ, Voorbij HA, De HM et al. Intrathyroidal dendritic cells. J Clin Endocrinol Metab 1988; 66:199-207. 210. Hutchings P, Rayner DC, Champion BR et al. High efficiency antigen presentation by thyroglobulin-primed murine spleen B cells. European Journal of Immunology 1987; 17:393-398. 211. Reiser H, Stadecker MJ. Costimulatory B7 molecules in the pathogenesis of infectious and autoimmune diseases. N Engl J Med 1996; 335:1369-1377. 212. Tandon N, Metcalfe RA, Barnett D et al. Expression of the costimulatory molecule B7/BB1 in autoimmune thyroid disease. Q J Med 1994; 87:231-236. 213. Matsuoka N, Eguchi K, Kawakami A et al. Lack of B7-1/BB1 and B7-2/B70 expression on thyrocytes of patients with Graves’ disease. Delivery of costimulatory signals from bystander professional antigen-presenting cells. J Clin Endocrinol Metab 1996; 81:4137-4143. 214. Heufelder AE, Smith TJ, Gorman CA et al. Increased induction of HLA-DR by interferon gamma in cultured fibroblasts derived form patients with Graves’ ophthalmopathy, dermopathy. J Clin Endocrinol Metab 1991; 73:307-313. 215. Phillips DI, Barker DJ, Rees SB et al. The geographical distribution of thyrotoxicosis in England according to the presence or absence of TSH-recepotr antibodies. Clin Endocrinol (Oxf) 1985; 23:283-287. 216. Cox SP, Phillips DIW, Osmond C. Does infection initiate Graves disease ?: a population based 10 year study. Autoimmunity 1989; 4:43-49. 217. Valtonen VV, Ruutu P, Varis K et al. Serological evidence for the role of bacterial infections in the pathogenesis of thyroid diseases. Acta Med Scand 1986; 219:105-111. 218. Gammon G, Sercarz E. How some T cells escape tolerance induction. Nature 1989; 342:183-185. 219. Mason D. Autoimmunity. Sci Prog 1992; 76:125-138. 220. Jones DE, Diamond AG. The basis of autoimmunity: an overview. Baillieres Clin Endocrinol Metab 1995; 9:1-24. 221. Ray SK, Putterman C, Diamond B. Pathogenic autoantibodies are routinely generated during the response to foreign antigen: A paradigm for autoimmune disease. Proc Natl Acad Sci USA 1996; 93:2019-2024. 222. Bech K, Larsen JH, Nerup J. Yersinia enterocolitica, infection and thyroid disorders. Lancet ii:1974; 951-952. 223. Bech K, Clemmensen O, Larsen JH et al. Thyroid disease and Yersinia. Lancet 1977; 1:1060-1061. 224. Takuno H, Sakata S, Miura K. Antibodies to Yersinia Enterocolitica serotype 3 in autoimmune thyroid disease. Endocrinologia Japonica 1990; 37:489-500. 225. Lidman K, Eriksson U, Norberg R et al. Indirect immunofluorescence staining of human thyroid by antibodies occurring in Yersinia enterocolitica infections. Clin Exp Immunol 1976; 23:429-435. 226. Gripenberg M, Miettinen A, Kurki P et al. Humoral immune stimulation and anti-epithelial antibodies in Yersinial infections. Arthritis Rheum 1978; 21:904-908. 227. Ingbar SH. A possible role for bacterial antigens in the pathogenesis of autoimmune thyroid disease. Plenum Press, New York 1987; 1:35-44.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

129

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.

130

Endocrine and Organ Specific Autoimmunity

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.

Insights into the Molecular Mechanisms of the Autoimmune Thyroid Diseases

131

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.

132

Endocrine and Organ Specific Autoimmunity

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.

Endocrine and Organ Specific Autoimmunity

134

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.

Insulin Autoimmune Syndrome (IAS, Hirata Disease)

135

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

Endocrine and Organ Specific Autoimmunity

136

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 <71.8pmpo/l and <5%, respectively.

Table 7.3a. Clinical Features in IAS Cases in Non-Japanese East Asians patient

Age

Sex

Disease/Drug

Race

Reference

1 2 3 4 5 6 7 8 9 10

52 48 31 18 67 28 26 24 21 11

M F F F F F F F F F

Vasculitis ? Graves’/MTZ Graves’/MTZ Graves’/MTZ — Graves’/MTZ Graves’/MTZ Graves’/MTZ Graves’/MTZ Hashimoto/

Chinese Chinese Korean Chinese Chinese Chinese Chinese Chinese Chinese Chinese

29 29 30 31 32 32 32 32 unpublished# unpublished*

unpublished#, by Lin from Taiwan. unpublished*, by Wacharasindhu from Thailand.

Insulin Autoimmune Syndrome (IAS, Hirata Disease)

137

Table 7.3b. Clinical Features in IAS Cases Out of Asia patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

Age

Sex

Disease/Drug

Race

42 58 43 3 26 48 5 82 61 55 44 84 74 NS NS NS NS NS 33 NS NS 63 57 5 57 45 48

M F F M F F M F F F M F M NS NS NS NS NS NS NS NS M F M F F F

Pulmonary TB/INH RA/NS — — — Brain damage — Lupus/hydralazine RA/asthma/penicillamine Graves’/carbimazole Pulmonary TB/INH RA/pyritinol Hypertention/Hydralazine — — — — — RA/penicillamine RA/pyritinol — — — — Malaria/NS — —

NS White Hispanic NS White White Hispanic White White White Morocco White Black White White White White White White White White White White White White White White

Reference 7 33 33 33 33 33 34 35 29 36 37 38 39 40 40 40 40 41 28 42 43 44 45 46 unpublished# 21 47

NS, not stated; RA, rheumatoid arthritis; INH, isoniatid; TB, tuberculosis. -, no associated disease. unpublished#, by Dozio from Italy.

showed monoclonal autoantibodies to human insulin.20 Recently, we reported 1 Netherlander IAS patient.21 However, 50 of 51 Japanese IAS patients so far examined, 2 Korean, and 1 Chinese IAS patients showed polyclonal autoantibodies to human insulin (Table 7.4).20 Recently a Thai IAS patient was shown to possess a polyclonal insulin autoantibody. It is likely that the incidence of polyclonal IAS is relatively high among East Asians, whereas monoclonal IAS is more prevalent in Caucasians.

Critical Amino Acids for IAS Polyclonal Responder and Importance of DR Gene Products in the Presentation of Human Insulin Antigen As reported in the study of serological typing of 27 Japanese IAS patients, Cw4/B62/ DR4 was a highly prevalent allelic combination.10 Table 7.4 shows the summary of clinical characteristics of IAS polyclonal responders at onset of IAS. Japanese IAS polyclonal responders (except patients 45 and 49) possessed HLA-DR4/DQ3, whereas the remaining two (patients 45 and 49) possessed DR9/DQ3 and not DR4; the American white polyclonal responder possessed DR4/DQ3 (Table 7.4). Ninety-six percent (48/50) of Japanese IAS

138

Endocrine and Organ Specific Autoimmunity

patients had DR4 (Odds ratio, 39.9, p<10-4). DR9 was positive in 12 (24%) Japanese IAS patients, though, this was not significant compared with Japanese healthy controls (Odds ratio, 0.8, p>0.65). The 48 DR4-positive Japanese IAS polyclonal responders consisted of 42 DRB1*0406positive, 5 DRB1*0403-positive, and 1 DRB1*0407-positive patients (Table 7.5). All 48 DR4positive Japanese IAS polyclonal responders possessed DQA1*0301/DQB1*0302 regardless of the differences in DR4 alleles. The two Korean and the Chinese IAS polyclonal responders were also positive for DRB1*0406/DQA1*0301/DQB1*0302. The phenotype of the American polyclonal responder was DRB1*0407/DQA1*0301/DQB1*0301. Thus, the DR4positive IAS polyclonal responders possessed DRB1*0406, DRB1*0403, or DRB1*0407 for DR4 alleles, and DQA1*0301/DQB10302 or DQA1*0301/DQB1*0301 for DQ3 alleles. For only DRB1*0406 individuals, incidence of B62/Cw4 was compared in Japanese IAS polyclonal responders and Japanese healthy controls. Sixty-seven percent (28/42) of Japanese IAS polyclonal responders had B62/Cw4, and 56% (5/9) of controls had (Odds ratio, 16; 95% confidence interval, 0.37-6.91). This indicates that the class I alleles are less important. The differences in DQβ1 alleles encoding DQ3 among the IAS polyclonal responders suggest that DQ αand DQ βchains are not important in the development of IAS. We showed that T cells from polyclonal IAS patients with DRB1*0406/DQA1*0301/DQB1*0302 alleles proliferated in the presence of autologous antigen-presenting cells that had been exposed to 40 µM human insulin.22 The proliferative response of T cells was completely blocked by anti-HLA-DR but not by anti-HLA-DQ monoclonal antibodies (Fig 7.2).23 Moreover, experiments with DRB1*0406 transfectants supported the view that DR gene products participate in the presentation of human insulin antigens (Table 7.6).23 The HLA-DRβ1-chains encoded by DRB1*0406, DRB1*0403, and DRB1*0407 share a sequence motif (Leu-Leu-Glu-Gln-Arg-Arg-Ala-Glu) that spans the amino acid residues 67-74 of the third hypervariable region. The two DR9/DQ3 Japanese IAS polyclonal responders (patients 45 and 49) were DRB1*0901/ DQA1*0301/DQB1*0303 homozygous. The products of DRB1*0406, DRB1*0403, and DRB1*0901 share the sequence motif ArgArg-Ala-Glu, corresponding to amino acid residues 71-74 of the DR β1-chain. Comparison of this region of the DRβ1-chain and other DRB1 allele products reveals that Arg71 and especially Glu74 may be important for polyclonal insulin autoantibody production in IAS, whereas residues 72 and 73 (Arg-Ala) are common in most DRB1 molecule (Table 7.4).

Patients with Graves’ Disease Who Developed IAS Possess HLA-B62/Cw4/DR4 Carrying DRB1*0406 As shown in Table 7.2, patients with Graves’ disease who developed IAS had received methimazole (MTZ). None of the IAS patients with Graves’ disease had received propylthiouracil before the onset of IAS. We examined differences in HLA class II genes between the patients with Graves’ disease treated with MTZ who developed IAS and those who had received MTZ but did not develop IAS.24 All 13 patients with Graves’ disease who developed IAS by MTZ therapy possessed a specific allelic combination, B62/Cw4/DR4 carrying DRB1*0406, whereas only one of 50 Graves’ disease patients without IAS had B62/Cw4/ DR4 and those 50 did not possess DRB1*0406. It is highly likely that patients with Graves’ disease develop IAS via treatment with MTZ when their B62/Cw4/DR4 carry DRB1*0406.

Different Amino Acids for IAS Monoclonal Responder The IAS monoclonal responder group consisted of 6 patients: 1 Japanese, 1 Norwegian, 1 Swiss, and 3 Italians (Table 7.7). Three of the 6 IAS monoclonal responders were DR4-positive and their class II phenotypes were DRB1*0405/DQA1*0301/DQB1*0401,

Insulin Autoimmune Syndrome (IAS, Hirata Disease)

139 Fig. 7.2. T cell responses (top from donor MI and bottom from donor SO) to human insulin blocked by anti-HLA DR monoclonal antibody (mAb). Results represent the mean of triplicates (SD<15% of the mean). R, enriched T cells; RI, R+ human insulin; A, antigen-presenting cells; DR, R+A+I+ anti-HLADRmAb; DQ, R+A+I+antiHLA-DQmAB; CI, R+A+ I+anti-HLA class I mAb; IgG1, R+A+I+mouse IgG1; IgG2a, R+A+I+mouse IgG2a.

DRB1*0401/DQA1*0301/DQB1*0301, and DRB1*0402/DQA1*0301/DQB1*0301 (Table 7.7). The remaining three IAS monoclonal responders had nonDR4 and nonDR9 phenotypes. Of the IAS monoclonal responders, not Glu74 but Ala74 as well as Asp57 and Gly86 on the DRB1 chain were shared. The finding suggests the mechanism of monoclonal insulin autoantibody production in IAS. One appropriate question that our data raises is whether antigen presentation efficiency of insulin peptides in patients with Ala74 in the DR β1-chain is indeed reduced compared with that in Glu74-positive patients.

Possible Role of the Specific Amino Acids on the DR β-chain in IAS Pathogenesis Based on the findings described above, we conclude that 1) DR4 is the dominant phenotype in terms of susceptibility to IAS, 2) DRB1*0406 is associated with the highest risk for the susceptibility to IAS, and 3) Glu74 in the DR4B1 chain is essential for polyclonal IAA production in IAS. Ser37 in the DR4 B1-chain may have a significant additive effect on polyclonal autoantibody production (Table 7.5). The three-dimensional structure of the HLA class II DR1 molecules determined by X-ray crystallography has shown an open-ended groove in which the peptides processed by antigen-presenting cells are bound as straight extended chains25 and an anchoring peptide side chain of the processed peptides was found to fit in a prominent nonpolar pocket near one end of the binding groove.25 Matsushita et al26 reported that peptide of human insulin α-chain (8TSICSLYQLE17) was shown to bind specifically to DRB1*0406 using its

1 2 3 4 5 6 7

8 9 10 11 12 13 14 15 16

17 18 19 20 21 22 23 24 25 26 27

Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese

Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese Japanese

Patient no.

Japanese Japanese Japanese Japanese Japanese Japanese Japanese

Ethnic Background

64 58 49 69 55 53 69 36 43 68 66

62 54 61 44 39 69 57 68 48

26 52 47 58 54 36 45

Age (years)

M F M F M F M F F M F

M M F M F M F F M

F F M M F M F

T-IRI Sex

4.6 12.0 11.9 167.2 0.9 35.0 47.0 192.5 23.0 5.4 21.0

12.6 4.4 5.1 3.0 2.7 12.3 2.8 11.7 11.1

114.3 54.1 23.4 0.8 60.0 8.2 7.2

I-insulin (pmol/l X 103)

82 81 48 67 48 55 65 79 57 64 57

69 81 72 70 69 65 51 68 81

69 73 79 48 70 59 32

Binding (%)

MPG — MPG TBM MPG — MTZ MTZ MTZ MPG MPG

— MPG MPG MPG MTZ — — MPG MTZ

MTZ GTG — — MTZ — MTZ

Drug Graves’ Bronchial asthma — — Graves’ — Graves’ Hashimoto’s IgA nephropathy — Liver dysfunction Cataract Dermatitis Graves’ — — Liver dysfunction Graves’ Drug-induced arthritis Dermatitis — Liver dysfunction NIDDM Liver dysfunction — Graves’ Graves’ Graves’ Liver dysfunction Cataract

Associated Disease

Table 7.4. Summary of Clinical Characteristics of IAS Polyclonal Responders at Onset of IAS

4, 13 4, 12 4, 12 4,— 4, 8 4, 9 4, 8 2, 4 4,— 4, 6 4, 9

4, 9 2, 4 4, 9 2, 4 4,— 4, 6 4, 9 4, 8 4,—

4,— 4, 13 4, 9 4,— 4,— 4, 9 4, 8

HLA-DR

140 Endocrine and Organ Specific Autoimmunity

49 54 70 67 50 52 74 54 79 49 59 66 84 42 71 70 65 64 49 71 81 77 71 61 31 18 61 F M F F F M M M M F M M F F F F M M F M M M F F F F F 18.0 3.4 46.8 120.0 1.7 13.3 5.2 3.0 3.1 1.4 24.3 6.2 5.0 2.5 5.1 4.5 1.1 9.3 5.4 3.0 1.4 0.7 7.4 12.0 8.9 36.4 4.8 83 67 94 80 64 38 78 76 70 88 69 91 73 24 67 84 50 48 75 68 92 64 80 90 62 82 1:64* MTZ MPG MTZ — GTT — — — — — MPG — MPG MPG — INF-α — Steroid — MPG — Aceglatone AHT drugs MTZ MTZ MTZ PNC Graves’ Liver dysfunction Graves’ — Urticaria — — — — NIDDM Liver dysfunction Hypertension Liver dysfunction Liver dysfunction — Renal cell carcinoma — Polymyositis — Liver cirrhosis — Urinary bladder carcinoma Hypertension Graves’ Graves’ Graves’ Rheumatoid arthritis 4,— 4,— 4,— 4, 8 1, 4 4, 6 4,— 4, 8 2, 4 4, 9 4, 8 4, — 4, 6 4, 9 4, 15 4, 8 4, 8 9, — 2, 4 4, 12 4, 14 9,— 4, 6 4,— 4, 9 4, — 4, 5

Hypoglycemic attacks occurred ª6 weeks after drug administration. All of the patients have remained healthy since the resolution of the hypoglycemic attacks.21 Abdominal computed tomography, abdominal ultrasound, abdominal angiography, and pancreaticocholangiography examinations performed in six patients failed to reveal the presence of a pancreatic tumor. Total immunoreactive insulin (T-IRI) (normal range, <5 mU/ml)10 and 125I-labeled human insulin binding (normal range, <5%)10 were measured as previously described. Methimazole (MTZ) was administered orally for treatment of Graves’ disease. Gold thioglucose (GTG) was administered intramuscularly for treatment of bronchial asthma. α-mercaptopropionyl glycine (MPG) was administered orally for treatment of liver dysfunction, cataracts, or dermatitis. Only one tablet (50 mg) of tolbutamide (TBM) was adminstered orally for treatment of non-insulin dependent diabetes (NIDDM) before the hypoglycemic attack. Glutathione (GTT) was administered intravenously for treatment of urticaria. Interferon-α (IFN-α) was administered intravenously for treatment of renal cell carcinoma.27 The anti-cancer drug aceglatone was administered orally for treatment of urinary bladder carcinoma. Japanese patient 50 was taking antihypertensive (AHT) drugs when IAS developed. Penicillamine (PNC) was administered orally for treatment of rheumatoid arthritis. Japanese patients 33, 36, 42, 46, and 48 possessed the DRB1*0403 allele. Japanese patient 37 and the white American patient possessed the RB1*0407 allele. The remaining patients, with the exception of Japanese patients 45 and 49 (DR9 homozygotes), possessed the DRB1*0406 allele. *1:64 was expressed as positive.28,29

Japanese 28 Japanese 29 Japanese 30 Japanese 31 Japanese 32 Japanese 33 Japanese 34 Japanese 35 Japanese 36 Japanese 37 Japanese 38 Japanese 39 Japanese 40 Japanese 41 Japanese 42 Japanese 43 Japanese 44 Japanese 45 Japanese 46 Japanese 47 Japanese 48 Japanese 49 Japanese 50 Korean 1 Korean 2 Chinese 1 White American 1

Insulin Autoimmune Syndrome (IAS, Hirata Disease) 141

106 40 (38) 29 (27) 9 (8) 7 (7) 2 (2) 70 (66) 74 (70) 26 (25) 23 (22)

48 (96) 12 (24) 42 (84) 5 (10) 1 (2) 50 (100) 50 (100) 48 (96) 48 (96)

Control

50

Data are n (%) or OR (95% confidence interval).

n DRB1 allele DR4 DR9 DRB1*0406 DRB1*0403 DRB1*0407 Glu74 in β-chain DBQ1 allele DQA1*0301 DQB1*0302 DQA1*0301/DQB1*0302

IAS Patients

44.1 (5.84-332) 73.8 (16.8-325 86.6 (18.8-380)

39.6 (9.12-171) 0.8 (0.39-1.82) 56.6 (20.4-156) 1.6 (0.47-5.22) 1.1 (0.09-12.0) 52.3 (6.95-393)

OR (95% confidence interval)

— — —

— Asn Ser Tyr Tyr —

37

— — —

— Glu Glu Glu Glu —

74

— — —

— Gly Val Val Gly —

86

DRB1 chain amino acid residue

Table 7.5. Incidence of DRB1 Alleles, Glu74 in DR B1-Chain and DBQ1 Alleles in Japanese IAS Polyclonal Responders and Control Subjects

142 Endocrine and Organ Specific Autoimmunity

1834 ± 23 2396 ± 199 247 ± 141 208 ± 25 328 ± 25 1563 ± 416

0406 0406

0406 0406 0405 0405

R Only

3711 ± 465 1618 ± 465 457 ± 4 2819 ± 251

905±79 5167 ± 263

R+L

3493 ± 572 1518 ± 391 ND 1309 ± 15

1554 ± 138 6104 ± 413

R+L+DTT

7280 ± 1,874 5727 ± 607 506 ± 46 572 ± 32

16,267 ± 306 ND

R+L+HI

11,854 ± 538 7492 ± 248 ND 630 ± 59

12,692 ± 374 15,218 ± 822

R+L+HI+DTT

a The 5 X 103 irradiated (80 Gy) L cell transfectants (L) were incubated with or without 40 mM human insulin (HI) in medium containing 2 mM DTT and cultured for 6 days with 5 X 104 enriched T cells (R, responder cells) [3H]thymidine incorporation of L cells alone were 3300 ± 97 for DRB1*0406 transfectant and 1586 ± 94 for DRB1*0405 transfectant, respectively.

IAS patients IS SM Healthy donors JJ KA NA ME

DRB1*

[3H]thymidine Incorporation (cpm)

Table 7.6. HLA-DRB1*0406-Transfected L Cells Can Present Human Insulin to Enriched T Cells from a Healthy Donor or an IAS Patient with-DRB1*0406 in the Presence of DTTa

Insulin Autoimmune Syndrome (IAS, Hirata Disease) 143

51 1 1 1 2 3

Patient no. 0405/0803 0401/— 0101/0601 1501/1502 0701/1501 0402/1101

DRB1 0301/0103 0301/— 0101/0102 0102/0103 0201/0102 0301/0501

DQA1 0401/0601 0301— 0501/0502 0601/0602 0201/0602 0301/0302

DQB1

Spontaneous remission Treatment Steroids Pancreatic surgery Plasmapheresis Azathioprine 6-Mercaptopurine 92 18 3 0 0 1

6 3 5 1 0

Female n=114

97

Male n=112

IAS patient

24(10.6%) 6(2.6%) 5(2.2%) 1(0.4%) 1(0.4%)

189(83.6%)

Total n=226

Table 7.8. Disease Course and Treatment in Japanese 226 IAS Patients

Japanese Norwegian Swiss Italian Italian Italian

Ethnic background Tyr Tyr Ser Ser Ser/Phe Tyr

37 Tyr Tyr Tyr Phe Phe/Tyr Phe/Tyr

47 Ser/Asp Asp Asp Asp Val/Asp Asp

57

Ala/leu Ala Ala Ala Ala/Gln Ala

74

DRB1 chain amino acid responders

Table 7.7. DRB1, DQA1 and DQB1 Alleles and Comparison of Amino Acid Residues in the DR B1 Chain in IAS Monoclonal Responders

Gly/Val Gly Gly Gly/Val Gly/Val Gly

86

144 Endocrine and Organ Specific Autoimmunity

Insulin Autoimmune Syndrome (IAS, Hirata Disease)

145

Fig. 7.3. Proposal of presentation and recognition of human insulin in insulin autoimmune syndrome.

10IxxLxxQ15 motif. The second anchor residue was reported to exhibit allele specificity in binding, especially with the amino acid residue 74 of DRB1 chain.26 However, the interaction of L (Leu) of insulin peptide with 74Glu in the DR4 β-chain has remained questionable because Leu was a hydrophobic residue and Glu was an acidic residue. Other portions of human insulin may be a candidate for presentation and recognition for T cells in IAS (in preparation). Although there are controversial points in the antigen-HLA-DRB1*0406 molecule-T cell receptor interaction, a reducing compound such as MTZ, MPG, or glutathione may cleave disulfide bonds in vivo and expose self-antigens such as insulin-derived peptides to DRB1*0406 molecules on antigen-presenting cells, resulting in insulin specific proliferating T cells. As mentioned previously, T cell recognition of human insulin in the context of DRB1*0406 molecules showed the highest risk for the susceptibility to IAS in the polyclonal IAS responders, whereas T cell recognition in the context of DRB1*0403 or DRB1*0407 did not. When human insulin-derived peptides were tested (for example, amino acids 8-17 of α chain), they were indeed recognized by T cells in the context of DRB1*0403 or DRB1*0407 (in preparation). Because polyclonal IAS patients exhibit typical polyclonal immunoglobulin G response to human insulin, the response may be an antigen-driven immune one with T cell help. Accordingly, typical HLA-and peptide-restricted recognition may contribute to the initiating event in the pathogenesis of IAS, in which Glu74 may act as the primary residue in the peptide-binding interaction (Fig. 7.3).

146

Endocrine and Organ Specific Autoimmunity

Natural History of IAS Table 7.8 shows the clinical course and IAS treatments. More than 80% of Japanese IAS patients had spontaneous remission. Spontaneous remission may have developed in less than 3 months. Some patients needed medication to stop the persistent hypoglycemic attacks; steroids, azathioprine, or 6-mercaptopurine. In five patients, plasmapheresis was performed to wash out insulin autoantibodies from sera. In the early 1970s, partial pancreas excision surgery was performed in 6 patients because of the misdiagnosis of insulinoma. Hyperplasia of the islet β cells in the excised part of the pancreas was reported in some IAS patients.5,6 The current recommended treatment is six or more small meals and to avoid sweets except at the time of hypoglycemic attacks. α-glucosidase inhibitors may sometimes play a helpful role decreasing immunoreactive insulin in the sera after taking a meal.21

A Novel Concept of Type VII Hypersensitivity Introduced by Insulin Autoimmune Syndrome (Hirata’ s Disease) Disorders resulting from aberrant, excessive or uncontrolled immune responses are called hypersensitivity diseases. Hypersensitivity diseases that are supposed to be due to immune responses against self antigens are called autoimmune diseases. Based on the principal criterion of the type of immune responses that leads to tissue injury, the conventional classification consists of type I (immediate hypersensitivity), type II (antibody-mediated), type III(immune complex-mediated), and type IV (T cell-mediated). Two new types V and VI have recently been derived from type II and proposed as “antibody-mediated hyperfunction of the target tissues” of which Graves’ disease is representative and “antibody-dependent cell-mediated cytotoxicity (ADCC)”, respectively. We propose a new concept of type VII hypersensitivity defined as immunologic diseases which are induced by the release of self-antigens from bound autoantibodies in serum. The selfantigens in type VII hypersensitivity are supposed to be located in the liquid phase, unlike the self-antigens on the cell membranes in types II and IV hypersensitivities. IAS is a type VII hypersensitivity disease.14

Abstract The insulin autoimmune syndrome (IAS), or Hirata’s disease, is characterized by the combination of fasting hypoglycemia, a high concentration of total serum immunoreactive insulin, and the presence of autoantibodies to native human insulin in serum. Since Hirata et al. first described the disease in 1970, there have been 226 reported cases reported in Japan. IAS is the third leading cause of spontaneous hypoglycemia in Japan. Only 26 cases in Caucasians and 10 cases in East Asians excluding Japanese have been reported in the past 26 years. Insulin autoantibody production is monoclonal or polyclonal, with the majority of IAS cases classified as polyclonal. We observed a striking association between human leukocyte antigen (HLA) class II alleles DRB1*0406/DQA1*0301/DQB1*0302 and Japanese IAS cases with polyclonal insulin autoantibodies, and T cell recognition of human insulin in the context of DRB1*0406 molecules. Moreover, glutamate at position 74 in the HLA-DR4 B 1-chain was shown to be essential to the production of polyclonal insulin autoantibodies in IAS, whereas alanine at the same position of the HLA-DR B 1-chain might be important in the production of monoclonal insulin autoantibodies.

References 1. Schlosstein L, Terasaki PI, Bluestone R et al. High association of an HL-A antigen B27 with ankylosing spondylitis. N Engl J Med 1973; 288:704-706.

Insulin Autoimmune Syndrome (IAS, Hirata Disease)

147

2. Park MS, Terasaki PI, Ahmed AR et al. HLA-DRw4 in 91% of Jewish pempemphigus vulgaris patients. Lancet 1979; 2:441-443. 3. Juji T, Satake M, Honda Y et al. HLA antigens in Japanese patients with narcolepsy. Tissue Antigens 1984; 24:316-319. 4. Prochazka EJ, Terasaki PI, Park MS et al. Association of primary sclerosing cholangitis with HLA-DRw52a. N Engl J Med 1990; 322:1842-1844. 5. Hirata Y, Ishizu H, Ouchi N et al. Insulin autoimmunity in a case of spontaneous hypoglycemia. J Jpn Diabetes Soc 1970; 13:312-320. 6. Hirata Y, Arimichi M. Insulin autoimmune syndrome-the second case. J Jpn Diabetes Soc 1972; 15:187-192. 7. Folling J, Norman N. Hyperglycemia, hypoglycemic attacks and production of anti-insulin autoantibodies without previous known immunization. Immunological and functional studies in a patient. Diabetes 1972; 21:814-826. 8. Hirata Y, Tominaga M, Ito J et al. Spontaneous hypoglycemia with insulin autoimmunity in Graves’ disease. Ann Int Med 1974; 81:214-218. 9. Oneda A, Matsuda K, Sato M et al. Hypoglycemia due to apparent autoantibodies to insulin. Characterization of insulin-binding protein. Diabetes 1974; 23:41-50. 10. Uchigata Y, Kuwata S, Tokunaga K et al. Strong association of insulin autoimmune syndrome with HLA-DR4. Lancet 1992; 339:393-394. 11. Uchigata Y, Eguchi Y, Takayama-Hasumi S et al. Insulin autoimmune syndrome (Hirata disease):Clinical features and epidemiology in Japan. Diabetes Res Clin Pract 1994; 22:89-94. 12. Takayama-Hasumi S, Eguchi Y, Sato A et al. Insulin autoimmune syndrome is the third leading cause of spontaneous hypoglycemic attacks in Japan. Diabetes Res Clin Pract 1990; 10:211-214. 13. Hirata Y. Methimazole and insulin autoimmune syndrome with hypoglycemia. Lancet ii 1983; 1037-1038. 14. Uchigata Y, Hirata Y, Omori Y. A nobel concept of type VII hypersensitivity induced by insulin autoimmune syndrome (Hirata’s disease). Autoimmunity 1995; 20:207-208. 15. Wasada T, Eguchi Y, Takayama-Hasumi S et al. Reverse phase high performance loquid chromatographic analysis of circulating insulin in the insulin autoimmune syndrome. J Clin Endocrinol Metab 1988; 66:153-158. 16. Eguchi Y, Uchigata Y, Yao K et al. Longitudinal changes of serum insulin concentration and insulin antibody features in persistent insulin autoimmune syndrome (Hirata’s disease). Autoimmunity 1994; 19:279-284. 17. Uchigata Y, Yao K, Takayama-Hasumi S et al. Human monoclonal IgG1 insulin autoantibody from insulin autoimmune syndrome directed at determinant at asparagine site on insulin B-chain. Diabetes 1989; 38:663-666. 18. Uchigata Y, Takayama-Hasumi S, Kawanishi K et al. Inducement of antibody that mimics insulin action on insulin receptor by insulin autoantibody directed at determinant at asparagine site on human insulin B chain. Diabetes 1991; 40:966-970. 19. Uchigata Y, Eguchi Y, Takayama-Hasumi S et al. The immunoglobulin class, the subclass and the ratio of κ:λ light chain of autoantibodies to human insulin in insulin autoimmune syndrome. Autoimmunity 1983; 3:289-297. 20. Uchigata Y, Tokunaga K, Nepom G et al. Differential immunologenetic determinants of polyclonal insulin autoimmune syndrome (Hirata’s disease) and monoclonal insulin autoimmune syndrome. Diabetes 1995; 44:1227-1232. 21. Schlemper RJ, Uchigata Y, Frolich M et al. Recurrent hypoglycemia caused by the insulin autoimmune syndrome: The first Dutch case. Netherlands J Med 1996; 48:188-192. 22. Uchigata Y, Omori Y, Nieda M et al. HLA-DR4 genotype and insulin-processing in insulin autoimmune syndrome. Lancet 1992; 340:1467. 23. Ito Y, Nieda M, Uchigata Y et al. Recognition of human insulin in the context of HLADRB1*0406 products by T cells of insulin autoimmune syndrome patients and healthy donors. J Immunol 1993; 15:5770-5776.

148

Endocrine and Organ Specific Autoimmunity

24. 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. 25. Brown JHG, Jardetzky TS, Gorga JC et al. Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1. Nature 1993; 364:33-39. 26. Matsushita S, Takahashi K, Motoki M et al. Allele specificity of structural requirement for peptides bound to HLA-DRB1*0405 and DRB1*0406 complexes: Implication for the HLAassociated susceptibility to methimazole-insuced insulin autoimmune syndrome. J Exp Med 1994; 180:873-883. 27. Akai H, Suzuki S, Hirai S et al. A case of insulin autoimmune syndrome developed during alpha-interferon therapy for renal cell cancer (Japanese) Diabetes J 1994; 22:17-20. 28. Benson EB, Healey LA, Barron EJ et al. Insulin antibodies in patients receiving penicillamine. Am J Med 1985; 78:857-861. 29. Benson EB, Ho P, Wang C et al. Insulin autoimmunity as a cause of hypoglycemia. Arch Intern Med 1984; 144:2351-2354. 30. Cho BY, Lee HK, Koh CS et al. Spontaneous hypoglycemia and insulin autoantibodies in patients with Graves’ disease. Diabetes Res Clin Prac 1987; 3:119-124. 31. Lee Y-J, Shin S-J, Torng J-K et al. Insulin autoimmune syndrome in a methimazole-treated Graves’ disease with polyclonal anti-insulin autoantibodies. J Formosa Med Assoc 1987; 867:164-170. 32. Chen C-H, Huang M-J, Huang B-Y et al. Insulin autoimmune syndrome as a cause of hypoglycemia—Report of four cases. Cang Guang Med 1990:42. 33. Goldman J, Baldwin D, Rubenstein A, Klink DD. Characterization of circulating insulin and proinsulin-binding antibodies in autoimmune hypoglycemia. J Clin Invest 1979; 1050-1059. 34. Rovira A, Valverde I, Escorihuela R et al. Autoimmunity to insulin in a child with hypoglycemia. Acta Pediatr Scand 1982; 71:343-345. 35. Blackshear P, Rhil D, Roner HE et al. Reactive hypoglycemia and insulin autoantibodies in drug-induced lupus erythematosus. Ann Intern Med 1983; 99:182-184. 36. Sklenar I, Wilkin TJ, Diaz J-L et al. Spontaneous hypoglycemia associated with autoimmunity specific to human insulin. Diabetes Care 1987; 10:152-159. 37. Tenn G, Eryssenlein V, Mellinghoff HU et al. Clinical and biochemical aspects of the insulin autoimmune syndrome (IAIS). Klin Wochenscher 1986; 64:929-934. 38. Archambeaud-Mouveroux F, Canivet B, Fressinaud C et al. Hypoglycemia autoimmune; responsibility du pyritinol? LaPresse Medicale 1988; 17:1733-11737. 39. Burth HB,Clement S, Sokol MS et al. Reactive hypoglycemic coma due to insulin autoimmune syndrome: Case report and literature review. Amer J Med 1992; 92:681-685. 40. Dozio N, Sodoyez-Goffaux F, Koch M. Are insulin autoantibodies different from exgenous insulin induced antibodies? Diabetologia 1987; 30(Supple):515A. 41. Anderson JH, Blackard WG, Goldman J et al. Diabetes and hypoglycemia due to insulin antibodies. Amer J Med 1978; 64:8686-873. 42. Faguer de Moustier B, Burgard M, Biotard C et al. Syndrome hypoglycemic auto-immun iduit par le pyritinol. Diabete & Metabolisme 1988; 14:423-429. 43. Discher T, Seipke G, Friedmann E et al. Recurrent hypoglycemia in the insulin autoimmune syndrome. Dtsch Med Wschr 1990; 115:1951-1955. 44. Sluiter WJ, Marrink J, Houwen B. Monoclonal gammopathy with an insulin binding IgG(κ) M-component, associated with severe hypoglycemia. British J Heamotology 1986; 62:679-687. 45. Scavini M, Dozio N, Sartori S et al. Effect of treatment on insulin bioavailability and 123I insulin biodistribution in insulin immune hypoglycemia. Diabetologia 1992; 35(Supple)-187A. 46. Meschi F, Dozio M, Bognetti E et al. An unusual case of recurrent hypoglycemia; 10-year follow up of a child with insulin auto-immunity. Eur J Pediatr 1992; 151:32-34. 47. Arnqvist H, Halban PA, Mathiesen UL et al. Hypoglycemia caused by atypical insulin antibodies in a patient with benign monoclonal gammopathy. J Intern Med 1993; 234;421-427.

CHAPTER 8

Type I Diabetes Mellitus Eiji Kawasaki, Ronald G. Gill and George S. Eisenbarth

Introduction to Diabetes Definition

D

iabetes mellitus is a series of disorders characterized by absolute or relative insulin deficiency that leads to hyperglycemia and altered glucose metabolism. These disorders are associated pathologically with micro- and macrovascular disease leading to accelerated arteriosclerosis, and various other characteristic long-term complications, including retinopathy, nephropathy, and neuropathy.1 The relative risks for blindness, renal failure, or amputation for people with diabetes are 20 to 40 times greater than for people without diabetes. Disease heterogeneity has important implications for research and for the clinical management of diabetes: first different genetic and environmental etiologic factors can result in similar diabetic phenotypes; and second the distinct disorders grouped together under the rubric diabetes may differ markedly in pathogenesis, natural history, and the responses to therapy and prophylactic measures. The recommended criteria for the diagnosis of diabetes are a casual plasma glucose concentration ≥ 200 mg/dl or a fasting plasma glucose ≥ 126 mg/dl or a two-hour glucose on 75 gram oral glucose tolerance (oral glucose tolerance testing is not recommended for routine clinical use) testing of ≥ 200 mg/dl. In the absence of unequivocal hyperglycemia with metabolic decompensation the criteria should be confirmed by testing on a different day. The diagnosis of gestational diabetes requires only two of four glucoses on a 100 gram glucose tolerance test fasting with glucose ≥ 105 mg/dl, 1 hour ≥ 190 mg/dl, 2 hour ≥ 165 mg/dl or 3 hour ≥ 145 mg/dl. Most patients with gestational diabetes have a return to normal glucose values postpregnancy, but a significant percentage progress to type 2 diabetes, and a smaller percentage (approximately 5%) are anti-islet autoantibody positive and have autoimmune type 1 diabetes. Multiple types of diabetes mellitus have been defined by the recent report of the American Diabetes Association Expert Committee on the diagnosis and Classification of Diabetes Mellitus (Table 8.1). The four major etiologic categories for disorders of glycemia are: I. Type 1 diabetes (β-cell destruction usually leading to absolute insulin deficiency). Type 1 diabetes is divided into two subcategories: A. Immune mediated and B. Idiopathic II. Type 2 diabetes (May range from predominantly insulin resistance to predominantly insulin secretory defect with insulin resistance). III. Other specific types of diabetes (e.g. Single gene mutations such as several MODY disorders, Type A insulin resistance, pancreatitis, endocrinopathies, drug induced diabetes, uncommon forms of diabetes such as those associated with “Stiff Man” syndrome or anti-insulin receptor antibodies, and genetic syndromes sometimes associated with diabetes such as a series of chromosome abnormalities) and IV. Gestational Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

150

Endocrine and Organ Specific Autoimmunity

Table 8.1. Classification of Type of Diabetes Classes of diabetes mellitus

Alternative terminology

Type 1 diabetes A. Immune mediated B. Idiopathic

Juvenile-onset diabetes Ketosis-prone diabetes, Brittle diabetes Insulin dependent diabetes

Type 2 diabetes

Adult-onset diabetes Maturity-onset diabetes Non-insulin dependent diabetes

Gestational diabetes mellitus Other specific types 1. Genetic defects β-cell function 2. Genetic defects insulin action 3. Exocrine pancreatic diseases 4. Endocrinopathies 5. Drug or chemical induced 6. Infections (Congenital rubella, CMV) 7. Uncommon immune-mediated 8. Other genetic sometimes DM associated

Secondary diabetes

diabetes mellitus (Defined as diabetes with onset or first recognition during pregnancy2,3 (1997 ADA Expert Committee Report). Type 1 diabetes is often characterized by an apparently rapid onset, a tendency of ketosis, and absolute dependence of insulin for survival and maintenance of health. Type 2 diabetes is characterized by an association with obesity, a lesser likelihood of ketoacidosis, and the absence of an absolute dependence on insulin for survival. Both forms of the disease may exist in the same family, and persons with either type are subject to the same long-term complications. Although this simple classification is useful, the classification of many patients with diabetes is not clear-cut. For example, about 20% of persons with the onset of diabetes after the age of 40 years require insulin for control of hyperglycemia but do not develop ketoacidosis if left untreated. Conversely, about 5% of persons with the onset of diabetes before the age 30 years have pathogenic and clinical features of type 2 diabetes.4 Many adults between the age 20 and 40 years have a form of diabetes that lingers in its clinical features between type 1 and type 2 for many years, defying conventional classification in the absence of laboratory evaluation.

Nomenclature Type 1/ Insulin Dependent Diabetes (IDDM) Type 1 diabetes is often associated with chronic and progressive autoimmune destruction of islet beta-cells with a long prodromal phase.5-7 The autoimmune phenomena associated with type 1 diabetes include lymphocytic infiltration of pancreatic islets8 and circulating serum antibodies to various islet cell antigens.9-11 The term IDDM came into use before the recent increase in knowledge concerning the autoimmune basis of type I diabetes and before the chronicity of the autoimmune disorder was recognized. The terms IDDM and NIDDM are descriptive clinical terms, while type 1 and type 2 diabetes are terms based

Type I Diabetes Mellitus

151

on etiological criteria. The autoimmune form of type 1 diabetes was thought to be a childhood disease but it is now recognized that type 1 autoimmune diabetes can present at any age.12 A significant percentage (5-10%) of adults presenting after age 40 with diabetes have an autoimmune etiology of their disorder.13,14 Not all diabetes presenting in children has an autoimmune etiology, and not even all insulin dependent diabetes of children is of autoimmune etiology.

Nonautoimmune Versus Autoimmune Causes of β-Cell Loss Several disorders other than type 1 diabetes lead to extensive pancreatic islet β-cell destruction. Within the past five years an increasing number of genetic disorders have been defined which are associated with IDDM.15 These disorders often have characteristic inheritance patterns or extrapancreatic disease manifestations. Several of the disorders listed in Table 8.2 can be diagnosed by methods which depend upon the sequencing of the relevant mutated gene. Diagnosis of these specific disorders can provide important prognostic information and often lead to the diagnosis of the disorder in relatives. For example, patients with MODY-2 (maturity-onset diabetes of the young) due to a mutation in glucokinase typically have very mild diabetes.16 These patients usually manifest hyperglycemia upon glucose tolerance testing in the absence of fasting hyperglycemia. The disorder appears not to be progressive and these individuals usually require no specific therapy. MODY-1 is caused by a mutation in the hepatocyte nuclear factor (HNF)-4α gene,17 MODY-2 by mutations in the glucokinase gene,18 and MODY-3 by mutations in the HNF-1α gene.19 MODY-1 and -3 can manifest in childhood or later in life, and many of these individuals behave as insulin dependent patients, even though these patients do not express anti-islet autoantibodies. With their insulin dependence and severe hyperglycemia, patients with MODY-1 and -3 are at risk for secondary diabetic complications. In contrast to MODY, patients with several of the other syndromes listed in Table 8.2 have severe extrapancreatic disease manifestations. Patients with Wolfram’s syndrome have a median survival of 30 years and develop progressive neurologic dysfunction 20-22 (DIDMOAD: diabetes insipidus, diabetes mellitus, optic atrophy, and deafness). The disorder has been linked to a gene on chromosome 4. The mutation apparently disrupts normal mitochondrial DNA gene replication, resulting in mitochondrial gene deletions.23,24 A maternally inherited diabetes mellitus is caused by a point mutation in the mitochondrial gene coding for tRNALEU(UUR) (especially nucleotide position at 3243), and is associated with nerve deafness and lower insulin secretory capacity.25 With a growing list of disorders associated with abnormalities of mitochondrial DNA, the typing of mitochondrial DNA should become increasingly available. The type “1.5” diabetes was initially described amongst African-American children.26 This form of diabetes is characterized by individuals who may require insulin therapy during an episode of ketoacidosis,27 but then may be able to discontinue insulin therapy for long periods of time. These individuals do not express anti-islet autoantibodies. It was originally thought that this form of diabetes was relatively rare. Recent studies of new onset patients indicates that as many as 1/2 of new onset African American or Hispanic American children presenting with diabetes lack all anti-islet autoantibodies and lack high risk HLA alleles. Two distinct autoimmune polyendocrine syndromes (APS), type I and type II, are recognized. APS-I is a rare disorder with autosomal recessive inheritance, caused by a currently unidentified gene that maps near the tip of the long arm of chromosome 21.28 A unifying characteristics within APS-II is the strong association with polymorphic genes of the HLA region. APS-II has also been known by various other names: Schmidt’s syndrome, the

Endocrine and Organ Specific Autoimmunity

152

Table 8.2. Examples of non-autoimmune causes of childhood diabetes Disorder

Insulin-dependent diabetes

Genetic abnormality

Extra-”pancreatic” disease manifestations

MODY-1

Progressive

Hepatocyte nuclear factor 4α

No

MODY-2

No

Glucokinase

No

MODY-3

Progressive

Hepatocyte nuclear factor 1α

No

Wolfram syndrome

Yes

Chromosome 4 mitochondrial DNA deletion

Neuronal

Kearns-Sayre

Yes

Mitochondrial DNA deletion

Neuronal

MELAS

Yes

Mitochondrial DNA mutation

Hearing loss

APS-I

Yes

Chromosome 21, autosomal recessive

Hypoparathroidism, candidiasis, etc.

APS-II

Yes

HLA DR3 and DR4, polygenic

Addison’s disease, etc.

Type 1 autoimmune

Yes

HLA, insulin gene, polygenic

Associated autoimmune disorders

Type 1.5

No, but ketoacidosis Unknown can occur

No

Type 2 diabetes

No

No

Unknown

polygrandular failure syndrome, organ-specific autoimmune disease, and polyendocrinopathy diabetes. APS-II occurs more often in females than males and is usually defined by the occurrence in the same individual of two or more of the following: Addison’s disease, Graves’ disease, autoimmune thyroiditis, type I diabetes, primary hypogonadism, myasthenia gravis or celiac disease. Among patients with Addison’s disease and APS-II reported by Neufeld and coworkers, type 1 diabetes (52%) and autoimmune thyroid disease (69%) were the most common coexisting conditions.29

What Are the Genes for Autoimmune Type 1 Diabetes? With long-term follow up the risk for development of diabetes of an identical twin of a patient with type I diabetes is approximately 70%, a figure much greater than “point” estimates of the past. This estimate of 70% is provided by a study which only evaluated

Type I Diabetes Mellitus

153

discordant identical twins with life table analysis of progression to diabetes30 and a population based study of monozygotic twins in Denmark.31 The majority of monozygotic twins who do not progress to diabetes express anti-islet autoantibodies, and thus the concordance for autoimmunity (diabetes or autoantibodies) is probably greater than 85%. In contrast approximately 13% of nonidentical twins are reported to progress to diabetes and approximately 6% of first degree relatives of patients with type I diabetes. For unknown reasons approximately 6% of offspring of fathers with type I diabetes versus 2% of offspring of mothers progress to diabetes.

Major Histocompatibility Complex (MHC) The major histocompatibility complex is located on the short arm of chromosome 6 and was initially named because polymorphic genes within this region are major determinants of the acceptance of grafted tissue between strains of mice and between patients. There are approximately 100 genes within the major histocompatibility complex with the majority of genes influencing immune function. Thus another name for many of the genes within this region are human leukocyte antigens (HLA), a name reflecting the utilization of leukocytes to serologically define alleles. They have been classified into HLA class I, II, and III genes. Class I and class II genes encode molecules involved in the presentation of antigen to the immune system. Class III MHC genes encode complement components C2, C4 and factor B+. At least 40% of the familial aggregation of type I diabetes is accounted for by HLA genes, and in particular the HLA class II molecules DQ and DR (termed IDDM1).32,33 The DP, DQ and DR genes are termed immune response genes. The DP, DQ, and DR molecules are found on the surface primarily of antigen presenting cells and function to present peptides to T cell receptors of CD4 positive T lymphocytes.34 The polymorphic amino acid residues of the two chains primarily line the peptide binding groove of DQ and each amino

Fig. 8.1. Life table analysis of “Identical” twin progression to diabetes.

154

Endocrine and Organ Specific Autoimmunity

Fig. 8.2. Transmission of DQα and DQβ haplotypes to affected children for the HBDI family repository HLA typed by Erlich and Noble.

n = the number of parents with a given allele for which transmission to diabetic offspring could be calculated. The letter D above the bars indicates DQB molecules with an aspartic acid at position 57 which is usually but not always associated with protection from diabetes.

acid chain variant is given a unique number (e.g. DQA1*0301, DQB1*0302). For DR molecules the α chain is not polymorphic, while the β chain is polymorphic and each unique variant needs to only be given a single DR number (e.g. DRB1*0401). Certain combinations of DR and DQ alleles are almost always found together on a chromosome and are said to be in linkage dysequilibrium. For example the DR3 allele DRB1*0301 is almost always associated with DQA1*0501, DQB1*0201 while the DR4 allele DRB1*0401 is commonly associated with DQA1*0301 and one of three DQB1 alleles, DQB1*0301, DQB1*0302, DQB1*0303. The highest risk for type I diabetes in the United States is associated with individuals expressing both DQA1*0501-DQB1*0201, and DQA1*0301-DQB1*0302 (with DRB1*0401 or 0402) and such individuals are often referred to as DR3/4 heterozygotes. Two percent of children born in the United States are such DR3/4 heterozygotes, and such individuals comprise approximately 40% of all children developing diabetes.35 In addition to these two high risk HLA haplotypes, there are less common HLA haplotypes associated with high diabetes risk. Though not as common in the general U.S. population high risk haplotypes such as DQA1*0401-DQB1*0402 are obviously of import to an individual or family in which this allele occurs (Fig. 8.2). For example DQB1*0402 is transmitted to 80% of diabetic children in families where one parent has DQA1*0401-DQB1*0402 without the DR3 or DR4 (DQB1*0302) high risk allele. The diabetes risk associated with DQ alleles, and in particular DQA1*0301-DQB1*0302 can be modified by associated DR alleles and in particular

Type I Diabetes Mellitus

155

DRB1*0403 with DQA1*0301, DQB1*0302 is not a high risk haplotype. If the DRB1 allele were *0401, 0402, or 0404 it would be a high risk haplotype. HLA DQ alleles are not only associated with diabetes risk but are also associated with dominant protection from type I diabetes. The most common and most protective HLA DQ allele is DQA1*0102-DQB1*0602.36 This allele occurs in more than 20% of individuals from many populations but less than 1% of children developing type I diabetes. There are rare individuals who develop autoimmune type I diabetes as children with these alleles but it is so rare that one should consider alternative causes of diabetes in such individuals (such as the disorders listed in Table 8.2) especially if autoantibodies are absent. The DQA1*0102DQB1*0602 allele appears to even be protective from progression to diabetes amongst relatives of patients with type I diabetes who express anti-islet autoantibodies.37 Approximately 7% of islet cell autoantibody positive relatives express DQB1*0102-DQB1*0602, and at present such individuals are excluded from trials for the prevention of diabetes. Table 8.3 lists a series of high risk and protective HLA haplotypes. The manner by which alleles of the major histocompatibility complex enhance or suppress the development of type I diabetes is currently unknown. The leading hypothesis is that the alleles determine risk by the autoantigenic peptides which they bind and present to T lymphocytes. Protective alleles are hypothesized to bind more avidly to autoantigenic peptides and present these peptides in a manner which does not activate autoimmunity. Alternatively MHC molecules may alter the T cell repertoire and thereby change the risk for diabetes and autoimmunity. With our current lack of knowledge there are additional hypotheses. The I-Ag7 DQ-like molecule of the NOD mouse is reported to be unstable and a poor presenter of peptides and thus might be defective in inducing central deletion. Class I molecules of the NOD mouse and man have also been reported to be defective.38 It is remarkable that the molecule DQA1*0102, DQB1*0602 appears to mediate dominant suppression of type I diabetes in all populations studied. With such dramatic suppression we favor the hypothesis that there is a dominant autoantigen whose recognition can be altered by changes induced in the T cell repertoire.

Table 8.3. High-Risk and Protective HLA Haplotypes HIGH RISK DR3: DR4:

DRB1*0301 DRB1*0401 DRB1*0402

DQA1*0501 DQA1*0301 DQA1*0302

DQB1*0201 DQB1*0302 DQB1*0302

MODERATE RISK DR8 DR2 DR1

DRB1*0801 DRB1*1501 DRB1*01

DQA1*0401 DQA1*0102 DQA1*0101

DQB1*0402 DQB1*0502 DQB1*0501

PROTECTIVE Strong protection DR2

DRB1*1501

DQA1*0102 DQA1*0101

DQB1*0602 DQB1*05031

Weak protection DR4 DR4

DRB1*0401 DRB1*0403

DQA1*0301 DQA1*0301

DQB1*0301 DQB1*0302

156

Endocrine and Organ Specific Autoimmunity

Genetics of Diabetes in the NOD Mouse and BB Rat Animal models of human diseases are extremely important for understanding pathogenesis and for the development of effective therapeutic approaches. The spontaneous animal models of type I diabetes include the Biobreeding (BB) rat and the nonobese diabetic (NOD) mouse. The diabetes-prone BB rat (DP-BB) differs from the human disease in that it has severe T cell lymphopenia. The lymphopenia is thought to be related to a defect of thymic maturation. The diabetes-resistant strain of BB rat (DR-BB) has the same MHC as the DP-BB, but does not have lymphopenia and does not develop diabetes if housed in specific pathogen free conditions. The lymphopenia trait is inherited in a simple autosomal recessive pattern, with 100% penetrance, segregating independently of the major histocompatibility complex. The NOD mouse derived from a subline of the CTS (cataract) strain on a Jcl-ICR background, during selective breeding searching for a new animal model of diabetes.39 Lymphocytic infiltration of the islet (insulitis) is detectable by electron microscopy as early as 2 weeks of age and by light microscopy around 4 weeks of age.40 The NOD mouse does not have lymphopenia. NOD mice differ from man in having sialadenitis and a marked female preponderance in diabetes incidence. Insulitis occurs in all females and in about 90% of males, although diabetes does not develop in all mice with insulitis. The mouse MHC on chromosome 17 (designated Idd-1) is necessary but not sufficient for the development of diabetes. The NOD mouse has a unique class II MHC, with no expression of I-E (the mouse homologue of HLA-DR) due to the deletion of the promoter region of the Eα gene41 and I-A of the NOD mouse termed I-Ag7 (homologous to DQ) is different from any known I-A molecules reported so far.42 Insulitis and diabetes do not develop in transgenic NOD mice expressing Eα, indicating that the defect in expression of Eα is essential for the occurrence of insulitis.43,44 Transgenic introduction of a “normal” I-A molecule also prevents diabetes.45 By 1990, mapping efforts in the NOD mouse had identified IDD-141 and provided evidence for linkage to the gene for Thy-1 (a T cell antigen) on mouse chromosome 9 (designated Idd-2).46 Further linkage studies in the NOD mouse have identified more than 10 susceptibility loci.47,48 Idd-3/Idd-10 on chromosome 3 and Idd-5 on chromosome 1 appear to have the important roles in determining susceptibility to diabetes and insulitis, while Idd-2 on chromosome 9 and Idd-4 on chromosome 11 have minor roles, principally affecting the timing of the onset of diabetes. Idd-9/Idd-11 on chromosome 4 appears to be of intermediate importance. Important progress has occurred for Idd-3 with creation of strains of mice congenic for a small region of chromosome 3.49 It appears that Idd-3 is due to a polymorphism of the gene for IL-2 and this polymorphism, while it does not influence IL-2 messenger RNA expression, influences glycosylation and the half-life of circulating Il-2. Mice congenic for the relevant normal loci have approximately a 1/3 reduction in the development of diabetes. Another locus of interest as defined by Claire-Salszer and coworkers appears to influence macrophage activation, with NOD macrophages expressing excessive cyclooxygenase. Mice congenic for the normal interval lack this phenotype. In contrast to NOD mice with their more than 15 IDDM loci, the genetics of type I diabetes in the BB rat model of type I diabetes appears much simpler. For a number of strain combinations, risk for type I diabetes segregates with two genetic regions: IDDM1 which is inherited in an autosomal recessive manner and determines with essentially 100% penetrance a severe T cell lymphopenia and IDDM 2 of the major histocompatibility complex. In the above crosses the great majority of rats which develop diabetes are homozygous for the lymphopenia locus (IDDM1) and also homozygous for class II alleles of the BB rat, though an important subset (approximately 10%) develops diabetes with one copy of the BB’s class II region. The gene underlying the severe lymphopenia of the BB rat is yet to be

Type I Diabetes Mellitus

157

identified. Therapies such as administration of monoclonal antibodies to the surface antigen RT6,50 can induce diabetes in nonlymphopenic (DR (diabetes resistant) BB rats.51,52

Non-MHC Genes It is clear that autoimmune type I diabetes is a multigenic disorder and that there is more than a single genetic syndrome giving rise to autoimmune beta cell destruction. In the NOD mouse model there are more than 10 genetic loci which appear to contribute to diabetes susceptibility.49,53 In man polymorphisms of the 5' flanking sequences of the insulin gene located on the short arm of chromosome 11 (termed IDDM2) are associated with diabetes risk.54,55 Polymorphisms of this region account for approximately 10% of the familial aggregation of type I diabetes. The influence of this region on diabetes risk is likely to be complex. The effect of the insulin gene polymorphism is dependent upon the parental (maternal versus paternal) inheritance of the insulin allele.55-60 Two studies from the United States indicate that “protective” insulin gene alleles are not protective if inherited from the mother.61,62 This finding may be related to maternal imprinting of the insulin gene at sites other than the pancreas in that there is no clear evidence of imprinting of insulin gene expression of islets.59 An alternative hypothesis is that the maternally imprinted IGF2 gene may contribute to diabetes susceptibility as this gene lies next to the insulin gene.59 In that insulin is a major autoantigen associated with type I diabetes, one hypothesis is that the insulin gene VNTR may influence risk by altering expression of insulin and lymphocyte development, perhaps in the thymus or yolk sac.63-65 Two studies provide evidence that the “protective” VNTR is associated with greater proinsulin message expression within the thymus.66,67 Multiple additional loci potentially associated with diabetes risk have been described (Table 8.4).56,68-71 These loci have primarily been detected by the means of analyzing large groups of sibling pairs for distorted sharing of microsatellite identified alleles. A major difficulty with these studies is the small magnitude of the distortions observed [often less than 55% with predicted sharing of 50% which converts into λs values representing sibling sharing close to 1.0 (Table 8.4)]. Probably related to these small distortions there is variability in confirming putative loci in different populations and even for data sets from the same population. It is likely that these loci may have different effects depending on alleles of the major histocompatibility complex. This is exemplified by IDDM 13 where its effect is apparently only seen amongst sibling pairs which do not share high risk HLA alleles (e.g., not HLA DR3 and DR4).72 Most studies to date have attempted to subdivide families in terms of HLA based solely on haplotype sharing of sibling pairs (e.g., sharing 2, 1 or 0 HLA haplotypes by descent amongst the diabetic sibling pairs). As recently demonstrated by Erlich and coworkers such analysis of sharing greatly underestimates the influence of HLA haplotypes in that more than 1/4th of siblings sharing no HLA haplotypes have the highest risk HLA genotype and are DR3, DR4(DQB1*0302) heterozygotes.73 It is likely that identification of non-MHC genes will facilitate understanding the pathogenesis of type I diabetes, but to date with only loci identified, each with a relatively small effect upon familial clustering, current knowledge of non-MHC diabetes susceptibility loci do not contribute to the diagnosis or prediction of type I diabetes.

Associated Disorders Type 1 diabetes is associated with other autoimmune diseases such as autoimmune thyroid disease, celiac disease, and Addison’s disease.74 It is likely that at least some of the genes underlying susceptibility will not be specific to type 1 diabetes but also will be involved in the susceptibility to these other autoimmune diseases. It is also likely that at least some of the genes contributing to diabetes susceptibility may cause beta-cell autoimmunity

Endocrine and Organ Specific Autoimmunity

158

Table 8.4. Putative Human IDDM loci Locus

Chromosome

Gene

λs

IDDM1 IDDM2 IDDM13 IDDM3 IDDM4 IDDM5 IDDM6 IDDM7 IDDM8 IDDM9 IDDM10 IDDM11 IDDM12 IDDM15

6p21 11P15.5 2q34 15q26 11q13 6q25 18q21 2q31 6q27 3q 10p11 -10q11 14q24.3-14q31 2q33 6p21

DR and DQ “VNTR” Insulin gene

1.7-4.2 1.6 1.2 <1.5 1.0-1.5 1.0-3.0 1.0-1.5 1.0-1.6 1.0-2.1 1.0-1.7 1.1-2.2 <1.5 <1.5 <1.5

that does not progress to overt diabetes during the person’s lifetime, but may be detected by the presence of autoantibodies. The high risk HLA haplotype associated with Graves’ disease and celiac disease are DR3, DQA1*0501-DQB1*0201, which is also one of the highest risk haplotypes for type I diabetes mellitus. Approximately 6% of individuals with type I diabetes when screened for celiac autoantibodies are positive for such autoantibodies and almost 12% of patients with type I diabetes with DR3 are positive for these anti-endomysial autoantibodies. The majority of patients with type 1 diabetes expressing anti-endomysial autoantibodies on biopsy have celiac disease even though the majority of such individuals are asymptomatic. Addison’s disease is associated with the same two high risk HLA haplotypes DR3 (DQA1*0501-DQB1*0201) and DR4 (DQA1*0301-DQB1*0302) as for type I diabetes mellitus. Similar to celiac disease a simple autoantibody screening test is available for Addison’s disease and 2% of individuals with autoimmune type 1 diabetes have anti-21 hydroxylase autoantibodies.75 Approximately 1/3 of these individuals have overt Addison’s disease when the autoantibodies are detected. Thyroid autoimmunity is very common in patients with type 1 diabetes, but only a subset of patients with anti-thyroid autoantibodies progress to overt hypothyroidism. Thus we currently screen for thyroid disease by testing for thyrotropin (TSH) levels. An autosomal recessive mutation on the distal long arm of chromosome 21 determines genetic susceptibility for APS-I. Approximately 15% of individuals with this syndrome (characterized by mucocutaneous candidiasis, hypoparathyroidism, and Addison’s disease) develop type I diabetes. In contrast to APS-II, neither Addison’s29 disease nor type I diabetes in patients with APS-I have an HLA association. For example, of approximately 400 individuals with type I diabetes we have studied, only two expressed DQA1*0102-DQB1*0602 (the strongly protective allele found in 20% of the general population). One of these two individuals with type I diabetes and DQB1*0602 had APS-I, making us suspect that DQA1*0102DQB1*0602 may not protect from type I diabetes with the chromosome 21 recessive mutation of APS-I.

Type I Diabetes Mellitus

159

The high risk HLA alleles for multiple sclerosis are the “protective” HLA alleles of type I diabetes, namely DQA1*0102-DQB1*0602. This may explain why multiple sclerosis infrequently occurs in individuals with type I diabetes, while it more frequently is found in siblings of patients with the disorder. One hypothesis is that the nonHLA genes contributing to the autoimmunity of type I diabetes contribute to the autoimmunity of multiple sclerosis.

Genetic Prediction of Type I Diabetes The technology for determining HLA alleles has improved dramatically during the past decade. DR and DQ alleles can be rapidly assessed following polymerase chain reaction amplification of DNA from minute quantities of blood or buccal smears.76 Drs. Rewers, Erlich and colleagues have initiated the Diabetes Autoimmunity Study in the Young (DAISY) with the screening of newborns from the large Denver City Hospital for HLA alleles associated with diabetes risk.35 More than 12,000 newborns have been HLA typed to date from cord blood samples. The PCR based typing system has proven to be highly reliable, with a discordance of approximately 0.5% on repeated blinded typing of duplicate samples. More than 90% of parents consented to the screening, including various ethnic groups and socioeconomic strata. Of the newborns, 2.4% were DR3/4, DQB1*0302, a high risk genotype for type I diabetes. It is estimated that this 2.4% of the population will comprise 40% of all individuals developing type I diabetes, and the absolute risk for such individuals from the general population is the same as if their father had type I diabetes (6-8%). A moderate risk group, DRX/4 (DQB1*0302), DR4/4 (DQB1*0302) and DR3/3, comprised 16.7% of those screened and is estimated to identify an additional 40% of children who develop diabetes. The majority of the remainder of individuals are at low risk for type I diabetes (< 1/300) except for certain “rare” genotypes such as DR1/4 and DR8/4. Together, the high and moderate risk group comprises 80% of all children developing diabetes. As discussed in a recent DAISY publication, “screening for genetic markers associated with (but not diagnostic for) a severe and currently incurable disease, such as IDDM raises important ethical issues”.35 At present such screening appears to be justified only for research purposes. Given the development of preventive therapies, such genetic stratification may aid the design of general population preventive trials.

What Are the Triggering/Preventive Factors? Long-term follow up studies from the United States and a population based study of identical twins from Denmark indicate that approximately 70% of identical twins of patients with type 1 diabetes will eventually become concordant for the disease.30,77 Of the monozygotic twins who are not yet diabetic, the majority appear to express anti-islet autoantibodies.30,31 Of note the above Danish twin study indicates that although only 13% of dizygotic twins develop diabetes on long term follow up, more than 50% express either cytoplasmic ICA, insulin or GAD65 autoantibodies.31 This is a very high rate and suggests environmental factors potentially acting in utero contribute to the activation of autoimmunity. Environmental factors which may either trigger or suppress the development of type I diabetes are poorly understood. In that in several countries the incidence of type I diabetes in childhood has more than doubled during the past two decades there are likely to be environmental factors which influence disease susceptibility.1,78 A number of viruses have been reported as diabetogenic and islet-cell antibodies have been described following viral infection.79,80 The one environmental factor clearly associated with the development of type I diabetes is congenital rubella infection.81 As many as 20% of children following congenital rubella infection will develop type I diabetes as teenagers or young adults. The manner by

160

Endocrine and Organ Specific Autoimmunity

which congenital rubella influences diabetes risk is currently unknown but is hypothesized to be related to dramatic alterations of T cell function following intrauterine infection.82 Abnormalities of T cell function would be concordant with the additional frequent occurrence of thyroiditis in patients with congenital rubella.83 An alternative hypothesis is that a peptide of the virus is homologous to an islet autoantigen and the infection triggers antiislet autoimmunity (molecular mimicry).84 In that T cell receptors are relatively degenerate in the range of peptides which they can recognize, the opportunity for molecular mimicry is large. A coxsackie B4 (CB4) virus was isolated from the pancreas of a child who died from viral encephalitis and diabetic ketoacidosis.85 Later reports of the pathological findings in the child’s pancreas indicate that beta-cell destruction may have preceded the virus infection.86 However, recently it has been shown that infection by E2 strain of CB4 virus induces long-term hyperglycemia and increases 64 kD islet-cell antigen expression in mice, suggesting a role of this viral infection in autoimmunity.87 Other viral infections during pregnancy have been associated with diabetes, and there are relatively weak associations of anti-viral antibodies and type I diabetes. With current knowledge of the natural history of type I diabetes many of these viral associations are likely to be fortuitous or result from triggering of hyperglycemia in individuals who have already destroyed the bulk of insulin producing cells.88,89 In spontaneous animal models of diabetes viral infections are primarily associated with the prevention of diabetes rather than its initiation with the exception of a strain of BB rats (nonlymphopenic strain) where infection with the Kilham rat virus triggers diabetes.90 A number of large-scale prospective studies are underway in which individuals are followed from birth for the appearance of anti-islet autoimmunity. It will be possible in these studies to define associations of enteroviral infections with development of autoimmunity, though current prospective data suggest that the ability to identify relevant viral triggers of autoimmunity will be difficult.89 Despite emphasis on viral infections a more likely trigger of autoimmunity (by analogy with celiac disease) is ingestion of dietary proteins.91-95 In both the BB rat and NOD mouse the dietary source of protein dramatically influences the development of diabetes.96 Circumstantial evidence has associated type 1 diabetes with the ingestion of bovine milk prior to three months of age.97 The epidemiologic studies linking milk ingestion to diabetes have primarily relied upon retrospective questioning of parents concerning infant feeding. In a current prospective study of the appearance of anti-islet autoimmunity there was no association between ingestion of bovine milk and the development of anti-islet autoantibodies.98 If important environmental factors could be identified, removal of these factors would be an approach for the prevention of diabetes. Trials are proposed in Finland aimed at the removal of bovine milk from infant diet.99 Vaccination for prevention of rubella is now a reality in many countries and congenital rubella infections have become rare.

What Are the Target Autoantigens? Humoral Autoimmunity Over the past decade, investigators have defined a large family of islet autoantigens. The first test for anti-islet autoantibodies consisted of the determination of antibodies reacting with frozen sections of human pancreas, termed cytoplasmic islet cell autoantibodies (ICA).100 This test has formed the basis for many studies of the natural history of the disease and the basis for several current large clinical trials of diabetes prevention. This test detects a subset (usually high titer) of glutamic acid decarboxylase (GAD) 65 and ICA512/IA-2 autoantibodies and fails to detect insulin autoantibodies.101 In addition there are one or

Type I Diabetes Mellitus

161

more additional autoantigens detected on frozen sections such as antibodies reacting with the molecule ICA69102,103 (blocking of anti-ICA69 monoclonal antibody staining of islets by patients sera) and a GM2-1 ganglioside.104 Despite the utility of the ICA test it has been difficult to standardize and has limited predictive potential in the absence of readily measurable biochemically detected anti-islet autoantibodies. Thus at present we reserve determination of cytoplasmic islet cell autoantibody determination for specific research questions and instead routinely determine GAD65, ICA512/IA-2 and insulin autoantibodies.9 Table 8.5 lists five islet autoantigens for which there currently exist autoantibody assays based upon recombinant DNA production of the autoantigen. The determination of anti-islet autoantibodies has been revolutionized by the cloning of a series of islet autoantigens and the ease of current autoantibody radio assays. For such radio assays many investigators simply take cloned cDNA of a given target autoantigen and in vitro transcribe and translate the cDNA to produced labeled autoantigen.105 This was first accomplished in the diabetes field for the enzyme GAD65 which is a cytoplasmic enzyme expressed in all islet cells of man and a series of neuroendocrine tissues. It was the association of autoantibodies to GAD65 with Stiff-Man syndrome, a rare neuromuscular disease characterized by muscle spasms, which first led to the identification of GAD65 as the elusive islet 64 kD autoantigen.106 Autoantibodies to GAD65 are now usually detected in a semiautomated 96-well format. The autoantigen ICA512 was originally discovered by Rabin and coworkers following the screening of an islet expression library with sera from patients with type I diabetes.107,108 The same molecule has been termed IA-2.109,110 Prior to the characterization of ICA512, Christie and coworkers had identified autoantibodies reacting with a 40 kD and 37 kD tryptic fragment of labeled islets.111 It is now understood that the 40 kD protein is ICA512/ IA-2 and the 37 kD molecule is a tryptic fragment of a molecule termed phogrin by Hutton and coworkers, IA-2β by Notkins and coworkers, and IAR by other investigators.112 ICA512 and phogrin are both associated with neuroendocrine secretory granules (e.g., the islet granules containing insulin). These molecules have domains which are homologous to tyrosine phosphatases, but to date enzymatic activity has not been demonstrated for either protein. The molecules are most homologous in their C-terminal intracytoplasmic domains, which are also the domain to which essentially all of the autoantibodies are directed. By utilizing the cytoplasmic domain, or other N terminal shortened constructs of ICA512 and phogrin,

Table 8.5. Recombinant Anti-Islet Autoantibody Assays Antigen

Sensitivity (Specificity)

Comment

Insulin

40-95% (99%)

Inversely Age Diabetes Onset Related

GAD65

70%

(99%)

Predominantly Age Independent

ICA512/IA-2

60%

(99%)

Islet Protein Tyrosine Phosphatase

Phogrin/IA-2β

55%

(99%)

Autoantibodies Predominantly Subset of ICA512/IA-2 Autoantibodies

Carboxypeptidase H 10%

(99%)

Low Sensitivity

162

Endocrine and Organ Specific Autoimmunity

assays for diabetes associated anti-islet autoantibodies can be improved (higher specificity) with little if any loss of sensitivity.113 With sera from children with new onset diabetes approximately 10% of such children express antibodies which react with ICA512 but fail to react with phogrin.114 To date we have only detected 1% of such children with phogrin autoantibodies who are negative for ICA512 autoantibodies. In addition the bulk of phogrin reactivity can be absorbed with synthetic ICA512 antigen, suggesting that the majority of autoantibodies directed against phogrin cross react with ICA512. Though insulin was the first autoantigen biochemically characterized,115 the assay for anti-insulin autoantibodies remains much less convenient compared to assays for GAD65 and ICA512. In fact we currently assay both GAD65 and ICA512 autoantibodies utilizing dual radioactive labeling of these molecules (3H-leucine for GAD65 and 35S methionine for ICA512) and can perform the assay for both autoantibodies in a single well requiring less than 10 µl of sera. Investigators have readily shared antigen clones with other clinical investigators and thus the GAD65 and ICA512 assays have been readily adopted in laboratories on four continents. In contrast the insulin autoantibody assay utilizes for duplicate determinations, with and without competition with unlabeled insulin, 600 µl of sera per determination. The insulin assay is performed in centrifuge tubes and is thus labor intensive. Recent workshop comparisons indicated that insulin autoantibody assays which utilized less than the above 600 µl were less sensitive except for a recent protein A assy. It will be important to improve the insulin autoantibody assay as insulin autoantibodies are one of the first to appear in a prediabetic individual and provide the most sensitive assay for detecting children less than age 10 developing type I diabetes. Attempts to measure insulin autoantibodies by standard ELISA techniques led to assays which could detect anti-insulin antibodies following subcutaneous insulin therapy but could not detect the autoantibodies of prediabetic and new onset patients.116 This lack of success of ELISA assays probably relates to the observation that insulin autoantibodies recognize a conformational determinant and are of very high affinity (1010) and very low capacity (10-12).117 Antibodies to carboxypeptidase H are too infrequent to contribute to a standard panel of autoantibodies. In addition to the antigens listed in Table 8.5, there are a series of other potential additional autoantigens101,118-124 with partially characterized molecules, or characterized molecules where the assay format does not allow determination of antibodies for thousands of sera samples. It is likely that further characterization of these molecules, as well as potentially unknown molecules, or refinements in assay methodology will provide additional autoantigens of value in the diagnosis and prediction of type I diabetes. The best autoantibody predictor of high diabetes risk is the expression of multiple “biochemically” determined autoantibodies. Figure 8.3 illustrates the progression to diabetes amongst a series of more than 600 relatives of patients with type I diabetes subdivided by the number of autoantibodies expressed testing for GAD65, insulin and ICA512bdc autoantibodies. Only one relative of more than 500 expressing none of these three autoantibodies progressed to diabetes. Expression of a single autoantibody was associated with an approximate 20% risk of diabetes with ten years of follow up and it is likely that many relatives expressing a single autoantibody will never progress to diabetes. Expression of multiple autoantibodies was associated with a very high risk of progression. The “combinatorial” analysis allowing ≥2 autoantibodies to be defined, independent of which two autoantibodies are expressed, gives approximately an 80% sensitivity for progression to diabetes with very high specificity. Less than 1/300 individuals from the general population express ≥2 autoantibodies, indicating expression of multiple autoantibodies approaches the risk of type 1 diabetes.

Type I Diabetes Mellitus

163

T Cell Autoimmunity In the NOD mouse, insulin-specific T cells comprise up to 50% of the infiltrating T cells which can be isolated from prediabetic NOD islets.125 Most importantly, insulin-reactive T cells can transfer diabetes to young NOD and NOD/scid recipients126 and transgenic expression of proinsulin prevents insulitis and diabetes.127 The majority of the islet-infiltrating clones respond to the mid-portion of the insulin B chain (B9-23). Molecular analysis of B9-23-specific T cell clones indicates heterogeneity in the selection of the TCRβ chain but marked restriction of both Vα and Jα usage.128 The combination of Vα13 and Jα45 or Jα34 is the dominant motif of B9-23 reactive T cell receptors and was utilized by 60% of the B9-23 reactive clones. In contrast to their presence in infiltrated islets, insulin-reactive T cells are quite difficult to detect in the peripheral lymphoid tissues of NOD mice.129 We believe this reflects the fact that these cells home to their target organ, the pancreas, and are in low frequency in peripheral tissues.128 Griffin and coworkers and Harrison and coworkers reported a peptide of proinsulin spanning the B-C chain junction recognized by rat T lymphocytes130 and human T lymphocytes respectively.131 Sherwin and coworkers reported an NOD T cell clone which prevents diabetes and specifically responds to islets132 which also reacts with the B:9-23 insulin peptide. T cell reactivity to GAD in the NOD mouse has recently been associated with the initiation and maintenance of autoimmune reactivity to islet β-cells, since administration of GAD early in disease limits β-cell destruction though it does not prevent insulitis.133,134

Fig. 8.3. Progression to diabetes of first degree relatives subdivided by the number of “biochemical” anti-islet autoantibodies at first determination.

164

Endocrine and Organ Specific Autoimmunity

T-cell responses to GAD have also been observed in a variable percentage of new-onset type 1 diabetics and prediabetics and controls.135,136 The level of T cell response to GAD was inversely correlated to the level of autoantibody response to this protein and directly correlated with diabetes risk in a population of relatives of parents with type 1 diabetes.135 These data indicate that GAD is an important autoantigen in type 1 diabetes and that autoimmunity to this antigen may have a pathogenic role in disease development. There are a series of additional autoantigens recognized by diabetogenic T cell clones.137 Of note, the T cell receptor of clone BDC2.5 (autoantigen unknown) when introduced transgenically into SCID mice is associated with markedly accelerated development of diabetes.138-140 A major difficulty of the studies of T cell reactivity in the animal models but particularly in man is the very low stimulation indexes. Probably related to such small stimulation is the lack of reproducibility between laboratories of detection of T cell responses.141-147 This is likely to relate to the low precursor frequency of T lymphocytes in peripheral blood or spleen reacting with any given autoantigen. Thus for the NOD mouse model many laboratories report stimulation which is greater in NOD mice compared to control strains while probably a similar number of laboratories are unable to distinguish T cell responses to glutamic acid decarboxylase of splenocytes of NOD mice as compared to control mouse strains. For T cell responses in man Bart Roep organized in 1996 an international workshop for the Immunology of Diabetes Society where more than 20 laboratories received “blinded” samples of a series of different islet autoantigens and peptides. The laboratories measured T cell responses in 10 controls and 10 individuals with new onset diabetes. Initial analysis of the results indicated that none of the laboratories, with none of the antigenic preparations, could distinguish T cell responses of controls versus patients. Further analysis with matching controls and patients for HLA alleles is necessary but the results of the workshop illustrate the difficulty of current T cell assays which utilize proliferation of T lymphocytes as measured by thymidine incorporation as their primary readout. Development of better T cell assays, perhaps utilizing determination of cytokine production is a high priority.

Is There a Primary Autoantigen? Though there is no consensus as to which of the autoantigens are central to the pathogenesis of type 1 diabetes,119 we have been particularly interested in the role of immunity directed against insulin.65,117,148-151 To date, insulin remains the only diabetes related β-cell specific autoantigen. For example, GAD is expressed within both β- and non-β-cells of human islets.152 The importance of insulin autoantibodies for prediction of progression to diabetes, independent of age, has recently been confirmed in the large ICARUS data set. In man, it is clear that polymorphisms of the variable nucleotide tandem repeat (VNTR) of the insulin gene influence disease risk.56,57,153 As described above, in studies of the NOD mouse, Wegmann and coworkers have discovered a predominance of insulin reactive T cell clones isolated from islet infiltrates.125,154 Moreover, oral insulin,155,156 nasal insulin,157 aerosol insulin158 and immunization with the B chain of insulin159-162 can delay or prevent the development of diabetes in NOD mice and recently, Wegmann and coworkers have found that intranasal or subcutaneous administration of insulin B9 to B23 peptide to NOD mice prevents the development of diabetes.157 Though both insulin and B chain immunization limit the development of diabetes in NOD mice, neither of these therapies suppress insulitis to the extent seen with administration of biologically active insulin. This suggests that the metabolic effect of intact insulin may synergize with an immunologic effect to prevent insulitis in NOD mice. A recent pilot trial149 provides evidence that prophylactic insulin therapy may prevent or delay the onset of type 1 diabetes in ICA-positive humans. This

Type I Diabetes Mellitus

165

intervention is now being tested by a large randomized trial organized by the National Institute of Health.

What Are the Effector Mechanisms? Lymphokines A large series of chemokines and other lymphokines have been implicated in the pathogenesis of type I diabetes. In particular interleukin-1 (IL-1) when combined with several other cytokines can directly inhibit insulin secretion and damage β-cells.163-166 IL-1 in part appears to act through induction of nitric oxide167 and blocking of free radical mediated killing can abrogate its effect. With transgenic technology a large series of lymphokine genes have been coupled to the rat insulin promoter and investigators have created strains of mice producing these lymphokines within islets. Interleukin-4 expression prevents diabetes.168 Frequently these molecules induce islet inflammation (e.g., interferon gamma) and this leads to islet destruction at times associated with islet directed autoimmune T lymphocytes.169-171 How representative these models are of the natural disease process is currently unknown.

Cytokine Involvement in Autoimmune Diabetes Cytokines have become a major focus for the study of both disease pathogenesis and protection.197 It has become increasingly clear that the expression of autoimmunity is more than the mere presence or absence of autoreactive cells but is also influenced by the pattern of cytokines produced by such cells. The variable cytokine-producing potential of activated CD4 T cells, as illustrated by Th1 and Th2 profiles, greatly influences the quality of immune responses.172,173 Th1 cytokines (e.g., IFNγ, TNF) are associated with pro-inflammatory reactivity while Th2 cytokines (e.g., IL-4, IL-5, IL-10) are associated with humoral reactivity. Most reports indicate that autoimmune islet damage in nonobese diabetic (NOD) mice is associated with pro-inflammatory Th1-like cytokines, such as IFNγ and TNF.174-177 Th1-like cytokines might be especially important for inflicting tissue damage since islets appear to be quite sensitive to inflammatory cytokines and other mediators such as oxygen free radicals.163,178-180 Consistent with this view that inflammatory cytokines contribute to the disease pathogenesis is the finding that systemic administration of Th1-promoting cytokines such as IL-12176 and TNFα181 can precipitate disease in NOD mice. Conversely, protection from disease is often associated with a shift of the cytokine balance towards Th2 cytokines, such as IL-4 and IL-10. This view is supported by studies showing that disease can be inhibited in NOD mice by administration of Th2 cytokines such as IL-4182 or IL-10.183 Transgenic NOD mice expressing IL-4 under control of the insulin promoter are also profoundly protected from disease onset.168 Therapeutic intervention leading to disease has been associated with a Th1-like to Th2-like shift in cytokine profiles as has been reported through adjuvant therapy174,184 or through antigen-specific immunization with insulin peptides.157 Compelling results implicating the significance of cytokines on disease pathogenesis was reported by Katz et al177 who showed that intentionally inducing IL-4 production from islet-specific T cell-receptor transgenic T lymphocytes greatly inhibited their subsequent ability to trigger islet damage. Other forms of tolerance, such as those induced by oral185 or neonatal tolerization186,187 are also associated with Th2-like cytokines. It is not clear whether Th2-like cytokines play an active role in regulating other cells as suggested by some reports188,189 or simply represent the absence of a destructive response, as implied by other studies.177 However, taken together, such studies are consistent with a counter-regulatory role of Th1 and Th2 cytokines in islet damage and protection, respectively.

166

Endocrine and Organ Specific Autoimmunity

There are notable exceptions to this general trend however. While anti-IFNγ treatment prevents disease onset in NOD mice,190 genetically IFNγ-deficient NOD mice continue to develop disease,191 albeit at a slower rate of onset. Similarly, while exogenous IL-10 administration prevents disease onset,183 transgenic NOD mice expressing IL-10 in islet cells develop accelerated disease.170 While expanding on the caveats concerning transgenic and genetargeted (‘knockout’) mice is beyond the scope of this discussion, such results challenge a strict cross-regulation of immunity and tolerance based solely on defined Th1 versus Th2 cytokines. Recently, D. Wegmann and colleagues have found that NOD mice treated with insulin peptides to prevent disease generate peptide-specific, IL-4 and/or IL-10 producing T cells.157 However, Th2-like clones isolated from treated animals are actually pathogenic, precipitating rapid disease onset, rather than being neutral or protective upon adoptive transfer to young NOD mice.192 Similar results have been generated studying allograft immunity in which alloreactive CD4 T cells intentionally driven to produce IL-4 triggered acute graft rejection following adoptive transfer into immune-deficient animals bearing established heart transplants.193 Such results question a strict correlation of Th1 cytokines with islet damage and Th2 cytokines with islet protection. Exceptions to the Th1/Th2 paradigm do not necessarily disprove the general tenet that cytokines are important factors in determining disease outcome. Rather, our definitions of cytokine patterns may be inadequate. There is a tendency to use a shorthand notation for cytokine deviation, with IFNγ being indicative of Th1-like responses and IL-4/IL-10 being indicative for Th2-like responses. However, these particular cytokines may only determine a portion of the outcome of the response; other cytokines or chemokines may be of equal or greater importance in determining disease outcome. For example, regulatory insulin-specific CD4 T cells have been described that may act through the activity of TGFβ,185,194 a cytokine known to inhibit a variety of immune functions. Other factors may also effect the role of cytokines in the response. The influence of particular cytokines may be exerted at different times during the course of the disease. IL-4 and IL-10, alone or in combination, are much more effective in preventing disease onset than in preventing disease recurrence in islets transplanted into recipients with active, primed disease.168,186,187,195,196 The microenvironment of cytokine production may be another important variable influencing disease. This concept is illustrated by the findings that systemic IL-10 administration inhibits disease in the NOD mouse,183 while IL-10 produced in the islet microenvironment in transgenic NOD mice appears to exacerbate the disease.170 Thus we may need to broaden our understanding of these varied features of cytokine patterns, timing, and anatomical distribution that are important for inducing or regulating disease.

Apoptosis It is known that both CD4 and CD8 T lymphocytes are important for the induction of anti-islet autoimmunity in NOD mice. Both CD4 and CD8 clones of T lymphocytes have been generated which are able to rapidly destroy β-cells and cause diabetes in SCID (Severe Combined Immunodeficiency) mice. CD4 clones reacting with insulin are able to destroy even human islets transplanted into SCID mice indicating that the killing cannot depend upon direct recognition on islets of class II molecules.198 The molecules which underlie the killing by both CD8 and CD4 T cells are the subject of intense investigation. For example islet cytotoxicity of a number of clones (both CD8 and CD4) appears to depend upon the expression by islet cells of intact Fas molecule.199 Thus for these clones a major pathway for islet destruction appears to be mediated by the clones expressing Fas ligand which engages the Fas receptor of islets and leads to apoptosis (see chapter 1). It is very likely that the Fas pathway is not the only operative pathway as T lymphocytes from transgenic mice with

Type I Diabetes Mellitus

167

BDC2.5 anti-islet T cell receptor from the clone of Haskins and coworkers can destroy islets which have the 1pr mutation in their Fas gene. A recent report suggests that the major mode for destruction of β-cells of the NOD mouse is by apoptosis.200 Apoptosis, exceeding that of NOD mice with the SCID gene is apparent as early as three weeks of age (Fig. 8.4). It is hypothesized that the amount of cell destruction from 3 to 12 weeks of age is considerable given the rapidity with which apoptotic cell bodies are removed in vivo. At approximately 12 weeks of age there is a major increase in apoptosis and the first mice become diabetic at approximately 15 weeks of age. The NOD mouse is characterized by extensive islet lymphocytic infiltrates (insulitis) even amongst animals which do not progress to diabetes (e.g., the majority of male NOD mice). Thus as Mathis and coworkers have hypothesized in the NOD mouse there appears to be several “check” points for progression to diabetes, one associated with the initiation of insulitis and another with marked β-cell destruction.201

Therapies for the Prevention of Beta Cell Destruction Animal Models Genetic Even though geneomic genetic manipulation is unlikely to be applied to man, the ability to introduce and delete specific genes in animal models of type I diabetes provides a very important test of pathogenic hypotheses, and provides information as to pathways for

Fig. 8.4. Insulin content (left panel) and β-cell apoptosis (right panel) of NOD mice. The “0” age control for apoptosis is from an immunodeficient SCID NOD mouse.

Endocrine and Organ Specific Autoimmunity

168

diabetes prevention. An important caveat is that genetic therapies which prevent diabetes may exacerbate other disorders. For example the DQ allele, DQB1*0602 which prevents type I diabetes is a high risk allele for multiple sclerosis. Thus it is likely that genetic knowledge will contribute to disease prevention by increased understanding rather than direct genetic manipulation of the genome. Table 8.6 lists a series (but not all) genetic manipulations which prevent diabetes of the NOD mouse. A remarkable number of genetic alterations of the NOD mouse have been studied. Many of the introduced genes such as cytokine genes were introduced with an insulin gene promoter directing synthesis of the specific gene to islet beta cells. A general but superficial summary of the results of the studies listed in Table 8.6 is that many but not all manipulations which interfere with both T and B lymphocyte function decrease disease while a few therapies prevent both diabetes and insulitis. The prevention of insulitis appears to be a more stringent test of the importance of specific pathways. A number of genetic manipulations do not prevent diabetes or insulitis and these experiments also provide important information. It is likely that the immune system has redundant mechanisms for targeting islets. For example introduction of a Vβ T cell receptor gene isolated from a T cell clone reacting with chicken ovalbumin (a nondiabetes relevant molecule) which is able to suppress more than 95% of endogenous Vβ chain usage does not influence the development of diabetes.202 This strongly suggests that the Vβ T cell repertoire is redundant in the targeting of essential diabetes relevant molecules and this may relate to the utilization of a shared Vα chain in targeting of the B:9-23 insulin peptide. Introduction of a proinsulin transgene into NOD mice to induce tolerance to proinsulin prevented diabetes and insulitis.127 Of interest, knocking out the enzyme “inductible” nitric oxide synthetase did not influence the devel-

Table 8.6. Genetic Prevention of Type I Diabetes in NOD Mice

Introduced gene I-E α I-A variants (pro 56, asp 57) IL-2 B6 variant Proinsulin on I-A promoter Heat shock protein Selected Vβ TCR

Human homologue

Comment

DR DQ Interleukin-2 Proinsulin Heat shock protein Vβ T cell receptor

Prevents diabetes and insulitis Decreases diabetes Decreases diabetes Prevents diabetes and insulitis Small decrease diabetes Only some Vβs suppress disease suggesting Vβ repertoire not always critical Prevents insulitis and diabetes

IL-4 insulin promoter “Knockout” gene Fas (From lpr mouse) RAG, SCID genes

IL-4 Fas, Apoptosis gene Immunocyte recombinases

Mu chain of immunoglobulin Beta 2 microglobulin

Mu chain Class I MHC gene

Class II genes Interferon gamma Inducible nitric oxide (INOS)

DP, DR, DQ Interferon gamma Nitric oxide synthetase

Prevents diabetes Prevents diabetes and insulitis and indicates T cell receptors or Ig essential for diabetes Contradictory results Prevents diabetes: class I and CD8 T cells important Class II genes essential Enhances diabetes No effect NOD diabetes

Type I Diabetes Mellitus

169

opment of diabetes of NOD mice, suggesting that this free-radical pathway is not central to disease pathogenesis. Dietary Interventions For both the BB rat and NOD mouse, a number of studies indicate that alteration of diet and especially the intake of specific proteins can result in marked suppression of diabetes. For example 62% of NOD mice fed a standard rodent diet developed diabetes at 48 weeks of age in contrast to 9% of mice fed a casein hydrolysate-based test formula (Nutramigen).203 Extensive studies of the BB rat demonstrate similar dramatic disease amelioration.95 Celiac disease which occurs in approximately 5% of patients with type I diabetes is a prototypic diet-influenced autoimmune disorder. In this disease both intestinal and the skin lesions of dermatitis herpetiformis respond to removing gliadin from the diet, and anti-endomysial autoantibodies disappear in individuals adhering to the restricted diet. Immunosuppression As would be expected for an autoimmune disorder, a series of immunosuppressive therapies prevent or delay diabetes of the NOD mouse and the BB rat including drugs such as cyclosporine A,204 mycophenolate mofetil, azathioprine and monoclonal antibodies reacting with key T cell molecules such as CD3 and CD4.205-207 For the BB rat model of type I diabetes where there is an autosomal recessive lymphopenia gene, and for the NOD mouse, bone marrow transplantation with marrow from normal rat or mouse strains prevents diabetes. The success of immunosuppressive therapies in the animal models led to trials of generalized immunosuppression in man (see below).204 Of note, short term immunosuppression with either monoclonals reacting with anti-CD3 or anti-CD4 can lead to long term reversal of diabetes. Therapy with anti-CD3 is paradoxically most effective if given at the time of diabetes onset in the NOD mouse model, suggesting that its long term effect may require immune activation associated with diabetes onset (Bach). Immune Deviation/Cytokine Therapies/Accessory Molecules Administration of either Bacillus Calmette-Guerin (BCG) or complete Freund’s advujant to female NOD mice dramatically inhibits the development of diabetes but does not block the development of insulitis.208 A nonrandomized pilot trial of BCG vaccination209 has been followed by a randomized placebo controlled trial at the Barbara Davis Center in the Barbara Davis Center Trial. BCG vaccinated individuals had if anything lower C-peptide upon follow up as compared to placebo vaccinated controls.210 Anti-Inflammatory Therapy Nicotinamide was originally studied because of its ability to block the development of diabetes caused by the drug streptozotocin. This observation was followed by reports of a delay in the development of diabetes in NOD mice treated with nicotinamide211 which was then followed by a pilot trial of nicotinamide in cytoplasmic islet cell antibody positiverelatives of patients with type I diabetes.209 In this pilot trial, controls were drawn from relatives in Denver and the bulk of the treated patients were from New Zealand, and an approximate 50% delay in the development of diabetes was reported with additional follow up of the treated cohort. In addition a population based trial is underway in New Zealand where approximately one half of school children in Auckland were screened and those with cytoplasmic ICA were offered nicotinamide therapy. In this trial, a large cohort of New Zealand school children were screened for expression of cytoplasmic islet cell antibodies while a matched group of school children were not screened. All ICA positive children were then treated with nicotinamide and compared to the nonscreened and nontreated children. Again

170

Endocrine and Organ Specific Autoimmunity

a 50% delay in progression to diabetes is claimed. Side effects of nicotinamide therapy have not been reported (a worry was the induction of tumors in animals treated with nicotinamide and streptozotocin). These nonrandomized trials have been followed by two “large” randomized trials in cytoplasmic ICA positive relatives. The “DENIS” trial from Germany has been stopped due to lack of effect upon progression to diabetes such that the trial is unlikely to show a delay in progression to diabetes with further follow up (25% of treated and placebo group progressed to diabetes). The larger European Nicotinamide Trial (ENDIT) is underway with planned follow up for several additional years. Antigen Specific Therapies A number of islet autoantigens when administered to NOD mice are able to delay or prevent the development of diabetes. One current hypothesis is that such therapies result in the creation of Th2 like T cells which suppress immunity at the site of antigen expression.212,213 As discussed previously autoantigens studied include, insulin, insulin B chain, the B9 to B23 peptide of insulin and peptides of GAD and heat shock protein 65.134,157,161,213,214 A very interesting model of disease amelioration is a model with transgenic expression of LCMV viral proteins in islets followed by infection with LCMV. In this model oral insulin is able to decrease the development of diabetes.215 Another transgenic model has utilized induced islet expression of ovalbumin with introduction of T lymphocytes with anti-ovalbumin T cell receptors. In this latter model the feeding of large amounts of ovalbumin enhances development of diabetes.216,217 It has been generally assumed that antigen specific therapies, especially utilizing molecules such as insulin which are only expressed within beta cells of the islets will not cause harm. An important caveat is that the modalities utilized to deviate an immune response may exacerbate the very immune response that one is attempting to suppress.

Man Immunosuppression A large number of trials of generalized immunosuppression have been carried out at the time of onset of diabetes or after pancreatic transplantation. Prednisone after diabetes onset provides little long term protection. Short courses of Anti-T cell antibody therapy with ATGAM (anti-thymocyte globulin),218 anti-CD3,219 and anti-CD5,220 had no permanent effect in terms of maintaining C-peptide. Trials of anti-CD3 were limited by acute inflammatory side effects and it is possible that newer anti-CD3 molecules may be able to avoid this problem. Azathioprine appears to have had relatively little efficacy in preventing progressive loss of C-peptide secretion after diabetes onset221 while cyclosporine A in large randomized blinded trials, while administered was very effective in preventing further loss of C-peptide secretion.204,222-224 Cyclosporine A suppressed anti-insulin autoantibodies but not cytoplasmic ICA. Following discontinuation of cyclosporine A the preservation of C-peptide secretion was rapidly lost. Therapy after onset of diabetes is unlikely to maintain a nondiabetic state even with the preservation of C-peptide secretion. In cyclosporine A treated patients hyperglycemia recurred in individuals who could transiently discontinue insulin therapy even though cyclosporine therapy continued. The recurrence of hyperglycemia appeared to be metabolically mediated in a setting where immunosuppression was instituted after the destruction of the majority of insulin secreting islet cells. The failure of immunosuppression to “cure” diabetes, and to preserve C-peptide secretion after discontinuing the drug has led to relatively little interest in generalized immunosuppression. The most efficacious drug, cyclosporine A, appeared to require relatively large

Type I Diabetes Mellitus

171

doses, and be associated with both renal toxicity (apparently no permanent toxicity from follow up of patients in these trials) and has with long term therapy a risk of malignancy. Major prevention trials are underway with administration of subcutaneous or oral insulin (DPT-1) and though the DENIS trial of nicotinamide has been stopped, the ENDIT nicotinamide trial is continuing. It is apparent that trials for the prevention of type I diabetes can be designed and executed. Antigen Specific Therapies The only antigen specific therapies in man have utilized insulin. The first nonrandomized pilot trial of insulin therapy was based on the findings in the BB rat model that subcutaneous insulin therapy prevented diabetes.225 In the BB rat model relatively large doses of insulin must be administered and it is thought that in this model insulin may effect diabetes development through feedback inhibition of beta cell function. To be able to suppress insulin secretion in man and to be able to give long term insulin therapy, the pilot trial utilized both intravenous insulin (given at 9 month intervals by continuous infusion) as well as low dose daily subcutaneous insulin.149 In retrospect with data that in the NOD mouse, insulin “vaccination” prevents diabetes, prevention of diabetes may be related to both beta cell rest and forms of immune regulation induced by insulin administration. After completion of the initial phase of the pilot trial, two extra arms were added to the pilot study, namely subcutaneous insulin alone, and intravenous insulin alone. For these pilot studies relatives of patients with type I diabetes at very high risk of progression to diabetes were identified by their expression of high titer cytoplasmic ICA, low first phase insulin secretion and/or expression of insulin autoantibodies. All eight nontreated individuals progressed to diabetes within 3 years of follow-up. In contrast 3/9 relatives treated with IV and subcutaneous insulin remain nondiabetic with 4.3 to 8 years of follow up. Of the group treated with subcutaneous insulin alone, six out of eight individuals remain nondiabetic with the shortest nondiabetic follow up now at 3 years and the longest 4.6 years. This preliminary trial suggests that if there is efficacy in diabetes delay/prevention, subcutaneous insulin alone may be effective. Pilot trials by Ziegler and coworkers in Germany and Vardi and coworkers in Israel have similar results.226 The above pilot trials, though statistically significant, are too small to conclude that insulin therapy will truly delay or for a subset prevent the development of diabetes. For example one of the individuals remaining nondiabetic in the IV/SQ therapy group who is now followed for 6 years expresses DQB1*0602, and though he expressed all three “biochemical” islet autoantibodies, one cannot conclude that he would not have remained nondiabetic without therapy. GAD65 and ICA512 autoantibodies were not altered by insulin therapy while antiinsulin antibodies dramatically increased and then surprisingly decreased after years of therapy in the longest nondiabetic child (8 years). This child was insulin autoantibody positive prior to the trial and is now insulin autoantibody negative for the past two years. Given the above pilot data, the National Institutes of Health has instituted the DPT-1 randomized trial of parenteral insulin therapy and a placebo controlled trial of oral insulin therapy with Jay Skyler at the University of Miami as the principal investigator. Entry criteria for the trial include the expression of ICA, lack of DQB1*0602, loss of first phase insulin secretion and/or expression of insulin autoantibodies. More than 40,000 relatives of patients with type I diabetes throughout the United States have been screened for participation in the trial and more than 150 entered into the parenteral portion of the trial and more than 80 in the oral portion of the trial. It will likely require two more years of recruitment to fill the projected trial numbers. The major outcome variable of the trial is the development of diabetes by adult National Diabetes Data Group criteria.

172

Endocrine and Organ Specific Autoimmunity

The major concern with insulin therapy in nondiabetic autoantibody positive individuals is the development of hypoglycemia. In the pilot trial with up to eight years of follow there have been no severe hypoglycemic episodes. Individuals in the trial eat what they desire, only occasionally monitor blood glucose (less than once per day) and when asked after three years of therapy whether they wanted to discontinue participation in the trial all have opted to continue. Receiving twice a day insulin injections is very different from treating insulin dependent diabetes with insulin, and this difference is readily recognized by the families participating in the trials. A recent report from Japan suggests that insulin therapy may help in preserving C-peptide secretion in islet autoantibody positive patients presenting with noninsulin dependent diabetes.227 In this small pilot study patients presenting as adults with diabetes were treated either with subcutaneous insulin or an oral hypoglycemic agent and were then followed for maintenance of C-peptide and for expression of cytoplasmic islet cell autoantibodies. The insulin treated patients appeared to lose expression of cytoplasmic ICA faster as compared to oral hypoglycemic treated patients. With current autoantibody assays approximately 5% of patients developing diabetes as adults appear to have slowly progressive autoimmune type I diabetes and thus if the results of this pilot study are confirmed in large randomized trials, a large number of patients may benefit from early insulin therapy.

Conclusion It is now possible to predict the development of autoimmune type 1 diabetes with a high degree of accuracy. It is also possible to prevent type 1 diabetes in a number of animal models with surprisingly benign therapies. It is also now possible to identify individuals at a high genetic risk for the progression to autoimmunity (expression of multiple autoantibodies early in life). With the above information, despite our incomplete knowledge of disease pathogenesis, trials for the prevention of type I diabetes are underway. Improved understanding of autoimmune type 1 diabetes will hopefully rapidly enhance the design and efficacy of preventive trials.

Acknowledgments This work was supported in part by grants from NIH (R37 DK32083 GSE); (R01 AI39213 GSE) and an American Diabetes Association Monitor based Fellowship Awards (GSE).

References 1. Rewers M, Norris JM. Epidemiology of type I diabetes. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York: Oxford University Press, 1996:172-208. 2. National Diabetes Data Group. Classification and diagnosis of diabetes mellitus and other categories of glucose intolerance. Diabetes 1979; 28:1039-1057. 3. World Health Organization. WHO Expert Committee on diabetes. Second report (Tech Rep Ser, No.626). Geneva: World Health Organization, 1980. 4. Tattersall RB, Fajans SS. A difference between the inheritance of classical juvenile-onset and maturity-onset diabetes. Diabetes 1975; 24:44-53. 5. Gorsuch AN, Spencer KM, Lister J et al. Evidence for a long prediabetic period in type I (insulin dependent) diabetes mellitus. Lancet 1981; 2:1363-1365. 6. Eisenbarth GS. Type I diabetes mellitus: A chronic autoimmune disease. N Engl J Med 1986; 314:1360-1368. 7. Verge CF, Eisenbarth GS. Prediction of type I diabetes: The natural history of the prediabetic period. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I diabetes: Molecular and cellular immunology. 1st ed. New York, NY: Oxford University Press, 1996:230-258.

Type I Diabetes Mellitus

173

8. Foulis AK, Liddle CN, Farwuharson MA et al. The histopathology of the pancreas in type I diabetes (insulin dependent) mellitus: A 25-year review of deaths in patients under 20 years of age in the United Kingdom. Diabetologia 1986; 29:267-274. 9. Verge CF, Gianani R, Kawasaki E et al. Prediction of type I diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2 autoantibodies. Diabetes 1996; 45:926-933. 10. Bingley PJ, Christie MR, Bonifacio E et al. Combined analysis of autoantibodies improves prediction of IDDM in islet cell antibody-positive relatives. Diabetes 1994; 43:1304-1310. 11. Roll U, Christie MR, Füchtenbusch M et al. Perinatal autoimmunity in offspring of diabetic parents: the German multi-center BABY-DIAB study: detection of humoral immune responses to islet antigens in early childhood. Diabetes 1996; 45:967-973. 12. Tuomi T, Groop LC, Zimmet PZ et al. Antibodies to glutamic acid decarboxylase reveal latent autoimmune diabetes in adults with a noninsulin-dependent onset of diabetes. Diabetes 1993; 42:359-362. 13. Lorenzen T, Pociot F, Hougaard P et al. Long-term risk of IDDM in first-degree relatives of patients with IDDM. Diabetologia 1994; 37:321-327. 14. Molbak AG, Christau B, Marner B et al. Incidence of insulin-dependent diabetes mellitus in age groups over 30 years in Denmark. Diabetic Med 1994; 11:650-655. 15. Hansen T, Eiberg H, Rouard M et al. Novel MODY3 mutations in the hepatocyte nuclear factor - 1a gene: Evidence for a hyperexcitability of pancreatic b-cells to intravenous secretagogues in a glucose-tolerant carrier of a P447L mutation. Diabetes 1997; 46:726-730. 16. Froguel P, Zouali H, Vionnet N et al. Familial hyperglycemia due to mutations in glucokinase: definition of a subtype of diabetes mellitus. N Engl J Med 1993; 328:697-702. 17. Yamagata K, Furuta H, Oda N et al. Mutations in the hepatocyte nuclear factor-4a gene in maturity-onset diabetes of the young (MODY1). Nature 1996; 384:458-459. 18. Froguel P, Vaxillaire M, Sun F et al. Close linkage of glucokinase locus on chromosome 7p to early-onset noninsulin-dependent diabetes mellitus. Nature 1992; 356:162-164. 19. Lehto M, Tuomi T, Mahtani M et al. Characterization of the MODY3 phenotype: earlyonset diabetes caused by an insulin secretion defect. J Clin Invest 1997; 99:582-591. 20. Rotig A, Cormier V, Chatelain P et al. Deletion of mitochondrial DNA in a case of earlyonset diabetes mellitus, optic atrophy, and deafness (Wolfram syndrome, MIM 222300). J Clin Invest 1993; 91:1095-1098. 21. Rando TA, Horton JC, Layzer RB. Wolfram syndrome: evidence of a diffuse neuro-degenerative disease by magnetic resonance imaging. Neurology 1992; 42:1220-1224. 22. Collier DA, Barrett TG, Curtis D et al. Linkage of Wolfram syndrome to chromosome 4p16.1 and evidence for heterogeneity. Am J Hum Genet 1996; 59:855-863. 23. Polymeropoulos MH, Swift RG, Swift M. Linkage of the gene for Wolfram syndrome to markers on the short arm of chromosome 4. Nat Genet 1994; 8:95-97. 24. Barrientos A, Volpini V, Casademont J et al. A nuclear defect in the 4p16 region predisposes to multiple mitochondrial DNA deletions in families with Wolfram syndrome. J Clin Invest 1996; 97:1570-1576. 25. Kadowaki T, Kadowaki H, Mori Y et al. A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA. N Engl J Med 1994; 330:962-968. 26. Winter WE, Maclaren NK, Riley WJ et al. Maturity-onset diabetes of youth in Black Americans. N Engl J Med 1987; 316:285-291. 27. Banerji MA, Chaiken RL, Huey H et al. GAD antibody negative NIDDM in adult black subjects with diabetic ketoacidosis and increased frequency of human leukocyte antigen DR3 and DR4. Diabetes 1994; 43:741-745. 28. Aaltonen J, Björses P, Sandkuijl L et al. An autosomal locus causing autoimmune disease: autoimmune polyglandular disease type I assigned to chromosome 21. Nat Genet 1994; 8:83-87. 29. Neufeld M, Maclaren NK, Blizzard RM. Two types of autoimmune Addison’s disease associated with different polyglandular autoimmune (PGA) syndromes. Medicine 1981; 60:355-362.

174

Endocrine and Organ Specific Autoimmunity

30. Verge CF, Gianani R, Yu L et al. Late progression to diabetes and evidence for chronic bcell autoimmunity in identical twins of patients with type I diabetes. Diabetes 1995; 44:1176-1179. 31. Petersen JS, Kyvik KO, Bingley PJ et al. Population based study of prevalence of islet cell autoantibodies in monozygotic and dizygotic Danish twin pairs with insulin dependent diabetes mellitus. BMJ 1997; 314:1575-1579. 32. Nepom GT. Immunogenetics and IDDM. Diabetes Rev 1993; 1:93-103. 33. Pugliese A, Eisenbarth GS. Human type I diabetes mellitus: genetic susceptibility and resistance. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:134-152. 34. Eisenbarth GS. Primer: immunology/autoimmunity. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:3-17. 35. Rewers M, Bugawan TL, Norris JM et al. Newborn screening for HLA markers associated with IDDM: Diabetes autoimmunity study in the young (DAISY). Diabetologia 1996; 39:807-812. 36. Baisch JM, Week T, Giles R et al. Analysis of HLA-DQ genotypes and susceptibility in insulin-dependent diabetes mellitus. N Engl J Med 1990; 322:1836-1841. 37. Pugliese A, Gianani R, Moromisato R et al. HLA-DQB1*0602 is associated with dominant protection from diabetes even among islet cell antibody-positive first degree relatives of patients with IDDM. Diabetes 1995; 44:608-613. 38. Faustman D, Li X, Lin HY et al. Linkage of faulty major histocompatibility complex class I to autoimmune diabetes. Science 1991; 254:1756-1761. 39. Makino S, Kunimoto K, Muraoka Y et al. Breeding of a nonobese, diabetic strain of mice. Exp Anim 1980; 29:1-13. 40. Fujita T, Yui R, Kusumoto Y et al. Lymphocytic insulitis in a “nonobese diabetic (NOD)” strain of mice: An immunohistochemical and electron microscope investigation. Biomed Res 1982; 3:429-443. 41. Hattori M, Buse JB, Jackson RA et al. The NOD mouse: Recessive diabetogenic gene within the major histocompatibility complex. Science 1986; 231:733-735. 42. Acha-Orbea H, McDevitt HO. The first external domain of the nonobese diabetic mouse class II I-A chain is unique. Proc Natl Acad Sci U S A 1987; 84:2435-2439. 43. Nishimoto H, Kikutani H, Yamamura K et al. Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature 1987; 328:432-434. 44. Uno M, Miyazaki T, Uehira M et al. Complete prevention of diabetes in transgenic NOD mice expressing I-E molecules. Immunol Lett 1991; 31:47-52. 45. Slattery RM, Kjer-Nielsen L, Allison J et al. Prevention of diabetes in nonobese diabetic I-Ak transgenic mice. Nature 1990; 345:724-726. 46. Prochazka M, Leiter EH, Serreze DV et al. Three recessive loci required for insulin-dependent diabetes in NOD mice. Science 1987; 237:286-289. 47. Todd JA, Aitman TJ, Cornall RJ et al. Genetic analysis of autoimmune type I diabetes mellitus in mice. Nature 1991; 351:542-547. 48. Wicker LS, Todd JA, Peterson LB. Genetic control of autoimmune diabetes in the NOD mouse. Ann Rev Immunol 1995; 13:179-200. 49. Denny P, Lord CJ, Hill NJ et al. Mapping of the IDDM locus Idd3 to a 0.35-cM interval containing the Interleukin-2 gene. Diabetes 1997; 46:695-700. 50. Moss J, Stevens LA, Cavanaugh E et al. Characterization of mouse Rt6.1 NAD: Arginine ADP-ribosyltransferase. J Biol Chem 1997; 272:4342-4346. 51. Like AA. Depletion of RT6.1+ lymphocytes alone is insufficient to induce diabetes in diabetes-resistant BB/WOR rats. Am J Pathol 1990; 136:565-574. 52. Gottlieb PA, Berrios JP, Mariani G et al. Autoimmune destruction of islets transplanted into RT6-depleted diabetes-resistant BB/Wor rats. Diabetes 1990; 39:643-646. 53. Todd JA, Farrall M. Panning for gold: Genome-wide scanning for linkage in type I diabetes. Hum Mol Genet 1996; 5:1443-1448.

Type I Diabetes Mellitus

175

54. Bell GI, Horita S, Karam JH. A polymorphic locus near the human insulin gene is associated with insulin dependent diabetes mellitus. Diabetes 1983; 33:176-183. 55. Undlien DE, Bennett ST, Todd JA et al. Insulin gene region-encoded susceptibility to IDDM maps upstream of the insulin gene. Diabetes 1995; 44:620-625. 56. Owerbach D, Gabbay KH. Localization of a type l diabetes susceptibility locus to the variable tandem repeat region flanking the insulin gene. Diabetes 1993; 42:1708-1714. 57. Lucassen AM, Julier C, Beressi J et al. Susceptibility to insulin dependent diabetes mellitus maps to a 4.1 kb segment of DNA spanning the insulin gene and associated VNTR. Nat Genet 1993; 4:305-310. 58. McGinnis RE, Spielman RS. Linkage disequilibrium in the insulin gene region: size variation at the 5’flanking polymorphism and bimodality among “class I” alleles. Am J Hum Genet 1994; 55:526-532. 59. Bennett ST, Lucassen AM, Gough SCL et al. Susceptibility to human type I diabetes at IDDM2 is determined by tandem repeat variation at the insulin gene minisatellite locus. Nat Genet 1995; 9:284-292. 60. Bui MM, Luo D, She JY et al. Paternally transmitted IDDM2 influences diabetes susceptibility despite biallelic expression of the insulin gene in human pancreas. J Autoimmun 1996; 9:97-103. 61. McGinnis RE, Spielman RS. Insulin expression: Is VNTR allele 698 really anomalous? Nat Genet 1995; 10:378-380. 62. Pugliese A, Awdeh ZL, Alper CA et al. The paternally inherited insulin gene B allele (1,428 FokI site) confers protection from insulin-dependent diabetes in families. J Autoimmun 1994; 7:687-694. 63. Kennedy GC, German MS, Rutter WJ. The minisatellite in the diabetes susceptibility locus IDDM2 regulates insulin transcription. Nat Genet 1996; 9:293-298. 64. Lucassen AM, Screaton GR, Julier C et al. Regulation of insulin gene expression by the IDDM associated, insulin locus haplotype. Hum Mol Genet 1995; 4:501-506. 65. Eisenbarth GS, Jackson RA, Pugliese A. Insulin autoimmunity: the rate limiting factor in pretype I diabetes. J Autoimmun 1992; 5 (suppl A):241-246. 66. Pugliese A, Zeller M, Fernandez A et al. The insulin gene is transcribed in the human thymus and transcription levels correlate with allelic variation at the INS VNTR-IDDM2 susceptibility locus for type I diabetes. Nat Genet 1997; 15:293-297. 67. Vafiadis P, Bennett ST, Todd JA et al. Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus. Nat Genet 1997; 15:289-292. 68. Hashimoto L, Habita C, Beressi JP et al. Genetic mapping of a susceptibility locus for insulin-dependent diabetes mellitus on chromosome 11q. Nature 1994; 371:161-164. 69. Luo D, Bui MM, Muir A et al. Affected-sib-pair mapping of a novel susceptibility gene to insulin-dependent diabetes mellitus (IDDM8) on chromosome 6q25-q27. Am J Hum Genet 1995; 57:911-919. 70. Davies JL, Kawaguchi Y, Bennett ST et al. A genome-wide search for human type 1 diabetes susceptibility genes. Nature 1994; 371:130-136. 71. Copeman JB, Cucca F, Hearne CM et al. Linkage disequilibrium mapping of type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33. Nat Genet 1995; 9:80-85. 72. Morahan G, Huang D, Tait BD et al. Markers on distal chromosome 2q linked to insulindependent diabetes mellitus. Science 1996; 272:1811-1813. 73. Noble JA, Valdes AM, Cook M et al. The role of HLA class II genes in insulin-dependent diabetes mellitus: molecular analysis of 180 caucasian, multiplex families. Am J Hum Genet 1996; 59:1134-1148. 74. Verge C, Eisenbarth GS. Autoimmune polyendocrine syndromes. In: Wilson JD, Foster DW, ed(s). Williams Textbook of Endocrinology. 9th ed. Philadephia: W.B. Saunders, 1997: 75. Brewer K, Parziale VS, Eisenbarth GS. Screening patients with insulin-dependent diabetes mellitus for adrenal insufficiency. N Engl J Med 1997; 337:202 76. Bugawan JL, Erlich HA. Rapid typing of HLA-DQB1 DNA polymorphism using nonradioactive oligonucleotide probes and amplified DNA. Immunogenetics 1991; 33:163-170.

176

Endocrine and Organ Specific Autoimmunity

77. Kyvik KO, Green A, Beck-Nielsen H. Concordance rates of insulin dependent diabetes mellitus: a population based study of young Danish twins. BMJ 1995; 311:913-917. 78. Padaiga Z, Tuomilehto J, Karvonen M et al. Incidence trends in childhood onset IDDM in four countries around the Baltic sea during 1983-1992. Diabetologia 1997; 40:187-192. 79. Helmke K, Otten A, Willems WR et al. Islet cell antibodies and the development of diabetes mellitus in relation to mumps infection and mumps vaccination. Diabetologia 1986; 29:30-33. 80. Bodansky HJ, Dean BM, Bottazzo GF et al. Islet cell antibodies and insulin autoantibodies in association with common viral infections. Lancet 1986; 2:1351-1353. 81. Ginsberg-Fellner F, Witt ME, Yagihashi S et al. Congenital rubella syndrome as a model for Type I (insulin-dependent) diabetes mellitus: Increased prevalence of islet cell surface antibodies. Diabetologia 1984; 27:87-89. 82. Rabinowe SL, George KL, Laughlin R et al. Congenital rubella: Monoclonal antibody defined T cell abnormalities in young children. Am J Med 1986; 81:779-782. 83. Clarke W, Shaver K, Bright GA et al. Autoimmunity in congenital rubella syndrome. J Pediatr 1984; 104:370-373. 84. Karounos DG, Wolinsky JS, Thomas JW. Monoclonal antibody to rubella virus capsid protein recognizes a b-cell antigen. J Immunol 1993; 150:3080-3085. 85. Yoon JW, Austin M, Onodera T et al. Virus-induced diabetes mellitus: isolation of a virus from the pancreas of a child with diabetic ketoacidosis. N Engl J Med 1979; 300:1173-1179. 86. Gepts W. The pathology of the pancreas in human diabetes. In: Andreani D, DiMario U, Federlin KF et al, Immunology in Diabetes. London: Kimpton Medical Publications, 1984:21-35. 87. Gerling I, Nejman C, Chatterjee NK. Effect of Coxsackie virus B4 infection in mice on expression of 64,000Mr autoantigen and glucose sensitivity of islets before development of hyperglycemia. Diabetes 1988; 37:1419-1425. 88. Foulis AK, McGill M, Farquharson MA et al. A search for evidence of viral infection in panreases of newly diagnosed patients with IDDM. Diabetologia 1997; 40:53-61. 89. Graves PM, Rewers M. The role of enteroviral infections in the development of IDDM: limitations of current approaches. Diabetes 1997; 46:161-168. 90. Ellerman KE, Richards CA, Guberski DL et al. Kilham rat virus triggers T cell-dependent autoimmune diabetes in multiple strains of rat. Diabetes 1996; 45:557-562. 91. Lachaux A, Bouvier R, Cozzani E et al. Familial autoimmune enteropathy with circulating anti-bullous pemphigoid antibodies and chronic autoimmune hepatitis. J Pediatr 1994; 125:858-862. 92. Elliott RB. Cow’s milk and the diabetes debate. Pediatrics 1995; 96:541 93. Gerstein HC. Cow’s milk exposure and type I diabetes mellitus—a critical overview of the clinical literature. Diabetes Care 1994; 17:13-19. 94. Virtanen SM, Saukkonen T, Savilahti E et al. Diet, cow’s milk protein antibodies and the risk of IDDM in Finnish children. Diabetologia 1994; 37:381-387. 95. Scott FW, Cloutier HE, Kleemann R et al. Potential mechanisms by which certain foods promote or inhibit the development of spontaneous diabetes in BB rats. Diabetes 1997; 46:589-598. 96. Scott FW. Food-induced type 1 diabetes in the BB rat. Diabetes Metab Rev 1996; 12:341-359. 97. Kostraba JN, Cruickshanks KJ, Lawler-Heavner J et al. Early exposure to cow’s milk and solid foods in infancy, genetic predisposition and risk of IDDM. Diabetes 1993; 42:288-295. 98. Graves PM, Rotbart HA, Norris JM et al. Apparent lack of association between enteroviral infections and prediabetic autoimmunity: Diabetes autoimmunity study in the young (DAISY). submitted 1996; 99. Ilonen J, Reijonen H, Herva E et al. Rapid HLA-DQB1 genotyping for four alleles in the assessment of risk for IDDM in the Finnish population. Diabetes Care 1996; 19:795-800. 100. Bottazzo GF, Florin-Christensen A, Doniach D. Islet cell antibodies in diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974; 2:1279-1283. 101. Dotta F, Gianani R, Previti M et al. Autoimmunity to the GM2-1 islet ganglioside before and at the onset of type I diabetes. Diabetes 1996; 45:1193-1196.

Type I Diabetes Mellitus

177

102. Martin S, Kardorf J, Schulte B et al. Autoantibodies to the islet antigen ICA 69 occur in IDDM and in rheumatoid arthritis. Diabetologia 1995; 38:351-355. 103. Pietropaolo M, Castano L, Babu S et al. Islet cell autoantigen 69 kDa (ICA69): Molecular cloning and characterization of a novel diabetes associated autoantigen. J Clin Invest 1993; 92:359-371. 104. Stassi G, Schloot N, Pietropaolo M. Islet cell autoantigen 69 kDa (ICA69) is preferentially expressed in the human islets of Langerhans than exocrine pancreas. Diabetologia 1997; 40:120-121. 105. Falorni A, Örtqvist E, Persson B et al. Radio immunoassays for glutamic acid decarboxylase (GAD65) and GAD65 autoantibodies using 35S or 3H recombinant human ligands. J Immunol Methods 1995; 186:89-99. 106. Kaprio J, Tuomilehto J, Koshenvuo M et al. Concordance for type 1 (insulin-dependent) and type 2 (noninsulin-dependent) diabetes mellitus in a population-based cohort of twins in Finland. Diabetologia 1993; 35:1060-1067. 107. Rabin DU, Pleasic SM, Palmer-Crocker R et al. Cloning and expression of IDDM-specific human autoantigens. Diabetes 1992; 41:183-186. 108. Gianani R, Rabin DU, Verge CF et al. ICA512 autoantibody radio assay. Diabetes 1995; 44:1340-1344. 109. Lan MS, LU J, Goto Y et al. Molecular cloning and identification of a receptor-type protein tyrosine phosphatase, IA-2, from human insulinoma. DNA Cell Biol 1994; 13:505-514. 110. Zhang B, Lan M, Notkins A. Autoantibodies to IA-2 in IDDM: location of major antigenic determinants. Diabetes 1997; 46:40-43. 111. Christie MR. Islet cell autoantigens in type I diabetes. Eur J Clin Invest 1996; 26:827-838. 112. Pietropaolo M, Hutton JC, Eisenbarth GS. Protein tyrosine phosphatase-like proteins: link with IDDM. Diabetes Care 1996; 20:208-214. 113. Kawasaki E, Yu L, Gianani R et al. Evaluation of islet cell antigen (ICA) 512/IA-2 autoantibody radio assays using overlapping ICA512/IA-2 constructs. J Clin Endocrinol Metab 1997; 82:375-380. 114. Kawasaki E, Eisenbarth GS, Wasmeier C et al. Autoantibodies to protein tyrosine phosphatase-like proteins in type I diabetes: overlapping specificities to phogrin and ICA512/ IA-2. Diabetes 1996; 45:1344-1349. 115. Greenbaum CJ, Palmer JP. Humoral immune markers: insulin antibodies. In: Palmer JP, ed. Prediction, Prevention, and Genetic Counseling in IDDM. Chichester, England: Wiley, 1996:63-76. 116. Greenbaum CJ, Wilkin TJ, Palmer JP. Fifth international serum exchange workshop for insulin autoantibody (IAA) standardization. Diabetologia 1992; 35:798-800. 117. Castano L, Ziegler AG, Ziegler R et al. Characterization of insulin autoantibodies in relatives of patients with type 1 diabetes. Diabetes 1993; 42:1202-1209. 118. Aanstoot H, Kang S, Kim J et al. Identification and characterization of glima 38, a glycosylated islet cell membrane antigen, which together with GAD65 and IA2 marks the early phases of autoimmune response in type I diabetes. J Clin Invest 1996; 97:2772-2783. 119. Atkinson MA, Maclaren NK. Islet cell autoantigens in insulin-dependent diabetes. J Clin Invest 1993; 92:1608-1616. 120. Gianani R, Eisenbarth GS. Humoral autoimmunity. In: Eisenbarth GS, Lafferty KJ, ed(s). Type I Diabetes: Molecular, Cellular, and Clinical Immunology. New York City, New York: Oxford University Press, 1996:209-229. 121. Martin S, Lampasona V, Dosch M et al. Islet cell autoantigen 69 antibodies in IDDM. Diabetologia 1996; 39:747 122. Elias D, Cohen IR. Treatment of autoimmune diabetes and insulitis in NOD mice with heat shock protein. Diabetes 1995; 44:1132-1138. 123. Mcevoy RC, Thomas NM, Ness J. Humoral immune markers: additional islet cell antigens--important clues to pathogenesis or red herrings? In: Palmer JP, ed. Prediction, prevention, and genetic counseling in IDDM. Chichester, England: Wiley, 1996:97-108. 124. Buschard K, Fredman P. Sulphatide as an antigen in diabetes mellitus. Diab Nutr Metab 1996; In press.

178

Endocrine and Organ Specific Autoimmunity

125. Wegmann DR, Norbury-Glaser M, Daniel D. Insulin-specific T cells are a predominant component of islet infiltrates in prediabetic NOD mice. Eur J Immunol 1994; 24:1853-1857. 126. Daniel D, Gill RG, Schloot N et al. Epitope specificity, cytokine production profile and diabetogenic activity of insulin-specific T cell clones isolated from NOD mice. Eur J Immunol 1995; 25:1056-1062. 127. French MB, Allison J, Cram DS et al. Transgenic expression of mouse proinsulin II prevents diabetes in nonobese diabetic mice. Diabetes 1996; 46:34-39. 128. Simone E, Daniel D, Schloot N et al. T Cell Receptor Restriction of Diabetogenic Autoimmune NOD T Cells. Proc Natl Acad Sci U S A 1997; 94:2518-2521. 129. Schloot N, Daniel D, Wegmann D. Limiting dilution analysis of islet infiltrating and peripheral T cells in prediabetic mice. Diabetologia 1994; 37:364 Abstract. 130. Griffin AC, Zhao W, Wegmann KW et al. Experimental autoimmune insulitis: induction by T lymphocytes specific for a peptide of proinsulin. Am J Pathol 1995; 147:845-857. 131. Rudy G, Stone N, Harrison LC et al. Similar peptides from two b cell autoantigens, proinsulin and glutamic acid decarboxylase, stimulate T cells of individuals at risk for insulindependent diabetes. Mol Med 1996; 1:625-633. 132. Zekzer D, Wong F, von Grafenstein H et al. An insulin-reactive T cell clone prevents IDDM in NOD mice by blocking homing of diabetogenic splenocytes to the Islet. Diabetes 1996; 45:189A Abstract. 133. Kaufman DL, Clare-Salzler M, Tian J et al. Spontaneous loss of T cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993; 366:69-72. 134. Tisch R, Yang X, Singer SM et al. Immune response to glutamic acid decarboxylase correlates with insulitis in nonobese diabetic mice. Nature 1993; 366:72-75. 135. Harrison LC, Honeyman MC, De Aizpurua HJ et al. Inverse relationship between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes. Lancet 1993; 341:1365-1369. 136. Atkinson MA, Kaufman DL, Campbell L et al. Response of peripheral-blood mononuclear cells to glutamate decarboxylase in insulin-dependent diabetes. Lancet 1992; 339:458-459. 137. Haskins K, Wegmann D. Diabetogenic T cell clones. Diabetes 1996; 45:1299-1305. 138. Bergman B, Haskins K. Autoreactive T cell clones from the nonobese diabetic mouse. Proc Soc Exp Biol Med 1997; 214:41-48. 139. McKeever U, Khandekar S, Newcomb J et al. Immunization with soluble BDC 2.5 T cell receptor-immunoglobulin chimeric protein: antibody specificity and protection of nonobese diabetic mice against adoptive transfer of diabetes by maternal immunization. J Exp Med 1996; 184:1755-1768. 140. Peterson JD, Pike B, Dallas-Pedretti A et al. Induction of diabetes with islet-specific T cell clones is age dependent. Immunology 1995; 85:455-460. 141. Atkinson MA, Bowman MA, Kuo-Jang K et al. Lack of immune responsiveness to bovine serum albumin in insulin-dependent diabetes. N Engl J Med 1993; 329:1853-1858. 142. Atkinson MA, Bowman MA, Campbell L et al. Cellular immunity to a determinant common to glutamate decarboxylase and Coxsackie virus in insulin-dependent diabetes. J Clin Invest 1994; 94:2125-2129. 143. Durinovic-Bello I, Hummel M, Ziegler AG. Cellular immune response to diverse islet cell antigens in IDDM. Diabetes 1996; 45:795-800. 144. Brooks-Worrell BM, Starkebaum GA, Greenbaum C et al. Peripheral blood mononuclear cells of insulin-dependent diabetic patients respond to multiple islet cell proteins. J Immunol 1996; 157:5668-5674. 145. Endl J, Otto H, Jung G et al. Identification of naturally processed T cell epitopes from glutamic acid decarboxylase presented in the context of HLA-DR alleles by T lymphocytes of recent onset IDDM patients. J Clin Invest 1997; 99:2405-2415. 146. Schloot NC, Roep BO, Wegmann DR et al. T cell reactivity to GAD65 peptide sequences shared with Coxsackie virus protein in IDDM patients and controls. Diabetologia 1997; In press.

Type I Diabetes Mellitus

179

147. Schloot N, Roep BO, Wegmann D et al. Altered immune response to insulin in newly diagnosed compared insulin-treated diabetic patients and healthy control subjects. Diabetologia 1997; 40:564-572. 148. Pugliese A, Bugawan T, Moromisato R et al. Two subsets of HLA-DQA1 alleles mark phenotypic variation in levels of insulin autoantibodies in first degree relatives at risk for insulin-dependent diabetes. J Clin Invest 1994; 93:2447-2452. 149. Keller RJ, Eisenbarth GS, Jackson RA. Insulin prophylaxis in individuals at high risk of type I diabetes. Lancet 1993; 341:927-928. 150. Ziegler AG, Ziegler R, Vardi P et al. Life table analysis of progression to diabetes of antiinsulin autoantibody positive relatives of individuals with type I diabetes. Diabetes 1989; 38:1320-1325. 151. Vardi P, Ziegler AG, Matthews JH et al. Concentration of insulin autoantibodies at onset of Type I diabetes: inverse log-linear correlation with age. Diabetes Care 1988; 11:736-739. 152. Petersen JS, Russel S, Marshall MO et al. Differential expression of glutamic acid decarboxylase in rat and human islets. Diabetes 1993; 42:484-495. 153. Julier C, Hyer RN, Davies J et al. Insulin-IGF2 region on chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility. Nature 1991; 354:155-159. 154. Wegmann DR, Gill RG, Norbury-Glaser M et al. Analysis of the spontaneous T cell response to insulin in NOD mice. J Autoimmun 1994; 7:833-843. 155. Zhang JZ, Davidson L, Eisenbarth GS et al. Suppression of diabetes in nonobese diabetic mice by oral administration of porcine insulin. Proc Natl Acad Sci USA 1991; 88:10252-10256. 156. Bergerot I, Fabien N, Maguer V et al. Oral insulin induces CD4+ regulatory cells that protect NOD mice from adoptive transfer of diabetes. J Immunol 1994; Abstract. 157. 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 U S A 1996; 93:956-960. 158. Harrison LC, Dempsey-Collier M, Kramer DR et al. Aerosol insulin induces regulatory CD8 yd T cells that prevent murine insulin-dependent diabetes. J Exp Med 1997; 184:2167-2174. 159. Maclaren N, Lafferty K. Perspectives in Diabetes: The 12th international immunology and diabetes workshop. Diabetes 1993; 42:1099-1104. 160. Muir A, Peck A, Clare-Salzler M et al. Insulin immunization of nonobese diabetic mice induces a protective insulitis characterized by diminished intraislet interferon-y transcription. J Clin Invest 1995; 95:628-634. 161. Ramiya VK, Shang X, Pharis PG et al. Antigen based therapies to prevent diabetes in NOD mice. J Autoimmun 1996; 9:349-356. 162. Muir A, Schatz D, Maclaren N. Antigen-specific immunotherapy: oral tolerance and subcutaneous immunization in the treatment of insulin-dependent diabetes. Diabetes Metab Rev 1993; 9:279-287. 163. Bendtzen K, Mandrup-Poulsen T, Nerup J et al. Cytotoxicity of human p17 interleukin-1 for pancreatic islets of Langerhans. Science 1986; 232:1545-47. 164. Bergmann L, Kroncke K, Suschek D et al. Cytotoxic action of IL-1B against islets is mediated via nitric oxide formation and is inhibited by NG-monomethyl-L-arginine. FEBS Lett 1992; 103-106. 165. Ling Z, In’t Veld PA, Pipeleers DG. Interaction of interleukin-1 with islet b-Cells: Distinction between indirect, aspecific cytotoxicity and direct, specific functional suppression. Diabetes 1993; 42:56-64. 166. Delaney CA, Green MHL, Lowe JE et al. Endogenous nitric oxide induced by interleukin1 b in rat islets of langerhans and HIT-T15 cells causes significant DNA damage as measured by the ‘comet’ assay. Feder Eur Biochem Soc 1993; 333:291-295. 167. Corbett JA, Wang JL, Sweetland MA et al. Interleukin 1b induces the formation of nitric oxide by b-cells purified from rodent islets of langerhans. J Clin Invest 1992; 90:2384-2391.

180

Endocrine and Organ Specific Autoimmunity

168. Mueller R, Krahl T, Sarvetnick N. Pancreatic expression of interleukin-4 abrogates insulitis and autoimmune diabetes in nonobese diabetic (NOD) mice. J Exp Med 1996; 184:1093-1099. 169. Sarvetnick N, Shizuru J, Liggitt D et al. Loss of pancreatic islet tolerance induced by b-cell expression of interferon-y. Nature 1990; 346:844-847. 170. Wogensen L, Lee M, Sarvetnick N. Production of interleukin 10 by islet cells accelerates immune-mediated destruction of b cells in nonobese diabetic mice. J Exp Med 1994; 179:1379-1384. 171. Sarvetnick N. IFN-y, IGIF, and IDDM. J Clin Invest 1997; 99:371-372. 172. Mosmann TR, Schumacher JH, Street NF et al. Diversity of cytokine synthesis and function of mouse CD4+ T cells. Immunol Rev 1991; 123:209-229. 173. Janeway CA, Jr., Bottomly K. Signals and signs for lymphocyte responses. Cell 1994; 76:275-285. 174. Rabinovitch A, Sorensen O, Suarez-Pinzon WL et al. Analysis of cytokine mRNA expression in syngeneic islet grafts of NOD mice: interleukin 2 and interferon gamma mRNA expression correlate with graft rejection and interleukin 10 with graft survival. Diabetologia 1994; 37:833-837. 175. Rabinovitch A. Roles of cytokines in IDDM pathogenesis and islet b-cell destruction. Diabetes Rev 1993; 1:215-240. 176. Trembleau S, Penna G, Bosi E et al. Interleukin 12 administration induces T helper type 1 cells and accelerates autoimmune diabetes in NOD mice. J Exp Med 1995; 181:817-821. 177. Katz JD, Benoist C, Mathis D. T helper cell subsets in insulin-dependent diabetes. Science 1995; 268:1185-1188. 178. Rabinovitch A, Sumoski W, Rajotte RV et al. Cytotoxic effects of cytokines on human pancreatic islet cells in monolayer culture. J Clin Endocrinol Metab 1990; 71(1):152-156. 179. Sumoski W, Baquerizo H, Rabinovitch A. Oxygen free radical scavengers protect rat islet cells from damage by cytokines. Diabetologia 1989; 32:792-796. 180. Pukel C, Bawuerizo H, Rabinovitch A. Destruction of rat islet cell monolayers by cytokines. Synergistic interactions of interferon-gamma tumor necrosis factor, lymphotoxin, and interleukin 1. Diabetes 1988; 37:133-36. 181. Yang XD, Tisch R, Singer SM et al. Effect of tumor necrosis factor alpha on insulin-dependent diabetes mellitus in NOD mice. I. The early development of autoimmunity and the diabetogenic process. J Exp Med 1994; 180:995-1004. 182. Rapoport MJ, Jaramillo A, Zipris D et al. Interleukin 4 reverses T cell proliferative unresponsiveness and prevents the onset of diabetes in nonobese diabetic mice. J Exp Med 1993; 178:87-99. 183. Pennline KJ, Rogue-Gaffney E, Monahan M. Recombinant human IL-10 prevents the onset of diabetes in the nonobese diabetic mouse. Clin Immunol Immunopathol 1994; 71:169-175. 184. Shehadeh NN, LaRosa F, Lafferty KJ. Altered cytokine activity in adjuvant inhibition of autoimmune diabetes. J Autoimmun 1993; 6:291-300. 185. Chen Y, Kuchroo VK, Inobe J et al. Regulatory T cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 1994; 265:1237-1240. 186. Powell TJ, Streilein JW. Neonatal tolerance induction by class II alloantigens activates IL4-secreting, tolerogen-responsive T cells. J Immunol 1990; 144:854-859. 187. Chen N, Field EH. Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. Transplantation 1995; 59:933-941. 188. MacKay P. Adoptive transfer of diabetes to and from old normoglycaemic BB rats. Diabetologia 1995; 38:145-152. 189. Saoudi A, Simmonds S, Hiutinga I et al. Prevention of experimental allergic encephalomyelitis in rats by targeting autoantigen to B cells: evidence that the protective mechanism depends on changes in the cytokine response and migratory properties of the autoantigenspecific T cells. J Exp Med 1995; 182:335-344.

Type I Diabetes Mellitus

181

190. Campbell IL, Kay TWH, Oxbrow L et al. Essential role for interferon-y and interleukin-6 in autoimmune insulin-dependent diabetes in NOD/Wehi mice. J Clin Invest 1991; 87:739-742. 191. Hultgren B, Huang X, Dybdal N et al. Genetic absence of y-interferon delays but does not prevent diabetes in NOD mice. Diabetes 1996; 45:812-817. 192. Wegmann D, Daniel D. Pathogenicity of insulin specific, Th2 T cell clones in NOD mice. Diabetes 1997; 46:40A Abstract. 193. VanBuskirk AM, Wakely ME, Orosz CG. Transfusion of polarized Th2-like cell populations into SCID mouse cardiac allograft recipients results in acute allograft rejection. Transplantation 1996; 62:229-238. 194. Zekzer D, Wong FS, Wen L et al. Inhibition of diabetes by an insulin-reactive CD4 T cell clone in the nonobese diabetic mouse. Diabetes 1997; 46:1124-1132. 195. Rabinovitch A, Suarez-Pinzon WL, Sorensen O et al. Combined therapy with interleukin4 and interleukin-10 inhibits autoimmune diabetes recurrence in syngeneic islet-transplanted nonobese diabetic mice. Transplantation 1995; 60:368-374. 196. Faust A, Rothe H, Schade U et al. Primary nonfunction of islet grafts in autoimmune diabetic nonobese diabetic mice is prevented by treatment with interleukin-4 and interleukin-10. Transplantation 1996; 62:648-652. 197. Coffman RL, Varkila K, Scott P et al. Role of cytokines in the differentiation of CD4+ T cell subsets in vivo. Immunol Rev 1991; 123:189-207. 198. Scholl M, Daniel D, Wegmann D et al. Autoimmune islet damage mediated by insulinspecific T cells. Transplant Proc 1996; In press. 199. Chervonsky AV, Wang Y, Wong FS et al. The role of Fas in autoimmune diabetes. Cell 1997; 89:17-24. 200. O’Brien BA, Harmon BV, Cameron DP et al. Apoptosis is the mode of b-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 1997; 46:750-757. 201. Katz JD, Wang B, Haskins K et al. Following a diabetogenic T cell from genesis through pathogenesis. Cell 1993; 74:1089-1100. 202. Lipes MA, Fenton RG, Zhou LJ et al. Autoimmunity occurs in transgenic T cell receptor beta gene nonobese diabetic (NOD) mice. Clin Res 1989; 37:572A 203. Karges W, Hammond-McKibben D, Cheung RK et al. Immunological aspects of nutritional diabetes prevention in NOD mice. Diabetes 1997; 46:557-564. 204. Carel J, Boitard C, Eisenbarth G et al. Cyclosporine delays but does not prevent clinical onset in glucose intolerant pretype I diabetic children. J Autoimmun 1996; 9:739-745. 205. Rossini AA, Greiner DL, Friedman HP et al. Immunopathogenesis of diabetes mellitus. Diabetes Rev 1993; 1:43-75. 206. Bowman MA, Leiter EH, Atkinson MA. Prevention of diabetes in the NOD mouse: implications for therapeutic intervention in human disease. Immunol Today 1994; 15:115-120. 207. Hayward AR, Shriber M, Cooke A et al. Prevention of diabetes but not insulitis in NOD mice injected with antibody to CD4. J Autoimmun 1993; 6:301-310. 208. Gazda LS, Baxter AG, Lafferty KJ. Regulation of autoimmune diabetes: characteristics of nonislet-antigen specific therapies. Immunol Cell Biol 1996; 74:401-407. 209. Shehadeh N, Calcinaro F, Bradley BJ et al. Effect of adjuvant therapy on development of diabetes in mouse and man. Lancet 1994; 343:706-707. 210. Klingensmith G, Allen H, Hayward A et al. Vaccination with BCG at diagnosis does not alter the course of IDDM. Diabetes 1997; 46:744 Abstract. 211. Yamada K, Nonaka K, Hanafusa T et al. Preventive and therapeutic effects of large-dose nicotinamide injections on diabetes associated with insulitis: An observation in nonobese diabetic (NOD) mice. Diabetes 1982; 31:749-753. 212. Solimena M, De Camilli P. From Th1 to Th2: Diabetes immunotherapy shifts gears. Nat Med 1996; 2:1311-1312. 213. Tian J, Clare-Salzler M, Herschenfeld A et al. Modulating autoimmune responses to GAD inhibits disease progression and prolongs islet graft survival in diabetes-prone mice. Nat Med 1996; 2:1348-1353.

182

Endocrine and Organ Specific Autoimmunity

214. Elias D, Reshef T, Birk OS et al. Vaccination against autoimmune mouse diabetes with a T cell epitope of the human 65-kDa heat shock protein. Proc Natl Acad Sci U S A 1991; 88:3088-3091. 215. von Herrath MG, Dyrberg T, Oldstone MBA. Oral insulin treatment suppresses virus-induced antigen-specific destruction of b cells and prevents autoimmune diabetes in transgenic mice. J Clin Invest 1996; 98:1324-1331. 216. Heath WR, Miller JFAD. Oral tolerance: feeding autoantigens can exacerbate rather than ameliorate autoimmune disease. J NIH Res 1997; 9:35-39. 217. Blanas E, Carbone FR, Allison J et al. Induction of autoimmune diabetes by oral administration of autoantigen. Science 1996; 274:1707-1709. 218. Eisenbarth GS, Srikanta S, Jackson RA et al. ATGAM and Prednisone immunotherapy of recent one type I diabetes mellitus. Diabetes Res 1985; 2:271-276. 219. Hayward AR, Shriber M. Reduced incidence of insulitis in NOD mice following anti-CD3 injection: requirement for neonatal injection. J Autoimmun 1992; 5:59-67. 220. Skyler JS, Lorenz TJ, Schwartz S et al. Effects of an anti-CD5 immunoconjugate (CD5Plus) in recent onset type I diabetes mellitus: a preliminary investigation. J Diabetes Complications 1993; 7:224-232. 221. Silverstein J, Maclaren N, Riley W et al. Immunosuppression with azathioprine and Prednisone in recent-onset insulin-dependent diabetes mellitus. N Engl J Med 1988; 319:599-604. 222. Mahon JL, Dupre J. Cyclosporine and azathioprine for IDDM. In: Palmer JP, ed. Prediction, Prevention, and Genetic Counseling in IDDM. Chichester, England: Wiley, 1996:257-272. 223. Bougneres PF, Carel JC, Castano L et al. Factors associated with early remission of type I diabetes in children treated with cyclosporine. N Engl J Med 1988; 318:663-670. 224. Chicz RM, Urban RG, Gorga JC et al. Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles. J Exp Med 1993; 178:27-47X. 225. Gotfredsen CF, Buschard K, Frandsen EK. Reduction of diabetes incidence of BB Wistar rats by early prophylactic insulin treatment of diabetes prone animals. Diabetologia 1985; 28:933-935. 226. Ziegler A, Schwertner R, Rabl W et al. Schwabing insulin prophylaxis (SIP) in relatives at high risk for type I diabetes. In: Baba S, Kaneko T, ed(s). Excerpta Medica. Elsevier Science, 1995:1080. 227. Kobayashi T, Nakanishi K, Murase T et al. Small doses of subcutaneous insulin as a strategy for preventing slowly progressive b-cell failure in islet cell antibody-positive patients with clinical features of NIDDM. Diabetes 1996; 45:622-626.

CHAPTER 9

Etiopathogenesis of Myasthenia Gravis (MG) Jean-François Bach, Ana Maria Yamamoto, Farid Djabiri and Henri-Jean Garchon

Introduction

M

yasthenia gravis (MG) is not one of the most common autoimmune disease (approximate prevalence of 0.1 per thousand in Western Europe) but it is certainly one of the most thoroughly studied autoimmune diseases. There is a very useful experimental animal model, induced by sensitization against the acetylcholine receptor (AChR). The autoimmune origin of the disease is established on the firm basis of antibody transfer experiments. More precisely, the disease is due to the blockade of neuromuscular conduction afforded by anti-AChR autoantibodies. The clinical presentation is heterogeneous and one must consider separately several clinical subsets even if these subsets share some common mechanisms including for all of them (except the so called seronegative form) the presence of AChR autoantibodies. - MG with thymus hyperplasia, usually observed in young females - MG with atrophic thymus - MG with thymoma - seronegative MG - drug-induced MG Experimental allergic myasthenia gravis (EAMG) is usually induced by immunizing normal animals (e.g., mice, rats or monkeys) by muscular preparations enriched in AChR derived from the homologous or heterologous species. The use of torpedo AChR is particularly attractive because of the high density of AChR in the electric plate of this fish species. MG can also be obtained by passive transfer of anti-AChR antibodies (see below). In any case, the disease resembles that of myasthenic patients at both the clinical level (neuromuscular fatigability) and at the electromyographic level. It is fair to recognize, however, that these manifestations remain relatively modest in intensity and transient. One regrets the absence in rodents of a spontaneous model of the disease that is presently only known in dogs.

Anti-AChR Autoantibodies The implication of anti-AChR antibodies in the pathogenesis of MG was described by Patrick and Lindstrom in Science.1 These authors fortuitously observed that rabbits immunized with affinity-purified AChR extracted from the electric organ of Electrophorus electricus developed muscular fatigability similar to that observed in human MG because the Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

Endocrine and Organ Specific Autoimmunity

184

anti-AChR antibodies formed cross-reacted with the rabbits’ own AChRs. Subsequently, they could show that the human disease was considerably (90%) and very specifically associated with the presence of anti-AChR antibodies.

Detection and Dosage The most currently used technique is a radioimmunoprecipitation assay in which a gross extract of human striated muscle is first incubated with radiolabeled 125 I α-bungarotoxin (bgt) (a high affinity ligand of the receptor). The complex thus formed is then incubated with the patient sera and ultimately precipitated with anti-human immunoglobulin antibodies. The amount of radioactivity in the precipitate provides an excellent quantitative assessment of the serum concentration of AChR antibodies.2-4 Table 9.1 presents the frequency of positive results found in a large series of patients evaluated in our laboratory. Other techniques have been developed using a similar assay with a rhabdomyosarcoma human cell line (TE671) as an AChR source (avoiding the laborious search for human muscle samples).5 Enzyme based immunoassays (ELISA) have also been developed but they have not been very much used because of the inferior capacity to quantitate the autoantibodies. Most laboratories used muscle from amputation or the TE671 line that expresses fetal AChR (γchain). Many patients present antibodies against adult AChR subtype (εchain).6-8 To overcome this problem, the TE671 line was recently transfected with the cDNA encoding the AChR ε subunit. This line improves the sensitivity of the reference immunoprecipitation test, especially in low titers and patients with ocular symptoms.9 It was disappointing to note that recombinant AChR (αchain) could not be used in place of the muscular extracts. Even if some monoclonal human anti-AChR autoantibodies recognize recombinant AChR, one must assume that the epitopes recognized by the patients’ anti-AChR antibodies are conformational and are not present in the recombinant α chain.

Table 9.1.Comparison Between AntiRACh Antibody Levels According to Sex and Age in Myasthenic Patients Men

Ocular myasthenia < 15 year old 15 to 40 year old > 40 year old Total Generalized myasthenia < 15 year old 15 to 40 year old > 40 year old Total

Women

Number of patients

Number of positive patients (%)

Number of Number of patients positive patients (%)

0 3 10 15

0 1 6 7 (53)

0 5 4 10

0 3 3 6 (70)

8 22 41 71

6 15 (72) 34 (83) 55 (80)

5 58 47 110

5 52 (89) 41 (87) 98 (92)

Etiopathogenesis of Myasthenia Gravis (MG)

185

Pathogenicity A myasthenic syndrome can be passively induced by the injection of myasthenic patients’ sera10 or immunoglobulins,11 or by anti-AChR monoclonal antibodies.12 Further evidence of the pathogenic role of anti-AChR autoantibodies is derived from the study of neonatal MG in babies born from myasthenic mothers.13 It is interesting though that the disease transmission is not constant even when mothers show high autoantibody serum titers. It is well known that transient neonatal MG may be transmitted by only 12% of mothers with MG. We have reported that women with antibodies directed to the embryos chain of the receptor (γchain) were significantly more prone to transmit the disease to their babies than women with anti-AChR autoantibodies directed against the adult form of the receptor.6

Mode of Action of Anti-AChR Autoantibodies Anti-AChR autoantibodies can block α-bgt binding to the receptor as demonstrated by the inhibition of α-bgt binding to AChR after preincubation of the receptor with the antibodies.7,14 This is probably not, however, the main mechanism of action since there is no good correlation between these blocking antibodies and disease activity. Another important mechanism is antibody-induced redistribution of the receptor, as an expression of antigenic modulation. The receptor turnover is accelerated and the amount of physiologically available receptor consequently decreased. This mechanism has been outlined in several in vitro studies using myoblast cultures.15

Clinical Correlations The heterogeneity of AChR antibodies and the probable restriction of the pathogenicity to a minor antibody subset explain the poor correlation observed between the antibody titer found in any of the assays used and the disease activity. The absence of any detectable antibodies in approximately 10% of patients (seronegative forms) is intriguing, the more so since immunoglobulins from some of these patients can transfer disease to mice16 and plasma exchange may be efficacious in these forms.17 Seronegative MG patients have similar symptoms and distribution of weakness to those with seropositive generalized MG, but proportionally more of them develop MG before puberty. Possibly seronegative MG patients have either low-affinity IgM antibodies that interfere directly with AChR function or IgM antibodies that indirectly affect function by binding to some other target at the neuromuscular junction. One may postulate that at least some of these cases are explained by autoantibodies directed to autoantigens other than AChR. The case of patients with restricted ocular MG is interesting inasmuch as the concentration of anti-α chain antibodies is usually lower than in the generalized form.

Genetics MG is not a hereditary disease as examplified by the low disease corcordance rate between siblings (< 2%). The observation of a number of concordant pairs of monozygotic twins (6/15)18 indicates however that there is an important genetic factor. This notion is also supported by the abnormally frequent presence of immunological abnormalities in family members of myasthenic patients and by the tight MHC control of experimental allergic MG.

186

Endocrine and Organ Specific Autoimmunity

Genetic Control of Susceptibility to Experimental Allergic Myasthenia Gravis (EAMG) The disease frequency in inbred mouse strain varies from mouse strain to strain but, surprisingly, this variation is not directly related to the presence or titer of anti-AChR antibodies. Studies of EAMG genetics reviewed by the main investigator in this field, Christadoss,19 have shown that the strain variability depends on H-2 haplotypes: the disease can easily be induced in H-2b, H-2r and H-2j mice, whereas H-2d, H-2k, H-2p and H-2s animals are resistant; H-2q and H-2f strains show intermediate susceptibility. Interestingly, anti-AChR antibody production is also H-2-linked but the linkage does not strictly overlap with susceptibility. Thus, the best anti-AChR antibody-producing strains are H-2b (like for disease) and H-2q, but H-2r, one of the other three highly susceptible strains, shows a low antibody titer suggesting that pathogenic autoantibodies represent a minor subset of global anti-AChR antibodies. It remains though that, in spite of some intra-H-2 variations, H-2b mice develop severe disease and high antibody titers, while H-2p and H-2k mice are neither susceptible to disease nor strong anti-AChR antibody producers. Importantly, studies of intra-H-2 recombinant and congenic mice have shown that the H-2 effect is exerted at the I-A locus for both autoantibody production and disease onset. The role of I-A (class II) is confirmed by the absence of disease and antibody in the C57 B6 C-H2bm12 strain with a mutant I-Aβ gene.20 Interestingly, C5-deficient mice with susceptibility H-2 genes are disease-resistant,21 suggesting a pathogenic role of complement. One should also mention that the presence of myoid cells in the thymus gland that could represent a sensitizing autoantigen source is apparently under genetic control.22 Different mouse strains are strikingly different in their capacity to seed for myogenic clones in thymic cultures with a sex dimorphism at the advantage of females. Interestingly, some of the genes in question appeared to be linked to the MHC complex.

Immunological Abnormalities in Relatives of MG Patients Immunological studies of family members of MG patients have provided conflicting data. On the one hand, Lefvert found low but significant titers of anti-AChR antibodies in 54% of unaffected relatives of MG patients.23 Additionally, Lefvert and Stalberg24 reported abnormal single-fiber electromyography (SF-PG) in 33-45% of asymptomatic siblings. On the other hand, Pascuzzi et al25 and Vincent et al4 observed no serologic or electromyographic abnormalities in unaffected siblings of MG patients. We have studied the sera of 240 first-degree relatives of MG patients and found no anti-AChR antibodies using the radioimmune precipitation assay employed by Pascuzzi and Vincent. However, using a more sensitive cell-ELISA we found that 17% had anti-AChR antibodies (our unpublished results). Another approach consists of looking for autoimmune diseases other than MG in the relatives of MG patients. Read et al26 reported the pedigrees of 44 MG patients: there was only one multiplex MG family but 13 patients had a total of 15 relatives with an autoimmune disease (hyperthyroidism in 6, RA in 4, IDDM in 4, and MG in 1).

HLA and MG

The HLA association with MG was discovered as early as 1972.27 The association was initially found for the B8 antigen, then extended to A1, DR3 Dw3 and complement factors. In fact, the association operates with the whole “ancestral” haplotype A1 B8 Cw7 C4AQ0 C4B1 C2C BfS DR3 DQ2 which is a fairly common extended haplotype (approximately 5% of all possible haplotypes). The corresponding genes have been transmitted en bloc over several generations, thus explaining the linkage disequilibrium. It is highest in female patients under 40 years of age at onset with high anti-AChR antibody titers and thymus hyper-

Etiopathogenesis of Myasthenia Gravis (MG)

187

plasia, but it also involves some males less than 40 years of age.28 Importantly, in some studies the HLA association was stronger (higher relative risk) for B8 (class I) than for DR3 (class II).29 Thus, a larger number of MG patients had the split haplotype A1 B8+ DR3than the opposite haplotype B8- DR3+. In a more thorough approach, Dawkins et al30 attempted to screen for recombinant haplotypes and to identify nonHLA genes closer to the putative predisposing locus than B8 or DR3. They have indicated that the gene in question could be located between the HLA-B and TNF loci.30 Sequence studies in progress should clarify this critical issue. The recent availability of precise DQ typing by means of sequencespecific oligonucleotide probes31,32 has confirmed the association with the class II region. On a large series of patients (n=114), we confirmed the association with the DR3 DQB 201 haplotype in young MG females. Additionally, we observed an even stronger association with DQB 604, an allele which is not in linkage disequilibrium with DR3. Interestingly, at variance with the DR3 haplotype which is associated with thymic hyperplasia (RR = 3.5, Pc < 0.01), the DQB 604 haplotype was significantly increased in the group of MG patients with thymoma (RR = 5.7, Pc < 0.05). The role of DQA1 alleles has also been emphasized by Khalil et al33 with data suggesting a possible role for a DQA1 DQB1 transcomplementation. MG with thymoma has also been associated with HLA-DR15 Dw234 and D-penicillamine-induced MG has been associated with DR1 and Bw35.35 Taken together, the finding of these associations with class II genes argues in favor of the existence of two predisposing genes within the MHC, one being a class II gene, the other in the central region, close to TNF.

NonHLA Genes An association with Ig heavy chain allotypes was first reported in Japanese patients, who showed a clear increase in the Gm1,2,21 haplotype, particularly those with thymoma and those with severe generalized MG.36 Attempts to reproduce these data in Caucasian MG patients have given ambiguous results. No association was found in Caucasians by Grosse-Wilde37 and Dondi,38 while a weak association was found with Gm1(1) by Smith.39,40 The Km allotypes were not associated either with the disease. However, the Km3 allotype was associated with high autoantibody titers.38 The role of TCR genes has been investigated by studying TCR polymorphism in MG patients and healthy controls. Negative results were first obtained for both α and β chains.41,42 However, further studies by the same group43,44 indicated a clear association with TCR α chain genes, in three patient populations from California, Australia and Italy. These results require confirmation because of the extreme variability of genotype frequencies between populations. The last accessible candidates are AChR genes. Two highly polymorphic microsatellites were recently characterized within the AChR α-subunit (CHRNA) gene in our laboratory. By means of linkage disequilibrium analysis, an allelic form of a polymorphic dinucleotide CA-repeat, located in the first intron of the gene and termed HB*14, was found to be closely associated with the disease.45 Combined analysis of these loci revealed a significant increase of DQA1*0101 alleles in HB*14+ vs HB*14- patients and of DQA1*0501 alleles in HB*14/DQA1*0101 patients.46 Importantly, the effect of DQA1*0101 was independent of allelically associated DQB1 and DRB1 genes. In contrast, the effect of DQA1*0501 could not be dissociated from that of DRB1*03 and DQB1*0201 on the extended DR3 haplotype. These results indicate that a combination of three genes, of which two are linked to HLA, contributes to disease susceptibility in a subgroup of MG patients. The HB*14 marker provides a powerful tool for subgrouping myasthenic patients and uncovering the role of HLA class II genes. The significance of the association of CHRNA and HLA-linked genes is currently unknown. It is however tempting to speculate that the microsatellite variant is in

Endocrine and Organ Specific Autoimmunity

188

linkage disequilibrium with a polymorphism of the coding region resulting in an abnormal presentation of an α-subunit peptide to autoreactive T lymphocytes by the HLA DQA1 gene product, leading to increased immunogenicity and facilitation of anti-self immunization. Alternatively, the association of HB*14 could reflect a regulatory anomaly of the CHRNA gene.

The Driving Role of the AChR Autoantigen Triggering Normal individuals may present low titers of anti-AChR autoantibodies in their serum (as well as natural autoantibodies to many other specificities). One may wonder whether the onset of MG symptoms results from an abnormally intense production of such antibodies from a conventional immune response driven by the AChR. Four sets of evidence argue in favor of the latter hypothesis. 1) Experimental sensitization against AChR induces a disease resembling human MG in many regards. 2) Genes coding for anti-AChR autoantibodies derived from lymphocytes of myasthenic patients show mutations in the complementarity determining regions (CDRs) and N additions highly suggestive of an antigen driven positive selection mechanism47 (Table 9.2). 3) Interesting data has been collected in Sweden and in France suggesting that T cells from myasthenic patients may use a restricted T cell repertoire suggesting the (possibly autoantigen driven) expansion of T cell clones. These data require confirmation and perhaps more importantly their significance must be elucidated. One would like in particular to determine the putative antigens (or epitopes) driving such expansion. 4) The disease is associated with the HLA complex and HLA molecules are known to present antigenic peptides to T cells. The apparent synergy observed between relative risks of AChR gene polymorphism (HB*14) and HLA-DQ alleles strengthens

Table 9.2. Somatic Mutations in Human Anti-Acetylcholine Receptor Monoclonal Autoantibodies Derived from a Myasthenic Patients Amino acid Replacements Antibody

Germline gene

Fws

CDR1 CDR2 CDR3

CDR/FW ratio

N additions

MH1

VH 1-18 vl Humlv 418

3 2

4 1

4 2

4 4

4 3.5

6

MH5

VH LI-1 Vl IGLV 21

9 5

3 3

4 1

2 3

1 1.4

14

MH6

VH HHG 19 Vl Humlv 11

7 1

2 2

6 2

4 2

1.71 6

5

MH7

VH MC-2 VK Vg

3 3

1 1

3 0

3 5

2.33 2

10

Etiopathogenesis of Myasthenia Gravis (MG)

189

this hypothesis even if the HLA complex may be involved in disease predisposition independently of any class II associated mechanisms (see section III).

Autoantigen Spreading a-Intramolecular Spreading Myasthenic patients show B and T cell autoreactivities to a multitude of AChR epitopes on the various chains of the receptor (intramolecular spreading). This response is polyclonal, heterogeneous and antigen driven. Using proliferation assays, it has been demonstrated that MG lymphocytes may recognize as many as 6 epitopes on the α chain. The stimulation index associated with these epitopes is often low but it appears highly significant nonetheless.48-52 Similarly, at the antibody level, several B cell epitopes have been outlined, notably by Tzartos,53 using a blocking assay in which patients’ serum antibody activity in the radioimmunoprecipitation assay is blocked by monoclonal anti-AChR antibodies specific of defined B epitopes. A dominant B cell epitope, the “main immunogenic region” (MIR), has been recognized but many other epitopes are also important. b-Intermolecular Spreading MG patients’ sera do not only contain autoantibodies to AChR. A wide variety of other antimuscle autoantibodies is found, notably against actinin, actin, myosin, ryanodine receptor, troponin, tropomyosin, rapsyn54,55 and titin.56 Antibodies against myofibrillar proteins are frequently present in sera from MG patients without thymoma. Interestingly, a positive correlation between the presence of antimyosin antibodies and disease severity has been observed.57 Some authors58,59 showed some immunologic similarities between the MIR and the myosin and troponin I proteins. Moreover, autoantibodies for different contractile elements of striated muscle are present in 80-90% of MG associated with thymoma (titin, ryanodine receptor).60,61 The latter antibodies (anti-titin) are found with a particular frequency in thymomatous patients and in late-onset MG patients. Recently, antibodies directed against a protein localized to the neuromuscular junction, the rapsyn, have been also shown in 30% of lupus patients. Autoantibodies may also be found against nonmuscular autoantigens (e.g. anti-thyroid) but with a much lower frequency suggesting a particular clustering of autoimmunity to the striated muscle. c-Interpretation Taken together, these data suggest that the primary immunization is driven by muscle cells, whatever is the primary autoantigen (AChR or another one). In any case, the autoimmune antibody response spreading to these muscle autoantigens might be secondary to the lesion created by the first autoimmune attack. This mechanism, which is very similar to that described in other autoimmune diseases such as experimental allergic encephalomyelitis or diabetes would lead to the important concept that anti-AChR autoantibodies need not be more prominent than the other antimuscle autoantibodies. Their clinical importance is linked to the fact that, at variance with other antimuscle autoantibodies, they bind to a functionally crucial molecule. It remains possible though that anti-AChR is indeed the primary autoantigen and that it is the anti-AChR autoimmune response which elicits the widespread antimuscle autoimmune response. The nonantimuscle autoantibodies noted in some patients probably exemplify the existence of an immune dysregulation, which itself could contribute to the exacerbation or the perpetuation of the anti-AChR autoimmune response.

190

Endocrine and Organ Specific Autoimmunity

Mechanisms of the Loss of Self Tolerance to AChR: The Role of the Thymus T cell Selection T cells are submitted to double selection during their passage in the thymus gland. At the contact of MHC molecules expressed at the surface of epithelial and dendritic cells as well as under the influence of a number of soluble mediators (cytokines, thymic peptide hormones...), they undergo first a positive selection which expands T cells recognizing self peptides (MHC or MHC + peptide). In a second stage, a majority of these thymocytes dies after encounter with the autoantigen (negative selection). It is apparent that MG patients’ T cells reactive to AChR (and other muscular autoantigens) have undergone positive selection and escaped negative selection in the thymus. This is not a unique behavior for autoreactive T cells since normal individuals usually show autoreactive cells to a wide array of autoantigens. The specific question in MG is to determine whether the AChR expressed in the thymus plays a role in stimulating a particular intensive positive selection or less likely may protect from negative selection. The thymic expression of AChR is at its best in myoid cells that are muscle-like cells present in small number in the normal thymus and possibly to some extent on epithelial cells. It is not absolutely clear whether such AChR expression shows a normal, decreased or increased level in MG thymus.62,63 The role of thymic soluble factors is also interesting to consider, notably that of thymic hormones (thymopoietin, thymulin) shown to be produced in exaggerated amount in MG.64,65 The question remains open, however, due to the uncertain functions of these hormones. This discussion of the role of the thymus in MG pathogenesis should take into account the presence of AChR specific autoreactive B cells shown to be functional in the thymus of myasthenic patients.66 The presence of such B cells is associated with that of germinal centers which importantly are not specific for MG (they are found in other autoimmune diseases such as systemic lupus erythematosus). Moreover thymic alterations are present in 90% of MG. Lastly, one may add that thymectomy has been reported to improve the disease course which represents evidence in favor of a positive role of the thymus. It should be noted, however, that mechanisms of this effect of thymectomy are not clear: Decreased generation of autoreactive AChR specific T or B cells? Improvement of immune dysregulation? Loss of thymic hormone production? The problem is complicated by the high variability of the clinical effect in terms of both efficacy and rapidity of onset. Incidentally, thymectomy may induce in some cases the occurrence of systemic lupus erythematosus.67 This observation is probably explained by an effect on regulatory T cells whose reduction leads to exacerbation of the prelupus state. It does not argue, however, by itself, for or against the role of autoantigen driving in the autoimmune process.68

Peripheral Rupture of Tolerance Whatever the role of the thymus in pathogenesis, one may assume that sensitization to AChR occurs in the periphery as is strongly suggested for other organ specific autoimmune diseases. The mechanisms of the rupture of self tolerance (or ignorance) remain obscure: local muscle inflammation increasing the immunogenicity of muscle autoantigens? autoantigen mimicry with microbial or viral antigens?

Etiopathogenesis of Myasthenia Gravis (MG)

191

References 1. Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science 1973; 180:871-872. 2. Lindstrom JM, Seybold ME, Lennon VA et al. Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates, and diagnostic value. Neurology 1976; 26:1054-1059. 3. Lindstrom J. An assay for antibodies to human acetylcholine receptor in serum from patients with myasthenia gravis. Clin Immunol Immunopathol 1977; 7:36-43. 4. Vincent A, Newsom-Davis J. Acetylcholine receptor antibody as a diagnostic test for myasthenia gravis: results in 153 validated cases and 2967 diagnostic assays. J Neurol Neurosurg Psychiatry 1985; 48:1246-1252. 5. Voltz R, Hohlfeld R, Fateh-Moghadam A et al. Myasthenia gravis: measurement of antiAChR autoantibodies using cell line TE671. Neurology 1991; 41:1836-1838. 6. Vernet Der Garabedian B, Lacokova M, Eymard B et al. Association of neonatal myasthenia gravis with antibodies against the fetal acetylcholine receptor. J Clin Invest 1994; 94:555-559. 7. Vernet Der Garabedian B, Eymard B, Bach JF et al. Alpha-bungarotoxin blocking antibodies in neonatal myasthenia gravis: frequency and selectivity. J Neuroimmunol 1989; 21:41-47. 8. Eymard B, Vernet Der Garabedian B, Berrih Aknin S et al. Anti-acetylcholine receptor antibodies in neonatal myasthenia gravis: heterogeneity and pathogenic significance. J Autoimmun 1991; 4:185-195. 9. Beeson D, Jacobson L, Newsom Davis J et al. A transfected human muscle cell line expressing the adult subtype of the human muscle acetylcholine receptor for diagnostic assays in myasthenia gravis. Neurology 1996; 47:1552-1555. 10. Toyka KV, Brachman DB, Pestronk A et al. Myasthenia gravis: passive transfer from man to mouse. Science 1975; 190:397-399. 11. Merlie JP, Heinemann S, Einarson B et al. Degradation of acetylcholine receptor in diaphragms of rats with experimental autoimmune myasthenia gravis. J Biol Chem 1979; 254:6328-6332. 12. Tzartos SJ, Lindstrom JM. Monoclonal antibodies used to probe acetylcholine receptor structure: localization of the main immunogenic region and detection of similarities between subunits. Proc Natl Acad Sci USA 1980; 77:755-759. 13. Lefvert AK, Osterman PO. Newborn infants to myasthenic mothers: a clinical study and an investigation of acetylcholine receptor antibodies in 17 children. Neurology 1983; 33:133-138. 14. Morel E, Vernet Der Garabedian B, Eymard B et al. Binding and blocking antibodies to the human acetylcholine receptor: Are they selected in various myasthenia gravis forms? Immunol Res 1988; 7:212-217. 15. Eymard B, de La Porte S, Pannier C et al. Effect of myasthenic patient sera on the number and distribution of acetylcholine receptors in muscle and nerve-muscle cultures from rat. Correlations with clinical state. J Neurol Sci 1988; 86:41-59. 16. Mossman S, Vincent A, Newsom-Davis J. Myasthenia gravis without acetylcholine-receptor antibody: a distinct disease entity. Lancet 1986; 1:116-119. 17. Miller RG, Milner Brown HS, Dau PC. Antibody-negative acquired myasthenia gravis: successful therapy with plasma exchange. Muscle & Nerve 1981; 4:255. 18. Namba T, Brunner NG, Brown SB et al. Familial myasthenia gravis. Report of 27 patients in 12 families and review of 164 patients in 73 families. Arch Neurol 1971; 25:49-60. 19. Christadoss P. Immunogenetics of experimental autoimmune myasthenia gravis. Crit Rev Immunol 1989; 9:247-278. 20. Bellone M, Ostlie N, Lei SJ et al. The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor. J Immunol 1991; 147:1484-1491. 21. Christadoss P. C5 gene influences the development of murine myasthenia gravis. J Immunol 1988; 140:2589-2592.

192

Endocrine and Organ Specific Autoimmunity

22. Wekerle H, Hohlfeld R, Ketelsen UP et al. Thymic myogenesis, T-lymphocytes and the pathogenesis of myasthenia gravis. Ann N Y Acad Sci 1981; 377:455-476. 23. Lefvert AK, Pirskanen R, Svanborg E. Anti-idiotypic antibodies, acetylcholine receptor antibodies and disturbed neuromuscular function in healthy relatives to patients with myasthenia gravis. J Neuroimmunol 1985; 9:41-53. 24. Stalberg E, Trontelj JV, Schwartz MS. Single-muscle-fiber recording of the jitter phenomenon in patients with myasthenia gravis and in members of their families. Ann NY Acad Sci 1976; 274:189-202. 25. Pascuzzi RM, Phillips Lh 2D, Johns TR et al. The prevalence of electrophysiological and immunological abnormalities in asymptomatic relatives of patients with myasthenia gravis. Ann NY Acad Sci 1987; 505:407-415. 26. Read AP, Kerzin-Storrar L, Dyer PA et al. Possible maternal effect in genetic susceptibility to myasthenia gravis. Lancet 1986; 2:167-168. 27. Pirskanen R, Tiilikainen A, Hokkanen E. Histocompatibility (HL-A) antigens associated with myasthenia gravis. A preliminary report. Ann Clin Res 1972; 4:304-306. 28. Safwenberg J, Hammarstrom L, Lindblom JB et al. HLA-A, -B, -C and -D antigens in male patients with myasthenia gravis. Tissue Antigens 1978; 12:136-142. 29. Dawkins RL, Christiansen FT, Kay PH et al. Disease associations with complotypes, supratypes and haplotypes. Immunol Rev 1983; 70:1-22. 30. Degli-Esposti MA, Andreas A, Christiansen FT et al. An approach to the localization of the susceptibility genes for generalized myasthenia gravis by mapping recombinant ancestral haplotypes. Immunogenetics 1992; 35:355-364. 31. Vieira ML, Caillat-Zucman S, Gajdos P et al. Identification by genomic typing of nonDR3 HLA class II genes associated with myasthenia gravis. J Neuroimmunol 1993; 47:115-122. 32. Spurkland A, Gilhus NE, Ronningen KS et al. Myasthenia gravis patients with thymus hyperplasia and myasthenia gravis patients with thymoma display different HLA associations. Tissue Antigens 1991; 37:90-93. 33. Khalil I, Berrih-Aknin S, Lepage V et al. Trans-encoded DQ alpha beta heterodimers confer susceptibility to myasthenia gravis disease. C R Acad Sci III 1993; 316:652-660. 34. Carlsson B, Wallin J, Pirskanen R et al. Different HLA DR-DQ associations in subgroups of idiopathic myasthenia gravis. Immunogenetics 1990; 31:285-290. 35. Garlepp MJ, Dawkins RL, Christiansen FT. HLA antigens and acetylcholine receptor antibodies in penicillamine induced myasthenia gravis. Br Med J Clin Res Ed 1983; 286:338-340. 36. Nakao Y, Matsumoto H, Miyazaki T et al. Gm allotypes in myasthenia gravis. Lancet 1980; 1:677-680. 37. Grosse-Wilde H, Toyka KV, Besinger UA et al. immunogenetics of myasthenia gravis. Significance of HLA-, complement and Cm gene systems for clinical and immunologic parameters. Dtsch Med Wochenschr 1983; 108:694-700. 38. Dondi E, Gajdos P, Bach JF et al. Association of Km3 allotype with increased serum levels of autoantibodies against muscle acetylcholine receptor in myasthenia gravis. J Neuroimmunol 1994; 51:221-224. 39. Smith CI, Grubb R, Hammarstrom L et al. Gm allotypes in Swedish myasthenia gravis patients. J Immunogenet 1983; 10:1-9. 40. Smith CI, Grubb R, Hammarstrom L et al. Gm allotypes in Finnish myasthenia gravis patients. Neurology 1984; 34:1604-1605. 41. Smith CI, Borgonovo L, Carlsson B et al. Molecular probing of disease susceptibility genes in myasthenia gravis patients: an analysis of T cell receptor and HLA class II genes using restriction fragment length polymorphism. Ann NY Acad Sci 1987; 388-397. 42. Oksenberg JR, Gaiser CN, Cavalli Sforza LL et al. Polymorphic markers of human T cell receptor alpha and beta genes. Family studies and comparison of frequencies in healthy individuals and patients with multiple sclerosis and myasthenia gravis. Hum Immunol 1988; 22:111-121. 43. Oksenberg JR, Sherritt M, Begovich AB et al. T cell receptor V alpha and C alpha alleles associated with multiple and myasthenia gravis. Proc Natl Acad Sci USA 1989; 86:988-992.

Etiopathogenesis of Myasthenia Gravis (MG)

193

44. Mantegazza R, Oksenberg JR, Baggi F et al. Increased incidence of certain TCR and HLA genes associated with myasthenia gravis in Italians. J Autoimmun 1990; 3:431-440. 45. Garchon HJ, Djabiri F, Viard JP et al. Involvement of human muscle acetylcholine receptor alpha-subunit gene (CHRNA) in susceptibility to Myasthenia Gravis. Proc Natl Acad Sci USA 1994; 91:4668-4672. 46. Djabiri F, Caillat-Zucman S, Gajdos P et al. Association of the AChR alpha-subunit gene (CHRNA), DQA1*0101, and the DR3 haplotype in myasthenia gravis. Evidence for a threegene disease model in a subgroup of patients. J Autoimmun 1997; 10:407-413. 47. Serrano MP, Cardona A, Vernet Der Garabedian B et al. Nucleotide sequences of variable regions of an human anti-acetylcholine receptor autoantibody derived from a myasthenic patient. Mol Immunol 1994; 31:413-417. 48. Bellone M, Tang F, Milius R et al. The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies. J Immunol 1989; 143:3568-3579. 49. Hohlfeld R, Kalies I, Kohleisen B et al. Myasthenia gravis: Stimulation of antireceptor autoantibodies by autoreactive T cell lines. Neurology 1986; 36:618-621. 50. Hohlfeld R, Toyka KV, Heininger K et al. Autoimmune human T lymphocytes specific for acetylcholine receptor. Nature 1984; 310:244-246. 51. Protti MP, Manfredi AA, Wu XD et al. Myasthenia gravis. T epitopes on the delta subunit of human muscle acetylcholine receptor. J Immunol 1991; 146:2253-2261. 52. Protti MP, Manfredi AA, Straub C et al. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis. Proc Natl Acad Sci USA 1990; 87:7792-7796. 53. Tzartos SJ, Seybold ME, Lindstrom JM. Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proc Natl Acad Sci USA 1982; 79:188-192. 54. Agius MA, Zhu S, Lin MY et al. Rapsyn antibodies in myasthenia gravis. (Abstract).IXth International Conference on Myasthenia Gravis and Related Disorders, Santa Monica, May 7-10. 1997; 28. 55. Buckel A, James E, Jacobson L et al. Associations between human acetylcholine receptor and RAPsyn. (Abstract).IXth International Conference on Myasthenia Gravis and Related Disorders, Santa Monica, May 7-10. 1997; P7. 56. Williams CL, Lennon VA. Thymic B lymphocyte clones from patients with myasthenia gravis secrete monoclonal striational autoantibodies reacting with myosin, alpha actinin, or actin. J Exp Med 1986; 164:1043-1059. 57. Sano M, Lennon VA. Enzyme immunoassay of anti-human acetylcholine receptor autoantibodies in patients with myasthenia gravis reveals correlation with striational autoantibodies. Neurology 1993; 43:573-578. 58. Mohan S, Barohn RJ, Krolick KA. Unexpected cross-reactivity between myosin and a main immunogenic region (MIR) of the acetylcholine receptor by antisera obtained from myasthenia gravis patients. Clin Immunol Immunopathol 1992; 64:218-226. 59. Mohan S, Barohn RJ, Jackson CE et al. Evaluation of myosin-reactive antibodies from a panel of myasthenia gravis patients. Clin Immunol Immunopathol 1994; 70:266-273. 60. Mygland A, Tysnes OB, Matre R et al. Anti-cardiac ryanodine receptor antibodies in thymoma-associated myasthenia gravis. Autoimmunity 1994; 17:327-331. 61. Gautel M, Lakey A, Barlow DP et al. Titin antibodies in myasthenia gravis: Identification of a major immunogenic region of titin. Neurology 1993; 43:1581-1585. 62. Raimond F, Morel E, Bach JF. Evidence for the presence of immunoreactive acetylcholine receptors on human thymus cells. J Neuroimmunol 1984; 6:31-40. 63. Wakkach A, Guyon T, Bruand C et al. Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis. J Immunol 1996; 157:3752-3760. 64. Vernet Der Garabedian B, Cardona A, Schmidt JM et al. Thymopoietin, a thymic hormone as cofactor of pathogenic antibodies in myasthenia gravis. J Autoimmun 1991; 4:XXXV.

194

Endocrine and Organ Specific Autoimmunity

65. Bach JF, Papiernik M, Levasseur P et al. Evidence for a serum-factor secreted by the human thymus. Lancet 1972; 2:1056-1058. 66. Melms A, Schalke BC, Kirchner T et al. Thymus in myasthenia gravis. Isolation of T-lymphocyte lines specific for the nicotinic acetylcholine receptor from thymuses of myasthenic patients. J Clin Invest 1988; 81:902-908. 67. Shoenfeld Y, Lorber M, Yucel T et al. Primary antiphospholipid syndrome emerging following thymectomy for myasthenia gravis: Additional evidence for the kaleidoscope of autoimmunity. Lupus 1997; 6:474-476. 68. Bach JF. Thymectomy and autoimmunity. Lupus 1997; 6:419.

CHAPTER 10

Multiple Sclerosis Konstantin Balashov and Howard L. Weiner

Introduction

M

ultiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) that affects CNS myelin. MS is characterized by multiple perivascular lesions found throughout the white matter in the brain and spinal cord, which are often periventricular. Although the cause and pathogenesis of MS are unknown, the most commonly held view is that it is an autoimmune disease directed against CNS myelin antigens related in some way to a viral infection. The cardinal histologic features of typical MS lesions are CNS inflammation and demyelination with relative axon preservation1,2 although recent studies suggest axons may be damaged.3 The inflammatory response in the CNS consists predominantly of activated T lymphocytes and macrophages accompanied by a local immune reaction with the secretion of cytokines and the synthesis of oligoclonal immunoglobulins within the CNS. A large number of clinical signs related to CNS damage may be observed in MS patients due to the widespread location of lesions. MS may be either relapsing-remitting or progressive with an unpredictable clinical course that generally spans 10-20 years, during which time neurological disability accumulates. The clinical course makes the assessment of therapeutic efficacy difficult. Advances in two areas have had a significant impact in understanding the disease and devising therapy: 1) magnetic resonance imaging (MRI) and 2) a clearer understanding of how the immune system functions and how it is regulated. MRI has shown MS to be a much more chronically active process that can be discerned clinically even in “stable” patients, and MRI has become an important modality for measuring outcome in clinical trials. As the function of the immune system has become better understood, the ways in which it can be manipulated and monitored have expanded greatly.4 There are two major hypothesis for the etiology of MS. In the first instance, the brain becomes infected by a virus or other infectious agent and cells that infiltrate that brain are targeted to the infectious agent. However, multiple attempts to isolate a MS specific virus have thus far failed.5 The alternative hypothesis is that the initial infiltrating cells are autoreactive T cells that recognize myelin antigens that are presented by local antigen-presenting cells in the CNS in the context of the major histocompatibility complex (MHC). Once the autoimmune cascade begins, it is likely that “epitope spreading” occurs, and T cells recognizing other organ-specific proteins are recruited to the CNS.

Epidemiology and Genetics Multiple sclerosis is approximately two-fold more common in females than males. The prevalence and incidence of MS appears to increase with distance from the equator. MS Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

196

Endocrine and Organ Specific Autoimmunity

affects 1-1.8 people per 1000,6 resulting in a prevalence of 250,000-350,000 cases in the United States.7 MS is unusual before adolescence, then steadily rises in incidence from the teens to age 35, and declines gradually thereafter. Epidemiologic studies support both genetic and environmental components of susceptibility. Reports of clusters, small “epidemics,” geographic variation in prevalence, and alteration of MS susceptibility by migration support an environmental factor or factors.8 The higher risk for MS in Europeans and in relatives of patients, especially in monozygotic versus dizygotic twins, adoption studies and the existence of MS-resistant ethnic groups support a genetic predisposition.9,10 For example, in several population-based studies the concordance rate for monozygotic twins varied from 5.9- 25.9 % (with average 17.8%) versus 0-3.6 % (with average of 2.0%) for dizygotic twins.11-17 A strong association in MS patients of European descent was found for HLA-DR2, that is, the extended haplotype HLA-DRB1*1501/DQA1*0102/DQB1*0602.18,19 In a recent series of studies, microsatellite markers were used in a genome screen to identify genomic regions potentially harboring MS susceptibility genes. However, no single region has been identified as a major influence on familial risk including T cell receptor genes.20-25 These results support a multifactorial etiology of MS, including both environmental and multiple genetic factors of a moderate effect. Currently, there is little evidence for a single or unique environmental agent in MS. An important question is the participation of environment factors in triggering exacerbations of MS or in influencing whether the course of MS is severe or mild. Correlations have been found between upper respiratory tract viral infections26-28 or both viral and significant bacterial infections29 and exacerbations of relapsing-remitting MS. How infections affect the course of MS is not clear. It may be connected with nonspecific polyclonal activation of T cells with increased T cell traffic into the CNS or with a virus-induced burst of IFN-γ that has been to be associated with exacerbation of the disease.30 It should be noted, however, that influenza immunization in MS patients was neither associated with an increased exacerbation rate in the postvaccination period nor a change in disease course over the subsequent 6 months.31

Magnetic Resonance Imaging (MRI) The MRI has made a major impact on our understanding of MS. It has provided a crucial diagnostic tool, demonstrated that the disease process is far more active than can be appreciated clinically, and has provided an important surrogate marker for clinical trials.32 Because there are silent areas where lesions occur, the correlation of MRI with disability has not been perfect. However with improved understanding of the disease process as viewed by MRI and newer imaging techniques, closer correlations with disability are occurring.33,34 In isolated neurologic symptoms, the presence of multiple lesions on MRI has predictive value of who is at risk for the development of MS.35 Gadolinium enhancement reflects breakdown of blood brain barrier, and we have found that the number of enhancing lesions correlates with changes in Expanded Disability Status Scale (EDDS) in relapsing remitting and relapsing progressive MS whereas T2 volume correlates with disability in progressive MS (Weiner et al, unpublished). Disability is also linked to damage as measured by T1 imaging and spinal cord atrophy.36,37 It has also been shown that gadolinium enhanced MRIs can be found even in clinically stable relapsing remitting MS patients.38 The use of contrastenhanced MRI imaging is now being widely used for evaluation of treatment responses in experimental trials. For example, it was shown that both interferon-beta39,40 or methylprednisolone41 treatments lead to reduction in the number of contrast-enhancing lesions. The presence of gadolinium-enhanced lesions positively correlates with increase of albumin in the CSF and number of CSF mononuclear cells in MS42 and with serum TNF-alpha levels.43

Multiple Sclerosis

197

Oligoclonal Immunoglobulins in the CSF The examination of cerebrospinal fluid (CSF) was the first laboratory test to support the clinical diagnosis of MS and continues to used in the diagnosis of MS and for monitoring its activity.44 Although, the CSF cell profile in MS appears to be of little clinical value,45 approximately 90% of MS patients have oligoclonal immunoglobulin bands reflecting oligoclonal B cell activation in the CNS. This finding is not unique to MS and can be detected in patients with other inflammatory diseases of the CNS especially infections. Investigations have yet to identify a specific CNS protein or infectious agent that reacts with the oligoclonal IgG-bands in the CSF of MS patients but not in other MS nonrelated diseases or controls.46,47 This may be related to the random antigen-nonrelated traffic of B cells via the blood-brain barrier into the CNS where they undergo activation, expansion and differentiation into plasma cells in MS or other local inflammatory processes. It is possible, however, that the specificity of oligoclonal IgG in the CSF could provide an important clue to the nature of immune reactivity in the CNS of MS patients.

Viruses

Despite many attempts, an infectious agent has not been identified in MS.5 Currently, the role of several viruses is being tested as being involved in MS: human herpes virus 6,48-53 herpes simplex virus,54 MS-associated retrovirus (MSRV) (previously called LM7),55 corona viruses,56, 57 Ebstein-Barr virus,58, 59 ERV9 retrovirus,60 human endogenous retrovirus ERV3,61 common population of reverse transcriptase containing viruses.62 Experimental allergic encephalomyelitis (EAE) is the primary animal model of MS induced by immunizing animals with myelin antigens such as MBP or PLP in adjuvant. In addition to EAE, another important animal model of a CNS demyelinating process is induced with live Theiler’s murine encephalomyelitis virus (TMEV). TMEV-specific cellular and humoral immunity and apoptosis of infected cells eliminate virus from the gray matter of the CNS during the acute phase of TMEV disease. However, during the chronic phase, TMEV persistently infects glial cells and/or macrophages in the white matter. During the chronic phase, recruitment of macrophages, TMEV-specific T cells and antibody, with the induction of apoptosis are harmful to the host, leading to inflammation and demyelination.63 These two murine models, in fact, are representative of the two major current hypothesis of CNS inflammation in MS: a cell-mediated autoimmune attack against myelin antigens or the presence of a persistent virus or infectious process within the nervous system against which the inflammatory response is directed.

Immunopathological Mechanisms (Fig. 10.1) Autoantigens and Autoreactive T cells MS is predominantly, although not exclusively a CNS disease. If MS is a T cell mediated autoimmune disease, the anatomic localization of the target autoantigens could theoretically influence the distribution of inflammatory lesions(Table 10.1). Myelin proteolipid protein (PLP), the most abundant protein of CNS myelin, and myelin-oligodendrocyte glycoprotein (MOG) are, therefore, attractive potential autoantigens in MS because they are found only in the CNS but not in peripheral nervous system (PNS). However, cases have been reported with MS-like lesions in the CNS of patients with PNS demyelinating diseases. Myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) are expressed in both the CNS and the PNS.1 The identification of MBP, PLP and MOG as potential target autoantigens in pathogenesis of human demyelinating disease have come from studies of experimental allergic encephalomyelitis (EAE). EAE is an autoimmune inflammatory disease of the CNS characterized by perivascular and subpial inflammatory infiltrates and

Endocrine and Organ Specific Autoimmunity

198

Fig. 10.1. Mechanisms of activation and differentiation of T cells in the CNS. The minimal requirement to induce inflammatory autoimmune disease in the CNS white matter is the activation of CD4+ T cells specific for a CNS-associated antigen. In MS, an inducing event, such as a bacterial superantigen or viral peptide (via molecular mimicry) activates autoreactive T cells. A second costimulatory molecule, such as B7.1 is required for activation. The interaction of T cells with APCs via CD40 ligand - CD40 molecule interaction is important for induction of IL-12 synthesis by APC and consequent Th1 cell generation in MS. Activated Th1 cells then migrate into the CNS where they recognize CNS antigens in the context of MHC, recruit effector cells and cause an inflammatory response. Oligodendrocytes can be lysed either directly via the cell to cell contact by cytotoxic T cells or via production of soluble cytotoxic mediators, such as TNF-α or nitric oxide.

Table 10.1. Potential Autoantigens in Multiple Sclerosis EAE-inducing autoantigens

Other potential autoantigens

proteolipid protein (PLP) myelin basic protein (MBP) myelin-oligodendrocyte glycoprotein s100-beta

myelin-associated glycoprotein (MAG) heat shock proteins alphaB crystallin oligodendroglial transaldolase oligodendrocyte myelin glycoprotein 2',3'-cyclic nucleotide 3' phospho-diesterase (CNP)

Multiple Sclerosis

199

lesions of demyelination. Disease is initiated by immunization with autoantigens together with adjuvants or by transfer of autoreactive T lymphocytes.64,65 Oligodendroglial transaldolase as a candidate antigen in pathogenesis of MS has also suggested.66 Other candidate autoantigens include 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) expressed by oligodendrocytes3 and oligodendrocyte myelin glycoprotein (OMgp).67 In the past, it was thought that an immune response against a single autoantigen could be viewed as the cause of MS. It is now clear that there are reactivities against more than one myelin autoantigen in MS.68,69 This is true of most inflammatory organ specific autoimmune diseases since inflammation in the organ releases antigens that leads to sensitization to additional antigens in the target organ. Immune responses against nonmyelin antigens can also induce CNS disease. Immunization with the proteins 100-beta, which is located in astrocytes, thymus and spleen results in T cell reactivity in rats and transfer of s100-beta -reactive T cells induces inflammation in the CNS of recipients.70, 71 Another family of proteins, heat shock proteins(HSP), can function as an immune target during infection, and there is a high likelihood of cross-reactive responses to epitopes shared by host and microbial HSP. These responses, if not properly regulated, could also theoretically contribute to the pathogenesis of multiple sclerosis. Increased specific immune responses to Mycobacteria tuberculosis HSP were detected in T cell lines72 and spinal fluid lymphocytes73 from MS patients compared to controls. Also, altered expression of several HSP were observed in areas of MS lesions on brain sections compared to normal myelin.74, 75 Furthermore, expression of the small heat shock protein alphaB crystalline by both oligodendrocytes and astrocytes in MS lesions and the capacity of this antigen to induce a specific T cell response76, 77 suggest that this nonmyelin antigen may also be considered an important potential autoantigen in MS. Although an inflammatory organ-specific autoimmune disease such as MS is presumably mediated by autoantigen-reactive T cells, such T cells are found at about the same frequency in normal individuals as in MS patients using conventional frequency assays. An increase in MBP and PLP reactive cells was observed, however, in the peripheral blood and CSF activated cells as measured by antigen-specific frequency among IL-2 receptor positive T cells69 (reviewed in ref. 78). As more is learned about the in vitro methods for estimating the frequency of myelin antigen-specific T cells, a better analysis of their frequency may be possible. For example, some methods include repeated stimulation of T cells with antigen in vitro that may lead to selective apoptosis of antigen-specific T cells79 and do not detect what proportion of autoantigen-specific cells were memory cells versus naive ones. Another interesting approach to study MBP reactivity in MS involves the estimation of frequency of MBP-specific T cell lines that carry the mutation of hypoxanthine-guanine phosphribosyl transferase (HPRT). 80 Investigators hypothesized that HPRT mutated cells represent actively dividing population of T cells, and they found an increased frequency of T cells reactive to MBP and MBP peptides in population of HPRT mutant cells in MS.80,81 In some susceptible strains of mice and rats there is restricted Vβ8.2 T cell receptor usage in response to encephalitogenic epitopes of MBP. This observation led to vaccination with synthetic peptides corresponding to the amino acid sequence of complementary determining region 2 (CDR2) or CDR3 of Vβ8.2 or monoclonal antibodies specific for Vβ8.2. These experimental treatment approaches for EAE have been successful (reviewed in ref. 64). This approach has been applied to multiple sclerosis using T cell or T cell receptor vaccination with Vβ5.2 determinants.82-87 Analysis of V-beta usage between human MBP-reactive T cell clones, clones isolated from MS brain (with undefined antigen specificity) and control clones was performed using meta-analysis at a recent international workshop on T cell receptor usage in human and experimental demyelinating diseases.88 The preferential expansion of T lymphocytes bearing gamma/delta T cell receptor in both MS lesions, CSF and

200

Endocrine and Organ Specific Autoimmunity

peripheral blood was observed by different laboratories in MS patients. HSP70 and bacterial superantigens have been suggested as major stimuli for these T cells in recent-onset MS but not in chronic MS or in other neurologic disease.89-94 This hypothesis is supported by finding that human oligodendrocytes and human fetal astrocytes express HSP and stimulate the preferential expansion of cytotoxic gamma delta T cells.95, 96

Altered Peptide Ligands, Molecular Mimicry, and Myelin-Specific T cell Responses The T cell receptor is capable of being stimulated by a large panel of peptides associated with a given MHC molecule.97 Although an individual T cell clone recognizes the “original” peptide used to generate the clone, there are other peptides that are capable of stimulating the same clone. These “variant” peptides can come from viral or bacterial proteins (molecular mimicry) or can be artificially synthesized by a simple substitution of one or another particular amino acid within the original peptide (altered peptide ligand or APL).98,99 Variant or altered peptides can induce a more or less potent response in T cells compared with the original peptide. Furthermore, altered peptide ligand can induce different patterns of cytokine secretion in T cell clones compared with the original peptide. The hypothesis of “molecular mimicry” suggests that the original induction of an immune response to an infectious agent may lead to a subsequent attack against an autoantigen in the target organ. For example, it has been shown that MBP-specific human T cell clones could recognize many T cell stimulatory peptides derived from microbial and self proteins.100-102 The exciting finding that the different affinity of an altered peptide ligand with the same T cell receptor could change the cytokine profile from Th1 type to Th2-type (see below) or decrease the Th1 proinflammatory cytokines has been exploited in a number EAE models for protection from the disease103-105 and is being tested as a treatment for MS patients.

Th1-Type vs. Th2-Type Cytokines in MS

Investigation of murine T cells has led to the broad division of CD4+ T cells into Th1 and Th2 types based on cytokine profile: Th1-type cells secrete IL-2, IFN-γ, and TNF-β whereas Th2-type cells secrete IL-4, IL-5, and IL-10. Th0 type cells have a mixed cytokine profile. Th1-type cytokines favor delayed-type hypersensitivity responses, whereas Th2-type cytokines favor antibody production and allergy-associated responses (reviewed in ref. 106). Furthermore, Th1- and Th2- type cytokines may cross regulate the expression of each other. Thus, in a number of murine models of organ-specific autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), Th1 cells contribute to the pathogenesis of the disease while Th2 cells can prevent disease (reviewed in ref. 107). However, there are differences regarding this division in murine and human systems. For example, in contrast to murine systems, IL-10 can be secreted by Th1 clones in humans108 and IL-12, a classical Th1-type differentiating factor produced by activated non-T cells, induces both IFN-γ and IL-10 secretion by human T cells.109-111 Based on results in EAE, attempts have been made to find an inverse correlation in prevalence of MS (a putative Th1-type disease) and allergic reactions and malignancies (that are often associated with Th2-type responses). In support of this, multiple sclerosis patients have been reported to have fewer allergic symptoms, a lower number of positive allergen-specific IgE test results, and lower composite allergy indexes than control subjects.112 IFN-γ, a cytokine that is the hallmark of Th1-type immune responses, plays an important role in MS disease pathogenesis as increased production of IFN-γ precedes clinical attacks113-115 and injection of MS patients with recombinant IFN-γ induced exacerbations of the disease.30 Furthermore, within the nervous system, the inflammatory process is characterized by increased IFN-γ expression.116 IL-12, a cytokine produced by non-T cells, is the most potent inducer of IFN-γ and Th1 type immune

Multiple Sclerosis

201

responses.117 We have found that endogenous IL-12 was responsible for raised IFN-γ secretion by activated peripheral blood mononuclear cells in progressive MS as anti-IL-12 antibodies reversed raised anti-CD3 induced IFN-γ in MS patients to normal levels. Furthermore, we found a marked increase in T cell receptor mediated IL-12 secretion in progressive MS patients vs. controls and vs. relapsing-remitting patients. Investigation of the cellular basis for raised IL-12 demonstrated that T cells from MS patients induced IL-12 secretion from non-T cells and that T cells from MS patients could even drive non-T cells from normal subjects to produce increased IL-12. Anti-CD40 ligand antibody completely blocked IL-12 secretion induced by activated T cells, and we found increased CD40 ligand expression by activated CD4+ T cells in MS patients vs. controls. The CD40 ligand-dependent Th1 type immune activation was observed in the progressive but not in the relapsing-remitting form of MS suggesting a link to disease pathogenesis and progression and providing a basis for immune intervention in the disease.118 Elevated serum IL-12 p70 has also been detected in chronic progressive MS119 and can be produced by activated microglial cells in vitro.120 Both IL-12p40, CD40 and CD40 ligand can be detected in MS lesions.121,122 In the experimental model EAE, animals treated with IL-12 in vivo have a more severe and prolonged form of EAE whereas anti IL-12 reduces the incidence and severity of adoptively transferred EAE.123 In addition, treatment of animals with anti CD40 ligand antibody completely prevented development of the disease 122 and CD40L-deficient mice failed to develop EAE.124 Recombinant IL-12 was also able to induce relapses in the EAE model in Lewis rats.125 Taken together, these studies provide a basis for future clinical trials in MS with agents that have anti-IL-12 or anti-CD40 ligand activity.

Activation of Autoreactive T cells Induction and exacerbation of immune-mediated demyelination in the CNS may be regulated by different mechanisms. For the induction of EAE a specific antigen (e.g., MBP) with appropriate costimulatory signals required. However, exacerbation of EAE can occur by superantigens, e.g., staphylococcal enteroxins B or A.126 Superantigens are potent T-cell activators and stimulate a large number of T cells bearing specific T cell receptor beta-chain variable regions. In addition, recombinant IL-12 can induce relapses in the EAE model in rats.125 In MS, IFN-γ (due to its antiproliferative capacity) and anti-TNF-α antibody (TNF-α is a potent cytokine that is cytotoxic to oligodendrocytes in vitro) were tested in MS. IFN-γ induced exacerbations in relapsing remitting MS patients and was discontinued from further trials.30 Unexpectedly, humanized anti-TNF-α antibody increased the number of gadolinium-enhancing lesions, CSF leukocyte count and IgG index in treated MS patients.127 It is possible that the anti-TNF-α treatment was associated with a burst of IFN-γ secretion.

Antigen-Nonspecific Defects of Immunoregulation in MS There are several defects of immunoregulation and marks of immune activation that have been described in MS. How they are involved in the pathogenesis of the disease is unclear but they are consistent with MS being a Th1 type autoimmune disease. It is beyond the scope of this chapter to review them in detail but they are listed below. 1. Defect of T suppressor function.128-130 2. Increased TNF-α receptor expression on peripheral T cells.131 3. IFN-γ induced influx of Ca2+ in T lymphocytes.132 4. Increased lymphocyte beta-adrenergic receptor density in progressive MS.133, 134 5. Abnormalities in the level of soluble ICAM-1(CD54) and VCAM-1.135-140 6. Increased number of B7.1(CD80) positive B cells in peripheral blood of active MS141 and in CSF of MS patients.142 B7.1 is also expressed in MS lesions.121

202

Endocrine and Organ Specific Autoimmunity

Immune-Mediated Mechanisms of Myelin Destruction There are two hypotheses to explain oligodendrocyte destruction in MS lesions: direct T cytotoxic cells (CTL) mediated lysis of oligodendrocytes or an indirect mechanism in which activated T cells induce toxic cytokine production (e.g., nitric oxide or TNF-α) by accessory cells. T cell produced cytokines may be also involved in lysis of oligodendrocytes. Recent studies have demonstrated that cytotoxicity mediated by CTLs may be secondary to the secretion of electron-dense cytoplasmic granules (perforin and serine esterase granzyme B) or a nonsecretory pathway based on the interaction of CD95L (Fas ligand) with the apoptosis-inducer CD95 (Fas) molecule expressed on target cells.143

Fas-Fas Ligand Although immunopathological pathways during the course of the demyelination in murine models of MS such as EAE and TMEV infection are not always the same, oligodendroglial apoptosis is observed in both models, suggesting that the demyelinating process may share a common terminal pathway and lead to a similar clinical and pathological picture.63 Apoptosis of oligodendroglial cells has also been observed in MS lesions.144,145 The role of Fas and Fas ligand, the molecules that are involved in one of the pathways of cytotoxic T cell mediated lysis of target cells has been studied in EAE. Results in the murine EAE model are not consistent at the present time. For example, EAE can be effectively induced146 or not147,148 in Fas-deficient mice. In humans, both Fas and Fas ligand positive cells, mainly oligodendrocytes and astrocytes, are found in MS lesions.144,149,150 However, dying oligodendrocytes did not exhibit evidence of apoptosis.149,150

Perforin The role of a perforin-mediated pathway in destruction of oligodendrocytes in MS has not been extensively studied. Oligodendrocytes are highly susceptible to attack by T cell perforin and respond similarly to sublethal attack by shedding membrane vesicles.151 A higher number of CD4+ T cells express perforin in peripheral blood of active MS patients152 and MBP-specific CD4+ T cells clones can mediate cytotoxicity via perforin-mediated cytotoxic mechanism.153 These findings suggest that perforin-mediated lysis of oligodendrocytes could be involved in the pathogenesis of MS. TNF-α and TNF-β (lymphotoxin alpha) have been considered as factors causing destruction of oligodendrocytes in vitro.154 TNF-α can be produced by T cells, astrocytes, and microglia cells and together with TNF-β may be found in MS lesions.154,155 In addition, TNF-α 113,115,156,157 and TNF-β 157 are increased prior to and during MS exacerbations. TNF-α has also been reported to be elevated in the serum and CSF in MS.158 Also, the number of TNF-α and TNF-β mRNA expressing cells are increased in the CSF of MS patients.159,160 Antibodies against TNF-α abrogated T cell transfer of EAE in mice154,161 whereas recombinant TNF-α augmented disease in Lewis rats.162 IFN-γ has been shown to be a potent inducer of apoptosis in oligodendrocytes163 something that was not shown earlier.154 Nitric oxide(NO) synthesis is associated with nonspecific immune-mediated cellular cytotoxicity and is felt to be involved in the pathogenesis of many chronic, inflammatory autoimmune diseases including MS. Inducible NO synthase mRNA has been detected in MS lesions but not in control brains.164,165 It has been suggested that NO may be a mechanism of death of oligodendrocytes166 although further studies are needed to define the role of NO in the pathology of MS.167

Multiple Sclerosis

203

Immunotherapy A number of immunotherapeutic approaches are being used to treat multiple sclerosis and are under active investigation. Beta-interferon 1a, beta-interferon 1b, and Copolymer 1 are approved specifically for use in multiple sclerosis in the treatment of relapsing remitting disease. Beta interferon is the most widely used drug and has been shown in clinical trials to decrease frequency of relapses, affect MRI lesions, and in some instances slow progression.11,168,169 The mechanism of action of the beta interferon drugs is not definitively known. However, they most likely function by decreasing IFN-γ secretion and increasing secretion of IL-4, IL-10 and TGF-β from lymphoid cells. They may also affect the trafficking of cells into the nervous system. Beta-interferon 1b is given by every other day subcutaneous injection and beta-interferon 1a by weekly intramuscular injection. Copolymer 1 is a random copolymer of four amino acids given as a daily injection which appears to work as a MBP analogue that induces Th2 type regulatory cells.170 Drugs used for treatment of inflammatory or malignant conditions have also found usefulness in MS. Intravenous methylprednisolone can speed recovery from acute attacks and patients with optic neuritis treated with IV methylprednisolone have a delay in onset of MS.171 Other therapies which have shown benefit in MS include cyclophosphamide, methotrexate, Cladribine, mitoxanthrone, and total lymph node irradiation.4 Over the past 15 years we have extensive experience using pulse cyclophosphamide for the treatment of progressive and relapsing progressive types of MS using protocols analogous to lupus nephritis protocols.4 We have recently found that although cyclophosphamide is a cytotoxic drug, it causes profound immune deviation. Specifically, patients treated with pulse cyclophosphamide have eosinophilia and increased IL-4 and IL-10 secretion.125 In addition, we recently found a marked decrease in IL-12 secretion by mononuclear cells in patients treated with cyclophosphamide (unpublished).172 Antigen-specific immune modulation of autoreactive cells in MS is being tested using a number of paradigms including oral tolerance, T cell receptor vaccination, and altered peptide ligands. It appears that the mechanism of action by which these approaches act is via bystander suppression.173 Because there is reactivity against multiple antigens in the target organ, the use of antigen specific therapy is problematic if one is inducing anergy or deletion. However, these approaches induce regulatory T cells that secrete cytokines such as IL-4, IL-10 and TGF-β when they reach the brain and suppress inflammation in an antigen nonspecific fashion. For oral tolerance, dose is important as antigen specific regulatory cells and bystander suppression is preferentially induced at lower doses.174 A recently completed phase III trial of a single dose bovine myelin in relapsing remitting MS did not show differences in terms of relapse rate between groups, although significant decrease in MRI lesion volume in oral myelin DR2+ males was observed. Future studies of oral tolerance and MS are planned and will involve recombinant human MBP plus adjuvants or synergists, such as orally administered IL-4 / IL-10 or conjugation of MBP to the cholera toxin B subunit (CTB) which serves as a mucosal adjuvant.175 Oral tolerance is also being tested in other autoimmune diseases including diabetes, rheumatoid arthritis, and uveitis, as well as in transplantation.174 Other therapies being studied in MS include the use of the anti-adhesion molecule anti-alpha 4 integrin, which has been shown to have suppress EAE. Paradoxically, a recent trial of anti-TNF antibody worsened MS, even though it has been of benefit in the EAE model and rheumatoid arthritis.127 This paradoxical affect may be explained by burst of IFN-γ which may occur with anti-TNF therapy. Future treatment in MS may involve targeting factors related to recruitment of cells to the nervous system (chemokines) and antibodies directed against costimulatory molecules such as anti-CD40 ligand and anti-B7.1. Also,

204

Endocrine and Organ Specific Autoimmunity

based on experimental data, anti-IL-12 components would be expected to be of benefit in MS. In summary, immunotherapy of MS involves 1) antigen specific approaches which induce regulatory cells that act via bystander suppression; 2) antigen nonspecific therapies which induce immune deviation or decrease IFN-γ secretion; and 3) drugs which affect lymphocyte trafficking or affect soluble products released by lymphocytes or mononuclear cells that cause CNS damage.

Remyelination Studies in both humans and experimental models demonstrate that myelin repair occurs in the CNS and is a normal physiologic response to myelin injury. Although remyelination in MS is often incomplete and limited, the extent of remyelination correlates well with the presence of oligodendrocytes in the lesions. The source and origin of the remyelinating cells is still unclear. They may be derived from either undifferentiated progenitor cells or, in part from mature oligodendrocytes that have escaped destruction during lesion activity. Complete remyelination of plaques is possible, leading to the formation of ‘shadow plaques’. No reliable data are available at present on the frequency of remyelination in different forms of MS. However, most studies agree that remyelination is especially prominent at the early stages of the disease, whereas it is sparse after several years of disease duration. In addition, very little remyelination is found in cases of primary progressive MS (reviewed in refs. 176,177). Furthermore, given that axonal loss occurs in MS, when significant axonal loss accumulates, irreversible damage to the nervous system has occurred and makes myelin repair not possible.

Conclusion Multiple sclerosis is a disease that affects young adults and generally runs its course over 10-20 years from disease onset. Unlike type I diabetes which becomes clinically evident once the majority of islet cell damage has occurred, the initial presentation in MS is with reversible clinical disability and minimal nervous system damage. Thus, the opportunity exists to prevent CNS damage and disability by early intervention, followed by more aggressive immunotherapy if the disease continues to be active. For example, we have found that there is a correlation between how long a person has been in the progressive phase and response to therapy with pulse cyclophosphamide. Thus, the future of therapy for MS is to find compounds that can be given early in the course of the disease and to treat aggressively if the disease is active either clinically or by MRI.

Acknowledgments This work was supported in part by a grant from the National Institutes of Health (NS23132), the National Multiple Sclerosis Society, and the Nancy Davis Center Without Walls.

References 1. Sobel RA. The pathology of multiple sclerosis. Neurologic Clinics 1995;13:1-21. 2. Prineas JW. The neuropathology of multiple sclerosis. In: Vinken PJ, Klawans HL, Koetsier JC, eds. Handbook of Clinical Neurology, Demyelinating Disease. Amsterdam and New York: Elsevier, 1985:213-257. 3. Trapp BD, Peterson J, Ransohoff RM et al. Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998; 338:278-285.

Multiple Sclerosis

205

4. Weiner HL, Hohol MJ, Khoury SJ et al. Therapy for multiple sclerosis. Neurologic Clinics 1995; 13:173-196. 5. Cook SD, Rohowsky-Kochan C, Bansil S, et al. Evidence for a viral etiology of multiple sclerosis. In: Cook SD, ed. Handbook of multiple sclerosis. New York: Marcel Dekker, Inc., 1996:97-118. 6. Wynn DR, Rodriguez M, O’Fallon WM, et al. A reappraisal of the epidemiology of multiple sclerosis in Olmsted County, Minnesota. Neurology 1990; 40:780-786. 7. Anderson DW, Ellenberg JH, Leventhal CM, et al. Revised estimate if the prevalence of multiple sclerosis in the United States. Ann Neurol 1992; 31:333-336. 8. Hogancamp WE, Rodriguez M, Weinshenker BG. The epidemiology of multiple sclerosis. Mayo Clinic Proceedings 1997; 72:871-878. 9. Ebers GC, Sadovnick AD, Risch NJ. A genetic basis for familial aggregation in multiple sclerosis. Canadian Collaborative Study Group. Nature 1995; 377:150-151. 10. Dyment DA, Sadnovich AD, Ebers GC. Genetics of multiple sclerosis. Human Mol Gene 1997; 6:1693-1698. 11. Hawkes CH. Twin studies in medicine--what do they tell us? Qjm 1997; 90:311-321. 12. Mumford CJ, Wood NW, Kellar-Wood H et al. The British Isles survey of multiple sclerosis in twins. Neurology 1994; 44:11-15. 13. Heltberg A, Holm NV. Concordance in twins and recurrance in sibships in multiple sclerosis. Lancet 1982; 1:1068. 14. Kinnunen E, Koskenvuo M, Kaprio J et al. Multiple sclerosis in a nationwide series of twins. Neurology 1987; 37:1627-1629. 15. Bobowick AR, Kurtzke JF, Brody JA et al. Twin study of multiple sclerosis: an epidemiology inquiry. Neurology 1978;28:978-987. 16. Ebers GC, Bulman DE, Sadovnick AD et al. A population study of multiple sclerosis in twins. NEJM 1986; 315:1638-1642. 17. Alperovitch A, Hors J, Lyon-Caen O. Multiple sclerosis in 54 twinships: concordance rate is independent on zygosity. Ann Neurol 1992; 32:724-727. 18. Olerup Q, Hillert J. HLA class II associated genetic susceptibility in multiple sclerosis. A critical evaluation. Tissue Ant 1991; 38:1-15. 19. Spurkland A, Tabira T, Ronningen KS et al. HLA-DRB1, -DQA1, DQB1, -DPA1 and DPB1 genes in Japanese multiple sclerosis patients. Tissue Ant 1991; 37:171-173. 20. Epplen C, Jackel S, Santos EJ et al. Genetic predisposition to multiple sclerosis as revealed by immunoprinting. Ann Neurol 1997; 41:341-352. 21. Droogan AG, Kirk CW, Hawkins SA et al. T cell receptor alpha, beta, gamma, and delta chain gene microsatellites show no association with multiple sclerosis. Neurology 1996; 47:1049-1053. 22. Haines JL, Ter-Minassian M, Bazyk A et al. A complete genomic screen for multiple sclerosis underscores a role for the major histocompatability complex. The Multiple Sclerosis Genetics Group [see comments]. Nat Gene 1996; 13:469-471. 23. Sawcer S, Jones HB, Feakes R et al. A genome screen in multiple sclerosis reveals susceptibility loci on chromosome 6p21 and 17q22. Nat Gene 1996; 13:464-468. 24. Kuokkanen S, Sundvall M, Terwilliger JD et al. A putative vulnerability locus to multiple sclerosis maps to 5p14-p12 in a region syntenic to the murine locus Eae2. Nat Med 1996; 13:477-480. 25. Ebers GC, Kukay K, Bulman DE et al. A full genome search in multiple sclerosis. Nat Gene 1996; 13:472-476. 26. Panitch HS. Influence of infection on exacerbations of multiple sclerosis. Ann Neurol 1994;36:S25-28. 27. Sibley WA, Bamford CR, Clark K. Clinical viral infections and multiple sclerosis. Lancet 1985; 1:1313-1315. 28. Andersen O, Lygner PE, Bergstrom T et al. Viral infections trigger multiple sclerosis relapses: a prospective seroepidemiological study. J Neurol 1993; 240:417-422. 29. Rapp NS, Gilroy J, Lerner AM. Role of bacterial infection in exacerbation of multiple sclerosis. Amer J Physic Med & Rehab 1995; 74:415-418.

206

Endocrine and Organ Specific Autoimmunity

30. Panitch HS, Hirsch RL, Schindler J et al. Treatment of multiple sclerosis with gamma interferon: Exacerbations associated with activation of the immune system. Neurology 1987; 37:1097-1102. 31. Miller AE, Morgante LA, Buchwald LY et al. A multicenter, randomized, double-blind, placebo-controlled trial of influenza immunization in multiple sclerosis. Neurology 1997; 48:312-314. 32. Francis GS, Evans AC, Arnold DL. Neuroimaging in multiple sclerosis. Neurologic Clinics 1995; 13:147-171. 33. Smith ME, Stone LA, Albert PS et al. Clinical worsening in multiple sclerosis is associated with increased frequency and area of gadopentetate dimeglumine-enhancing magnetic resonance imaging lesions. Ann Neurol 1993; 33:480-489. 34. Khoury SJ, Guttmann CR, Orav EJ et al. Longitudinal MRI in multiple sclerosis: correlation between disability and lesion burden. Neurology 1994; 44:2120-2124. 35. Filippi M, Horsfield MA, Morrissey SP et al. Quantitative brain MRI lesion load predicts the course of clinically isolated syndromes suggestive of multiple sclerosis. Neurology 1994; 44:635-641. 36. Losseff NA, Wang L, Lai HM et al. Progressive cerebral atrophy in multiple sclerosis. A serial MRI study. Brain 1996; 119:2009-2019. 37. van Walderveen MA, Barkhof F, Hommes OR et al. Correlating MRI and clinical disease activity in multiple sclerosis: relevance of hypointense lesions on short-TR/short-TE (T1weighted) spin-echo images. Neurology 1995; 45:1684-1690. 38. McFarland HF, Frank JA, Albert PS et al. Using gadolinium-enhanced magnetic resonance imaging lesions to monitor disease activity in multiple sclerosis. Ann Neurol 1992; 32:758-766. 39. Calabresi PA, Stone LA, Bash CN et al. Interferon beta results in immediate reduction of contrast-enhanced MRI lesions in multiple sclerosis patients followed by weekly MRI. Neurology 1997; 48:1446-1448. 40. Stone LA, Frank JA, Albert PS et al. The effect of interferon-beta on blood-brain barrier disruptions demonstrated by contrast-enhanced magnetic resonance imaging in relapsingremitting multiple sclerosis. Ann Neurol 1995; 37:611-619. 41. Gasperini C, Pozzilli C, Bastianello S et al. The influence of clinical relapses and steroid therapy on the development of Gd-enhancing lesions: a longitudinal MRI study in relapsing-remitting multiple sclerosis patients. Acta Neurol Scand 1997; 95:201-207. 42. Trojano M, Avolio C, Simone IL et al. Soluble intercellular adhesion molecule-1 in serum and cerebrospinal fluid of clinically active relapsing-remitting multiple sclerosis: Correlation with Gd-DTPA magnetic resonance imaging-enhancement and cerebrospinal fluid findings. Neurology 1996; 47:1535-1541. 43. Spuler S, Yousry T, Scheller A et al. Multiple sclerosis: prospective analysis of TNF-alpha and 55 kDa TNF receptor in CSF and serum in correlation with clinical and MRI activity. J Neuroimmunol 1996; 66:57-64. 44. Whitaker JN, Benveniste EN. Cerebrospinal fluid. In: Cook SD, ed. Handbook of multiple sclerosis. New York: Marcel Dekker, Inc., 1996:295-316. 45. Polman CH, deGroot CJA, Koetsier JC et al. Cerebrospinal fluid cells in multiple sclerosis and other neurological disease: and immunocytochemical study. J Neurol 1987; 234:19-22. 46. Kaiser R, Obert M, Kaufmann R et al. IgG-antibodies to CNS proteins in patients with multiple sclerosis. Euro J Med Res 1997; 2:169-172. 47. Cortese I, Tafi R, Grimaldi LM et al. Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries. Proc Natl Acad Sci USA 1996; 93:11063-11067. 48. Liedtke W, Malessa R, Faustmann PM, et al. Human herpesvirus 6 polymerase chain reaction findings in human immunodeficiency virus associated neurological disease and multiple sclerosis. J Neurovirol 1995;1:253-258. 49. Braun DK, Dominguez G, Pellett PE. Human herpesvirus 6. Clinical Microbiology Reviews 1997; 10:521-567.

Multiple Sclerosis

207

50. Nielsen L, Larsen AM, Munk M et al. Human herpesvirus-6 immunoglobulin G antibodies in patients with multiple sclerosis. Acta Neurol Scand 1997; 169:76-78. 51. Martin C, Enbom M, Soderstrom M et al. Absence of seven human herpesviruses, including HHV-6, by polymerase chain reaction in CSF and blood from patients with multiple sclerosis and optic neuritis. Acta Neurol Scand 1997; 95:280-283. 52. Sanders VJ, Felisan S, Waddell A et al. Detection of herpesviridae in postmortem multiple sclerosis brain tissue and controls by polymerase chain reaction. J Neurovirol 1996; 2:249-258. 53. Herndon RM. Herpesviruses in multiple sclerosis. Arch Neurol 1996;53:123-124. 54. Sanders VJ, Waddell AE, Felisan SL et al. Herpes simplex virus in postmortem multiple sclerosis brain tissue. Arch Neurol 1996; 53:125-133. 55. Perron H, Garson JA, Bedin F et al. Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis. The Collaborative Research Group on Multiple Sclerosis. Proc Natl Acad Sci USA 1997; 94:7583-7588. 56. Murray RS, Brown B, Brian D et al. Detection of coronavirus RNA and antigen in multiple sclerosis brain. Ann Neurol 1992; 31:525-533. 57. Cristallo A, Gambaro F, Biamonti G et al. Human coronavirus polyadenylated RNA sequences in cerebrospinal fluid from multiple sclerosis patients. New Microbiol 1997; 20:105-114. 58. Munch M, Hvas J, Christensen T et al. The implications of Epstein-Barr virus in multiple sclerosis--a review. Acta Neurol Scand 1997; 169:59-64. 59. Vaughan JH, Riise T, Rhodes GH et al. An Epstein Barr virus-related cross reactive autoimmune response in multiple sclerosis in Norway. J Neuroimmunol 1996; 69:95-102. 60. Perron H, Firouzi R, Tuke P et al. Cell cultures and associated retroviruses in multiple sclerosis. Collaborative Research Group on MS. Acta Neurol Scand 1997; 169:22-31. 61. Rasmussen HB, Heltberg A, Lisby G et al. Three allelic forms of the human endogenous retrovirus, ERV3, and their frequencies in multiple sclerosis patients and healthy individuals. Autoimmunity 1996; 23:111-117. 62. Hackett J, Jr., Swanson P, Leahy D et al. Search for retrovirus in patients with multiple sclerosis. Ann Neurol 1996; 40:805-809. 63. Tsunoda I, Fujinami RS. Two models for multiple sclerosis: experimental allergic encephalomyelitis and Theiler’s murine encephalomyelitis virus. J Neuropathol Exp Neurol 1996; 55:673-686. 64. Martin R, McFarland HF, McFarlin DE. Immunological aspects of demyelinating diseases. Annu Rev Immunol 1992; 10:153-187. 65. Bernard CC, Johns TG, Slavin A et al. Myelin oligodendrocyte glycoprotein: a novel candidate autoantigen in multiple sclerosis. J Mol Med 1997; 75:77-88. 66. Colombo E, Banki K, Tatum AH et al. Comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis. J Clin Invest 1997; 99:1238-1250. 67. Mikol DD, Stefansson K. A phosphatidylinositol-linked peanut agglutinin-binding glycoprotein in central nervous system myelin and on oligodendrocytes. J Cell Biol 1988; 106:1273-1279. 68. Kerlero de Rosbo N, Milo R, Lees MB et al. Reactivity to myelin antigens in multiple sclerosis. Peripheral blood lymphocytes respond predominantly to myelin oligodendrocyte glycoprotein. J Clin Invest 1993; 92:2602-2608. 69. Zhang J, Markovic-Plese S, Lacet B et al. Increased frequency of interleukin 2-responsive T cells specific for myelin basic protein and proteolipid protein in peripheral blood and cerebrospinal fluid of patients with multiple sclerosis. J Exp Med 1994; 179:973-984. 70. Linington C, Waksman B. The T cell receptor in multiple sclerosis. Proc Natl Acad Sci USA 1992; 756:294-304. 71. Kojima K, Berger T, Lassmann H et al. Experimental autoimmune panencephalitis and uveoretinitis transferred to the Lewis rat by T lymphocytes specific for the S100 beta molecule, a calcium binding protein of astroglia. J Exp Med 1994; 180:817-829.

208

Endocrine and Organ Specific Autoimmunity

72. Salvetti M, Ristori G, Buttinelli C et al. The immune response to mycobacterial 70-kDa heat shock proteins frequently involves autoreactive T cells and is quantitatively disregulated in multiple sclerosis. J Neuroimmunol 1996; 65:143-153. 73. Birnbaum G, Kotilinek L, Albrecht L. Spinal fluid lymphocytes from a subgroup of multiple sclerosis patients respond to mycobacterial antigens. Ann Neurol 1993; 34:18-24. 74. Aquino DA, Capello E, Weisstein J et al. Multiple sclerosis: Altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin. J Neuropathol Exp Neurol 1997; 56:664-672. 75. Raine CS, Wu E, Ivanyi J et al. Multiple sclerosis: a protective or a pathogenic role for heat shock protein 60 in the central nervous system? Laboratory Investigation 1996; 75:109-123. 76. van Noort JM, van Sechel A, Boon J et al. Minor myelin proteins can be major targets for peripheral blood T cells from both multiple sclerosis patients and healthy subjects. J Neuroimmunol 1993; 46:67-72. 77. Bajramovic JJ, Lassmann H, van Noort JM. Expression of alphaB-crystallin in glia cells during lesional development in multiple sclerosis. J Neuroimmunol 1997; 78:143-151. 78. Hafler DA, Weiner HL. Immunologic mechanisms and therapy in multiple sclerosis. Immunol Rev 1995; 144:75-77. 79. Bieganowska KD, Ausubel LJ, Modabber Y et al. Direct ex vivo analysis of activated, Fassensitive autoreactive T cells in human autoimmune disease. J Exp Med 1997; 185:1585-1594. 80. Allegretta M, Nicklas JA, Sriram S et al. T cells responsive to myelin beasic protein in patients with multiple sclerosis. Science 1990; 247:718-721. 81. Lodge PA, Johnson C, Sriram S. Frequency of MBP and MBP peptide-reactive T cells in the HPRT mutant T cell population of MS patients. Neurology 1996; 46:1410-1415. 82. Zhang J, Stinissen P, Medaer R et al. T cell vaccination: clinical application in autoimmune diseases. J Mol Med 1996; 74:653-662. 83. Vandenbark AA, Chou YK, Whitham R et al. Treatment of multiple sclerosis with T cell receptor peptides: results of a double-blind pilot trial. Nat Med 1996; 2:1109-1115. 84. Chou YK, Morrison WJ, Weinberg AD et al. Immunity to TCR peptides in multiple sclerosis. II. T cell recognition of V beta 5.2 and V beta 6.1 CDR2 peptides. J Immunol 1994; 152:2520-2529. 85. Bourdette DN, Whitham RH, Chou YK et al. Immunity to TCR peptides in multiple sclerosis. I. Successful immunization of patients with synthetic V beta 5.2 and V beta 6.1 CDR2 peptides. J Immunol 1994; 152:2510-2519. 86. Wilson DB, Golding AB, Smith RA et al. Results of a phase I clinical trial of a T cell receptor peptide vaccine in patients with multiple sclerosis. I. Analysis of T cell receptor utilization in CSF cell populations. J Neuroimmunol 1997; 76:15-28. 87. Gold DP, Smith RA, Golding AB et al. Results of a phase I clinical trial of a T cell receptor vaccine in patients with multiple sclerosis. II. Comparative analysis of TCR utilization in CSF T cell populations before and after vaccination with a TCRV beta 6 CDR2 peptide. J Neuroimmunol 1997; 76:29-38. 88. Hafler DA, Saadeh MG, Kuchroo VK et al. TCR usage in human and experimental demyelinating disease. Immunol Today 1996; 17:152-159. 89. Shimonkevitz R, Colburn C, Burnham JA et al. Clonal expansions of activated gamma/ delta T cells in recent-onset multiple sclerosis. Proc Natl Acad Sci USA 1993; 90:923-927. 90. Stinissen P, Vandevyver C, Raus J et al. Superantigen reactivity of gamma delta T cell clones isolated from patients with multiple sclerosis and controls. Cell Immunol 1995; 166:227-235. 91. Stinissen P, Vandevyver C, Medaer R et al. Increased frequency of gamma delta T cells in cerebrospinal fluid and peripheral blood of patients with multiple sclerosis. Reactivity, cytotoxicity, and T cell receptor V gene rearrangements. J Immunol 1995; 154:4883-4894. 92. Liedtke W, Meyer G, Faustmann PM et al. Clonal expansion and decreased occurrence of peripheral blood gamma delta T cells of the V delta 2J delta 3 lineage in multiple sclerosis patients. Int Immunol 1997; 9:1031-1041.

Multiple Sclerosis

209

93. Battistini L, Salvetti M, Ristori G et al. Gamma delta T cell receptor analysis supports a role for HSP 70 selection of lymphocytes in multiple sclerosis lesions. Mol Med 1995; 1:554-562. 94. Battistini L, Selmaj K, Kowal C et al. Multiple sclerosis: limited diversity of the V delta 2-J delta 3 T cell receptor in chronic active lesions. Ann Neurol 1995; 37:198-203. 95. Freedman MS, Bitar R, Antel JP. gamma delta T cell-human glial cell interactions. II. Relationship between heat shock protein expression and susceptibility to cytolysis. J Neuroimmunol 1997; 74:143-148. 96. Freedman MS, D’Souza S, Antel JP. gamma delta T cell-human glial cell interactions. I. In vitro induction of gammadelta T cell expansion by human glial cells. J Neuroimmunol 1997; 74:135-142. 97. Nanda NK, Arzoo KK, Geysen HM et al. Recognition of multiple peptide cores by a single T cell receptor. J Exp Med 1995; 182:531-539. 98. Evavold BD, Allen PM. Separation of IL-4 production from Th cell proliferation by an altered T cell receptor ligand. Science 1991; 252:1308-1310. 99. Evavold BD, Sloan-Lancester J, Wilson JB et al. Specific T cell recognition of minimally homologous peptides: evidence for multiple endogenous ligands. Immunity 1995; 2:655-705. 100. Wucherpfennig KW, Strominger JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 1995; 80:695-705. 101. Hemmer B, Fleckenstein BT, Vergelli M et al. Identification of high potency microbial and self ligands for a human autoreactive class II restricted T cell clone. J Exp Med 1997; 185:1651-1659. 102. Talbot PJ, Paquette JS, Ciurli C et al. Myelin basic protein and human coronavirus 229E cross-reactive T cells in multiple sclerosis. Ann Neurol 1996; 39:233-240. 103. Prabhu Das M, Nicholson LB, Greer JM et al. Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein. J Exp Med 1997; 186:867-876. 104. Nicholson L, Greer J, Sobel R et al. An altered peptide ligand mediates immune deviation and prevents autoimmune encephalomyelitis. Immunity 1995; 3:397-405. 105. Brocke S, Gijbels K, Allegretta M et al. Treatment of experimental encephalomyelitis with a peptide analogue of myelin basic protein. Nature (London) 1996; 379:343-346. 106. Mosmann TR, Coffman RL. TH1 and TH2 cells: different pattern of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989; 7:145-173. 107. Liblau RS, Singer SM, McDevitt HO. Th1 and Th2 CD4+ T cells in the pathogenesis of organ-specific autoimmune diseases. Immunol Today 1995; 16:34-38. 108. Del Prete G, De Carli M, Almerigogna F et al. Human IL-10 is produced by both type 1 helper (Th1) and type 2 helper (Th2) T cell clones and inhibits their antigen-specific proliferation and cytokine production. J Immunol 1993; 150:353-360. 109. Gerosa F, Paganin C, Peritt D et al. Interleukin-12 primes human CD4 and CD8 T cell clones for high production of both interferon-γ and interleukin-10. J Exp Med 1996; 183:2559-2569. 110. Jeannin P, Delneste Y, Seveso M et al. IL-12 synergizes with IL-2 and other stimuli in inducing IL-10 production by human T cells. J Immunol 1996; 156:3159-3165. 111. Windhagen A, Anderson D, Carrizosa A et al. IL-12 induces human T cells secreting IL-10 with IFN-g. J Immunol 1996; 1996:1127-1131. 112. Oro AS, Guarino TJ, Driver R et al. Regulation of disease susceptibility: decreased prevalence of IgE-mediated allergic disease in patients with multiple sclerosis. J Allergy Clin Immunol 1996; 97:1402-1408. 113. Beck J, Rondot P, Catinot L et al. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: Do cytokines trigger off exacerbations? Acta NeurolScand 1988; 78:318-323. 114. Dettke M, Scheidt P, Prange H et al. Correlation between interferon production and clinical disease activity in patients with multiple sclerosis. J Clin Immunol 1997; 17:293-300.

210

Endocrine and Organ Specific Autoimmunity

115. Philippe J, Debruyne J, Leroux-Roels G et al. In vitro TNF-alpha, IL-2 and IFN-gamma production as markers of relapses in multiple sclerosis. Clin Neurol Neurosurg 1996; 98:286-290. 116. Woodroofe MN, Cuzner ML. Cytokine mRNA expression in inflammatory multiple sclerosis lesions: detection by nonradioactive in situ hybridization. Cytokine 1993; 5:583-588. 117. Trinchieri G. Interleukin-12: A proinflammatory cytokine with immunoregulatory functions that bridge innate resistance and antigen-specific adaptive immunity. Annu Rev Immunol 1995; 13:251-276. 118. Balashov KE, Smith DR, Khoury SJ et al. Increased interleukin 12 production in progressive multiple sclerosis: induction by activated CD4+ T cells via CD40 ligand. Proc Natl Acad Sci USA 1997; 94:599-603. 119. Nicoletti F, Patti F, Cocuzza C et al. Elevated serum levels of interleukin-12 in chronic progressive multiple sclerosis. J Neuroimmunol 1996; 70:87-90. 120. Becher B, Dodelet V, Fedorowicz V et al. Soluble tumor necrosis factor receptor inhibits interleukin 12 production by stimulated human adult microglial cells in vitro. J Clin Invest 1996; 98:1539-1543. 121. Windhagen A, Newcombe J, Dangond F et al. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD86), and interleukin 12 cytokine in multiple sclerosis lesions. J Exp Med 1995; 182:1985-1996. 122. Gerritse K, Laman JD, Noelle RJ et al. CD40-CD40 ligand interactions in experimental allergic encephalomyelitis and multiple sclerosis. Proc Natl Acad Sci USA 1996; 93:2499-2504. 123. Leonard JP, Waldburger KE, J GS. Prevention of experimental autoimmune encephalomyelitis by antibodies against interleukin 12. J Exp Med 1995; 181:381-386. 124. Grewal IS, Foellmer HG, Grewal KD et al. Requirement for CD40 ligand in costimulation induction, T cell activation, and experimental allergic encephalomyelitis. Science 1996; 273:1864-1867. 125. Smith T, Hewson AK, Kingsley CI et al. Interleukin-12 induces relapse in experimental allergic encephalomyelitis in the Lewis rat. Am J Pathol 1997; 150:1909-1917. 126. Schiffenbauer J, Johnson HM, Butfiloski EJ et al. Staphylococcal enterotoxins can reactivate experimental allergic encephalomyelitis. Proc Natl Acad Sci USA 1993; 90:8543-8546. 127. van Oosten BW, Barkhof F, Truyen L et al. Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2. Neurology 1996; 47:1531-1534. 128. Antel JP, Arnason BGW, Medof ME. Suppressor cell function in multiple sclerosis. Ann Neurol 1978; 129C:159-170. 129. Morimoto C, Hafler DA, Weiner HL et al. Selective loss of the suppressor-inducer T cell subset in progressive multiple sclerosis. Analysis with anti-2H4 monoclonal antibody. NEJM 1987; 316:67-72. 130. Balashov KE, Khoury SJ, Hafler DA et al. Inhibition of T cell responses by activated human CD8+ T cells is mediated by interferon-gamma and is defective in chronic progressive multiple sclerosis. J Clin Invest 1995; 95:2711-2719. 131. Bongioanni P, Meucci G. T cell tumor necrosis factor-alpha receptor binding in patients with multiple sclerosis. Neurology 1997; 48:826-831. 132. Martino G, Clementi E, Brambilla E et al. Gamma interferon activates a previously undescribed Ca2+ influx in T lymphocytes from patients with multiple sclerosis. Proc Natl Acad Sci USA 1994; 91:4825-4829. 133. Karaszewski JW, Reder AT, Anlar B et al. Increased lymphocyte beta-adrenergic receptor density in progressive multiple sclerosis is specific for the CD8+, CD28- suppressor cell. Ann Neurol 1991; 30:42-47. 134. Karaszewski JW, Reder AT, Anlar B et al. Sympathic skin responses are decreased and lymphocyte beta-adrenergic receptors are increased in progressive multiple sclerosis. Ann Neurol 1990; 27:366-372. 135. Hartung HP, Michels M, Reiners K et al. Soluble ICAM-1 serum levels in multiple sclerosis and viral encephalitis. Neurology 1993; 43:2331-2335.

Multiple Sclerosis

211

136. Droogan AG, McMillan SA, Douglas JP et al. Serum and cerebrospinal fluid levels of soluble adhesion molecules in multiple sclerosis: predominant intrathecal release of vascular cell adhesion molecule-1. J Neuroimmunol 1996; 64:185-191. 137. Giovannoni G, Lai M, Thorpe J et al. Longitudinal study of soluble adhesion molecules in multiple sclerosis: correlation with gadolinium enhanced magnetic resonance imaging. Neurology 1997; 48:1557-1565. 138. Calabresi PA, Tranquill LR, Dambrosia JM et al. Increases in soluble VCAM-1 correlate with a decrease in MRI lesions in multiple sclerosis treated with interferon beta-1b. Ann Neurol 1997; 41:669-674. 139. Rieckmann P, Altenhofen B, Riegel A et al. Soluble adhesion molecules (sVCAM-1 and sICAM-1) in cerebrospinal fluid and serum correlate with MRI activity in multiple sclerosis. Ann Neurol 1997; 41:326-333. 140. Mossner R, Fassbender K, Kuhnen J et al. Vascular cell adhesion molecule--a new approach to detect endothelial cell activation in MS and encephalitis in vivo. Acta Neurol Scand 1996; 93:118-122. 141. Genc K, Dona DL, Reder AT. Increased CD80(+) B cells in active multiple sclerosis and reversal by interferon beta-1b therapy. J Clin Invest 1997; 99:2664-2671. 142. Svenningsson A, Dotevall L, Stemme S et al. Increased expression of B7-1 costimulatory molecule on cerebrospinal fluid cells of patients with multiple sclerosis and infectious central nervous system disease. J Neuroimmunol 1997; 75:59-68. 143. Moretta A. Molecular mechanisms in cell-mediated cytotoxicity. Cell 1997; 90:13-18. 144. Dowling P, Shang G, Raval S et al. Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain. J Exp Med 1996; 184:1513-1518. 145. Dowling P, Husar W, Menonna J et al. Cell death and birth in multiple sclerosis brain. J Neurol Sci 1997; 149:1-11. 146. Marusic S, Tonegawa S. Tolerance induction and autoimmune encephalomyelitis amelioration after administration of myelin basic protein-derived peptide. J Exp Med 1997; 186:507-515. 147. Waldner H, Sobel RA, Howard E et al. Fas- and FasL-deficient mice are resistant to induction of autoimmune encephalomyelitis. J Immunol 1997; 159:3100-3103. 148. Sabelko KA, Kelly KA, Nahm MH et al. Fas and fas ligand enhance the pathogenesis of experimental allergic encephalomyelitis, but are not essential for immune privilege in the central nervous system. J Immunol 1997; 159:3096-3099. 149. Bonetti B, Raine CS. Multiple sclerosis: Oligodendrocytes display cell death-related molecules in situ but do not undergo apoptosis. Ann Neurol 1997; 42:74-84. 150. D’Souza SD, Bonetti B, Balasingam V et al. Multiple sclerosis: Fas signaling in oligodendrocyte cell death. J Exp Med 1996; 184:2361-2370. 151. Scolding NJ, Jones J, Compston DA et al. Oligodendrocyte susceptibility to injury by T-cell perforin. Immunology 1990; 70:6-10. 152. Rubesa G, Podack ER, Sepcic J et al. Increased perforin expression in multiple sclerosis patients during exacerbation of disease in peripheral blood lymphocytes. J Neuroimmunol 1997; 74:198-204. 153. Vergelli M, Hemmer B, Muraro PA et al. Human autoreactive CD4+ T cell clones use perforin- or Fas/Fas ligand-mediated pathways for target cell lysis. J Immunol 1997; 158:2756-2761. 154. Selmaj K, Raine CS, Farooq M et al. Cytokine cytotoxicity against oligodendrocytes. J Immunol 1991; 147:1522-1529. 155. Hoffman FM, Hinton DR, Johnson K et al. Tumor necrosis factor identified in multiple sclerosis brain. J Exp Med 1989; 170:607-612. 156. Sharief MK, Hentges R. Association between tumor necrosis factor-alpha an disease progression inpatients with multiple sclerosis. NEJM 1991; 325:467-472. 157. Rieckmann P, Albrecht M, Kitze B et al. Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing-remitting multiple sclerosis is associated with disease activity. Ann Neurol 1995; 37:82-88.

212

Endocrine and Organ Specific Autoimmunity

158. Rentzos M, Nikolaou C, Rombos A et al. Tumor necrosis factor alpha is elevated in serum and cerebrospinal fluid in multiple sclerosis and inflammatory neuropathies. J Neurol 1996; 243:165-170. 159. Matusevicius D, Navikas V, Soderstrom M et al. Multiple sclerosis: the proinflammatory cytokines lymphotoxin-alpha and tumor necrosis factor-alpha are upregulated in cerebrospinal fluid mononuclear cells. J Neuroimmunol 1996; 66:115-123. 160. Navikas V, He B, Link J et al. Augmented expression of tumor necrosis factor-alpha and lymphotoxin in mononuclear cells in multiple sclerosis and optic neuritis. Brain 1996; 119:213-223. 161. Ruddle NH, Bergman CM, McGrath M et al. An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J Exp Med 1990; 172:1193-1200. 162. Kuroda Y, Shimamoto Y. Human tumor necrosis factor-alph augments experimental allergic encephalomyelitis in rats. J Neuroimmunol 1991; 34:159-164. 163. Vartanian T, Li Y, Zhao M et al. Interferon-gamma-induced oligodendrocyte cell death: implications for the pathogenesis of multiple sclerosis. Mol Med 1995; 1:732-743. 164. Bagasra O, Michaels FH, Zheng YM et al. Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis. Proc Natl Acad Sci USA 1995; 92:12041-12045. 165. Bo L, Dawson TM, Wesselingh S et al. Induction of nitric oxide synthase in demyelinating regions of multiple sclerosis brains. Ann Neurol 1994; 36:778-786. 166. Merrill JE, Ignarro LJ, Sherman MP et al. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol 1993; 151:2132-2141. 167. Parkinson JF, Mitrovic B, Merrill JE. The role of nitric oxide in multiple sclerosis. J Mol Med 1997; 75:174-186. 168. Jacobs LD, Cookfair DL, Rudick RA et al. Intramuscular interferon beta-1a for disease progression in relapsing multiple sclerosis. The Multiple Sclerosis Collaborative Research Group (MSCRG) [see comments]. Ann Neurol 1996; 39:285-294. 169. Anonymous. Interferon beta-1b in the treatment of multiple sclerosis: final outcome of the randomized controlled trial. The IFNB Multiple Sclerosis Study Group and The University of British Columbia MS/MRI Analysis Group. Neurology 1995; 45:1277-1285. 170. Johnson KP, Brooks BR, Cohen JA et al. Copolymer 1 reduces relapse rate and improves disability in relapsing-remitting multiple sclerosis: Results of a phase III multicenter, doubleblind placebo-controlled trial. The Copolymer 1 Multiple Sclerosis Study Group [see comments]. Neurology 1995; 45:1268-1276. 171. Beck RW, Cleary PA, Trobe JD et al. The effect of corticosteroids for acute optic neuritis on the subsequent development of multiple sclerosis. The Optic Neuritis Study Group [see comments]. NEJM 1993; 329:1764-1769. 172. Comabella M, Balashov K, Isaazadeh S et al. Elevated interleukin-12 in progressive multiple sclerosis correlates with disease activity and is normalized by pulse cyclophosphamide therapy. J Clin Invest 1998; 102:671-678. 173. Miller A, Lider O, Weiner HL. Antigen-driven bystander suppression following oral administration of antigens. J Exp Med 1991; 174:791-798. 174. Weiner HL. Oral tolerance: Immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997; 18:335-343. 175. Sun JB, Rask C, Olsson T et al. Treatment of experimental autoimmune encephalomyelitis by feeding myelin basic protein conjugated to cholera toxin B subunit. Proc Natl Acad Sci USA 1996; 93:7196-7201. 176. Lassmann H, Bruck W, Lucchinetti C et al. Remyelination in multiple sclerosis. Multiple Sclerosis 1997; 3:133-136. 177. Lucchinetti CF, Noseworthy JH, Rodriguez M. Promotion of endogenous remyelination in multiple sclerosis. Multiple Sclerosis 1997; 3:71-75.

CHAPTER 11

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy Aaron I. Vinik, Gary L. Pittenger, Zvonko Milicevic, Jadranka Knezevic-Cuca

Introduction

O

ur knowledge of the physiology and pathophysiology of immune responses in the central (CNS) and peripheral nervous systems (PNS) has grown over the last decade. This new information has facilitated advances in experimental and clinical investigations and the application of new therapies to neurologic diseases with presumed autoimmune pathogenesis. We now know that immune mechanisms are involved in the initiation and/or perpetuation of tissue destruction in several neurologic diseases.1-6 Furthermore, evidence has been adduced for a primary, or contributing, role of immune effectors in the pathogenesis of a large group of peripheral neurological disorders. Multiple sclerosis and myasthenia gravis are examples of MHC class II—and probably TCR— Vb gene-related disorders. In both conditions there is a well-defined role for the immune system in their pathogenesis. Autoantibodies to specific antigens during disease initiation and T cell activation have been identified. Chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain- Barré syndrome (GBS), multiple motor polyneuropathy (MMP) and polyneuropathies associated with monoclonal gammopathies of undetermined significance (MGUS) are a group of peripheral neuropathies with clearly established immune pathogenesis.7-9 Table 11.1 presents a comparison of the clinical profiles of these peripheral nerve disorders compared with the typical diabetic polyneuropathy profile. The similarities between the autoimmune neuropathies and that which occurs in diabetes suggests that there may be an overlap in the immune-mediated neuropathies and the syndromes encountered in the heterogeneous diabetic neurologic syndromes.10 Diabetic neuropathy encompasses a wide range of clinical syndromes. This is likely to be due to more than one pathogenic process leading to the neurologic disorder in these individuals.11-14 The recognition of the heterogeneity of the clinical neurologic syndromes and the awareness of the multiplicity of possible destructive factors directed against peripheral neurons in diabetic patients with neuropathy has resulted in a recent intensification of the research endeavor to define the relative importance of each of these causative factors in various neuropathic syndromes. Thus, progress has been made, with a shift in focus from the traditional theories of the role of glycemia, increase in polyol pathway activity and accumulation of advanced glycemic endproducts (AGEs) to the notion that oxidative stress as well as autoimmune mechanisms may be relevant to the destructive forces operative in the nervous system in diabetes.11-14 Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

Endocrine and Organ Specific Autoimmunity

214

Table 11.1. The Distribution (%) of Signs and Symptoms of Neuropathy in Different Conditions

DSPN Motor/Sensory Distal Asymmetric Multifocal

Vasculitis

CIDP

MGUS

Diabetes

3 27 70

91 9 0

100 0 0

67 0 33

DSPN = Distal Symmetric Polyneuropathy; CIDP = Chronic Inflammatory Demyelinating Polyneuropathy; MGUS = Monoclonal Gammopathy of Undetermined Significance.

One of the research fields that has gained significant attention is the role of immune mechanisms in diabetic neuropathy. Three different syndromes that occur in diabetes display clear evidence of immune system involvement in mediation of the neurologic deficit: (a) proximal diabetic neuropathy (PDN);15,16 (b) autonomic neuropathy in patients with type 1 (insulin-dependent) diabetes mellitus;17-20 and (c) certain forms of distal symmetric diabetic polyneuropathy (DSPN).21 The immunopathogenic and clinical characteristics, as well as the response to treatment, of each of these types of diabetic neuropathy will be discussed in this chapter.

The Normal Barrier to Immune-Mediated Nerve Destruction Although the CNS has long been considered an immunologically privileged site, recent studies showed dynamic, albeit tightly regulated, interaction between lymphoid cells, the CNS vasculature, and parenchyma. Normally, the circulating T cells are in a state of selftolerance.22,23 This means that although potentially autoreactive, they are inactivated or ignorant of their putative autoantigens. Nonstimulated T cells cannot cross the blood-brain and blood-nerve barrier.24 After activation, trans-endothelial transport can happen only if surface molecules on T cells match those on the endothelial cells.25 The specific activation of the immune response begins outside the nervous system, in the circulation and the lymphoid tissues. T cell activation requires the engagement of both antigenic and costimulatory signals between antigen-specific T cells and other cells, such as antigen-presenting cells (APCs) or target cells.26,27 In the absence of all necessary signals, exclusive stimulation of the T cells through the complex of T cell receptor (TCR) and CD3 (e.g., partial activation) induces T cell anergy.28,29 Apart from the need for antigenic recognition, autoimmune disease of the nervous system requires breakdown of the normal bloodnerve barrier. The endothelial cells of blood vessels form a barrier between blood and the CNS and PNS, termed the blood-brain and blood-nerve barriers, respectively. Consequently, they are critical cells in regulating vascular permeability and trapping or exudation of lymphoid cells and immunoglobulins into target tissues. The first step in antigen-specific recruitment and attachment of T lymphocytes to endothelial cells is binding of T cell antigen receptors (TCRs) and major histocompatibility complex (MHC) molecule receptors (CD4 and CD8) on T lymphocytes to antigen in the antigen-presenting groove of MHC molecules on endothelial cells.30,31 The activated T cells, macrophages and infectious agents secrete a variety of lymphokines or cytokines such as interferon gamma (IFN-γ) the interleukins-1, -2, -4, and -6, and tumor necrosis factor (TNF-α).32 On the endothelial cells of the blood vessels at the blood-brain and blood-nerve barriers these molecules induce a variety of adhesion

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

215

molecules that allow the traffic of T cells from the circulation to their respective tissue organs.33,34 The main adhesion molecules involved in leukocyte-endothelial interaction and regulation of leukocyte migration are classified by structure into three families: selectins, integrins, and immunoglobulin supergene families.35,36 They are involved in three sequential steps of adhesion: tethering (slowing and rolling), activation and strong adhesion, and migration. Chemokines, displayed on or released from the endothelium, further attract leukocytes and activate integrin adhesiveness.37 Endothelial cells also respond to and secrete cytokines which are important in recruitment of immunocytes. Specific properties in terms of display signals (e.g., molecules expressed at the cell surface) vary with size and location of endothelial cells.38 The presence of organ-specific antigens on endothelial cells is an area of active investigation. It must be noted that CNS vasculature contrasts with the PNS vasculature. The physiologic characteristics of the blood-brain barrier provide better protection than the bloodnerve barrier of the PNS vasculature to minimize immune-mediated damage to the cerebral endothelium and blood-brain barrier itself. Furthermore, there are specific areas, such as the hypothalamus and the dorsal root ganglia, where the blood-nerve barrier is quite frivolous, and transport of proteins into the nervous system is unimpeded, making these regions of the nervous system prime targets for autoimmune processes.

Pathogenic Immune Mechanisms in Neurologic Diseases

Dalakas39 has proposed the following criteria for a disease to be defined as an autoimmune condition: (a) the antigen must be known; (b) there must be a specific antibody response against the autoantigen; (c) the diseases or the pathological process must be transferable to experimental animals with the patients serum that contains the pathogenic autoantibody; (d) the serum immunoglobulin, when bound to the antigen, can cause in situ destruction of the target tissue, impairing function, (e) the removal of the autoantibodies or suppression of the B cells responsible for their production must be associated with clinical improvement.40,41 Based on these requirements the immune-mediated neurological conditions may be liberally divided into two major groups: one in which the autoimmune pathogenesis is well characterized [definite autoimmune diseases; myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome (LEMS), and probably the anti-myelin-associated glycoprotein (MAG) IgM demyelinating polyneuropathies are in the first group], and a second in which putative autoimmune causes are implicated [possible or probable autoimmune diseases; peripheral neuropathies including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy and some other conditions]42,43 (Table 11.2). There are, however, a variety of means whereby the nervous system may be targeted by immune mechanisms. The Pathogenesis of Immune-mediated Nerve Damage (See Table 11.3) 1. Cellular immune mechanisms 2. Humoral immune mechanisms 3. Antigenic targets in the nervous system 4. Mechanism of cell death-apoptosis 1. Cellular Immune Mechanisms Several mechanisms of neural tissue damage mediated by the immune system have been proposed (Fig. 11.1): (a) cytokine induced autoimmunity;44,45 (b) molecular mimicry triggered by an infection;46.47 and c) autoimmunity induced by superantigens,48,49 Podusl, et al50 showed increased permeability of blood-nerve and blood-brain barrier due to increased

216

Endocrine and Organ Specific Autoimmunity

Table 11.2. Major Autoimmune Neuropathy Groups Neurologic conditions with definite autoimmune pathogenesis -Myasthenia gravis -Lambert-Eaton myasthenic syndrome (LEMS) -Anti-myelin associated glycoprotein (MAG) IgM demyelinating polyneuropathies Neurologic conditions with putative autoimmune pathogenesis -Guillain-Baré syndrome -Chronic inflammatory demyelinating polyneuropathy -Multifocal motor neuropathy -Multiple sclerosis -Inflammatory myopathies -Stiff-man syndrome -Isaac’s syndrome -Paraneoplastic syndromes -Neurologic conditions associated with systemic autoimmune disease -Vasculitides

glycation of immunoglobulins. This could be a new mechanism involved in the breakdown of tolerance of nervous antigens in diabetic patients. Activated T cells or specific antibodies, initiated and produced in the periphery, upon reaching the PNS and CNS recognize the antigens to which they have been sensitized and, under certain conditions, engage in damage of the targeted cell (Table 11.4). The location of the inflammation, type of inflammatory infiltrate, and persistence of vascular inflammation are determined by spatial and temporal dispersion of adhesion molecules, cytokines, leukotrienes, activated components in the complement cascade, and microbial products.51 The final effectors of the nerve damage could be: (a) T cells, as occurs in demyelinating diseases of the CNS and PNS, in which T cells recognize the autoantigen presented to them by the MHC class II-expressing cells (macrophages, astrocytes or possibly Schwann cells);52 (b) autoantibodies, for example antibodies generated against myelin components, such as myelin glycoprotein or glycolipids, may exert a direct myelinotoxic effect;53,54 (c) macrophages, which play a primary role in Fc receptor-mediated phagocytosis or via specific antibodies in antibody-dependent, cell-mediated cytotoxicity;55,56 (d) membranolytic attack complex and complement-fixing antibody, as occurs in the monoclonal gammopathies (MG), where specific anti-acetylcholine receptor antibodies are generated and act both directly and by induction of the lytic component of the complement pathway.57 Vasculitis is also a frequent cause of neuronal damage58,59 and may be a manifestation of diverse diseases. Vasculitis is a specific form of immune-mediated tissue damage. It can be a multi-organ or organ-specific disease that shares the central feature of inflammation of blood vessels. Immunopathogenic mechanisms that have been found in various vasculitides include vascular inflammation that is a consequence of: (a) immune complex deposition (heterologous or autoantigen induced); (b) autoantibody mediation; and (c) T cellmediated vascular inflammation (reviewed in Moore).60 It may be primary (idiopathic) or secondary. The latter are those provoked by antigens from microbial, toxic and neoplastic antigens. The histological features, the clinical characteristics, and the presence of any underlying cause defines the individual diseases. There may be specific types of leukocytes (neutrophils, T lymphocytes, eosinophils) predominant in the vascular infiltrates of specific diseases. Vasculitis has recently been described in some patients with diabetic

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

217

Fig. 11.1. Mechanisms involved in the immune cell activation in the immune-mediated neurologic diseases.

-demyelination -axon deg. -multifocal -IgM, IgG, complement -T cells

-motor -sensory

-chronic

-GM1 Ab -asialo-GM1-Ab -sulfatide

-PLA

Pathology

Lesion distribution

Time course

Antigen -derived Ab

Natural Ab

Distal symmetric diabetic polyneuropathy

-unknown

-unknown

-acute or subacute

-motor -sensory

-demyelination -axon deg. -diffuse inflammation vasculitis, perivasculitis

Proximal diabetic polyneuropathy

-PLA

-GM1α Ab -GD1b Ab -anti-glycolipids P0 and P2 Ab

-acute -relapsing

-motor -sensory

-demylination -axon deg. -inflammation ++ APC, T&B cells Ig, complement -multifocal

Guillain-Barré syndrome

-chronic

-motor -sensory

-demylination -axon deg. -inflammation +

Multifocal motor neuropathy

-PLA

-no

-myelin associated -GM1 Ab glycoprotein -GD1b Ab (MAG) Ab -anti-glycosphingolipids and glycolipids P0 and P2 Abs -anti-tubulin Ab

-subacute -chronic -relapsing

-motor -sensory

-demylination -axon deg. -inflammation

CIDP

Table 11.3. A Comparison of the Clinical Features of Neuropathies with an Autoimmune Component

-PLA

-MAG Ab -sulfatide

-chronic

-motor -sensory

-demylination -axonal deg.

Polyneuropathy associated with MGUS

218 Endocrine and Organ Specific Autoimmunity

-no associated lesion

-sympath. tests ↓ -parasym. tests ↓

-prolonged or non-evokable

-no data

Autonomic function tests

Sensory evoked potentials

Immunotherapy

-mild, rarely -no data

-proximal conduction block -reduced amplitude -generalized slowing

-no data

-no data

-motor↓+++ -sensory↓++

-no data

-no data

-motor↓+++ -sensory→

-steroids -IVIg -immunosuppressive drugs -interferon-α 2A

-steroids -IVIg -immunosuppressive drugs

-refractory period -no data increase -reduced amplitude

-no data

-vibratory sen. ↓ -pinprick sen. ↓

-motor↓+++ -sensory↓+

NSS↓+++, sensory NSS↓++, sensory, NSS↓++, sensory NDS↓+, NDS↓+ MRCS↓+++ NDS↓+, MRCS↓+++ MRCS↓+++

-small fibers

-steroids -IVIg -immunosuppressive drugs

-reduced amplitude -slowing

-no data

-vibratory sen. ↓+++ -position sens↓+ -pain sens. ↓+ -light touch ↓+

-motor↓+++ -sensory↓+++

NSS↓++, sensory NDS↓++, MRCS↓++

-no data

Modified from Pittenger et al, Diabetics Mellitus: A fundamental and clinical text. LeRolth, Taylor, Olefsky, eds. Lippincott-Raven. Philadelphia, PA

-steroids -IVIg -immunosuppressive drugs

-no data

-no data

Quantitative -vibratory sen. ↓+++ sensory -pinprick sen. ↓+++ testing -light touch sen. ↓+++ -position sen. ↓+++

NSS↓+++, sensory NDS++↓, MRCS+++↓

-sensory↓+++ -motor↓+++

NSS↓++, sensory NDS↓++, MRCS+↓

Clinical findings

-no

Electroneuro- -sensory↓+++ graphi -motor↓++ findings

-myelinating fibers

Associated autonomic neuropathy

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy 219

Endocrine and Organ Specific Autoimmunity

220

Table 11.4. Autoantigens are associated with a number of both central and peripheral neuropathic syndromes. This is a partial list of the most common autoimmune neuropathy syndromes and the antigens that have been associated with their pathogenesis. Autoantigens Associated with Autoimmune Neuropathies Neuropathy Syndrome

Autoantigen

Amyotrophic lateral sclerosis Eaton Lambert CIDP Guillain Barré Stiff Man Sensory Paraneoplastic Diabetic

Voltage-gated calcium channels Synaptotagmin MAG, P0,P2, Tubulin GM1, GD1, GT1b, P0,P2 GAD Sulfatide, GD1b, MAG Hu, P Anca, C Anca GM1, GT1b,GAD, PLA, MGUS

Fig. 11.2. Cellular vascular infiltrate involving the vasa nervorum in a patient with proximal diabetic neuropathy in a biopsy of the obturator nerve.

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

221

neuropathy,15,61 especially the proximal type, and is also thought to be responsible for the mononeuropathies and the mononeuritis multiplex complex (Fig. 11.2). 2. Humoral Immune Mechanisms Contrasting with the proximal neuropathies or the mononeuritides, there is fairly strong evidence to support both tissue and humoral immune mechanisms in the pathogenesis of diabetic somatic and autonomic neuropathy. We have examined a large cohort of patients with diabetic polyneuropathy and found antibodies to a multitude of different antigens (Table 11.5), as well as antibody binding in various sites in peripheral nerves (Fig. 11.3). Organ-specific, complement-fixing autoantibodies against unknown antigens from adrenal medulla (CF-ADM) and sympathetic ganglia (CF-SG) have been demonstrated in IDDM patients.62-66 With duration of diabetes greater than 5 years, CF-ADM occurs in both ICA+ and ICA- patients, suggesting that the antigenic targets in adrenal medulla and pancreatic islets are different. Because these experiments were done as cross-sectional studies, it is not known whether the generation of antibody preceded neuronal destruction. Observations by Zanone et al on the prevalence of CF-SG antibodies or at least one of the autoantibodies directed to autonomic nervous system structures (CF-SG, CF-ADM or complement-fixing vagal (CF-V) autoantibody) in patients with diabetic autonomic neuropathy strongly support the relationship between autoantibodies to autonomic structures and autonomic neuropathy in IDDM.64 Recent studies have demonstrated that there is an immunoglobulin in the serum of patients with IDDM and clinical neuropathy that is able to inhibit the growth, and in some cases induce death, of an adrenergic neuroblastoma cell clone, an in vitro model for sympathetic neurons.20,67 Sera from patients with IDDM without signs of clinical neuropathy exert no consistent effect, although some of these sera also inhibit cell proliferation, and it has been suggested that these might be preneuropathic patients. In contrast, sera from NIDDM patients with neuropathy showed little effect on

Fig. 11.3. Representative direct immunofluorescence photomicrographs made using anti-human IgG and IgM in sural nerve biopsies from type I diabetic patients with neuropathy showing perineurial staining for IgM (A) and IgG (B), axonal IgG staining (C) and nodal IgG staining.

Endocrine and Organ Specific Autoimmunity

222

Table 11.5. The Reported Frequency of Antibodies to Particular Antigens in Diabetic Neuropathies Antigen Phospholipid Neuroblastoma (N1E-115) GAD 65/67 GM1 (monosialoganglioside) Monoclonal gammopathy

Frequency 88% 66% 35% 14% 8%

Publication Vinik, et al.70 Pittenger, et al.20,67 Vinik, et al. 70 Milicevic, et al.21,175 Milicevic, et al.21,175

cell proliferation, suggesting that the development of neuropathy in IDDM and NIDDM might occur via different mechanisms. The differences in the presentation of neuropathy observed by various investigators may simply reflect different pathogenesis of chronic neurologic complications of IDDM, perhaps generated by different antigen-antibody complexes. A number of autoantigens have been described in IDDM patients that might induce immune system responses resulting in nerve damage, including phospholipids, glycolipids and glutamic acid decarboxylase (GAD). Rabinowe et al have reported anti-ganglioside GT1b IgG antibody in IDDM and correlated the antibody to changes in orthostatic blood pressure,68 suggesting a role in autonomic neuropathy. It appears that anti-GT1b autoantibody recognizes sympathetic ganglia and adrenal medullary antigens. There are probably other, as yet unrecognized, antigens associated with IDDM that may participate in neuronal recognition and destruction. Immunogenetic analyses done in IDDM patients showed a significantly higher frequency of the heterozygous genetic constellation HLA-DR3/DR4 in those with mild and severe autonomic diabetic neuropathy of the cardiovascular system, compared with those who did not have any signs of autonomic nervous system disturbances.69 This observation suggests a relationship between diabetic autonomic neuropathy and immune response genes within MHC, which is in accordance with other evidence of autoimmunity generated against sympathetic nervous system structures in IDDM patients. Recent studies of the prevalence of antiphospholipid antibodies (PLA), a family of closely related immunoglobulins that interact with one or more negatively charged phospholipids (constituents of nervous tissues), have also shown an increased incidence in IDDM, with a further increase in prevalence in IDDM patients with neuropathy. It does not appear that there is a direct correlation between specific features of neuropathy and PLA, but we have found that sera with high titers of IgG PLA inhibited cell growth and differentiation of a neuroblastoma cell line.70 PLA have been described in a number of autoimmune, neurologic and hematologic disorders. In these disorders PLA appearance increases the risk of arterio-venous thrombosis, thrombocytopenia, hemolytic anemia and fetal loss. These features, occurring by themselves or commonly together, have been described as the antiphospholipid syndrome, found in PLA positive persons without clinical or serological signs of any other disease. Thus, the possibility that PLA might contribute to diabetic neuropathy, either by direct neuronal toxicity or by compromise of the neuronal vessels, must be considered. In our study PLA were found in 88% of a diabetic population with neuropathy, compared to 32% in those diabetic patients without neurologic complications and 2% in the general population. Most of the PLA+ patients were positive for the IgG fraction. Correlation between PLA level and warm perception threshold suggests that small, C-fibers contain a specific antigen recognized by PLA. Results obtained from experimental

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

223

investigations in humans as well as other species demonstrate that autoimmunity may be caused not only by functional immunocyte disorders, but also by the changed antigenicity of target cells. It is reported that there is a change in the phospholipid composition of the extracellular leaflet of the plasma membrane of erythrocytes, with an increase in phosphatidylserine in response to hyperglycemia in diabetes.71 It is as yet unclear whether this “phospholipid asymmetry” is a common cellular effect of diabetes. In light of the increased prevalence of phospholipid autoantibodies in patients with diabetic neuropathy, it must be considered that these events acting in combination may be important in neuronal destruction. It has been argued that PLA are of no pathological consequence, but are an epiphenomenon consequent upon tissue damage and the generation of an immune response. Considerable circumstantial evidence supports the notion that PLA may indeed be capable of inflicting injury, and that the damage may be selective for specific parts of the nervous system as well as other tissues.72,73 In addition, concurrent vascular changes may lead to significant interaction between the two pathogenetic mechanisms. Future studies should help resolve questions about the role of PLA autoantibodies in development of neuropathic syndromes. Glycolipid autoantibodies may also play a significant role in diabetic neuropathy. Autoantibodies to the gangliosides sialo- and asialo-GM1 have been described in diabetic patients.74 In addition, Pestronk et al have characterized GM1 autoantibodies to be regulated by T cell independent B cells.75 However, Bansal et al have determined that while anti-GM1 IgM is clearly associated with multifocal motor neuropathy, there is no correlation with anti-GM1 titer in patients with diabetic peripheral neuropathy.76 However, these authors did not divide the diabetic patient population according to type of neuropathy (e.g., motor, sensorimotor, autonomic) and may have oversimplified their analysis. As described previously, anti-GM1 is most often associated with lower motor neuropathies and therefore may not play a major role in diabetic neuropathies, which for the most part do not have a large motor component. The 67 kDa form of GAD, which is a product of a different gene than GAD-65, is the predominant form in neurons.77 Antibodies to GAD are found in the neurologic disorder Stiff-man syndrome78-80 in which there is a high prevalence of type 1 diabetes. Harrison et al reported the presence of GAD antibodies in type 1 diabetes and a progressive decline in anti-GAD titer with increasing duration of diabetes.81 In 9 patients with autonomic neuropathy these antibodies persisted, suggesting a role in the pathogenesis of neuropathy. This has, however, been contested82,83 and is not compatible with observations by other investigators.70,83 Furthermore, GAD is a cytoplasmic enzyme in neurons, and access of the autoantibody to the protein in live cells is problematic. Thus, the role of anti-GAD antibodies in diabetic neuropathy remains elusive. Other target antigens associated with diabetes and/or neuropathy, such as growth factors, must also be considered. Insulin autoantibodies are a common feature of both IDDM and NIDDM. There are structural and biochemical similarities between the insulin family of peptides and NGF.84,85 NGF was discovered nearly 50 years ago. It has been known since the pioneering work of Levi-Montalcini et al that neural crest-derived cells, sympathetic neurons and dorsal root ganglion neurons (DRG) are developmentally dependent on NGF.86 More recently, it has been shown that adult DRG and sympathetic neurons, both populations of neurons affected in diabetic neuropathy, are dependent on NGF either for their maintenance87 or survival.88 It has been suggested that antibodies to insulin may cross-react with NGF and contribute to an effective reduction in NGF available to nerves, thereby contributing to the development of neuropathy.89 This is significant, because transgenic mice that have an inactive low affinity NGF receptor, p75, present a similar neuropathic

224

Endocrine and Organ Specific Autoimmunity

condition to that seen in diabetic neuropathy, with loss of sensory (especially temperature perception) and autonomic innervation.90 Thus, autoimmunity may play a role in the NGF deficiency reported in diabetes by mechanisms related to immune neutralization of available NGF. What may be relevant to this argument is the recent demonstration that NGF treatment of diabetic patients with neuropathy improves small nerve fiber function.91 If this turns out to be due to reversal of immune-mediated NGF deficiency, it would add considerable support to the theory that loss of neuropeptidergic support is a viable pathogenetic mechanism for certain types of diabetic neuropathies. In another study we tested the hypothesis that serum from patients with diabetic neuropathy would affect sensory and autonomic, but not motor neurons.92 Serum was collected from 39 patients, 25 with type 1 and 14 with type 2 diabetes, with varying presentations of neuropathy. The age, type and duration of diabetes, HbA1c levels, NDS, NSS, vibration perception threshold (VPT), ratios of cardiac rate on lying/standing and inspiration/expiration (QAFT) and peroneal motor nerve conduction velocity (PMNCV) were recorded. Sera were applied to VSC4.1 (motor cell line) and N1E-115 (sensory/autonomic cell line) cells in vitro, and the number of viable cells counted. MANOVA was performed using cell numbers as the dependent variable and clinical measures as independent variables. None of the sera from any patients tested were cytotoxic to VSC4.1 motor neurons. In contrast, sera from patients with known autoimmune motor neuropathy (e.g., CIDP) were toxic to VSC4.1 cells. N1E-115 cytotoxicity (sensory/autonomic) occurred more frequently in response to type 1 sera than type 2 or control sera (p<0.02). There was an inverse correlation between N1E cytotoxicity and PMNCV (p<0.03) and VPT (p<0.01), but not QAFT, reflecting a correlation with abnormal myelinated sensory fiber function in diabetic neuropathy. Age (p<0.02), duration of diabetes (p<0.02) and HbA1c (p<0.03) were also correlated with N1E cytotoxicity, suggesting that glycation may alter the immunogenicity of neural proteins, promoting immune-mediated nerve destruction. This study supported the hypotheses that: 1) autoimmunity may contribute to diabetic neuropathy by acting in concert with hyperglycemia to damage sensory/autonomic neurons, 2) there is a correlation between the specific nerve fiber dysfunction in the patient (e.g., motor or sensory) and the type of neuron that exhibits in vitro serum cytotoxicity, not only for diabetic neuropathy, but also for other forms of autoimmune neuropathies. Failure of Neurotrophic Support as a Mechanism for the Pathogenesis of Diabetic Neuropathy There is a growing body of evidence that neurotrophic factors (NTFs) may play a role in the pathogenesis and repair of diabetic neuropathy. IGF-1 promotes the proliferation and regeneration of neurons in diabetic rats, probably modulated by the IGF-binding proteins (IGFBP). A deficiency of IGF-1 is associated with sensory diabetic neuropathy. The cytokine, IL-6, has also been shown to be neurotrophic, promoting the growth and differentiation of both sensory and autonomic neurons. Hyperglycemia and advanced glycosylation end products also have effects on NTFs. Both have been shown to be able to stimulate IL-6 production by monocytes and Schwann cells in the peripheral nerves. Thus, a compensatory increase in IL-6 may be found in patients with diabetes. In other studies we tested whether NTFs were altered in patients with diabetes and the relationship to nerve fiber dysfunction.93 Serum was collected from 27 patients with diabetes, 6 IDDM patients and 21 NIDDM patients, and 10 control subjects. The groups were similar in age, height and gender ratio. The diabetic subjects all had symptomatic peripheral neuropathy and relatively poor glycemic control (HbA1c, 9.1±0.4%). The sera were tested by either ELISA or radioimmunoassay for cytokines (IL-1b, IL-2, IL-4, IL-6, IL-10, IL-12 and TNF-a) and growth factors (IGF-1, IGF-2, IGFBP-1, and IGFBP-3). The patients were tested for sensory

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

225

abnormalities (vibration, cold and warm perception thresholds). IGF-1 (193±18 pg/ml) and IGFBP-3 (3.89±0.2 pg/ml) were significantly (p<0.05) reduced in the diabetic group compared to healthy controls (269±27 and 5.2±0.4 pg/ml), but only one serum had abnormal (>x±3s) levels of IGF-1. IGFBP-3 levels correlated with vibration perception in diabetic subjects (r=0.39, p<0.05). IL-6 was not significantly different in control (0.2±0.2 pg/ml) vs. patient (25.9±16.0) sera. However, sera from nine patients (four IDDM, and five NIDDM) were found to have elevated (>4 pg/ml) IL-6 measures. These nine patients also had significantly elevated vibration perception (p<0.001), cold perception (p<0.001) and warm perception thresholds (p<0.05) compared to control subjects and to patients with normal IL-6 levels. Only one subject’s serum showed measurable IL-4 and none showed measurable IL-1β or TNF-α. No significant differences were detected in IL-2, IL-10 or IL-12. The results of our study93 suggest that there is a selective failure of neurotrophic support in patients with diabetic neuropathy that may contribute to the sensory nerve fiber loss. This deficit is reflected in the reduced levels of IGF-1 and IGFBP-3 correlating with sensory dysfunction. In contrast, higher levels of neurotrophic IL-6 were found in patients with sensory diabetic neuropathy. This may represent an appropriate compensatory increase of the anti-inflammatory cytokine with diabetic neuropathy in response to hyperglycemia and AGEs and may serve to rescue damaged neurons. In selective patients high levels of the pro-inflammatory cytokines, IL-2, IL-4 and TNFα were found but this was not a consistent finding. Moreover, high levels of IL-6 were found predominantly in type 2 diabetes, more in keeping with a compensatory mechanism than an autoimmune process, but this needs to be resolved. Recent reports have suggested that both growth factor and immune therapy are successful in treating diabetic neuropathy, but the role of these cytokines and of the IGFs have not been evaluated in diabetic neuropathies. These findings suggest that restoration of normal neurotrophic support may be a possible mechanism of action and therapeutic approach to certain neuropathies in the context of diabetes. 3. Antigenic Targets in the Nervous System In 1991, Anand et al described a 30 year old woman with long-standing dizziness who was found to have severe orthostasis and a reduced skin-flare response.94 Autonomic tests indicated a selective impairment of adrenergic nerve function. Plasma levels of norepinephrine, epinephrine, dopamine and dopamine β-hydroxylase were undetectable. Skin biopsy showed loss of tyrosine hydroxylase and neuropeptide Y, markers of adrenergic sympathetic fibers and the sensory neuropeptides, substance P and CGRP. Sural nerve biopsy showed depletion of small, unmyelinated C-fibers, and assay showed reduced NGF content. Since NGF selectively induces tyrosine hydroxylase and dopamine β-hydroxylase is necessary for the survival of sympathetic nerve fibers and is required for the expression of substance P and CGRP in adult sensory neurons, it is apparent that immune neutralization of NGF could generate a clinical syndrome similar to that found in diabetic neuropathy. Guy et al95 found an association between diabetic autonomic neuropathy and iritis, suggesting an immunological background. Since iritis itself is an immunologically-mediated disorder with circulating immune complexes, they speculated that the associated small fiber damage which results in autonomic neuropathy might have been due to autoimmunity. Furthermore, the iris has a high NGF content and it must be considered that, due to the homology between NGF and the insulin family of proteins, insulin autoantibodies might recognize epitopes on NGF in the iris resulting in iritis. Thus, insulin autoantibody formation, which is universal in people with diabetes who are treated with insulin, and also can occur as a primary phenomenon, may be contributing not only to diabetic neuropathy but also other autoimmune conditions such as iritis through interaction with NGF.

226

Endocrine and Organ Specific Autoimmunity

The insulin-like growth factors (IGF) are widely distributed throughout the nervous system and exert profound effects on developing neurons.96 Summarizing briefly, whether acting via paracrine, autocrine or endocrine mechanisms, the rationale for implicating IGFs in the pathogenesis of diabetic neuropathy are: 1) IGFs in nervous tissue are regulated by insulin, 2) IGF receptors are present in the appropriate tissues (e.g., neurons, Schwann cells, ganglia) involved in diabetes-associated nerve disorders,97 3) IGFs exert numerous effects on nerve tissue growth and function, including indirect effects such as those mediated through NGF, and 4) IGF binding proteins are present in the nervous system, are regulated by insulin and glycemic state,98 and have been shown to modulate IGF action in nervous tissue. IGFs are proteins that, like NGF, have significant homology with insulin and IGF-I can even cross react with the insulin receptor.99 Thus, the possibility that autoimmune processes based on recognition of similar peptide structures in insulin and the IGFs exists. However, as yet there is no data supporting a role for autoimmune processes acting on IGFs in the neuronal deficits of diabetic neuropathy. The only autoimmune condition experimentally related to IGF autoimmunity thus far is autoimmune thyroiditis,100 or Grave’s disease. We have however recently reported that IGF1 and IGFBP3 levels are low in diabetic neuropathy93 and this correlates with impaired vibration perception. Although this has not been shown to be due to immune neutralization, the data support the notion that there may be fiber specific autoimmune processes giving rise to specific clinical disorders within the spectrum of diabetic neuropathies. 4. Mechanism of Neuronal Cell Death-Apoptosis Recent studies indicate that dysregulation of apoptosis, a process of cell involution, is involved in the development of autoimmune diseases, including IDDM, and many neurodegenerative diseases.101,102 IgM in serum from IDDM patients causes an increase in the voltage-dependent L-type calcium channel activity of insulin-producing cells leading to an increase in the concentration of free cytoplasmic Ca2+, associated with DNA fragmentation and apoptotic cell death.103 Fas-mediated cellular apoptosis requires activation of Fas, a type II membrane-bound 40 kDa protein which shows structural homology with tumor necrosis factor-α (TNF-α receptor and the low-affinity nerve growth factor (NGF) receptor, p75, by Fas ligand (FasL). FasL may also circulate in a soluble form and act in concert with antibodies specific to target tissue antigens. Apoptosis may be protective or harmful depending upon the target cell. For example, Garchon et al reported that activated T lymphocytes from nonobese diabetic mice were resistant to induction of apoptosis and remained free to cause destruction of β cells.104 As a corollary, the testis is a privileged site for islet transplantation because the Sertoli cell expresses FasL, which induces apoptosis of T cells, thereby protecting transplanted β cells.105 However, little was known about the relevance of apoptosis and disruptions of the regulation of apoptosis to diabetes and its neuropathic complications. We have carried out a series of studies designed to elucidate the possibility that there may be specific autoimmune processes giving rise to specific clinical syndromes. The studies reported here support the hypothesis that IDDM serum exerts cytotoxic effects on N1E115 neuroblastoma cells by activation of an apoptotic mechanism. N1E-115 Neuroblastoma Cell as a Model for the Study of Neuronal Apoptosis as a Mechanism for the Development of Neuropathy The N1E-115 murine neuroblastoma cell line (kindly provided by Dr. E. Richelson, Gainesville, Florida) is an adrenergic cell clone derived from the C-1300 murine neuroblastoma tumor. These cells have the characteristics of sympathetic neuroblasts and can be

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

227

induced to differentiate and extend neurites by removal of serum from the media. Our previous studies demonstrating a positive correlation between the toxicity of IDDM serum and the clinical presentation of diabetic neuropathy20 makes it an excellent model in which to study diabetic neuropathy. Previous reports demonstrated that the characteristics of DMSO-induced apoptosis in N1E-115 neuroblastoma cells include cytoplasmic condensation, chromatin clumping, and DNA “laddering” or DNA fragmentation.106 Similarly, the morphological changes caused by IDDM serum in these studies include cytoplasmic condensation, segmentation, cell shrinkage and lifting. These changes were observed as early as 2-4 h after exposure to IDDM serum. There is an important difference in the two methods for induction of apoptosis in NB cells, however. DMSO first causes differentiation of N1E-115 cells, followed 8 d later by cell death, whereas serum treatment inhibits differentiation of the cells and can cause cell death within 24 h.20 For most, but not all, forms of apoptosis and hypoxic-ischemic neuronal injury, a rise in intracellular calcium has been hypothesized to be an early primary event.107 In these studies, we showed that cytoplasmic free calcium increased as early as 10 s after exposure to IDDM serum and continued rising until at least 10 minutes after application. The rise in cytoplasmic free calcium in N1E-115 cells preceding DNA fragmentation and cell death has precedent in reports on other cell types. There is evidence from studies with pancreatic β cells that there is an IgM autoantibody in IDDM serum that interacts with L-type Ca2+ channels to increase Ca2+ influx and cause apoptosis.103 Furthermore, a motor cell-neuroblastoma cell hybrid has been used to describe autoantibodies to the alpha subunit of Ca2+ channels in sera from patients with amyotrophic lateral sclerosis.108,109 However, which Ca2+ channels might be involved in neuronal apoptosis in response to IDDM serum needs to be resolved. Suggestive of a role for L-type channels in the destruction of neurons in diabetes is the observation that nimodipine, a blocker of L-type Ca2+ channels, may reverse the slowing of nerve conduction velocities in streptozotocin diabetic rats (Kappelle et al, 1992). Whatever the case, there is increasing evidence that calcium entry can activate latent enzymes that contribute to the structural changes of apoptosis. The earliest time that DNA fragmentation was detected in N1E-115 cells after exposure to serum was at 2 h, peaking between 4-8 h after adding the IDDM serum to the cell culture, while morphologically the dying cells maintained the integrity of their plasma membranes. This is consistent with Ca2+ activation of endonuclease digestion within the nucleus. It is thought that the DNA ladder is formed by the action of these endonucleases at internucleosomal spaces where the DNA is vulnerable to enzyme activity. The combination of DNA laddering, intracellular calcium increase and morphological changes consistent with apoptosis all suggest a coordinated response to the application of IDDM serum. Our previous studies indicate that the effect occurs through the action of an autoantibody,67 resulting in apoptosis of the adrenergic neuron cell line. Therefore, it is important to identify the antigen recognized by the autoantibody and its relationship to induction of apoptosis. Apoptosis is regulated by many extrinsic and intrinsic cellular signals, and the threshold of apoptotic cell death is also dynamically regulated by multiple inducers and inhibitors of gene products (Fig. 11.4110). Several apoptosis-related oncogene products are also expressed and regulated in neurons. Bcl-2, a cell-death suppressor, is found in the mitochondrial membrane, the nucleus and the endoplasmic reticulum. A high level of expression of bcl-2 in sympathetic neurons prevents cell death induced by deprivation of nerve growth factor.111 However, we were unable to detect bcl-2 in N1E-115 cells by immunofluorescence. The apparent lack of bcl-2 in the neural cell line might simply reflect the lack of bcl-2 in neuroblasts, or it may result from gene loss during tumorigenesis. Furthermore, the lack of bcl-2 may be associated with an increased propensity for apoptosis in this cell line. Because we were unable to show the presence of bcl-2 in resting N1E-115 cells, it was

228

Endocrine and Organ Specific Autoimmunity

Fig. 11.4. Apoptosis, the physiological removal of cells from tissues, is regulated through a number of pathways. This schematic diagram shows some of the mechanisms shown to promote apoptosis (light arrows) or inhibit apoptosis (dark arrows) in a number of cell types. Extracellular signals (e.g., fas) binding to their ligands trigger an intracellular signaling response (e.g., ceramide) which activates nuclear factors leading to programmed cell death. A number of intracellular signals can block progression to apoptosis (e.g., bcl-2). Thus, the regulation of apoptosis occurs through a balance between pro-apoptotic and anti-apoptotic signals.

unlikely that bcl-2 participated in the regulation of the apoptotic response studied here. Hence, we sought an alternative antigen. Fas (APO-1, CD95) is a cell-surface receptor with a molecular weight of 35-40 kDa, dependent on the source species, belonging to the TNF/NGF receptor superfamily. Fas is expressed in many cell lines, but the largest body of research shows that Fas mediates apoptosis in susceptible T-lymphocyte target cells.112 Fas-mediated apoptosis may be antibody dependent. When Fas ligand or anti-Fas antibodies bind to the Fas receptor, the target cell undergoes apoptosis. The apoptotic signal through Fas requires the crosslinking and trimerization of Fas receptors.113 The trimerized Fas complex can then be activated by antibody action, resulting in the transduction of the signal for induction of apoptosis. Polymerization of Fas can be accomplished either with Fas ligand or with antibodies to Fas recognizing specific epitopes.114 Consistent with this hypothesis, immunofluorescence revealed a clustering of Fas on the NB-cell surface in response to IDDM serum, in contrast to its normal diffuse distribution on the cell membrane.10 This clustering appears to be a consequence of molecular crosslinking of Fas by a factor in IDDM serum. According to our previous study, the cytotoxic factor is likely to be an autoantibody.67 The cytotoxicity of patients’ sera on neuroblastoma cells was a dose-dependent and time-dependent effect (Fig. 11.5a). The morphological changes of NB cells after culture in medium with IDDM serum included an acentric condensation of cytoplasm, cell shrinkage and lifting from the dish, suggesting an apoptotic cell death (Fig. 11.5b). In addition, the total viable cell number was

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

229

A

Fig. 11.5. Cell proliferation (neuroblastoma NIE 115 ) is inhibited by sera from patients with insulin-dependent diabetes and neuropathy or nondiabetic autoimmune neuropathies (A). The cells treated with toxic sera exhibit morphological characteristics of apoptosis (B).

B

significantly reduced (p<0.05) in cultures treated with patients´ sera,20 while the cell number increased in control serum-treated cultures. The loss of viable cells may have been underestimated due to the exclusion of trypan blue by cells undergoing apoptotic cell death but with intact cell membranes. A DNA fragmentation ladder, characteristic of apoptotic cell death, was revealed as early as 2 h, in some cases, after exposure of the cells to “toxic” serum from IDDM patients with neuropathy and further fragmentation with time. N1E115 cells treated with dimethyl sulfoxide, which is known to cause apoptosis in these cells, showed DNA laddering similar to that seen with IDDM serum.106 Two out of the three

230

Endocrine and Organ Specific Autoimmunity

completely toxic sera of patients with IDDM and neuropathy caused the onset of DNA laddering in the N1E-115 cells within 2 h, indicating that the cells treated with patient serum were undergoing apoptosis. A requisite for activation of apoptosis is a rise in intracellular Ca2+. Five of the six IDDM sera tested caused a sustained increase of intracellular Ca2+, starting within 10 s after initial exposure to the serum and increasing up to 600 s, while control sera did not induce a rise, but instead caused no change or even a decrease. The initial deviation in all tests was an artifact due to exposure to outside light with the addition of serum to the cuvette. In searching for candidate apoptotic factors, we examined the N1E-115 cells for expression of bcl-2 and Fas. Bcl-2 was not expressed, but Fas protein was expressed on the cell surface in a characteristic heterogeneous staining pattern. The surface staining was totally blocked by the three cytotoxic IDDM sera suggesting that Fas might be one surface antigen recognized by autoantibodies from IDDM serum. Fas staining on these neural cells showed a cluster pattern after culture with IDDM serum from patients with neuropathy for 24 h. As before, the cell numbers were reduced within 24 h compared to control serumtreated cultures. Further confirming a role of Fas in neuroblastoma cell apoptosis, electrophoresis of proteins immunoprecipitated with RK8 anti-Fas antibody revealed a protein band at 40 kDa, similar to the reported molecular weight of Fas. Treatment of the cells with RK8 antibody, which has been shown to activate the Fas mechanism in other cells,115 caused the loss of neural cells in a dose-dependent manner within 3 d of culture, confirming the presence of membrane-bound Fas on N1E-115 cells and supporting a role for Fas-mediated immune destruction of neural cells. Our finding that IDDM serum blocks the Fas cell-surface immunofluorescence using a Fas-specific antiserum suggests competition of the IgG in IDDM serum and the rabbit anti-Fas antibody, indicating that Fas might be one of the membrane antigens recognized by autoantibodies in IDDM serum. Further supporting this hypothesis is the observation that treatment with anti-Fas monoclonal antibody caused NB-cell death in a dose-dependent fashion, just as did the IDDM immunoglobulin in serum.10 Cytotoxicity of IDDM serum might be enhanced by the expression of Fas or an increase in circulating FasL. It remains to be determined whether Fas expression in NB cells is altered by exposure to IDDM serum and whether circulating FasL is increased in IDDM serum. Furthermore, there is no evidence in the literature for or against the presence of Fas or Fas ligand on cells in peripheral nerves. However, we cannot exclude the possibility that other unknown antigens or death factors, such as glycolipids, the low affinity NGF receptor (p75),116,117 the TNF-α receptor or other as yet unknown regulators of apoptosis, may also be involved. The roles of these regulators in apoptotic neuronal death and whether they might contribute to the development of diabetic neuropathy remain to be elucidated. Figure 11.4 is a schematic outline of the possible players in the apoptosis-mediated impairment of neuronal cell function. Thus far we have only explored the Fas-mediated mechanisms. While these seem to account for a significant proportion of the toxicity of serum there remains a significant contribution by factors that have yet to be determined. Studies of the second messengers involved in the pathways of programmed cell death, such as CREB and P38 kinase, should shed much light on the process and provide new insights into therapeutic strategies in the future.

Diabetic Neuropathy. An Overview of Conditions That May Be a Consequence of Autoimmunity Diabetes mellitus is associated with a whole array of neurologic syndromes, which differ in their etiology, pathogenesis, clinical characteristics, and response to treatment. Distal

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

231

symmetric polyneuropathy, the most frequently seen sensorimotor deficit in diabetic patients, is now considered the most common peripheral neuropathy in the western world.118,119 It has a significant impact on morbidity in patients with diabetes. When autonomic neuropathy occurs in diabetic patients, a significant increase in mortality follows.74,120 Classification and Pathology According to the San Antonio Convention, the main groups of neurological disturbances in diabetes mellitus include: (a) subclinical neuropathy determined by abnormalities in electrodiagnostic and quantitative sensory testing; (b) diffuse clinical neuropathy such as distal symmetric sensorimotor and autonomic syndromes; and (c) focal syndromes.121 Proximal diabetic neuropathy is classified as a focal neuropathy, but detailed analysis reveals the presence of overlapping features between PDN and DSPN with many similarities between the multifocal and the diffuse syndromes. This has added a need to be cautious in ascribing a universal concept of the pathogenesis of any single form of neuropathy. Nonetheless there are certain pointers. Pathologic and immunopathogenic studies of the nerve biopsies taken from diabetic patients with neuropathy that have been done over the last 15 years have yielded numerous crucial observations important for our understanding of causative factors in diabetic neuropathy.6,10,61,122-124 A significant pathologic variability exists even within clinically defined syndromes, especially in patients with DSPN. In patients with DSPN and PDN, two common peripheral neuropathic syndromes in diabetes, the following pathologic patterns have been described: (a) multifocal axonal loss (b) primary demyelination (c) secondary demyelination (d) occlusion of small blood vessels (e) infiltration of immune cells and (f) deposits of antibodies and complement.15,125,126 We believe that these pathologic patterns, which may occur in various combination in these patients, reflect differences in the etiology and pathogenesis of the neurologic deficit. For example, in patients with predominantly axonal loss and secondary demyelination, a metabolic etiology must be suspected. In contrast, immune-mediated syndromes are more likely in those people with primary demyelination, multifocal changes, and immune cell infiltration. Pathogenesis Figure 11.6 shows our current view on the pathogenesis of diabetic neuropathy. The figure depicts multiple etiologies, including metabolic, vascular, autoimmune, and neurohormonal growth factor deficiency. The metabolic hypothesis is the prevailing theory as to the primary cause of diabetic neuropathy, and implicates persistent hyperglycemia as the primary factor responsible for the nerve damage in diabetes mellitus. The results of the Diabetes Control and Complications Trial127 endorse the importance of glycemic control in prevention of neuropathy. Recent observations emphasize the role of oxidative stress, a consequence of increased polyol activity, depletion of NADPH and NADH thereby compromising the ability to synthesize nitric oxide (NO) from its precursor, arginine.128 A number of functional disturbances have been demonstrated in the microvasculature of the nerves of diabetic subjects, in addition to the histopathologic abnormalities of small blood vessels (microvascular hypothesis) likely to be due to this oxidative stress and NO and PGI2 depletion.129 Studies have demonstrated decreased neural blood flow, increased vascular resistance, decreased pO2 and altered vascular permeability characteristics, such as a loss of the anionic charge barrier and decreased charge selectivity.130 A clear relationship between microvascular insufficiency and neuropathy has not been established as yet. It is possible that ischemia precedes neuropathy, or it may be that both conditions are the result of separate processes caused by the same etiologic factor(s). It may even be that loss of the nerve reduces the requirement for nutritional

232

Endocrine and Organ Specific Autoimmunity

Fig. 11.6. Theoretical framework for the pathogenesis of diabetic neuropathy. Most people with diabetes will develop subclinical or clinical neuropathy given sufficient time. The risk factors include genetic predisposition, excessive consumption of alcohol, and smoking. The prevailing concept is that multiple factors, including metabolic, microvascular, autoimmune, and neurotrophic, play roles in the pathogenesis of diabetic neuropathic syndromes. EDRF-endothelium-derived relaxing factor; GF-growth factor; GLA-γ-linolenic acid; IGF-insulin-like growth factor; NO-nitric oxide; PGI2-prostaglandin I2; TRK-high-affinity nerve GF receptor.

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

233

delivery with consequent vascular “shut down.” Our own noninvasive studies of cutaneous blood flow in the feet and hands of patients with diabetes show impairment of blood flow prior to the development of clinically overt neuropathy, suggesting that a vasculopathy may be causative in the development of neuropathy.131,132 Apart from the metabolic and vascular factors involved in the pathogenesis of neuropathy, there are data to support a role for growth factor deficiency (neurotrophic hypothesis).118,133-136 Many of the neuronal changes characteristic of diabetic neuropathy are similar to those observed following either removal of target-derived growth factors by axotomy, or depletion of endogenous growth factors by experimental induction of growth factor autoimmunity. Since neuronal growth factors can promote the survival, maintenance and regeneration of neurons subject to the noxious effects of diabetes, the success of diabetic patients in maintaining normal nerve morphology and function may ultimately depend on the expression and efficacy of these factors. It has recently become evident that the immune system plays an important role in the pathogenesis of certain diabetic neuropathic syndromes. The observations in support of the immune hypothesis include: (a) epidemiologic observations; (b) pathologic and immunopathologic observations; (c) clinical and electrophysiologic evidence; and finally (d) effect of immunotherapies in certain subgroups of patients with diabetic neuropathy. Data from several, large epidemiologic studies have shown that in up to 11 % of diabetic neuropathic patients neurologic signs precede the diagnosis of diabetes.118 Both DSPN and PDN, as well as autonomic neuropathy, may be found prior to the diagnosis of diabetes or early in the course of the disease.137 This may indicate that the neuropathogenic process in some diabetic patients is independent of the metabolic changes caused by long-term hyperglycemia. As was discussed above, the pathological and immunopathological findings suggest a possible role of the immune system in mediating nerve damage in a subset of diabetic neuropathic patients. These observations, as well as some recent immunoserologic, immunogenetic and clinical observations which indicate the importance of immune system in the pathogenesis of diabetic neuropathy, will be discussed here. Some limited experiences with immunotherapies will also be presented.

Proximal Diabetic Neuropathy (PDN) Clinical Presentation and Epidemiology. PDN can be clinically identified based on proximal muscle weakness and muscle wasting. It may be symmetric or asymmetric in distribution, and is frequently associated with pain in the anterolateral aspect of the thighs. The condition is readily recognizable clinically with prevailing weakness of the muscles innervated by femoral and obturator nerves, including quadriceps, iliopsoas, obturator and adductor muscles. The strength of the gluteus maximus and minimus, and hamstrings is relatively preserved. Those affected have great difficulty rising out of chair unaided and often climb up their bodies. PDN can present as acute or chronic condition. The neurologic deficit is frequently associated with significant weight loss. Subramony and Wilbourn, who performed the first detailed electrophysiologic study, described reduced nerve conduction velocity of the femoral nerve, together with paraspinal denervation, and denervation of the pelvifemoral muscle group.138 It is far less frequent than DSPN. However, PDN is a progressive neuropathy, which may cause severe motor deficit and permanent disability in more than 50% of affected individuals. Some recent immunopathologic discoveries have created new insights into the pathogenesis of PDN. Accordingly, new therapeutic modalities that are currently being evaluated in several centers in the world, may give new hope for patients with this disabling form of proximal neuropathy.

234

Endocrine and Organ Specific Autoimmunity

Pathology and Immunopathology of PDN The pathologic characteristics of PDN have been analyzed in a limited number of case report studies. Sural nerve biopsies from three patients with PDN were evaluated in a study by Bradley et al from 1984.139 The authors showed inflammatory response around the epineurial arterioles together with axonal loss. The appropriateness of the use of sural nerve biopsies in studies of PDN have been contested by others, since the distribution of the deficit in this neuropathy is still debated. Said et al reported on the results of their study which included 10 patients with various clinical forms of PDN.140 Four patients had sensory neuropathy, while six others had sensory and motor deficits. These authors were the first to use a proximal nerve, the cutaneous nerve of the thigh, to define the pathology of PDN. This is a sensory nerve, frequently affected in PDN patients. In addition to the typical findings of axonal loss and demyelination, various patterns of inflammation were also described. Krendel et al described almost identical pathology in another group of patients with PDN.141 It is not known which population of immune cells is involved in this process, or which cell type is predominant in the affected tissue. Another important question relates to the distribution of the inflammatory response, and consequently, to the distribution of neurologic deficit in PDN. Antigens and Immunopathogenesis of PDN Two patterns of inflammation have been found in the nerves taken from patients with PDN: (a) vasculitis and perivasculitis and (b) diffuse inflammation distributed across the endoneurium and epineurium15,61 (see Fig. 11.2). The origin of the antigens involved or their structure are unknown. In our study of 10 patients with PDN done in the Diabetes Institutes of Eastern Virginia Medical School, we performed a biopsy of the branch of the obturator nerve supply to the gracilis muscle. We chose this nerve, since morphologic evaluation of a proximal motor nerve may be a better indicator of the pathological processes that underlies PDN, than evaluation of a proximal sensory nerve, since a motor deficit predominates in these individuals. Immunopathologic evaluation using antibodies against IgG, IgM and C3 component of complement was done. We did not find antibody deposits in any of the nerves studied. We did however, find evidence of a vasculitis. These findings indirectly suggest that cell-mediated immunity is more important in the pathogenesis of vasculitis and proximal deficit than humoral mechanisms. Immunogenetics of PDN No study has been performed to evaluate if an association between immune response genes and PDN exists. Immunotherapy for PDN The therapeutic approach to PDN has changed over the last several years. In the era that preceded these recent advances in our understanding of its pathogenesis, the mainstay of treatment was physical therapy and symptomatic measures directed towards pain. In 1995, Krendel et al published their case report on 23 patients with various forms of diabetic neuropathy, who they treated with immunomodulatory regimen or immunosuppression.141 Despite these encouraging clinical results of IVIg treatment described in proximal diabetic neuropathies, as well as in other autoimmune diseases, including several autoimmune-mediated peripheral neuropathies (Guillain-Barré syndrome, CIDP, multifocal motor neuropathy), its mechanism(s) of action are not well understood.3,142-144 It is believed that these may include interference with the pathophysiologic cascade of the immune reaction at several different sites: (a) immunomodulation by Fc receptor blockade, (b) idiotypic/antiidiotypic interactions, (c) down-regulation of autoantibody production, (d) direct effect on

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

235

antibody-binding site on nerve, muscle, or neuromuscular junction, (e) solubilization of immune complexes, (f ) possible action on natural killer or suppressor cells, (g) immuno-modulatory role of soluble class I and class II HLA antigens.145,146 Recent evidence suggests that the primary mode of action of IVIg in antibody-mediated autoimmune disorders is through idiotypic/anti-idiotypic interactions.147 In cases that fail to ameliorate with IVIg alternative treatments with plasmapheresis, prednisone and azothioprine could be considered, but there have been no controlled clinical trials comparing the different regimens. To re-evaluate these initial observations described in the literature, we first conducted a pilot study on a small group of PDN patients, who we treated with IVIg. This study was designed as a prospective evaluation of clinical and electrophysiological status of patients with motor form of PDN. The responses were variable, but the majority of patients showed improvement. Based on this initial experience, we initiated two prospective studies on PDN. One is a multicenter, placebo-controlled, clinical study, in which the effects of IVIg are being assessed. In the second, IVIg is being compared to an immunosuppressive regime that includes steroids and azathioprine. Both studies are currently underway, but no results are yet available.

Autonomic Diabetic Neuropathy Clinical Presentation and Epidemiology The function of the autonomic nervous system (ANS), estimated by cardiovascular testing, is impaired in both type 1 and type 2 (noninsulin-dependent) diabetes mellitus.148-152 Dysfunction of adrenergic as well as cholinergic pathways occurs in these individuals, affecting cardiovascular, gastrointestinal and genitourinary systems and eyes. Although clinical features of autonomic neuropathy generally occur only in patients with diabetes mellitus of long duration, it has become evident that subclinical neuropathy, mainly in the form of cardiac autonomic neuropathy, evolves early in the course of diabetes, and in the absence of other microvascular complications.137,153 Estimates of the prevalence of abnormal cardiovascular autonomic reflexes range from 8% in recently diagnosed type 1 diabetes to 90% in candidates for pancreas transplantation.154 It has been reported that autonomic neuropathy increases cardiovascular mortality in patients with diabetes.120,149,155,156 A full understanding of the mechanisms by which cardiovascular autonomic neuropathy leads to excess cardiovascular mortality is still lacking. The mechanisms by which autonomic neuropathy has been most frequently postulated to increase mortality include increased susceptibility to fatal ventricular arrhythmias and increased propensity to cardiovascular events.19 It has been suggested that autonomic imbalance in patients with neuropathy results in QTc interval prolongation that predisposes to cardiac arrhythmias.157 The available evidence favors a multiplicity of factors involved in the damage of the autonomic nervous system in diabetes. Metabolic disturbances and lack of growth factors or their effects on the target tissues has gained more attention than others. More recently, autoimmunity involving sympathetic nervous system destruction in type 1 diabetes mellitus has been promoted.20,158-160 This approach has been based on numerous pathologic, immunopathologic and serologic observations. Pathology and Immunopathology of Autonomic Neuropathy The first indication that certain components of ANS may be targeted by an autoimmune reaction came from the report by Duchen et al158 who described lymphoplasmocytic infiltrates in sympathetic ganglia and in and around bundles of unmyelinated nerve fibers of five type 1 diabetic patients studied at autopsy. In this and other reports sympathetic

236

Endocrine and Organ Specific Autoimmunity

myelinated and vagal nerves examination showed loss of myelinated fibers and marked excess of collagen, but with no clear infiltration. In another report by Brown et al159 the high frequency of adrenal medullary fibrosis in patients with long standing disease have been described. In one subsequent retrospective autopsy study using formalin fixed tissue and monoclonal antibodies specific for anti-human leukocyte antigen, anti-B-lymphocyte antigen and anti-CD45 antigen, signs of moderate to severe adrenal medullary infiltration were detected in 20% of type 1 diabetic patients and mild to severe fibrosis in another 52%.160 Accordingly, one can argue that inflammatory infiltration is present only in the autonomic nervous system structures of type 1 patients, specifically in the sympathetic part (adrenal medulla and ganglia), not seen elsewhere in the nervous system. These differences in histopathologic findings may be the results of different pathogenesis of certain chronic neurologic complications of the disease. To be more precise, it is possible that lesions of certain parts of the sympathetic nervous system (ganglia, adrenal medulla and nonmyelinated fibers) are a consequence of cellular autoimmune process. Other sympathetic structures may be prone to the deleterious effects of hyperglycemia and humoral immune mechanisms. Antigens and Immunopathogenesis of Autonomic Neuropathy The autoimmune hypothesis is strongly supported by the demonstration of organspecific, complement-fixing autoantibodies against unknown antigens from adrenal medulla (CF-ADM) and sympathetic ganglia (CG-SG).64,66 Further studies showed frequent CF-SG presence in ICA+ and ICA- first degree relatives, and in as much as 60% newly diagnosed ICA+ diabetic patients.62 With duration of diabetes greater than 5 years CF-ADM occurs in both ICA+ and ICA- patients, suggesting that the antigenic targets of adrenal medulla and pancreatic islets are different.161 Glutamic acid decarboxylase (GAD) and gangliosides have been evaluated as possible targets of an autoimmune reaction.70,162,163 The potential importance of a link between anti-GAD antibodies and autonomic neuropathy lies in the fact that GAD is abundant in autonomic nervous structures, including ganglia.164 Zanone et al83 in a recent report did not find correlation between antibodies against GAD65 and autonomic diabetic neuropathy. Some others have presumed gangliosides as a possible target for specific autoantibodies. Rabinowe et al have reported anti-ganglioside GT1b IgG antibody in type 1 diabetes68 and correlated the autoantibody to changes in orthostatic blood pressure, suggesting a role in autonomic neuropathy. Anti-GT1b autoantibody recognizes sympathetic ganglia and adrenal medullary antigens. Immunogenetics of Autonomic Neuropathy Analyses that have been done in type 1 diabetic patients showed significantly higher frequency of the heterozygous genetic constellation, HLA-DR3/DR4, in those with mild and severe autonomic neuropathy of the cardiovascular system, compared with those who did not have any sign of dysfunction.69 This observation is of great importance because of the possible pathogenic link between autonomic neuropathy and type II polyendocrine autoimmune syndrome (PAS II), which is also characterized by a high frequency of HLADR3/DR4 heterozygotes and autoimmune processes in various tissues. In this PAS type 1 diabetes is associated with lymphocytic thyroiditis, adrenalitis and pernicious anemia. This immunogenetic observation suggests that concomitant presence of diabetes and autonomic diabetic neuropathy (e.g., ganglionitis and medullitis) may be one of the forms of (incompletely expressed) type II PAS. Therapy for Autonomic Neuropathy The preventive effects of intensive insulin treatment on autonomic nervous system disturbances reported by the DCCT Group was only partial165 and could be explained by an

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

237

inability to affect the autoimmune process, present in a subgroup of patients, by improved metabolic status. A possible preventive effect of immunotherapy on autonomic neuropathy has not been evaluated as yet. In other clinical situations, when organ dysfunction due to autonomic neuropathy is already present, a number of symptomatic measures are available. The discussion of these measures is beyond the scope of this chapter and has been described in detail elsewhere.166

Distal Symmetric Polyneuropathy (DSPN) Clinical Presentation and Epidemiology DSPN is the most common clinical neurologic syndrome in diabetes. The small fibers, large fibers, or both can be damaged. Small nerve fiber dysfunction usually (although not always) occurs early and is often present before objective signs, or electrophysiologic evidence of large fiber deficit.162 It is manifested first in the lower limbs, with pain and hyperalgesia and is followed by loss of thermal sensitivity, and reduced light touch and pin prick sensation. Large fiber neuropathies may involve sensory and/or motor nerves.12,74 They are manifested by reduced vibration (often the first objective evidence of neuropathy) and position sense, weakness, muscle wasting and depressed tendon reflexes. Most patients with DSPN have a “mixed” variety with both large and small nerve fiber involvement. In the case of DSPN, a “glove and stocking” distribution of sensory loss is almost universal. Distal muscle weakness may accompany the sensory loss. In some patients motor deficit may display progressive course, with severe functional loss and disability.162 Pathology and Immunopathology of DSPN The most prominent pathological change in patients with DSPN is the loss of myelinated and unmyelinated axons.15,167,168 Another feature of DSPN is segmental and paranodal demyelination.169 Demyelination may be primary, due to a direct effect of the disease process on myelin or Schwann cell function, or secondary to axonal loss. Endoneurial and epineurial blood vessel basement membrane thickening and obstruction of vasa nervorum are the main pathohistologic microvascular changes in diabetic nerve.170,171 It has been suggested by some, that the degree of endoneurial vessel disease may be important, since it was correlated with the severity of nerve fiber pathology and clinical symptoms.15,167,172 Another important observation is a multifocal distribution of tissue changes, which indicate the importance of the nonmetabolic factors in the pathogenesis of DSPN.173,174 If the metabolic factors were the only ones involved in the nerve damage, then diffuse changes should be present. In fact, the lesions described in diabetic patients are very similar to those found in biopsies of nerves from nondiabetic subjects with vasculitis or inflammatory neuropathies. It is particularly interesting that immune cell infiltration has recently been shown in sural nerve biopsies taken from patients with DSPN, in addition to the classic pathologic features described above. We have also shown that deposits of antibodies and complement are present in the axons and perineurium of sural nerves taken from diabetic patients with neuropathy126 (Fig. 11.3). Antigens and Immunopathogenesis of DSPN Available evidence suggests two possible immunopathogenic mechanisms are operating in some patients with DSPN: (a) complement-fixing autoantibodies and generation of lytic complex and (b) T cell mediated cytotoxicity. Our preliminary investigations also showed that circulating anti-neuronal antibodies are present in diabetic serum, in addition to the tissue deposits of immune effectors. We were able to find circulating autoantibodies directed against motor and sensory nerve structures using an indirect immunofluorescence

238

Endocrine and Organ Specific Autoimmunity

technique (Fig. 11.3). Several antigens have been considered as presumed targets of the immune response, including gangliosides, glutamic acid decarboxylase 65 kDa (GAD65) and phospholipids.70,76,83 In the study conducted in the Diabetes Institutes of Eastern Virginia Medical School, we have screened 67 patients with DSPN and found 14 patients with raised titers of antiganglioside GM1 antibodies.175 Since anti-ganglioside GM1 antibodies are the most frequently reported neuroimmunologic abnormality in immune-mediated, nondiabetic motor neuropathies, we examined the prevalence of these antibodies in 67 diabetic patients with distal symmetric polyneuropathy and various degrees of motor deficit. We found that 14 patients (20.8%) had titers above the normal limit. Duration of neuropathy and duration of diabetes did not differ between the anti-GM1 positive and antiGM1 negative group. Clinical motor deficits were variable within each of the two groups, however, the mean Medical Research Council score in the anti-GM1 positive group was significantly higher then in anti-GM1 negative patients. This suggests that there is a subset of diabetic patients who clinically resemble distal symmetric polyneuropathy, but who are anti-GM1 antibody positive. Higher motor disability scores in this group of patients may indicate a possible role of immune mechanisms in the pathogenesis of motor neuropathy in this subgroup of patients. Detailed neurologic evaluations revealed a frequent association of anti-GM1 antibodies with DSPN characterized by a more severe emphasis on a motor deficit. Similarly, antiGMl antibodies are associated with several chronic, nondiabetic motor neuropathic syndromes that are treatable with immunotherapy.15,176 It has been argued that anti-GM1 antibodies are not pathogenic but passively reflect cellular destruction. However, a number of observations suggest that they may have pathogenic potential.175,177-180 Possible association of anti-GAD65 antibodies with DSPN has been evaluated in several prospective and cross sectional studies.70,181 These antibodies were first found in patients with another neurologic disease, stiff man syndrome. The results of the available studies are inconsistent. Long-term prospective investigations are needed for this uncertainty to be resolved. We have also found anti-phospholipid antibodies (PLA) in 88% of our diabetic population with DSPN, as compared to 32% in diabetic patients without apparent neurologic complications, and 2% in the general population.70 There is evidence that PLA may be injurious to neural tissue and that damage may be selective for specific parts of the nervous system.72,74,182 Since PLA are associated with a tendency to vascular thrombosis, their presence may provide a link between the immune and vascular theories of causation of neuropathy. Therapy of DSPN The modern treatment of distal symmetric neuropathy includes various measures which have been described in detail:183-185 (a) measures directed at the pathogenic process; (b) pain treatment and (c) other symptomatic measures. A controlled clinical evaluation of immunotherapy is still lacking, since these immunopathologic and immunoserologic observations have only been recently reported. Carefully designed, prospective studies, should be planned and conducted with a goal to assess: (a) the association between neurologic dysfunction and immunologic abnormalities and (b) full scientific characterization of the beneficial effects of immunotherapy.

Summary and Conclusions The autoimmune theories for the pathogenesis of diabetic neuropathies have been the Cinderella of all likely theories. There have, however, been major strides that are beginning

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

239

to lend credence to their veracity, not the least of which has been the observation in the DCCT that even with the most rigorous control of blood glucose one can only entertain a 50% reduction in the likelihood of developing neuropathy or abrogating its relentless course. While some contend that hyperglycemia engenders an “oxidative stress” with a concomitant reduction in the availability of essential growth factors necessary for the maintenance of the health and integrity of neurons, there are now proponents that consider that autoimmune processes may prevail, especially under certain circumstances. There is now almost irrefutable evidence that the proximal neuropathies have a cellular form of vasculitis at the root of their etiopathogenesis. Moreover, these have been shown to respond to treatment with immunotherapy. Also on the horizon is discovery of the relationship between autonomic neuropathies and humoral mechanisms for the destruction of neurons. What is less clear is the role of autoimmunity in peripheral somatic nerve damage. There is now data that supports both a humoral and cellular form of autoimmune nerve destruction. What has not been resolved is whether the nervous system is the culprit or the victim of the process. Without careful longitudinal studies this question may go unanswered for a long while. Thus, it is feasible that there is a primary autoimmune process similar to that which occurs in the pancreas. The similarity between neurons and the pancreatic β cell has not escaped the attention of investigators in the field. Similar to β cell destruction is the cosegregation of patients with neuropathy and certain HLA configurations and the possibility that hyperglycemia or some other assault may damage the nervous system, attracting the attention of the immune surveillance system at the expense of the nervous system. The normal barrier to immune-mediated processes in the nervous system may also be broken by glycation of barrier proteins creating a “leaky” barrier that permits access of humoral factors to otherwise occult areas. What are these antigens? This question is actively being pursued. Some overlap with recognized autoimmune neuropathies has allowed some headway to be made. However, we still must ascertain whether there are antigens that are unique to the diabetic condition. Whatever these might be, there is now accumulating evidence that immunoglobulins can exert their toxicity in conjunction with complement and that the apoptotic process may be implicated with its cadre of players. These are exciting times in the neuropathy arena and we can look forward to an escalation of interest in this neglected area. Furthermore, as has occurred with the proximal neuropathies, we can anticipate novel therapeutic approaches based upon immunologic principles. This is a ride we would like to share with our patients who are afflicted with this most life-spoiling of the diabetic complications as well as one that abrogates longevity.

References 1. Pourmand R. Myasthenia gravis. Dis Mon 1997; 43:65-109. 2. Bernard CC, Johns TG, Slavin A et al. Myelin oligodendrocyte glycoprotein: A novel candidate autoantigen in multiple sclerosis. J Mol Med 1997; 75:77-88. 3. Dalakas MC. Intravenous immune globulin therapy for neurologic disorders. Ann Intern Med 1997; 126:721-730. 4. Kalaria RN, Cohen DL, Premkumar DR. Cellular aspects of the inflammatory response in Alzheimer’s disease. Neurodegeneration 1996; 5:497-503. 5. van der Meche FG. The Guillain-Barré syndrome; pathogenesis and treatment. Rev Neurol (Paris) 1996; 152:355-358. 6. Prineas JW. Pathology of inflammatory demyelinating neuropathies. Bailliere Clin Neurol 1994; 3:1-24. 7. Feasby TE. Inflammatory-demyelinating polyneuropathies. Neurol Clin 1992; 10:651-670. 8. Davidson DLW, O’Sullivan AF, Morley KD. HLA antigens in familial Guillain-Barré syndrome. J Neurol Neurosurg Psychol 1992; 1282:7684-7711.

240

Endocrine and Organ Specific Autoimmunity

9. Parry GJ, Sumner AJ. Multifocal motor neuropathy. Neurol Clin 1992; 10:671-684. 10. Pittenger GL, Milicevic Z, Vinik AI. Autoimmune mechanisms of diabetic neuropathy. In: LeRoith D, Olefsky JM, Taylor S, eds. Diabetes Mellitus: A Fundamental and Clinical Text. Philadelphia: J.B. Lippincott-Raven 1996:751-758. 11. Stevens MJ, Feldman EL, Greene DA. The aetiology of diabetic neuropathy: the combined roles of metabolic and vascular defects. Diabetic Med 1995; 12:566-579. 12. Vinik AI, Newlon PG, Lauterio TJ et al. Nerve survival and regeneration in diabetes. Diabetes Metab Rev 1995; 3:139-157. 13. Vinik AI, Milicevic Z, Pittenger G. Beyond glycemia. Diabetes Care 1995; 18:1037-1041. 14. Tomlinson DR, Fernyhough P, Diemel LT et al. Deficient neurotrophic support in the aetiology of diabetic neuropathy. Diabetic Med 1996; 13:679-681. 15. Said G, Goulon-Goreau C, Lacroix C et al. Nerve biopsy findings in different patterns of proximal diabetic neuropathy. Ann Neurol 1994; 35:559-569. 16. Krendel DA. Diabetic inflammatory vasculopathy. Muscle Nerve 1997; 20:520-521. 17. Brown FM, Watts M, Rabinowe SL. Aggregation of subclinical autonomic nervous system dysfunction and autoantibodies in families with type 1 diabetes. Diabetes 1991; 40:1611-1614. 18. Rabinowe SL. Immunology of diabetic and polyglandular neuropathy. Diabetes Metab Rev 1990; 6:169-188. 19. Zola BE, Kahn JK, Juni JE et al. Abnormal cardiac function in diabetic patients with autonomic neuropathy in the absence of ischemic heart disease. J Clin Endocrinol Metab 1986; 63:208-214. 20. Pittenger GL, Liu D, Vinik AI. The toxic effects of serum from patients with type I diabetes mellitus on mouse neuroblastoma cells: A new mechanism for development of autonomic neuropathy. Diabetic Med 1993; 10:925-932. 21. Milicevic Z, Newlon PG, Pittenger GL et al. The prevalence of anti-ganglioside GM1 antibodies in patients with diabetic neuropathy. Diabetologia 1997;(In Press) 22. Kaneko H, Nakajima A, Azuma M. Manipulation of costimulatory pathways in autoimmune disease. Nippon Rinsho 1997; 55:1531-1536. 23. Guerder S, Flavell RA. Costimulation in tolerance and autoimmunity. Int Rev Immunol 11995; 3:135-146. 24. Griffin DE, Hess JL, Moench TR. Immune responses in the central nervous system. Toxicol Pathol 1987; 15:294-302. 25. Miller SD, McRae BL, Vanderlugt CL et al. Evolution of the T cell repertoire during the course of experimental immune-mediated demyelinating diseases. Immunol Rev 1995; 144:225-244. 26. Croft M. Activation of naive, memory and effector T cells. Curr Opin Immunol 1994; 6:431-437. 27. Nickoloff BJ, Turka LA. Immunological functions of nonprofessional antigen-presenting cells: New insights from studies of T cell interaction with keratinocytes. Immunol Today 1994; 15:464-469. 28. Ota H, Yanagida H, Dobashi H et al. Clonal deletion and clonal anergy as the mechanisms of self-tolerance induction. Nippon Rinsho 1997; 55:1331-1336. 29. Fields P, Fitch FW, Gajewski TF. Control of T lymphocyte signal transduction through clonal anergy. J Mol Med 1996; 74:673-683. 30. Weenink SM, Gautam AM. Antigen presentation by MHC class II molecules. Immunol Cell Biol 1997; 75:69-81. 31. Monaco JJ. Pathways for the processing and presentation of antigens to T cells. J Leuko Biol 1995; 57:543-547. 32. Merrill JE, Benveniste EN. Cytokines in inflammatory brain lesions: helpful and harmful. Trends Neurosci 1996; 19:331-338. 33. Stoll G, Jander S, Archelos J et al. Macrophages and endothelial cells express intercellular adhesion molecule-1 in immune mediated demyelination but not in Wallerian degeneration of the rat peripheral nervous system. Lab Invest 1993; 68:637-644.

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

241

34. Oka N, Akiguchi I, Nagao M et al. Expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) in chronic inflammatory demyelinating polyneuropathy. Neurology 1994; 44:946-950. 35. McMurray RW. Adhesion molecules in autoimmune disease. Semin Arthritis Rheum 1996; 25:215-233. 36. Linington C, Lassmann H, Ozawa K et al. Cell adhesion molecules of the immunoglobulin supergene family as tissue-specific autoantigens. Induction of experimental allergic neuritis (EAN) by PO protein-specific T cell lines. Eur J Immunol 1992; 22:1813-1817. 37. Weber C, Alon R, Moser B et al. Sequential regulation of alpha 4 beta 1 and alpha 5 beta 1 integrin avidity by CC chemokines in monocytes: Implication for transendothelial chemotaxis. J Cell Biol 1996; 134:1063-1073. 38. Ahmed N, Christou N. Systematic inflammatory response syndrome: interactions between immune cells and the endothelium. Shock 1996; 6:S39-S42. 39. Dalakas MC. Basic aspects of neuroimmunology as they relate to immunotherapeutic targets: present and future prospects. Ann Neurol 1995; 37:S2-S13. 40. Weiner HL, Hauser SL. Neuroimmunology I: Immunoregulation in neurological disease. Ann Neurol 1982; 11:437-449. 41. Newson-Davis J. Autoimmunity in neuromuscular disease. Ann NY Acad Sci 1988; 540:25-38. 42. Wucherpfennig K, Weiner HL. Immunologic mechanisms in chronic demyelinating diseases of the central and peripheral nervous system. Res Publ Assoc Res Nerv Ment Dis 1990; 68:105-116. 43. Martin R, McFarland HF, McFarlin DE. Immunological aspects of demyelinating diseases. Annu Rev Immunol 1992; 10:153-187. 44. Price P. Autoimmunity and the highway to diabetes. Immunol Cell Biol 1997; 75:1-6. 45. Miossec P. Cytokine induced autoimmune disorders. Drug Saf 1997; 17:93-104. 46. von Herrath MG, Oldstone MB. Virus induced autoimmune disease. Curr Opin Immunol 1996; 8:878-885. 47. Hou J, Said C, Franchi D et al. Antibodies to glutamic acid decarboxylase and P2-C peptides in sera from coxsackie virus B4-infected mice and IDDM patients. Diabetes 1994; 43:1260-1266. 48. Conrad B, Weldman E, Trucco G et al. Evidence for superantigen involvement in insulindependent diabetes mellitus aetiology. Nature 1994; 371:351-355. 49. Quiros E, Maroto MC. Superantigens: Concept and applications in the pathogenesis and treatment of infections and autoimmune diseases. An Med Interna 1996; 13:347-352. 50. Poduslo JF, Curran GL. Glycation increases the permeability of proteins across the bloodnerve and blood-brain barriers. Mol Brain Res 1994; 23:157-162. 51. Luscinskas FW, Gimbrone MAJ. Endothelial dependent mechanisms in chronic inflammatory leukocyte recruitment. Annu Rev Med 1996; 47:413-421. 52. Yu RK, Ariga T, Kohriyama T et al. Autoimmune mechanisms in peripheral neuropathies. Ann Neurol 1990; 27(Suppl1):S30-S35. 53. van den Berg L, Hays AP, Nobile Orazio E et al. Anti-MAG and anti-SGPG antibodies in neuropathy. Muscle Nerve 1996; 19:637-643. 54. Gabriel JM, Erne B, Miescher GC et al. Selective loss of myelin-associated glycoprotein from myelin correlates with anti-MAG antibody titre in demyelinating paraproteinaemic polyneuropathy. Brain 1996; 119:775-787. 55. Ozawa K, Saida T, Saida K et al. In vivo CNS demyelination mediated by antigalactocerebroside antibody. Acta Neuropathol Berl 1989; 77:621-628. 56. Mosley K, Cuzner ML. Receptor-mediated phagocytosis of myelin by macrophages and microglia: Effect of opsonization and receptor blocking agents. Neurochem Res 1996; 21:481-487. 57. Scheife RT, Hills JR, Munsat TL. Myasthenia gravis: Signs, symptoms, diagnosis, immunology and current therapy. Pharmacotherapy 1981; 1:39-54. 58. Kissel JT, Riethman JL, Omerza J et al. Peripheral nerve vasculitis: Immune characterization of the vascular lesions [see comments]. Ann Neurol 1989; 25:291-297.

242

Endocrine and Organ Specific Autoimmunity

59. Nicolai A, Bonetti B, Lazzarino LG et al. Peripheral nerve vasculitis: A clinico-pathological study. Clin Neuropathol 1995; 14:137-141. 60. Moore PM. Neurological manifestation of vasculitis: Update on immunopathogenic mechanisms and clinical features. Ann Neurol 1995; 37(Suppl. 1):S131-41. 61. Dyck PJ, Giannini. Pathologic alterations in the diabetic neuropathies of humans: A review. J Neuropathol Exp Neurol 1996; 55:1181-1193. 62. Rabinowe S, Brown F, Watts M et al. Anti-sympathetic ganglia antibodies in IDDM subjects of varying duration and patients at high risk of developing IDDM. Diabetes Care 1989; 12:1-6. 63. Brown FM, Vinik AI, Ganda OP et al. Different effects of duration on prevalence of antiadrenal medullary and pancreatic islet cell antibodies in type I diabetes mellitus. Horm Metab Res 1989; 21:434-437. 64. Zanone MM, Peakman M, Purewal T et al. Autoantibodies to nervous tissue structures are associated with autonomic neuropathy in Type 1 (insulin-dependent) diabetes mellitus. Diabetologia 1993; 36:564-569. 65. Brown FM, Brink SJ, Freeman R et al. Anti-sympathetic nervous system autoantibodies. Diminished catecholamines with orthostasis. Diabetes 1989; 38:938-941. 66. Sundkvist G, Lind P, Bergstrom B et al. Autonomic nerve antibodies and autonomic nerve function in type 1 and type 2 diabetic patients. J Intern Med 1991; 229:505-510. 67. Pittenger GL, Liu D, Vinik AI. The neuronal toxic factor in serum of type 1 diabetic patients is a complement-fixing autoantibody. Diabetic Med 1995; 12:380-386. 68. Rabinowe SL, Myerov A, Brown F. Anti-ganglioside GT1b IgG antibodies in Type I diabetes: orthostatic blood pressure and autonomic antibodies. Clin Res 1991; 39:364 (Abstract). 69. Barzilay J, Warram JH, Rand LI et al. Risk for cardiovascular autonomic neuropathy is associated with the HLA-DR3/DR4 phenotype in type I diabetes mellitus. Ann Intern Med 1992; 116:544-549. 70. Vinik AI, Pittenger GL, Stansberry KB et al. Phospholipid and glutamic acid decarboxylase autoantibodies in diabetic neuropathy. Diabetes Care 1995; 18:1225-1232. 71. Wilson MJ, Richter Lowney K, Daleke DL. Hyperglycemia induces a loss of phospholipid asymmetry in human erythrocytes. Biochemistry 1993; 32:11302-11310. 72. Lockshin MD, Qamar T, Druzin ML et al. Antibody to cardiolipin, lupus anticoagulant, and fetal death. J Rheumatol 1987; 14:259-262. 73. Lockshin MD, Druzin ML, Goei S et al. Antibody to cardiolipin as a predictor of fetal distress or death in pregnant patients with systemic lupus erythematosus. N Engl J Med 1985; 313:152-156. 74. Vinik AI, Holland MT, LeBeau J et al. Diabetic neuropathies. Diabetes Care 1992; 15:1926-1975. 75. Pestronk A, Adams RN, Kuncl RW et al. Differential effects of prednisone and cyclophosphamide on autoantibodies in human neuromuscular disorders. Neurology 1989; 39:628-633. 76. Bansal AS, Abdul-Karim B, Malik RA et al. IgM ganglioside GM1 antibodies in patients with autoimmune disease or neuropathy, and controls. J Clin Pathol 1994; 47:300-302. 77. Kaufman DL, Houser CR, Tobin AJ. Two forms of the gamma-aminobutyric acid synthetic enzyme glutamate decarboxylase have distinct intraneuronal distributions and cofactor interactions. J Neurochem 1991; 56:720-723. 78. Baekkeskov S, Aanstoot H-J, Christgau S et al. Identification of the 64K autoantigen in insulin-dependent diabetes as the GABA-synthesizing enzyme glutamic acid decarboxylase. Nature 1990; 347:151-156. 79. Solimena M, Folli F, Denis-Donini S et al. Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type 1 diabetes mellitus. N Engl J Med 1988; 318:1012-1020. 80. Solimena M, Folli F, Aparisi A et al. Autoantibodies to GABA-ergic neurons and pancreatic beta cells in stiff-man syndrome. N Engl J Med 1990; 322:1555-1560.

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

243

81. Harrison LC, Honeyman MC, DeAizpurua HJ et al. Inverse relation between humoral and cellular immunity to glutamic acid decarboxylase in subjects at risk of insulin-dependent diabetes. Lancet 1993; 341:1365-1369. 82. Tuomi T, Zimmet PZ, Rowley MJ et al. Persisting antibodies to glutamic acid decarboxylase in type 1 (insulin-dependent) diabetes mellitus are not associated with neuropathy. [Letter]. Diabetologia 1993; 36:685-680. 83. Zanone MM, Petersen JS, Pekman M et al. High prevalence of autoantibodies to glutamic acid decarboxylase in long-standing IDDM is not a marker of symptomatic autonomic neuropathy. Diabetes 1994; 43:1146-1151. 84. Frazier WA, Hogue-Angeletti R, Bradshaw RA. Nerve growth factor and insulin, structural similarities indicate an evolutionary relationship reflected by physiological action. Science 1972; 176:482-488. 85. Mobley WC. Nerve growth factor, 2nd of 3 parts. N Engl J Med 1977; 297:1149-1158. 86. Levi-Montalcini R, Booker B. Destruction of the sympathetic ganglia in mammals by an antiserum to the nerve-growth promoting factor. Proc Natl Acad Sci USA 1960; 46:384-390. 87. Matheson SF, Gold B, Mobley WC. Somatofugal axonal atrophy in intact adult sensory neurons following injection of nerve growth factor (NGF) antiserum. Soc Neurosci Abstr 1989; 15:707. 88. Rich KM, Luszynski JR, Osborne PA, Johnson EM, Jr. Nerve growth factor protects adult sensory neurons from cell death and atrophy caused by nerve injury. J Neurocytol 1987; 16:261-268. 89. Bennett T. Physiological investigation of diabetic autonomic failure. In: R. Bannister, ed. Autonomic Failure. A Textbook of Clinical Disorders of the Autonomic Nervous System. Oxford: Oxford University Press 1983; 406-436. 90. Smeyne RJ, Klein R, Schnapp A et al. Severe sensory and sympathetic neuropathies in mice carrying a disrupted Trk/NGF receptor gene. Nature 1994; 368:246-249. 91. Apfel SC, Arezzo JC, Brownlee M et al. Nerve growth factor administration protects against experimental diabetic sensory neuropathy. Brain Res 1994; 634 (1):7-12. 92. Pittenger GL, Burcus N, Malik RA et al. Cytotoxicity of serum on sensory/autonomic and motor neural cells in vitro predicts sensory neuropathy. Diabetes 1997; 46:125A (Abstract). 93. Pittenger GL, Stansberry KB, Vinik AI. Failure of neurotrophic support as a mechanism for the pathogenesis of diabetic neuropathy. Peripheral Nerve Society (Abstract)1997. 94. Anand P, Rudge P, Mathias CJ et al. New autonomic and sensory neuropathy with loss of adrenergic sympathetic function and sensory neuropeptides. Lancet 1991; 337:1253-1254. 95. Guy RJC, Richards F, Edmonds ME et al. Diabetic autonomic neuropathy and iritis: an association suggesting an immunological cause. Brit Med J 1984; 289:343-345. 96. Ishii DN. Implications of insulin-like growth factors in the pathogenesis of diabetic neuropathy. Brain Res Rev 1995; 20:47-67. 97. Cheng HL, Feldman EL. Insulin-like growth factor-I (IGF-I) and IGF binding protein-5 in Schwann cell differentiation. J Cell Physiol 1997; 171:161-167. 98. Quattrin T, Thrailkill K, Baker L et al. Dual hormonal replacement with insulin and recombinant human insulin-like growth factor I in IDDM. Effects on glycemic control, IGFI levels, and safety profile. Diabetes Care 1997; 20:374-380. 99. Mynarcik DC, Williams PF, Schaffer L et al. Identification of common ligand binding determinants of the insulin and insulin-like growth factor 1 receptors. Insight into mechanisms of ligand binding. J Biol Chem 1997; 272:18650-18655. 100. Kohn LD, Kosugi S, Ban T et al. Molecular basis for the autoreactivity against thyroid stimulating hormone receptor. Int Rev Immunol 1992; 9:135-165. 101. Carson DA, Ribeiro JM. Apoptosis and disease. Lancet 1993; 341:1251-1254. 102. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267:1456-1462. 103. Juntti-Berggren L, Larsson O, Rorsman P et al. Increased activity of L-type Ca2+ channels exposed to serum from patients with Type 1 diabetes. Science 1993; 261:86-90.

244

Endocrine and Organ Specific Autoimmunity

104. Garchon HJ, Luan JJ, Eloy L et al. Genetic analysis of immune dysfunction in nonobese diabetic (NOD) mice: Mapping of a susceptibility locus close to the bcl-2 gene correlates with increased resistance of NOD T cells to apoptosis induction. Eur J Immunol 1994; 24:380-384. 105. Bellgrau D, Gold D, Selawry H et al. A role for CD95 ligand in preventing graft rejection. Nature 1995; 377:630-632. 106. Kruman II, Kostenko MA, Gordon RY et al. Differentiation and apoptosis of murine neuroblastoma cells N1E-115. Biochem Biophys Res Commun 1993; 191:1309-1318. 107. Kirino T. Cerebral ischemia and neuronal death. No To Hattatsu 1994; 26:130-135. 108. Kimura F, Smith RG, Delbono O et al. Amyotrophic lateral sclerosis patient antibodies label Ca2+ channel a1 subunit. Ann Neurol 1994; 35:164-171. 109. Smith RG, Alexianu ME, Crawford G et al. Cytotoxicity of immunoglobulins from anyotrophic lateral sclerosis patients on a hybrid motoneuron cell line. Proc Natl Acad Sci USA 1994; 91:3393-3397. 110. Steller H. Mechanism and genes of cellular suicide. Science 1995; 267:1445-1449. 111. Garcia I, Marinou I, Tsujimoto Y et al. Prevention of programmed cell death of sympathetic neurons by the bcl-2 proto-oncogene. Science 1992; 258:302-304. 112. Weller M, Frei K, Groscurth P et al. Anti-Fas/APO-1 antibody-mediated apoptosis of cultured human glioma cells. Induction and modulation of sensitivity by cytokines. J Clin Invest 1994; 94:954-964. 113. Peitsch MC, Tschopp J. Comparative molecular modeling of the Fas-ligand and other members of the TNF family. Mol Immunol 1995; 32:761-772. 114. Nagata S, Golstein P. The Fas death factor. Science 1995; 267:1449-1456. 115. Ogasawara J, Watanabe Fukunaga R, Adachi M et al. Lethal effect of the anti-Fas antibody in mice. Nature 1993; 364:806-809. 116. Jensen LM, Zhang Y, Shooter EM. Steady-state polypeptide modulations associated with nerve growth factor (NGF)-induced terminal differentiation and NGF deprivation-induced apoptosis in human neuroblastoma cells. J Biol Chem 1992; 267:19325-19333. 117. Rabizadeh S, Oh J, Zhong LT et al. Induction of apoptosis by the low-affinity NGF receptor. Science 1993; 261:345-348. 118. Vinik AI, Mitchell BD, Leichter SB et al. Epidemiology of the complications of diabetes. In: Leslie RDG and Robbins DC, eds. Diabetes: Clinical Science in Practice. Cambridge United Kingdom: Cambridge University Press, 1995:221-287. 119. Boulton AJ, Knight G, Drury J et al. The prevalence of symptomatic, diabetic neuropathy in an insulin- treated population. Diabetes Care 1985; 8:125-128. 120. Levitt NS, Stansberry KB, Wychanck S et al. Natural progression of autonomic neuropathy and autonomic function tests in a cohort of IDDM. Diabetes Care 1996; 19:751-754. 121. Consensus statement: Report and recommendations of the San Antonio conference on diabetic neuropathy. American Diabetes Association American Academy of Neurology. Diabetes Care 1988; 11:592-597. 122. Brown F. Sural nerve biopsies in Guillain-Barré syndrome: axonal degeneration and macrophage-associated demyelination and absence of cytomegalovirus genome. Muscle Nerve 1993; 16:112. 123. Khalili-Shirazi A, Atkinson P, Gregson N et al. Antibody response to P0 and P2 myelin proteins in Guillain-Barré syndrome and chronic idiopathic demyelinating polyradiculopathy. J Neuroimmunol 1993; 46:245-251. 124. Segal P, Teitelbaum D, Ohry A. Cell-mediated immunity to nervous system antigens in diabetic patients with neuropathy. Isr J Med Sci 1983; 19:7-10. 125. Younger DS, Rosoklija G, Hays AP. Peripheral nerve immunohistochemistry in diabetic neuropathy. Semin Neurol 1996; 16:139-142. 126. Milicevic Z, Pittenger G, Liuzzi JF et al. Evidence for the presence of immunoglobulin-G (IgG) and -M (IgM) in sural nerve biopsies of patients with diabetic sensorimotor neuropathy. Diabetes 1995; 44:63A-(Abstract).

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

245

127. DCCT Research Group. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin dependent diabetes mellitus. N Engl J Med 1993; 329:977-986. 128. Stevens MJ. Nitric Oxide as a Potential Bridge Between the Metabolic and Vascular Hypotheses of Diabetic Neuropathy. Diabetic Med 1995; 12:292-295. 129. Hill MA, Colen LB, Vinik AI. Microvascular and compression in the etiology of diabetic neuropathy. In: LeRoith D, Taylor SI, Olefsky JM, eds. Diabetes Mellitus. A Fundamental and Clinical Text. Philadelphia: J.B. Lippincott Company 1996:759-766. 130. Tuck RR, Schmelzer JD, Low PA. Endoneurial blood flow and oxygen tension in the sciatic nerves of rats with experimental diabetic neuropathy. Brain 1984; 107:935-950. 131. Stansberry KB, Shapiro BS, Hill MA et al. Impaired peripheral vasomotion in diabetes. Diabetes Care 1996; 19:715-721. 132. Stansberry KB, Hill A, Shapiro SA et al. Impairment of peripheral blood flow responses in diabetes resembles an enhanced aging effect. Diabetes Care 1997;(In Press). 133. Liuzzi FJ, Depto AS. Neuropathy: Growth Factors and Nerve Regeneration. In: LeRoith D, Olefsky JM, Taylor SI, eds. Diabetes Mellitus: A Fundamental and Clinical Text. Philadelphia: Lippincott-Raven 1996:766-771. 134. Anand P, Terenghi G, Warner G et al. The role of endogenous nerve growth factor in human diabetic neuropathy. Nature Med 1996; 2:703-707. 135. Zanone MM, Banga JP, Peakmen M et al. An investigation of antibodies to nerve growth factor in diabetic autonomic neuropathy. Diabetic Med 1994; 11:378-383. 136. Brewster WJ, Fernyhough P, Diemel LT et al. Diabetic neuropathy, nerve growth factor and other neurotrophic factors. Trends Neurosci 1994; 17:321-325. 137. Ziegler D, Dannehl K, Muhlen H et al. Prevalence of cardiovascular autonomic dysfunction assessed by spectral analysis, vector analysis, and standard tests of heart rate variation and blood pressure responses at various stages of diabetic neuropathy. Diabetic Med 1992; 9:806-814. 138. Subramony SH, Wilbourn AJ. Diabetic proximal neuropathy. Clinical and electromyographic studies. J Neurol Sci 1982; 53:293-304. 139. Bradley WG, Chad D, Verghese JP et al. Painful lumbosacral plexopathy with elevated erythrocyte sedimentation rate: A treatable inflammatory syndrome. Ann Neurol 1984; 15:457-464. 140. Said G, Slama G, Selva J. Progressive centripetal degeneration of axons in small fiber type diabetic polyneuropathy: A clinical and pathological study. Brain 1983; 106:791-807. 141. Krendel DA, Costigan DA, Hopkins LC. Successful treatment of neuropathies in patients with diabetes mellitus. Arch Neurol 1995; 52:1053-1061. 142. Lockwood CM. New treatment strategies for systemic vasculitis: the role of intravenous immune globulin therapy. Clin Exp Immunol 1996; 104 Suppl 1:77-82. 143. Hall PD. Immunomodulation with intravenous immunoglobulin. Pharmacotherapy 1993; 13:564-573. 144. Wolf HM, Eibl MM. Immunomodulatory effect of immunoglobulins. Clin Exp Rheumatol 1996; 14(Suppl 15):S17-25. 145. Kaveri S, Vassilev T, Hurez V et al. Antibodies to a conserved region of HLA class I molecules, capable of modulating CD8 T cell-mediated function, are present in pooled normal immunoglobulin for therapeutic use. J Clin Invest 1996; 97:865-869. 146. Buelow R, Burlingham WJ, Clayberger C. Immunomodulation by soluble HLA class I. Transplantation 1995; 59:649-654. 147. Kaveri S, Prasad N, Vassilev T et al. Modulation of autoimmune response by intravenous immunoglobulin. Multiple Sclerosis 1997; 3:121-128. 148. Low PA, Walsh JC, Huang CY et al. The sympathetic nervous system in diabetic neuropathy. A clinical and pathological study. Brain 1975; 98:341-356. 149. Zola BE, Vinik AI. Effects of autonomic neuropathy associated with diabetes mellitus on cardiovascular function. Coronary Artery Disease 1992; 3:33-41.

246

Endocrine and Organ Specific Autoimmunity

150. Ewing DJ, Campbell IW, Clarke BF. The natural history of diabetic autonomic neuropathy. QJ Med 1980; 49:95-108. 151. Pfeifer MA, Weinberg CR, Cook DL et al. Autonomic neural dysfunction in recently diagnosed diabetic subjects. Diabetes Care 1984; 7:447-453. 152. O’Brien IA, O’Hare JP, Lewin IG et al. The prevalence of autonomic neuropathy in insulin-dependent diabetes mellitus: A controlled study based on heart rate variability. Q J Med 1986; 61:957-967. 153. Barkai L, Madacsy L, Kassay L. Investigation of subclinical signs of autonomic neuropathy in the early stage of childhood diabetes. Horm Res 1990; 34:54-59. 154. Kennedy WR, Navarro X, Sutherland DE. Neuropathy profile of diabetic patients in a pancreas transplantation program. Neurology 1995; 45:773-780. 155. Vinik AI, Mitchell B. Clinical aspects of diabetic neuropathies. Diabetes Metab Rev 1988; 4:223-253. 156. Zola BE, Vinik AI. Effect of autonomic neuropathy associated with diabetes mellitus on cardiovascular function. Current Science. Coronar Artery Disease 1992; 3:33-41. 157. Kahn JK, Sisson JC, Vinik AI. QT interval prolongation and sudden cardiac death in diabetic autonomic neuropathy. J Clin Endocrinol Metab 1987; 64:751-754. 158. Duchen LW, Anjorin NA, Watkins PJ et al. Pathology of autonomic neuropathy in diabetes mellitus. Ann Intern Med 1980; 92:301-303. 159. Brown FM, Zuckerman M, Longway S et al. Adrenal medullary fibrosis in IDDM of long duration. Diabetes Care 1989; 12:494-497. 160. Brown FM, Smith AM, Longway S et al. Adrenal medullitis in type I diabetes. J Clin Endocrinol Metab 1990; 71:1491-1495. 161. Brown FM, Vinik AI, Ganda OP. Different effects of duration on prevalence of anti-adrenal medullary and pancreatic islet cell antibodies in type I diabetes mellitus. Horm Metab Res 1988; 21:434-437. 162. Björk E, Velloso LA, Kämpe O et al. GAD autoantibodies in IDDM, stiff-man syndrome, and autoimmune polyendocrine syndrome type I recognize different epitopes. Diabetes 1994; 43:161-165. 163. Daw K, Ujihara N, Atkinson M et al. Glutamic acid decarboxylase autoantibodies in stiffman syndrome and insulin-dependent diabetes mellitus exhibit similarities and differences in epitope recognition. J Immunol 1996; 156:818-825. 164. Jessen KR, Mirsky R, Dennison ME et al. GABA may be a neurotransmitter in the vertebrate peripheral nervous system. Nature 1979; 281:71-74. 165. The DCCT Research Group. Factors in development of diabetic neuropathy. Baseline analysis of neuropathy in feasibility phase of Diabetes Control and Complications Trial (DCCT). Diabetes 1988; 37:476-481. 166. Vinik AI, Suwanwalaikorn S, Holland M et al. Diagnosis and management of diabetic autonomic neuropathy. In: DeFronzo R, ed. Current Management of Diabetes Mellitus. St. Louis: Mosby-Year Book, Inc. 1997. 167. Behse F, Buchthal F, Carlsen F. Nerve biopsy and conduction studies in diabetic neuropathy. J Neurol Neurosurg Psychiatry 1977; 40:1072-1082. 168. Veves A, Malik RA, Lye RH et al. The relationship between sural nerve morphometric findings and measures of peripheral nerve function in mild diabetic neuropathy. Diabetic Med 1991; 8:917-921. 169. Thomas PK, Beamish NG, Small JR et al. Paranodal structure in diabetic sensory polyneuropathy. Acta Neuropathol Berl 1996; 92:614-620. 170. Malik RA, Tesfaye S, Thompson SD et al. Endoneurial localization of microvascular damage in human diabetic neuropathy. Diabetologia 1993; 36:454-459. 171. Malik RA. The pathology of human diabetic neuropathy. Diabetes 1997; 46:S50-S53. 172. Britland ST, Young RJ, Sharma AK et al. Relationship of endoneurial capillary abnormalities to type and severity of diabetic polyneuropathy. Diabetes 1990; 39:909-913. 173. Dyck PJ, Karnes JL, O’Brien P et al. The spatial distribution of fiber loss in diabetic polyneuropathy suggests ischemia. Ann Neurol 1986; 19:440-449.

Autoimmune Mechanisms in the Pathogenesis of Diabetic Neuropathy

247

174. Dyck PJ, Lais A, Karnes JL et al. Fiber loss is primary and multifocal in sural nerves in diabetic polyneuropathy. Ann Neurol 1986; 19:425-439. 175. Milicevic Z, Pittenger GL, Stansberry KB et al. Raised anti-ganglioside GM1 antibody (GM1 Ab) titers in a subset of patients with distal symmetric polyneuropathy (DSPN). Diabetes 1997; 46:125A (Abstract). 176. Visser LH, van der Meche FG, van Doorn PA et al. Guillain-Barré syndrome without sensory loss (acute motor neuropathy). A subgroup with specific clinical, electrodiag-nostic and laboratory features. Dutch Guillain-Barré Study Group. Brain 1995; 118:841-847. 177. Santoro M, Thomas FP, Fink ME. IgM deposits at nodes of Ranvier in a patient with amyotrophic lateral sclerosis, anti-GM1-antibodies and multifocal conduction block. Ann Neurol 1990; 28:373-377. 178. Minuk J, Latov N, Cashman N et al. Upper and lower motor neuron disease in a patient with anti-GM1 ganglioside antibody. Neurology 1990; 40(Suppl 1):183-180. 179. Adams D, Steck AJ, Perruisseau G. Predictive value of anti-GM1 antibodies in neuromuscular diseases. Neurology 1990; 40(Suppl 1):299-290. 180. Roberts M, Willison HJ, Vincent A et al. Multifocal motor neuropathy human sera block distal motor nerve conduction in mice. Ann Neurol 1995; 38:111-118. 181. Roll U, Nuber A, Schroder A et al. No association of antibodies to glutamic acid decarboxylase and diabetic complications in patients with IDDM. Diabetes Care 1995; 18:210-215. 182. Harris EN, Englert H, Derue G et al. Antiphospholipid antibodies in acute Guillain-Barré syndrome. Lancet 1983; 2:1361-1362. 183. Vinik AI, Milicevic Z. Preventive measures and treatment options for diabetic neuropathy. Contemp. Int Med 1994; 6:41-55. 184. Vinik AI, Milicevic Z, Colen LB et al. Histopathological and electrophysiologic heterogeneity in patients with proximal diabetic neuropathy (PDN). 1996;(Unpublished abstract). 185. Vinik AI, Milicevic Z. Recent advances in the diagnosis and treatment of diabetic neuropathy. The Endocrinologist 1996; 6:443-461.

248

Endocrine and Organ Specific Autoimmunity

Ocular Autoimmunity

249

CHAPTER 12

Ocular Autoimmunity Luiz V. Rizzo and Robert B. Nussenblatt

Introduction

U

veitis is a term originally used to describe an inflammatory process of the uvea (the median layer of the eye or tunica vasculosa bulbi, which includes the iris, ciliary body and choroid).1 This term however, has been used loosely to describe any intraocular inflammation affecting the uvea as well as retina, sclera and vitreous humor.1,2 Uveitis can be characterized in several different ways. Anatomically, it might be anterior (encompassing the anterior segment of the eye, i.e., the iris and ciliary body), intermediate (when involving the vitreous and pars plana), posterior (when the choroid and retina are involved), or panuveitis (when all segments are involved). Clinically, uveitis can be acute or chronic. In terms of its etiology, exogenous uveitides are those in which an exogenous agent can be identified as the cause of the inflammation (such is the case in viral, bacterial or parasitic uveitis). Uveitis is said to be endogenous when the triggering agent for the inflammatory process is unknown or when uveitis is of well-established autoimmune nature.1 All uveitides contribute to 10% of severe ocular handicap cases in the USA and appear at a rate of 70,000 new cases a year representing an important concern in ophthalmology but also as well as public health.1 The concept that endogenous uveitis my be caused by an autoimmune phenomenon was first introduced in the beginning of the century. Uhlenhuth in 1903, showed the existence of antibodies recognizing crystallin antigens.3 Elschnig in 1910 introduced the idea that autoantigens could be involved in the development of uveitis.4 Uveitides can be a single entity affecting only the eye, as is the case of birdshot chorioretinopathy or sympathetic ophthalmia which represents the prototypical organ-specific autoimmune disease. Uveitis may also be a part of a systemic syndrome affecting other organs as is the case for the uveitis associated with ankylosing spondilitis, juvenile rheumatoid arthritis, Voght-Koyanagi-Harada syndrome (uveoencephalitis), Behçet’s disease, and sarcoidosis.1 In other instances, uveitis with autoimmune characteristics may develop as a consequence of a known agent or entity without direct ocular involvement, as is the case in the paraneoplasic syndromes or septic shock.1 The possible generation of T cells or antibodies that recognize self-antigens is a consequence inherent to the immune system because of the random nature by which immune molecules responsible for target recognition (T cell receptor and immunoglobulins) are assembled. There are four classic mechanisms described that are employed by the immune system to avoid the development of autoimmunity: clonal selection,5,6 active suppression,7,8 immune privilege9-11 and clonal inactivation.7,8 In addition, if the “danger theory” proposed by Matzinger and Fuchs is correct some of these mechanisms may not be necessary, or even exist.12 Their theory suggests that there is no particular differentiation by the immune system between self and nonself antigens. Alternatively, the development of an Endocrine and Organ Specific Autoimmunity, edited by George S. Eisenbarth. ©1999 R.G. Landes Company.

250

Endocrine and Organ Specific Autoimmunity

immune response against a target (self or nonself) is guided by the perception by the immune system that the target represents a threat to the organism’s homeostasis. Negative selection (clonal selection) of putatively autoaggressive T cell clones occurs in the thymus and it is dependent on the presence of the target autoantigens found there.6 However, not all self-antigens are present in the thymus in sufficient concentrations to induce deletion of the respective T cells bearing antigen receptors capable of recognizing such antigens with high affinity.13-17 In the case of ocular-specific antigens this becomes particularly important since during ontogeny the eye becomes a sequestered organ before the thymus is fully developed. Consequently, some ocular antigens may never reach the thymus and the T cells bearing antigen receptors capable of recognizing such antigens may never be deleted.18,19 The importance of thymic presentation of ocular antigens in terms of the development of uveitis has been confirmed recently by experiments showing that experimental autoimmune uveoretinitis (EAU)-susceptible rat strains do not express either arrestin (S-Ag) or interphotoreceptor retinoid binding protein (IRBP) in their thymi and consequently are easily induced to respond to such antigens and generate an autoimmune reaction in the eye. On the other hand, mice that do express such antigens in the thymus and therefore would undergo negative selection of T cells reacting with high affinity to these retinal antigens, require doses 500-2,000-fold higher of protein immunization than those used in rats to develop uveitis.20 It is known that ocular-antigen-specific T cells will reach the blood at a frequency of 1 to 5 precursors per million cells.21,22 In spite of the presence of such autoreactive cells in the blood most people do not develop uveitis. Part of the protection against autoreactive cells in the periphery comes from the eye being a privileged immune site. In the eye, the anterior chamber, cornea and retina are considered immune privilege sites. The concept of immune privilege was first defined by Medawar, even though the phenomenon had been known for more than 50 years at the time of his observations.23 According to his theory the lack of lymphatic drainage would restrict autoantigens of immune privilege sites from being recognized and attacked by the immune system. Years later however, it was shown that antigens placed inside the eye (an immunoprivileged site) are capable of inducing an immune response.24,25 Thus the concept of immune privilege has changed. Immunoprivileged sites are no longer considered inaccessible. However, because of particular local circumstances in these organs, immune responses there develop differently than they would in the rest of the body. It has been shown that Fas ligand (FasL)-expressing cells encircle the eye forming a protective barrier against the deleterious effects of activation of T lymphocytes in the eye.26 FasL expression has been shown to be expressed on the corneal epithelium and endothelium, iris, ciliary body and throughout the retina. The expression of FasL in such tissues allows them to induce apoptosis of any activated T lymphocyte that expresses Fas. Several other characteristics contribute to the immunoprivileged site status of the eye. The absence of efferent lymphatic drainage and consequent drainage of lymph directly to the blood, results in immune suppression.27,28 Soluble substances which can suppress the immune response are also part of the mechanisms involved in maintenance of immune privilege. TGF-β, PGE2 and neuropeptides (melanocyte stimulating hormone-α, vasoactive intestinal peptide, and calcitonin related gene) all contribute to the generation of a deviant immune response in the sites where these substances are secreted.10,29-34 In the eye, substances attached to the cellular membrane act as inhibitors of the complement cascade.35,36 Also, cells with suppressor activity such as Müller cells and retinal pigment epithelial cells (RPE) play a role in modifying the response inside the eye to curtail inflammation.37-42 Another important component of immune privilege is the local tissue barrier. In the eye cells from the parenchyma (RPE cells) have tight junctions. In addition, the expression of

Ocular Autoimmunity

251

hyaluronic acid on the cellular surface and reduced expression of MHC class I and II complete the array of physical constituents of what is called the blood-retinal barrier.19,33,37-42 An additional mechanism for controlling autoimmunity in the eye is the phenomenon of anterior chamber associated immune deviation (ACAID).24,25,43-45 In response to the introduction of antigens to the anterior chamber of the eye a state of systemic tolerance to that antigen results. ACAID is a unique aspect of immune privilege in the eye and it may play a role in the development of tolerance against ocular antigens.46-49 Autoimmunity will develop only if all the safe-guard mechanisms described above, as well as others not described in this chapter, fail. In experimental models of disease the point at which tolerance is broken and autoimmunity ensues is easily determined because disease induction is produced by active immunization with a specific target antigen. Nevertheless, it is often necessary to use one or more adjuvants to break the state of tolerance.50-52 In the case of uveitis, a break in the blood-retinal barrier generally represents the beginning of the autoimmune process1 and sometimes may constitute its precipitating factor.53

The Animal Model of Uveitis Animal models help to understand the mechanisms involved in disease development, risk factors, as well as factors that precipitated or ameliorate disease progress. Animal models will also serve as a template for the study of new therapeutic strategies.54-56 The first animal model for autoimmune uveitis was described in 1965 by Wacker and Lipton. It showed that it was possible to induce uveitis in guinea pigs by intradermal injections of retinal antigens extract.57 Arrestin, or retinal S antigen (S-Ag), was localized at the external layer of the photoreceptors and implicated as the uveitogenic antigen.58 Because the term uveitis represents a complex group of diseases, as described above, the scientific literature contains descriptions for many animal models for it. These models include: crystallin-induced experimental uveitis, in which intraocular inflammation occurs after immunization with crystallin antigens followed by a lesion to the animal’s lens, induced by puncture with a needle;59 experimental inflammatory uveitis (EIU), induced by the injection of lipopolysaccharide from gram-negative bacteria;60 immunogenic uveitis, induced by the immunization with heterologous albumin followed by intraocular injection of the immunizing antigen;51,61-63 experimental anterior allergic uveitis (EAAU), induced by immunization with melanin or melanin-associated antigen;64-68 and experimental autoimmune uveitis (EAU) which can be induced in rodents and primates by immunization with S-Ag,58 Phosducin,69 Rhodopsin,70-72 recoverin,73 or IRBP.74,75 The most important characteristic of some of these models is that they resemble the human disease in its clinical manifestations as well as in its histopathological presentation.

Immunopathogenesis Most of the studies in immunopathogenesis of endogenous uveitis have been carried out in the animal models of the disease. The rat and mouse models of EAU74,76 have been especially useful because of the availability of reagents to study their immune response and because genetically defined strains of mice and rats allowed more sophisticated analysis of the genetic component to susceptibility and resistance to disease development (see below). We have learned from the animal models that uveitogenic T cells can be elicited in the periphery by immunization with one of several retinal antigens. The diseases induced by different uveitogenic proteins share their histopathological features and cellular mechanisms, suggesting that they also share the immunopathogenic mechanisms necessary for disease induction and progression. The presence of many retinal antigens that may act as a target for the autoimmune process in uveitis may account for the difficulty to isolate a single

252

Endocrine and Organ Specific Autoimmunity Fig. 12.1. A) Normal histological architecture of the human retina. B) severe destruction of the retina in sympathetic ophthalmia. Retinal detachment and subretinal hemorrhage, retinal folds, destruction of the photoreceptor layer and lymphocytic infiltrates throughout the retina. Courtesy of Dr. Chi-Chao Chan, Immunopathology Section, NEI-NIH.

candidate target in human uveitides. Interestingly, peripheral blood lymphocytes from patients with intermediate and posterior uveitis were shown to proliferate to S-Ag.77,78 Once T cells that recognize uveitogenic epitopes are activated they will migrate to the eye and initiate a cascade of events leading to the destruction of the photoreceptor layer and loss of vision (Fig. 12.1). It has been reported that any activated T cell can penetrate the retina, however only cells recognizing pathogenic epitopes will remain in the retina after 12 hours.79 These cells will proceed to break the blood-retinal barrier through the secretion of various cytokines which induce expression of adhesion molecules and MHC class II throughout the retina and its draining blood vessels. This breach allows the recruitment of other T cells as well as monocytes and polymorphonuclear cells from the blood. In the rat and mouse models of uveitis CD4+ T cells that recognize retinal antigens have been used to adoptively transfer disease to naive recipients confirming the nature of the autoimmune phenomenon.59,80 Uveitogenic cells have also been isolated from the uveitic eye in murine EAU and shown to behave in a similar fashion to those found in the periphery suggesting that they constitute the same population at different stages of differentiation and that cells will migrate from the periphery into the eye and mediate its autoimmune destruction.19,81 In the rat model of EAU, it has been shown that infiltrating T lymphocyte populations will change from mostly CD4+ to predominately CD8+ as disease progresses.82 The same dynamics in the infiltrating cell population was shown in a case of sympathetic ophthalmia

Ocular Autoimmunity

253

Table 12.1. Association Between HLA and Disease Disease Acute anterior uveitis Ankylosing spondylitis Behçet’s Disease Birdshot retinochoroidopathy Ocular-Cutaneous Pemphigoid Ocular Histoplasmosis Reiter’s syndrome Sympathetic ophthalmia Vogt-Koyanagi-Harada syndrome

HLA

Relative risk

HLA-B27 (W) HLA-B8 (AA) HLA-B27 (W) HLA-B7 (AA) HLA-B51 (O) HLA-A29 HLA-B12 (W) HLA-B7 (W) HLA-B27 (W) HLA-A11 (M) MT-3 (O)

10 5 100 4-6 50-225 3-4 40 4 75

AA, African-American; M, mixed ethnicity; o, oriental; W, white Caucasian

Table 12.2. Relationship Between Genetic Background, MHC Genes and Susceptibility to EAU Mouse strain

Non-MHC Genetic background

H-2

Susceptibility to EAU

B10.A B10.BR B10.RIII B10.M B10.PL B10.Q B10.S B10.D2 B10.SM C57BL/10 C57BL/6 A/J A.CA A.SW AKR LP.RIII BALB/c DBA/1 NZW

C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/10 C57BL/6 A/J A/J A/J AKR LP BALB/c DBA/1 NZW

a k r f u q s d v b b a f s k r d q z

High High High Low Low Low Low Low to Medium Low Medium Medium Medium Low Low Low Medium Low Low Low

Mice were immunized with 100 µg of IRBP, subcutaneously, emulsified in complete Freund’s adjuvant containing 2.5 µg of Mycobacterium tuberculosis in addition to an intraperitoneal dose of 1 µg of pertussis toxin.

254

Endocrine and Organ Specific Autoimmunity

in which both eyes were enucleated from a patient at different time points during the course of disease. The eye enucleated early showed mainly CD4+ cells, whereas the eye enucleated a year later was infiltrated mostly with CD8+ cells.83 It becomes apparent that T cell dynamics in human uveitis parallels that seen in the animal model. The responses of ocular tissues in human uveitis and in the animal models also seem to parallel each other. The expression of cell membrane markers is one of the earliest events seen when uveitis is induced experimentally. MHC class II molecules are expressed on many resident ocular cells, including the RPE, Müller cells and vascular endothelium. Recently, adhesion molecules have also been shown to be expressed as a result of experimental uveitis induction and in patients with endogenous disease.84 It remains to be resolved whether lymphocyte proliferative responses to retinal proteins in patients with uveitis, as well as the other local changes described above, are reflective of an autoimmune process or whether they are a secondary phenomenon due to the release of autoantigens that have no implications for the pathogenesis of uveitis. Nevertheless, based on the similarities between the disease in the animal models and in humans, it seems reasonable to extrapolate the findings in these models to human disease which would suggest that endogenous uveitides are caused by autoimmune aggression mediated by CD4+ T cells recognizing ocular antigens.

Immunogenetics Strong HLA associations have been observed in many types of human uveitic diseases (See Table 12.1 for specific associations). Also in the murine model of EAU the association between disease susceptibility and H-2 haplotype has been described (See Table 12.2 for specific associations). The association with antigens from the major histocompatibility complex1,65,78,85-87 suggests that the capacity to recognize certain epitopes expressed in ocular antigens and/or antigens that mimic such epitopes is associated with disease development. This finding also suggests that defects in the clonal selection of T cells (be it the negative selection of autoreactive clones or the lack of positive selection of suppressor clones) may be involved in disease development. The latter possibility was recently reinforced by the finding that uveitis develops in nude mice transplanted with embryonic thymi from susceptible rat strains.88 One of the strongest association between HLA and disease occurs in birdshot retinochoroidopathy and HLA-A29, the relative risk for this association is between 50 and 225. It is noteworthy that particularly in birdshot retinochoroidopathy most patients will present peripheral blood lymphocyte proliferation to S-Ag,78 making this one of the few uveitic conditions in which a putative target antigen is known. An increased frequency of certain types of uveitis in specific ethnic groups has been reported in the literature. It is interesting to note that most of the associations between human uveitis and HLA are to class I antigen in spite of the putative role of CD4+ class II-restricted cells in the pathogenic process. This discrepancy may signify that the participation of CD8+ cells is more important in humans than in the animal models, either as effector cells or as regulatory cells. Genetic susceptibility to the development of experimental uveitis in the rodent models is a well characterized fact.89 Studies in the mouse model of uveitis have suggested that susceptibility is controlled by both MHC and non-MHC genes.86,90 Studies using the mouse model of EAU have revealed that the primary control of susceptibility is determined by MHC genes of the class II I-A subregion (equivalent to the human HLA-DR), suggesting that the ability to recognize uveitogenic epitopes in the target ocular antigen plays a major part in the development of uveitis, as discussed above. Thus the presence of certain H-2 haplotypes was shown to be a sine qua non condition for the establishment of disease and the genes involved were labeled “permissive” to the development of disease. Interestingly,

Fig. 12.2. Immunopathogenesis of Uveitis. Activated T cells recognizing ocular antigens break the blood retinal barrier and through the secretion of cytokines mediate the recruitment of other lymphocytes, PMN and macrophages from the peripheral blood. Increased expression of adhesion molecules on the retinal blood vessels facilitates the recruitment process and enhanced expression of MHC class I and II molecules in ocular resident cells enhances the targeting of these cells by lymphocytes.

Ocular Autoimmunity 255

256

Endocrine and Organ Specific Autoimmunity

Fig. 12.3. Cytokine balance in the immune response. Th1-type cytokines generally work as mediators of inflammation whereas Th-2 type cytokines often act as anti-inflammatory cytokines. These groups of cytokines exert cross-regulatory activities towards one another and often the presence of high levels of one group of cytokines will cause the inhibition of the other.

expression of I-E gene products (equivalent to the human HLA-DQ) was shown to lessen the severity of disease. Secondary control of susceptibility appears to be linked to nonMHC genes, with some backgrounds completely preventing the development of disease, irrespective of the MHC haplotype these strains expressed. The genes involved were termed “conducive” to the development of disease. Non-MHC influences on susceptibility can be due to diverse mechanisms including differences in hormonal responses to stress, mast cell numbers, vascular response to cytokines, and lymphokine response patterns. All these factors have been suggested to play a role in susceptibility to autoimmunity in general. Recent data from the animal models suggest that genetic susceptibility to EAU is linked to the ability to mount a Th1-type response against the retinal antigen used to immunize the animals.90,91 On the other hand, resistance to disease is not linked to a Th2 response but rather to a “null” profile. In resistant strains the response towards the immunizing antigen never includes any of the Th1-type response characteristics (high IgG2a and IFN-γ secretion in response to the immunizing antigen), and may or may not include Th2-type response characteristics (high IgG1 production and secretion of IL-4, IL-5 and IL-10).

Immunotherapy Induction of ACAID (Anterior Chamber Associated Immune Deviation) to Retinal Antigens The fact that injecting both particulate and soluble antigens into the anterior chamber results in a deviant immune response with diminished DTH, has been recognized for many

257

Fig. 12.4. Immunotherapeutic approaches in uveitis. Different immunotherapeutic strategies and their targets during the development of an autoimmune response. The design is courtesy of Dr. Rachel R. Caspi, Section on Immunoregulation, NEINIH.

Ocular Autoimmunity

years9,24,25,33,44,45,76,92,93 (see Fig. 12.4 for a summary of immunotherapeutic approaches to uveitis). The recent finding that ACAID can be induced by antigen-pulsed macrophages incubated in vitro with TGF-β29,30 has opened the possibility to exploit this phenomenon for immunotherapy of autoimmune diseases. Mice infused with splenic macrophages, pulsed with IRBP in the presence of supernatants from cultured iris/ciliary body cells, are protected from subsequent induction of EAU.46 Because infusion of ACAID-induced suppressor cells can inhibit the effector stage of EAU and abort ongoing disease,47 the therapeutic potential of this form of immunotherapy is obvious. Furthermore, investigators have shown

258

Endocrine and Organ Specific Autoimmunity

the regulatory cells induced by injecting alloantigens in the anterior chamber can persist for at least 6 months, suggesting that suppression induced by injecting antigen in the anterior chamber is a long lasting phenomenon.94

Intravenous (iv) Injection of Antigen Intravenous (iv) injection of an antigen before immunization is a potent way to induce suppression at the T cell level. This approach has been used successfully to prevent the development of hen egg lysozyme (HEL)-induced arthritis, neuritis and uveitis.27,95 It has been shown that the tolerogenic stimulus is more potent when the triggering antigen is given iv coupled to syngeneic cells.95-97 Alternatively, the targeting of antigen to B cells by covalently linking a uveitogenic peptide to anti-IgD antibody inhibits EAU in rats.98 This procedure targets B cells as the preferential APCs for the antibody-linked antigen and ameliorates uveitis through the preferential induction of Th2 cells to the antigen (see below for further explanation on the putative mechanism). Unfortunately, in most instances tolerance induced by iv injection of antigen was much more effective in preventing disease when given prior to the pathology-inducing antigenic challenge27,28,96 which would significantly impair the use of such approaches in clinical medicine. Because humans are an outbred population, it is likely that autoimmunity in different individuals is elicited by different epitopes or even different antigens. Hence, it becomes important to determine if induction of tolerance against one antigen can result in tolerance to other antigens present in the same organ. This phenomenon is called bystander suppression and it is based on the fact that although a suppressor cell that secretes anti-inflammatory cytokines does so in response to stimulation by specific antigen, these cytokines are not antigen-specific and will affect any cell in the vicinity. It has been reported that animals tolerized by iv immunization do not present bystander suppression.99,100

T cell Vaccination, TCR-Peptide Vaccination An approach used to target the antigen-specific pathogenic T cells has been to immunize susceptible animals with syngeneic attenuated pathogenic T cells or their cloned receptors. This approach was successfully employed in EAE and was the focus of great interest.101 In EAU, injections of sub-pathogenic doses of a T helper (Th) cell line specific to a major epitope of IRBP was able to protect rats partially from subsequent challenge with a uveitogenic dose of the same cells.102 Vaccination against T cell-receptor (TCR) epitopes of the Vβ8.2 family, which has been implicated as a pathogenic T cell clonotype in EAU was minimally effective in protecting from S-Ag-induced EAU and was ineffective in protecting from IRBPinduced EAU.103-109 In contrast, an identical protocol was shown to suppress both the afferent and the efferent phases of EAE.110,111 This might be due to the recruitment of unrelated T cells having a more crucial role in the development of pathology in EAU as compared to EAE.112-114 This variability in the outcome of the same vaccination protocol underscores the caution that must be used when attempting to manipulate delicately balanced immunological systems.

T cell Targeting Drugs The macrolide antibiotics cyclosporin A (CsA) and FK506 are agents that act primarily by suppressing an early stage in T cell activation, by blocking nuclear binding factors involved in lymphokine gene transcription and other early activation genes.115 A new drug entering this group of anti-T cell macrolides is rapamycin. Unlike CsA and FK506, rapamycin targets a later stage of T cell activation, arresting T cells prior to entry into the S-phase by inhibiting the IL-2-stimulated expression of p34cdc2, a serine/threonine kinase required for cells to progress through the cell cycle.116 These three drugs are effective either alone or

Ocular Autoimmunity

259

in certain combinations (CsA + Rapamycin) in the treatment of EAU as well as human uveitis.117-121 Some studies have shown that all three drugs can induce long lasting tolerance to allograft rejection even after discontinuation of the drug treatment. In most cases it has been shown that such tolerance is dependent upon the persistence of the antigen.122-129 In uveitis, there are reports suggesting that FK506 and CsA might induce antigen-specific suppressor cells.130,131 However, several other reports suggest that disease recurs, and may even rebound, when therapy is suspended or the drug doses are rapidly reduced.131-134 The downside to any of these drugs is the systemic toxicity associated with therapeutic doses and the fact that they do not specifically target the pathogenic cells. The fact that rapamycin acts on a different stage of disease than CsA or FK506 can, however, be exploited to design tolerance-inducing protocols that would also require lesser doses of the combined drugs.125

Perturbation of Th1/Th2 Balance It has been proposed that, in T cell-mediated autoimmune diseases, the pathogenic cell is of the Th1 phenotype. Given the known cross-regulatory mechanisms that are involved in the in vivo relationship between these two subsets of Th cells18,19,135,136 it is conceivable that enhancement of the Th2 component of the response against the antigen involved in the triggering of autoimmunity should control the pathogenic process. It has been reported that giving recombinant IL-4 to animals afflicted with EAE resulted in amelioration of clinical disease, induction of MBP-specific Th2 cells, diminished demyelination, and inhibition of the synthesis of inflammatory cytokines in the central nervous system.137 We have shown that IL-10 when given in the afferent phase of disease is effective in blocking uveitis development and that the combination with IL-4 is even more effective (manuscript in preparation). Nevertheless, in our hands IL-4 was ineffective in protecting against the development of EAU in mice. It is important to point out that the treatment of rats with IL-4 has been shown to increase disease scores in EAU.138 Taken together these results suggest that although the use of the so called anti-inflammatory cytokines may present some promise in the treatment of uveitis we should proceed with caution because of the intricacies of the cytokine network.

Idiotypic Network Manipulation

The idiotypic network theory proposed by Jerne in 1984139 suggests that each new antigen-recognizing molecule induced by an immune response comprises a new antigen never seen by the immune system and elicits an anti-idiotypic response that in turn will also induce an anti-anti-idiotypic response with similar characteristics to the initial antigenspecific response. Evidence for idiotypic regulation of ocular autoimmunity has been presented based on the ability of preimmunization with monoclonal antibodies (id) directed against a particular epitope of S-Ag to suppress S-Ag-induced uveitis.123,124 A similar effect could also be elicited by preimmunization with polyclonal anti-id antibodies.124 Protection could be transferred from antibody-treated donors with T cells but not with the γ-globulin fraction of the sera;122 and lymph node cells from antibody-treated mice were able to inhibit the proliferation of S-Ag primed cells in vitro. Interestingly, the target epitope of the antibody used to elicit the anti-idiotypic response is a nonpathogenic epitope located at the opposite end of the S-Ag molecule from the currently known pathogenic epitopes.

Oral Administration of Retinal Antigens Oral tolerance is the phenomenon by which contact with antigen through the mucosal tissues results in decreased responses to the same antigen when presented later by parenteral immunization. The ability of the gut associated lymphoid tissue (GALT) to effect this regu-

260

Endocrine and Organ Specific Autoimmunity

latory function has been recognized for many years.140 It is noteworthy that this property is not restricted to the gut mucosa but inherent to any mucosal surface in the body such as the one surrounding the respiratory tract and the genitourinary tract.141-144 The ability of orally induced tolerance to prevent the development of autoimmune diseases was first explored in EAE and the findings were expanded to EAU. 9,145-147 The intragastric administration S-Ag or IRBP was able to prevent or attenuate subsequent development of EAU after a parenteral challenge with pathogenic doses of the corresponding proteins and a phase I/II clinical trial in the treatment of uveitis by oral administration of retinal antigens have just been completed with encouraging results.148 The mechanisms by which oral tolerance is achieved are unclear. It has been suggested that feeding of small amounts of protein would result in tolerance by active suppression, whereas a high dose would result in anergy or deletion in a similar fashion to tolerance induced by iv injection of antigen.146,149 Other factors such as the frequency of administration, the nature of the antigen, and the use of substances that prevent or retard protein degradation may also help to determine which of the two mechanisms will develop.147,150,151 It was also proposed that inhibition of systemic DTH after feeding proteins is due to active suppression whereas inhibition of systemic humoral immunity may result from T cell anergy.150,152,153 The existence and nature of the suppressor cells involved in oral tolerance is still under investigation. In the rat depletion of CD8+ cells has been shown to affect the ability to adoptively transfer protection from orally tolerized animals to naive animals.154 However, more recently, we have shown that oral tolerance can develop in the absence of CD8+ cells.155 We have reported that anti-inflammatory cytokines IL-4, IL-10 and TGF-β may be involved in the development of oral tolerance in EAU,147 suggesting that cells of the Th2 subset may act as the regulatory cells in this system. Interestingly, the use of oral tolerance was first proposed as an approach to diminish allergic reactions and to decrease IgE production156,157 which is contradictory with the hypothesis that Th2 type responses are preferentially elicited when antigens are introduced per os. As stated above, it is likely that in different individuals different antigens may be responsible for the induction of autoimmunity. Consequently, the use of oral tolerization in the clinic would only be possible under either one of two conditions: the eliciting antigen is known (in this case treatment may need to be customized for each patient) or, feeding of an antigen present in the target organ and likely to be exposed during the inflammatory process can generate suppressor cells secreting “antigen-nonspecific” anti-inflammatory cytokines which acting locally would decrease inflammation. This phenomenon (bystander suppression) is of utmost importance from the clinical point of view since in most cases the triggering antigen in human autoimmune diseases is unknown. Although bystander suppression has been shown in different models of EAE,158 we and others have been unable to show the same phenomenon in EAU.19,146

Anti-IL-2 Receptor Therapy The concept behind anti-IL-2 receptor (CD25) therapy is similar to that of the anti-T cell drugs described above, the idea being to disturb the expansion of activated autoreactive cells and interrupt the autoimmune process. Because all activated T cells express the CD25, the α-chain of the tri-chain receptor for IL-2, targeting this molecule offers the possibility to permanently eliminate all autoreactive cells that are contributing to disease pathology at the time of treatment. This can be achieved through cytotoxic anti-CD25 antibodies or through toxins coupled to the anti-CD25 antibodies or IL-2 itself. Potential limitations of such therapy are the lack of complement binding, high immunogenicity of the anti-CD25 antibody and consequent toxicity. These undesirable effects can be overcome by the proper molecular

Ocular Autoimmunity

261

manipulation of such compounds.159 Two variants of anti-CD25 targeting therapy were tested in the Lewis rat model of uveitis, a recombinant chimeric toxin (IL-2-PE40) constructed of the IL-2-binding domain and pseudomonas exotoxin and the anti-CD25 monoclonal antibody ART18.160,161 In both cases treatment was effective in protecting against the development of uveitis in the efferent stage of diseases. Results suggest that the uveitogenic lymphocytes were eliminated by treatment. The combination of subtherapeutic doses of CsA and ART18 antibody was particularly effective. Recently the genetically engineered anti-CD25 and anti-CD122 (anti-TAC and anti-Micβ) were tested successfully in a primate model of uveitis162 and are currently being tested in a phase I/II clinical trial at the uveitis clinic of the National Eye Institute.

References 1. Nussenblatt RB, Withcup SM, Palestine AG. Uveitis: Fundamentals and Clinical Practice. 2nd ed. St. Louis: Mosby, 1996. 2. Whitcup SM, Nussenblatt RB. Treatment of autoimmune uveitis. Ann N Y Acad Sci 1993; 696:307-18. 3. Uhlenhuth PT. Zur lehre von der unterscheidung verschiedener eiweissarten mit hilfe spezifixher sera. Fetschritf zum 60 geburstag von Robert Koch. Jena: Fisher, Germany, 1903:49-74. 4. Elschnig A. Albrecht von Graefes Arch. Ophthalmol 1910; 76:509-46. 5. Nossal GJ. Negative selection of lymphocytes. Cell 1994; 76(2):229-39. 6. Pardoll D, Carrera A. Thymic selection. Curr Opin Immunol 1992; 4(2):162-5. 7. Robey E, Fowlkes BJ. Selective events in T cell development. Annu Rev Immunol 1994; 12:675-705. 8. Fowlkes BJ, Ramsdell F. T cell tolerance. Curr Opin Immunol 1993; 5(6):873-9. 9. Streilein JW. Unraveling immune privilege [comment]. Science 1995; 270(5239):1158-9. 10. Streilein JW. Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments. Curr Opin Immunol 1993; 5(3):428-32. 11. Barker C, Billingham R. Immunological privileged sites. Adv Immunol 1977; 25:1-54. 12. Matzinger P. Tolerance, danger, and the extended family. Annu Rev Immunol 1994; 12:991-1045. 13. Jones DE, Diamond AG. The basis of autoimmunity: an overview. Baillieres Clin Endocrinol Metab 1995; 9(1):1-24. 14. Roitt IM. The role of autoantigens in the driving of autoimmune diseases. Immunol Ser 1993; 59:119-29. 15. Schwartz RS, Datta SK. Autoimmunity and Autoimmune diseases. In: Paul WE, ed. Fundamental Immunology. Third ed. New York: Raven Press, 1995: 819-866. 16. Steinman L. Escape from horror autotoxicus: pathogenesis and treatment of autoimmune disease. Cell 1995; 80(1):7-10. 17. Theofilopoulos AN. The basis of autoimmunity: Part I. Mechanisms of aberrant self-recognition. Immunol Today 1995; 16(2):90-8. 18. Rizzo LV, Caspi RR. Immunotolerance and prevention of ocular autoimmune disease. Curr Eye Res 1995; 14(9):857-64. 19. Rizzo LV. Estudos sobre a imunonopatogenese e imunoterapia da uveite experimental autoimune. Ph.D.: University of Sao Paulo, 1997. 20. Charukamnoetkanok P, Fukushima A, Whitcup SM, Gery I, Egwagu CE. Correlation between thymic expression of immunopathogenic retinal antigens and resistance to autoimmune disease. Tolerance and Autoimmunity 1997, Keystone, Colorado. 21. Hirose S, Tanaka T, Nussenblatt RB, et al. Lymphocyte responses to retinal-specific antigens in uveitis patients and healthy subjects. Curr Eye Res 1988; 7(4):393-402.

262

Endocrine and Organ Specific Autoimmunity

22. Hirose S, McAllister C, Mittal K, Vistica B, Shinohara T, Gery I. A cell line and clones of lymphocytes from a healthy donor with specificity to S-antigen. Invest Opht Vis Sci 1988; 29:1636-1641. 23. Medawar PB. Immunity to homologous grafted skin. III. The fate of skin homografts transplanted to the brain, the subcutaneous tissue, and to the anterior chamber of the eye. Br. J. Exp. Pathol. 1948; 29:58-69. 24. Kaplan HJ, Streilein JW. Immune response to immunization via the anterior chamber of the eye. I. F. lymphocyte-induced immune deviation. J Immunol 1977; 118(3):809-14. 25. Kaplan HJ, Streilein JW. Immune response to immunization via the anterior chamber of the eye. II. An analysis of F1 lymphocyte-induced immune deviation. J Immunol 1978; 120(3):689-93. 26. Griffith TS, Brunner T, Fletcher SM, Green DR, Ferguson TA. Fas ligand-induced apoptosis as a mechanism of immune privilege [see comments]. Science 1995; 270(5239):1189-92. 27. Jacobs MJ, van den Hoek AE, van de Putte LB, van den Berg WB. Anergy of antigenspecific T lymphocytes is a potent mechanism of intravenously induced tolerance. Immunology 1994; 82(2):294-300. 28. Jacobs MJ, van den Hoek AE, van de Putte LB, van den Berg WB. Suppression of hen egg lysozyme-induced arthritis by intravenous antigen administration: No role in this for antigen-driven bystander suppression. Clin Exp Immunol 1994; 96(1):36-42. 29. Wilbanks GA, Mammolenti M, Streilein JW. Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta. Eur J Immunol 1992; 22:165-173. 30. Wilbanks GA, Streilein JW. Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-beta. Eur J Immunol 1992; 22:1031-1036. 31. Wilbanks GA, Streilein JW. Studies on the induction of anterior chamber-associated immune deviation (ACAID). 1. Evidence that an antigen-specific, ACAID-inducing, cell-associated signal exists in the peripheral blood. J Immunol 1991; 146(8):2610-7. 32. Taylor AW, Streilein JW, Cousins SW. Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor. J Immunol 1994; 153(3):1080-6. 33. Streilein JW. Ocular immune privilege and the Faustian dilemma. The Proctor lecture. Invest Ophthalmol Vis Sci 1996; 37(10):1940-50. 34. Streilein JW. Immunological nonresponsiveness and acquisition of tolerance in relation to immune privilege in the eye. Eye 1995; 9(Pt 2):236-40. 35. Shimada K. The complement components and their inactivators in the intraocular fluids of the guinea pig. Invest Ophthalmol 1970; 9(4):307-15. 36. Bora NS, Gobleman CL, Atkinson JP, Pepose JS, Kaplan HJ. Differential expression of the complement regulatory proteins in the human eye. Invest Ophthalmol Vis Sci 1993; 34(13):3579-84. 37. Roberge FG, Caspi RR, Chan CC, Nussenblatt RB. Interactions between retinal glial cells and immune mononuclear cells. Yen Ko Hsueh Pao 1986; 2(2):101-2. 38. Roberge FG, Caspi RR, Chan CC, Nussenblatt RB. Inhibition of T lymphocyte proliferation by retinal glial Muller cells: Reversal of inhibition by glucocorticoids. J Autoimmun 1991; 4(2):307-14. 39. Liversidge J, McKay D, Mullen G, Forrester JV. Retinal pigment epithelial cells modulate lymphocyte function at the blood-retina barrier by autocrine PGE2 and membrane-bound mechanisms. Cell Immunol 1993; 149(2):315-30. 40. Helbig H, Gurley RC, Palestine AG, Nussenblatt RB, Caspi RR. Dual effect of ciliary body cells on T lymphocyte proliferation. Eur J Immunol 1990; 20(11):2457-63. 41. Caspi RR, Roberge FG. Glial cells as suppressor cells: Characterization of the inhibitory function. J Autoimmun 1989; 2(5):709-22. 42. Caspi RR, Roberge FG, Nussenblatt RB. Organ-resident, nonlymphoid cells suppress proliferation of autoimmune T-helper lymphocytes. Science 1987; 237(4818):1029-32.

Ocular Autoimmunity

263

43. Kaplan HJ, Streilein JW. Do immunologically privileged sites require a functioning spleen? Nature 1974; 251(5475):553-4. 44. Kaplan HJ, Streilein JW, Stevens TR. Transplantation immunology of the anterior chamber of the eye. II. Immune response to allogeneic cells. J Immunol 1975; 115(3):805-10. 45. Kaplan HJ, Stevens TR, Streilein JW. Transplantation immunology of the anterior chamber of the eye. I. An intraocular graft-vs-host reaction (immunogenic anterior uveitis). J Immunol 1975; 115(3):800-4. 46. Hara Y, Caspi RR, Wiggert B, Dorf M, Streilein JW. Analysis of an in vitro-generated signal that induces systemic immune deviation similar to that elicited by antigen injected into the anterior chamber of the eye [published erratum appears in J Immunol 1992 Dec 15; 149(12):4116]. J Immunol 1992; 149(5):1531-8. 47. Hara Y, Caspi RR, Wiggert B, Chan CC, Streilein JW. Use of ACAID to suppress interphotoreceptor retinoid binding protein-induced experimental autoimmune uveitis. Curr Eye Res 1992; 11 Suppl:97-100. 48. Hara Y, Caspi RR, Wiggert B, Chan CC, Wilbanks GA, Streilein JW. Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber-associated immune deviation with interphotoreceptor retinoid-binding protein. J Immunol 1992; 148(6):1685-92. 49. Mizuno K, Clark AF, Streilein JW. Induction of anterior chamber associated immune deviation in rats receiving intracameral injections of retinal S antigen. Curr Eye Res 1988; 7(6):627-32. 50. Kleinau S, Lorentzen J, Klareskog L. Role of adjuvants in turning autoimmunity into autoimmune disease. Scand J Rheumatol Suppl 1995; 101:179-81. 51. Sasaki K, Sanui H, Inomata H. Uveitis induced by various cross-reactive antigens in guinea pigs. Ophthalmic Res 1990; 22(5):330-6. 52. Weiner HL, Zhang ZJ, Khoury SJ et al. Antigen-driven peripheral immune tolerance. Suppression of organ-specific autoimmune diseases by oral administration of autoantigens. Ann N Y Acad Sci 1991; 636:227-32. 53. Wacker WB, Rao NA, Marak G, Jr. Experimental sympathetic ophthalmia. pp. 121-6. In: Silverstein Am, O’Connor Gr, ed. Immunology and immunopathology of the eye. New York, Masson, 1979. 54. Parish NM, Cooke A. Animal models of autoimmune endocrine disease and their uses in developing new methods of intervention. Baillieres Clin Endocrinol Metab 1995; 9(1):175-98. 55. Signore A, Procaccini E, Chianelli M, Pozzilli P. Retroviruses and diabetes in animal models: hypotheses for the induction of the disease. Diabete Metab 1995; 21(3):147-55. 56. Swanborg RH. Experimental autoimmune encephalomyelitis in rodents as a model for human demyelinating disease [see comments]. Clin Immunol Immunopathol 1995; 77(1):4-13. 57. Wacker WB, Lipton MM. Experimental allergic uveitis: Homologous retina as uveitogenic antigen. Nature 1965; 206(981):253-4. 58. Wacker WB, Donoso LA, Kalsow CM, Yankeelov J, Jr., Organisciak DT. Experimental allergic uveitis. Isolation, characterization, and localization of a soluble uveitopathogenic antigen from bovine retina. J Immunol 1977; 119(6):1949-58. 59. Gery I, Streilein JW. Autoimmunity in the eye and its regulation. Curr Opin Immunol 1994; 6(6):938-45. 60. Howes E, Jr., McKay DG, Aronson SB. An ultrastructural study of the ciliary process in the rabbit following systemic administration of bacterial endotoxin. Lab Invest 1971; 24(3):217-28. 61. Bonnet P, Faure JP, Le Douarec JC. Standardization of an experimental immune uveitis in the rabbit for topical testing of drugs. Mod Probl Ophthalmol 1976; 16:285-304. 62. Gamble CN, Aronson SB, Brescia FB. Experimental uveitis. I. The production of recurrent immunologic (Auer) uveitis and its relationship to increased uveal vascular permeability. Arch Ophthalmol 1970; 84(3):321-30. 63. Uusitalo H. An experimental uveitis induced by bovine serum albumin. A transmission and scanning electron microscopic study. Acta Ophthalmol (Copenh) 1984; 62(3):413-24.

264

Endocrine and Organ Specific Autoimmunity

64. Bora NS, Kim MC, Kabeer NH et al. Experimental autoimmune anterior uveitis. Induction with melanin-associated antigen from the iris and ciliary body. Invest Ophthalmol Vis Sci 1995; 36(6):1056-66. 65. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune anterior uveitis (EAAU): induction by melanin antigen and suppression by various treatments. Pigment Cell Res 1993; 6(1):1-6. 66. Broekhuyse RM, Kuhlmann ED. Experimental autoimmune anterior uveitis. The preparation of uveitogenic ocular melanin. Invest Ophthalmol Vis Sci 1993; 34(3):698-700. 67. Francois J, Ringoir S, Matton-Van Leuven MT, Da Silva DM, Albarran E. [Melanin of the uvea and autoimmunity]. Bull Mem Soc Fr Ophtalmol 1970; 83:125-38. 68. Rao NA, Marak GE, Jr. Experimental granulomatous uveitis: An electron microscopic study of pigment containing giant cells. Invest Ophthalmol Vis Sci 1985; 26(9):1303-5. 69. Dua HS, Lee RH, Lolley RN, et al. Induction of experimental autoimmune uveitis by the retinal photoreceptor cell protein, phosducin. Curr Eye Res 1992; 11 Suppl:107-11. 70. Schalken JJ, Winkens HJ, van Vugt AH, Bovee-Geurts PH, de Grip WJ, Broekhuyse RM. Rhodopsin-induced experimental autoimmune uveoretinitis: Dose-dependent clinicopathological features. Exp Eye Res 1988; 47(1):135-45. 71. Schalken JJ, van Vugt AH, Winkens HJ, Bovee-Geurts PH, De Grip WJ, Broekhuyse RM. Experimental autoimmune uveoretinitis in rats induced by rod visual pigment: Rhodopsin is more pathogenic than opsin. Graefes Arch Clin Exp Ophthalmol 1988; 226(3):255-61. 72. Schalken JJ, Winkens HJ, Van Vugt AH, De Grip WJ, Broekhuyse RM. Rhodopsin-induced experimental autoimmune uveoretinitis in monkeys. Br J Ophthalmol 1989; 73(3):168-72. 73. Gery I, Chanaud NPr, Anglade E. Recoverin is highly uveitogenic in Lewis rats. Invest Ophthalmol Vis Sci 1994; 35(8):3342-5. 74. Gery I, Mochizuki M, Nussemblatt RB. Retinal specific antigens an immunopathogenic processes they provoke. Prog Retinal Res 1986; 5:75-109. 75. Fox GM, Kuwabara T, Wiggert B et al. Experimental autoimmune uveoretinitis (EAU) induced by retinal interphotoreceptor retinoid-binding protein (IRBP): Differences between EAU induced by IRBP and by S-antigen. Clin Immunol Immunopathol 1987; 43(2):256-64. 76. Caspi RR, Roberge FG, Chan CC et al. A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J Immunol 1988; 140(5):1490-5. 77. Nussenblatt RB, Gery I, Ballintine EJ, Wacker WB. Cellular immune responsiveness of uveitis patients to retinal S-antigen. Am J Ophthalmol 1980; 89(2):173-9. 78. Nussenblatt RB, Mittal KK, Ryan S, Green WR, Maumenee AE. Birdshot retinochoroidopathy associated with HLA-A29 antigen and immune responsiveness to retinal S-antigen. Am J Ophthalmol 1982; 94(2):147-58. 79. Predengast RA, Coskuncan NM, Lutty GA, McLeod DS, Caspi RR. T cell traffic and the pathogenesis of experimental autoimmune uveoretinitis. 6th International Symposium on the Immunology and Immunopathology of the Eye 1994, Bethesda, USA: 59-62. 80. Nussenblatt RB, Gery I. Experimental autoimmune uveitis and its relationship to clinical ocular inflammatory disease. J Autoimmun 1996; 9(5):575-85. 81. Rizzo LV, Silver P, Wiggert B et al. Establishment and characterization of a murine CD4+ T cell line and clone that induce experimental autoimmune uveoretinitis in B10.A mice. J Immunol 1996; 156(4):1654-1660. 82. Chan CC, Mochizuki M, Palestine AG, BenEzra D, Gery I, Nussenblatt RB. Kinetics of Tlymphocyte subsets in the eyes of Lewis rats with experimental autoimmune uveitis. Cell Immunol 1985; 96(2):430-4. 83. Chan CC, Benezra D, Rodrigues MM et al. Immunohistochemistry and electron microscopy of choroidal infiltrates and Dalen-Fuchs nodules in sympathetic ophthalmia. Ophthalmology 1985; 92(4):580-90. 84. Whitcup SM, Chan CC, Li Q, Nussenblatt RB. Expression of cell adhesion molecules in posterior uveitis. Arch Ophthalmol 1992; 110(5):662-6. 85. Broekhuyse RM, Kuhlmann ED, Winkens HJ. Experimental autoimmune posterior uveitis accompanied by epitheloid cell accumulations (EAPU). A new type of experimental ocular

Ocular Autoimmunity

265

disease induced by immunization with PEP-65, a pigment epithelial polypeptide preparation. Exp Eye Res 1992; 55(6):819-29. 86. Caspi RR, Grubbs BG, Chan CC, Chader GJ, Wiggert B. Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes. J Immunol 1992; 148(8):2384-9. 87. Wetzig RP, Chan CC, Nussenblatt RB, Palestine AG, Mazur DO, Mittal KK. Clinical and immunopathological studies of pars planitis in a family. Br J Ophthalmol 1988; 72(1):5-10. 88. Ichikawa T, Taguchi O, Takahashi T et al. Spontaneous development of autoimmune uveoretinitis in nude mice following reconstitution with embryonic rat thymus. Clin Exp Immunol 1991; 86:112-117. 89. Caspi RR. Immunogenetic aspects of clinical and experimental uveitis. Reg Immunol 1992; 4(5):321-30. 90. Caspi RR, Silver PB, Chan CC et al. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response. J Immunol 1996; 157(6):2668-75. 91. Sun B, Rizzo LV, Sun S-H, Caspi RR. Genetic susceptibility to experimental autoimmune uveitis involves more than a predisposition to generate a Th1-like or a Th2-like response. J. Immunol 1997; in press. 92. Streilein JW. Peripheral tolerance induction: Lessons from immune privileged sites and tissues. Transplant Proc 1996; 28(4):2066-70. 93. Streilein JW, Niederkorn JY. Induction of anterior chamber-associated immune deviation requires an intact, functional spleen. J Exp Med 1981; 153(5):1058-67. 94. Bando Y, Ksander BR, Streilein JW. Incomplete activation of lymphokine-producing T cells by alloantigenic intraocular tumors in anterior chamber-associated immune deviation. Immunology 1993; 78(2):266-72. 95. Dua HS, Gregerson DS, Donoso LA. Inhibition of experimental autoimmune uveitis by retinal photoreceptor antigens coupled to spleen cells. Cell Immunol 1992; 139(2):292-305. 96. Gregorian SK, Rostami A. Delayed-type hypersensitivity response in experimental autoimmune neuritis treated with peptide-coupled spleen cells. J Neuroimmunol 1994; 51(1):69-75. 97. Gregorian SK, Lee WP, Beck LS, Rostami A, Amento EP. Regulation of experimental autoimmune neuritis by transforming growth factor-beta 1. Cell Immunol 1994; 156(1):102-12. 98. Kozhich AT, Whitcup SM, Gery I. Amelioration of cell-mediated autoimmune eye disease by targeting the uveitogenic antigen to B cells. Invest Ophthalmol Vis Sci 1997; 38(4):S438. 99. Eynon EE, Parker DC. Small B cells as antigen-presenting cells in the induction of tolerance to soluble protein antigens. J Exp Med 1992; 175(1):131-8. 100. Eynon EE, Parker DC. Parameters of tolerance induction by antigen targeted to B lymphocytes. J Immunol 1993; 151(6):2958-64. 101. Lider O, Reshef T, Beraud E, Ben-Nun A, Cohen IR. Anti-idiotypic network induced by T cell vaccination against experimental autoimmune encephalomyelitis. Science 1988; 239:181-183. 102. Beraud E, Kotake S, Caspi RR, et al. Control of experimental autoimmune uveoretinitis by low dose T cell vaccination. Cell Immunol 1992; 140(1):112-22. 103. Merryman CF, Donoso LA, Zhang XM, Heber-Katz E, Gregerson DS. Characterization of a new, potent, immunopathogenic epitope in S-antigen that elicits T cell expressing V beta 8 and V alpha 2-like genes. J Immunol 1991; 146:75-80. 104. Gregerson DS, Fling SP, Merryman CF, Zhang XM, Li XB, Heber-Katz E. Conserved T cell receptor V gene usage by uveitogenic T cells. Clin Immunol Immunopathol 1991; 58:154-161. 105. Egwuagu CE, Chow C, Beraud E et al. T cell receptor beta-chain usage in experimental autoimmune uveoretinitis. J Autoimmun 1991; 4(2):315-24. 106. Egwuagu CE, Bahmanyar S, Mahdi RM, Nussenblatt RB, Gery I, Caspi RR. Predominant usage of V beta 8.3 T cell receptor in a T cell line that induces experimental autoimmune uveoretinitis (EAU). Clin Immunol Immunopathol 1992; 65(2):152-60.

266

Endocrine and Organ Specific Autoimmunity

107. Egwuagu CE, Mahdi RM, Nussenblatt RB, Gery I, Caspi RR. Evidence for selective accumulation of V beta 8+ T lymphocytes in experimental autoimmune uveoretinitis induced with two different retinal antigens. J Immunol 1993; 151(3):1627-36. 108. Kawano Y, Sasamoto Y, Kotake S, Thurau SR, Wiggert B, Gery I. Trials of vaccination against experimental autoimmune uveoretinitis with a T cell receptor peptide. Curr Eye Res 1991; 10(769-795). 109. Kawano Y, Sasamoto Y, Vacchio MS, Hodes RJ, Gery I. Immune responses against selfTCR peptides. Cell Immunol 1994; 159(2):235-45. 110. Burns FR, Li XB, Shen N et al. Both rat and mouse T cell receptors specific for the encephalitogenic determinant of myelin basic protein use similar V alpha and V beta chain genes even though the major histocompatibility complex and encephatlitogenic determinants being recognized are different. J Exp Med 1989; 169:27-39. 111. Vanderbark AA, Hashim G, Offner H. Immunization with a synthetic T cell receptor Vregion peptide protects against experimental autoimmune ecephalomyelites. Nature 1989; 341:541-544. 112. Sedgwick J, Brostoff S, Mason D. Experimental allergic encephalomyelitis in the absence of a classical delayed-type hypersensitivity reaction. Severe paralytic disease correlates with the presence of interleukin 2 receptor-positive cells infiltrating the central nervous system. J Exp Med 1987; 165:1058-1075. 113. Caspi RR, Chan CC, Fujino Y et al. Recruitment of antigen-nonspecific cells plays a pivotal role in the pathogenesis of a T cell-mediated organ-specific autoimmune disease, experimental autoimmune uveoretinitis. J Neuroimmunol 1993; 47(2):177-88. 114. Kim MK, Caspi RR, Nussenblatt RB, Kuwabara T, Palestine AG. Intraocular trafficking of lymphocytes in locally induced experimental autoimmune uveoretinitis (EAU). Cell Immunol 1988; 112(2):430-6. 115. Schreiber SL, Crabtree GR. The mechanism of action of cyclosporin A and FK 506. Immunol Today 1992; 13:136-142. 116. Flanagan WM, Crabtree GR. Rapamycin inhibits p34cdc2 expression and arrests T lymphocyte proliferation at the G1/S transition. Ann N Y Acad Sci 1993; 696:31-37. 117. Nussenblatt RB, Salinas-Carmona M, Waksman BH, Gery I. Cyclosporin A: alterations of the cellular immune response in S-antigen-induced experimental autoimmune uveitis. Int Arch Allergy Appl Immunol 1983; 70(4):289-94. 118. Nussenblatt RB, Palestine AG, Chan CC. Cyclosporin A therapy in the treatment of intraocular inflammatory disease resistant to systemic corticosteroids and cytotoxic agents. Am J Ophthalmol 1983; 96(3):275-82. 119. Nussenblatt RB, Palestine AG, Rook AH, Scher I, Wacker WB, Gery I. Treatment of intraocular inflammatory disease with cyclosporin A. Lancet 1983; 2(8344):235-8. 120. Fujino Y, Chan CC, de Smet MD et al. FK 506 treatment of experimental autoimmune uveoretinitis in primates. Transplant Proc 1991; 23(6):3335-8. 121. Roberge FG, Xu D, Chan CC, de Smet MD, Nussenblatt RB, Chen H. Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibitor of lymphocyte growth factor signal transduction. Curr Eye Res 1993; 12:197-203. 122. de Kozak Y, Mirshahi M, Boucheix C, P. FJ. Modulation of experimental autoimmune uveoretinitis by adoptive transfer of cells from rats immunized with anti-S antigen monoclonal antibody. Reg Immunol 1989; 2:311-320. 123. de Kozak Y, Mirshahi M. Experimental autoimmune uveoretinitis: idiotypic regulation and disease suppression. Int Ophthalmol 1990; 14:43-56. 124. de Kozak Y. Regulation of retinal autoimmunity via the idiotypic network. Curr Eye Res 1990; 9(Suppl):193-200. 125. Chavin KD, Qin L, Woodward JE, Lin J, Bromberg JS. Anti-CD2 monoclonal antibodies synergize with FK506 but not with cyclosporine or rapamycin to induce tolerance. Transplantation 1994; 57(5):736-740. 126. Granger DK, Matas AJ, Jenkins MK, Moss AA, Chen SC, Almond PS. Prolonged survival without post-transplant immunosuppression in a large animal model. Surgery 1994; 116(2):236-241.

Ocular Autoimmunity

267

127. Chen H, Luo H, Daloze P, Xu D, Wu J. Rapamycin-induced long-term allograft survival depends on persistence of alloantigen. J Immunol 1994; 152(6):3107-3118. 128. Strasser S, Alejandro R. Cyclosporine-induced immune unresponsiveness in dogs with intrasplenic islet allografts. Transplantation 1992; 52(5):916-918. 129. Martin DF, DeBarge LR, Nussenblatt RB, Chan CC, Roberge FG. Synergistic effect of rapamycin and cyclosporin A in the treatment of experimental autoimmune uveoretinitis. J Immunol 1995; 154(2):922-7. 130. Fujino Y, Okumura A, Nussenblatt RB, Gery I, Mochizuki M. Cyclosporine-induced specific unresponsiveness to retinal soluble antigen in experimental autoimmune uveoretinitis. Clin Immunol Immunopathol 1988; 46(2):234-48. 131. Kawashima H, Fujino Y, Mochizuki M. Antigen-specific suppressor cells induced by FK506 in experimental autoimmune uveoretinitis in the rat. Invest Ophthalmol Vis Sci 1990; 31:2500-2507. 132. Sai P, Maugendre D, Loreal O, Maurel C, Pogu S. Effects of cyclosporin on autoimmune diabetes induced in mice by streptozocin: Beta cell-toxicity and rebound of insulinitis after cessation of treatment. Diabetes Metab 1988; 14:455-462. 133. Nussenblatt RB, Rodrigues MM, Salinas-Carmona MC, Gery I, Cevario S, Wacker W. Modulation of experimental autoimmune uveitis with cyclosporin A. Arch Ophthalmol 1982; 100(7):1146-9. 134. Fite KV, Pardue S, Bengston L, Hayden D, Smyth JJ. Effects of cyclosporine in spontaneous, posterior uveitis. Curr Eye Res 1986; 5:787-796. 135. Rizzo LV, DeKruyff RH, Umetsu DT, Caspi RR. Regulation of the interaction between Th1 and Th2 T cell clones to provide help for antibody production in vivo. Eur J Immunol 1995; 25(3):708-16. 136. Rizzo LV. The subsets of T helper cells involved in the development of experimental autoimmune uveoretinitis. Ophthalmol McGill 1994; 6(3):5-8. 137. Racke MK, Bonomo A, Scott DE et al. Cytokine-induced immune deviation as a therapy for inflammatory autoimmune disease. J Exp Med 1994; 180(5):1961-6. 138. Ramanathan S, de Kozak Y, Saoudi A et al. Recombinant IL-4 aggravates experimental autoimmune uveoretinitis in rats. J Immunol 1996; 157(5):2209-15. 139. Jerne NK. Idiotypic networks and other preconceived ideas. Immunol Rev 1984; 79:5-24. 140. Dakin R. Remarks on a cutaneous affection, produced by certain poisonous vegetables. Am J Med Sci (old series) 1829; 4:98-100. 141. Kagnoff MF. Immunology of the intestinal tract. Gastroenterol 1993; 105:1275-1280. 142. Keren DF. Antigen processing in the mucosal immune system. Semin Immunol 1992; 4(4):217-26. 143. Laissue JA, Borisch Chappuis B, Müller C, Reubi JC, Gebbers J-O. The intestinal immune system and its relation to disease. Dig Dis 1993; 11:298-312. 144. Scott H, Halstensen TS, Brandtzaeg P. The immune system of the gastrointestinal tract. Pediatr Allergy Immunol 1993; 4 (suppl 3):7-15. 145. Nussenblatt RB, Caspi RR, Mahdi R et al. Inhibition of S-antigen induced experimental autoimmune uveoretinitis by oral induction of tolerance with S-antigen. J Immunol 1990; 144(5):1689-95. 146. Gregerson DS, Obritsch WF, Donoso LA. Oral tolerance in experimental autoimmune uveoretinitis. Distinct mechanisms of resistance are induced by low dose vs high dose feeding protocols. J Immunol 1993; 151(10):5751-61. 147. Rizzo LV, Miller-Rivero NE, Chan C-C, Wiggert B, Nussenblatt RB, Caspi RR. Effect of Interleukin-2 on the induction of oral tolerance in experimental autoimmune uveoretinitis. In: Nussenblatt RB, Whitcup SW, Caspi RR, Gery I, eds. Advances in Ocular Immunology. Proceedings of the 6th Congress on Immunology and Immunopathology of the Eye. Bethesda, MD: Elsevier Science B. V., 1994. 148. Nussenblatt RB, Gery I, Weiner HL et al. Treatment of uveitis by oral administration of retinal antigens: results of a phase I/II randomized masked trial [see comments]. Am J Ophthalmol 1997; 123(5):583-92.

268

Endocrine and Organ Specific Autoimmunity

149. Melamed D, Friedman A. Modification of the immune response by oral tolerance: antigen requirements and interaction with immunogenic stimuli. Cell Immunol 1993; 146(2):412-20. 150. Hancock WW, Sayegh MH, Kwok CA, Weiner HL, Carpenter CB. Oral, but not intravenous, alloantigen prevents accelerated allograft rejection by selective intragraft Th2 cell activation. Transplantation 1993; 55(5):1112-8. 151. Hancock WW, Sayegh MH, Zhang ZJ, Kwok CA, Weiner HL, Carpenter CB. Oral immunization with allogeneic splenocytes inhibits development of accelerated but not acute rejection of cardiac grafts: analysis of intragraft effector mechanisms. Transplant Proc 1992; 24(1):250-1. 152. Chandler C, Passaro E, Jr. Transplant rejection. Mechanisms and treatment. Arch Surg 1993; 128(3):279-83. 153. Friedman A, Weiner HL. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc Natl Acad Sci U S A 1994; 91(14):6688-92. 154. Lider O, Santos LMB, Lee CSY, Higgins PJ, Weiner HL. Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein II. Suppression of disease and in vitro immune responses is mediated by antigen-specific CD8+ T lymphocytes. J Immunol 1989; 142(3):748-752. 155. Vistica BP, Chanaud NPr, Felix N et al. CD8 T cells are not essential for the induction of “low-dose” oral tolerance. Clin Immunol Immunopathol 1996; 78(2):196-202. 156. Chase MW. Inhibition of experimental drug allergy by prior feeding of the sensitizing agent. Proc Soc Exp Biol Med (NY) 1946; 61:257-259. 157. Wells HG, Osborne TB. Studies on the chemistry of anaphylaxis. J Infect Dis 1911; 8:66. 158. 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; 12:809-37. 159. Waldmann TA, Pastan IH, Gansow OA, Junghans RP. The multichain interleukin-2 receptor: A target for immunotherapy. Ann Inter Med 1992; 116(4):148-60. 160. Roberge FG, Lorberboum-Galski H, LeHoang P et al. Selective immunosuppression of activated T cells with the chimeric toxin IL-2-PE-40. Inhibition of experimental autoimmune uveoretinitis. J Immunol 1989; 143(8):3498-502. 161. Higuchi M, Diamantstein T, Osawa H, Caspi RR. Combined anti-interleukin-2 receptor and low-dose cyclosporine therapy in experimental autoimmune uveoretinitis. J Autoimmun 1991; 4(1):113-24. 162. Guex-Crosier Y, Raber J, Chan CC et al. Humanized antibodies against the alpha-chain of the IL-2 receptor and against the beta-chain shared by the IL-2 and IL-15 receptors in a monkey uveitis model of autoimmune diseases. J Immunol 1997; 158(1):452-8.

Index

269

INDEX 21-hydroxylase 45, 47, 49, 50 γδ (gamma-delta) 91

A Acetylcholine receptor 44, 49, 67, 183, 216 Adrenocortical failure 23, 34, 35 Alopecia 20, 23, 31, 33, 34, 41, 53 Ankylosing spondylitis 4, 133 Apoptosis 8, 76, 113-115, 132, 166, 167, 197, 199, 202, 215, 226-230, 247, 250 APS-II (autoimmune polyendocrine syndrome type II) 41, 43-48, 50, 51, 54, 151, 152, 158 Autoantibodies 1, 2, 10-12, 24, 25, 28-31, 33, 35, 44, 45, 47-54, 63, 67, 68, 70-76, 85, 89, 90, 92, 98-100, 104, 105, 108, 115-117, 133, 135, 137-139, 146, 149, 151, 153, 155, 158-162, 164, 169-172, 183-189, 213, 215, 216, 221-223, 225, 227, 228, 230, 234, 236, 237 Autoantigen 2, 11, 24-26, 28-31, 33, 36, 48, 65, 69, 70, 72, 73, 85, 90, 92, 98, 101, 103, 108, 109, 112, 114, 116-118, 155, 157, 160-162, 164, 170, 185, 186, 188-190, 197, 199, 200, 214-216, 222, 249, 250, 254

B BB rat 156, 157, 160, 169, 171

C CD4+ T cell 3-5, 7, 14, 24, 33, 165, 166 CD8+ 3-5, 24, 33, 77, 91, 109, 118, 166, 201, 214, 252, 254, 260

Celiac disease 2, 9, 41, 43, 45-48, 52, 54, 85-92, 152, 157, 158, 160, 169 Central nervous system 63, 69, 195 CTLA-4 100, 101, 112 Cytokine 24, 53, 76, 91, 103, 109, 112, 113, 164-166, 168, 169, 190, 195, 200-203, 214-216, 224, 225, 252, 256, 258-260

D Demyelination 195, 197, 199, 201, 202, 231, 234, 237, 259 Dermatomyositis 64, 72 Diabetes mellitus 2, 4, 8, 10-14, 19, 23, 28, 30, 41, 43, 45-54, 70, 76, 86, 87, 101, 117, 118, 149-172, 189, 203, 204, 213, 214, 221, 223-227, 230, 231, 233-238 DQ 3, 4, 29, 34, 42, 45-48, 50, 54, 70, 86-89, 91, 92, 100, 137-139, 146, 153-159, 168, 171, 186-188, 196, 254 DR 3-6, 29, 34, 45-48, 50, 54, 69, 70, 86, 87, 91, 100, 101, 133, 137-139, 145, 146, 153-159, 186-188, 196, 199, 203, 222, 223, 236, 254 DRB1*0406 138, 139, 145, 146

E EAMG 183, 186 Ectodermal dystrophy 19, 22, 34 Epidemiology 195, 233, 235, 237 Experimental autoimmune uveitis (EAU) 250-252, 254, 256-260

F Fas L (Fas ligand) 8, 9, 166, 202, 226, 228, 230, 250

270

Endocrine and Organ Specific Autoimmunity

G

101, 150, 151, 153, 156, 157, 159, 186, 221-230 Immunogenetic 222, 233, 234, 236, 254 Immunoregulation 24, 201 Immunotherapy 117, 118, 202, 203, 204, 234, 237, 238, 239, 256, 257 Insulin 1, 2, 7, 9, 11, 12, 14, 28, 29, 45, 48, 51-53, 70, 86, 133, 135, 137-139, 145, 146, 149-151, 157, 159-166, 168, 170-172, 214, 223, 225, 226, 235, 236 Insulin autoimmune syndrome 9, 133, 146 Insulin receptor 52, 135, 149 Integrin 29, 203, 215 Interleukin 7, 48, 90, 91, 165, 214 Intestinal malabsorption 23

GAD65 (glutamic acid decarboxylase) 26, 28, 29, 33, 35, 51, 52, 70, 160, 164, 222, 236, 238 Ganglioside 161, 222, 223, 236, 238 Gene 2-6, 9, 11, 12, 21-25, 28, 30, 31, 33, 34, 36, 41, 45-50, 53, 54, 64, 65, 67-79, 86-88, 90-92, 99-101, 103, 105, 107-111, 113-116, 118, 137-139, 145, 149, 151-154, 156-160, 162-170, 172, 183, 185-190, 195-197, 199-202, 204, 213, 215, 216, 221-227, 230, 231, 233-239, 249-251, 254, 256, 258, 260, 261 Gliadin 2, 9, 47, 52, 85, 86, 88-92, 169 Gluten 43, 48, 85, 86, 88-92 Graves’ disease 10, 19, 41, 43, 44, 47-49, 87, 97, 99-101, 103-114, 116-118, 134, 135, 138, 146, 152, 158

H Haplotype 23, 45-47, 70, 87, 154, 155, 157, 158, 186, 187, 196, 254, 256 Hepatitis 12, 21, 23, 30, 36, 54, 135 Hirata Disease 133 HLA 3, 4, 28, 29, 33, 34, 45-48, 50, 54, 70, 86-89, 100, 101, 109, 110, 112, 114, 133, 135, 137-139, 145, 146, 151, 153-159, 164, 186-189, 196, 222, 235, 236, 239, 254 HLA-DR4 100, 133, 137, 146 Humoral autoimmunity 28, 49, 52, 67, 74, 160, Hypoglycemia 42, 52, 133-135, 146, 172 Hypoparathyroidism 19, 22, 23, 25, 33, 34, 41, 45, 50, 52, 53, 158 Hypothyroidism 10, 12, 19, 29, 41, 45, 48, 49, 52, 97, 98, 106, 116, 158

I ICA512 (IA-2) 52, 70, 160-162, 171 IDDM 19, 28, 29, 31, 33-36, 50, 70, 71,

K Kearns-Sayre syndrome 53 Keratopathy 22, 23, 34

L Lymphoma 64, 72, 86

M Magnetic resonance imaging 195, 196 Methimazole 103, 117, 135, 138 MHC (major histocompatibility complex) 3-6, 34, 45, 46, 67, 71, 73-77, 79, 86, 87, 92, 101, 103, 107, 110, 112, 113, 115, 153, 155-157, 185-187, 190, 195, 200, 213, 214, 216, 222, 251, 252, 254, 256 MODY 149, 151 Molecular mimicry 33, 113, 114, 160, 200, 215 Monoclonal autoantibodies 137 Multiple sclerosis 4, 115, 117, 118, 159, 168, 195, 199, 200, 202-204, 213 Myasthenia gravis 9, 10, 41, 44, 47-50, 53, 64, 65, 67, 68, 74-77, 117, 152, 183, 186, 213, 215

Index

Myelin 195, 197, 199-204, 213, 215, 216, 224, 225, 231, 234-237, 259

N Neuropathy 45, 53, 64, 71, 75, 149, 213-216, 221-227, 229-231, 233, 234-239 NK1.1 T lymphocyte 9 NOD mouse 8, 11, 14, 155-157, 160, 163, 164, 166-169, 171 Non-MHC gene 157, 254, 256

O Ophthalmopathy 43, 49, 97, 99 Opsoclonus-myoclonus syndrome 71 Oral tolerance 117, 203, 259, 260

P Paraneoplastic autoimmune disorder 65, 69, 72 Paraneoplastic encephalomyelitis 64, 71 Pemphigus 64, 72, 133 Penicillamine 9, 47, 48, 135, 187 POEMS syndrome 53

R Retina 53, 249-252, 254, 256, 259, 260 Retinopathy 64, 69, 74, 76, 149, 249 Rubella 48, 159, 160

S

271

110, 111, 115, 118, 145, 153, 160, 163, 164, 167, 168, 170, 196, 199, 200, 201, 203, 214, 249 Th1 7, 8, 24, 99, 109, 165, 166, 200, 201, 256, 259 Thymoma 9, 10, 44, 47, 53, 64, 65, 67, 68, 72, 74, 75, 183, 187, 189 Thymus 3, 4, 6-8, 44, 73, 157, 183, 186, 190, 199, 250 Thyroglobulin 29, 48, 49, 98, 101, 102, 108, 116 Thyroid peroxidase 29, 33, 49, 98, 101-103, 108 Thyroid stimulating hormone 49 Thyroiditis 43, 45, 47-49, 53, 97, 99, 101, 103, 104, 106-108, 112, 113, 115-118, 152, 160, 226, 236 Titin 53, 67, 189 TNF (tumor necrosis factor) 7, 8, 24, 53, 87, 91, 109, 165, 187, 196, 200-203, 214, 224-226, 228, 230 Tolerance 6-8, 28, 29, 48, 51, 73, 90, 101, 113, 116, 117, 135, 149, 151, 165, 166, 168, 190, 203, 214, 216, 251, 258-260 Transglutaminase 2, 48, 52, 85, 89, 90, 92 TSH receptor 49, 101, 102, 109, 111, 114 Twins 12, 86, 88, 99, 100, 152, 153, 159, 185, 196 Type 1 diabetes 11, 12, 41, 45-52, 54, 87, 149-152, 157-160, 162, 164, 172, 223, 235, 236

S-Ag 250-252, 254, 258-260 Sensory neuropathy 64, 71, 75, 234 Stiff-Man syndrome 53, 161, 223

U

T

Uveitis 117, 203, 249-252, 254, 257-261

T cell 1-10, 12, 14, 24, 33, 47-49, 52, 67, 68, 71-77, 79, 86, 88-91, 99, 101, 109-118, 135, 138, 145, 146, 153, 155, 156, 160, 163-170, 188-190, 195-197, 199-203, 213-216, 223, 226, 237, 249-252, 254, 258-260 T cell receptor 1, 2, 5-7, 9, 14, 48, 88, 91,

V Vasculitis 21, 216, 234, 237, 239 Virus 1, 48, 74, 75, 90, 107, 112, 114, 118, 159, 160, 195-197

Y Yo antigen 74